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WO2008028250A1 - Therapeutic agents, targets and diagnostics - Google Patents

Therapeutic agents, targets and diagnostics Download PDF

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Publication number
WO2008028250A1
WO2008028250A1 PCT/AU2007/001328 AU2007001328W WO2008028250A1 WO 2008028250 A1 WO2008028250 A1 WO 2008028250A1 AU 2007001328 W AU2007001328 W AU 2007001328W WO 2008028250 A1 WO2008028250 A1 WO 2008028250A1
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WO
WIPO (PCT)
Prior art keywords
cxs
seq
nos
diabetes
nucleic acid
Prior art date
Application number
PCT/AU2007/001328
Other languages
French (fr)
Inventor
Gregory Royce Collier
Kenneth Russell Walder
David Harry Segal
Kiymet Bozaoglu
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Autogen Research Pty Ltd
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Publication of WO2008028250A1 publication Critical patent/WO2008028250A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the field of therapeutic agents and targets. More particularly, the present invention is directed to the identification ' of molecules or antagonists or agonists thereof for use in medical treatment and/or diagnostic protocols. The present invention further contemplates a screening protocol for potential therapeutic targets.
  • Diabetes represents a significant and debilitating disease.
  • the incidence of diabetes is increasing rapidly. It has been estimated that there were about 700,000 persons with diabetes in Australia in 1995 while in the US, the prevalence of diabetes increased from 4.9% in 1990 to 6.9% in 1999 (Mokdad Diabetes Care 24(2):A ⁇ 2, 2001).
  • Type I diabetes There are two main types of diabetes referred to as Type I and Type II diabetes.
  • Type I diabetes also known as insulin-dependent diabetes mellitus (IDDM)
  • IDDM insulin-dependent diabetes mellitus
  • Type II diabetes occurs later in life and is sometimes known as late-onset diabetes or non- insulin-dependent diabetes mellitus (NIDDM), because insulin treatment is not always needed.
  • Type II diabetes develops when the body becomes resistant to insulin. This happens when the body's tissues, such as muscle, do not respond fully to the actions of insulin, so cannot make use of glucose in the blood. The pancreas responds by producing more insulin.
  • the liver where glucose is stored, releases more glucose to try to increase the amount of glucose available. Eventually, the pancreas becomes less able to produce enough insulin and the tissues become more resistant to insulin. As a result, blood glucose levels slowly start to rise.
  • Mitochondrial dysfunction refers to any illness resulting from a deficiency of any mitochondrial-located protein which is involved in energy metabolism. Therefore, deficiencies of the respiratory (electron transport) chain, either resulting from a deficiency in one or more of the mitochondrial or nuclear-encoded proteins, are mitochondrial disorders. Also, by definition, disorders of the fatty acid (beta) oxidation, Krebs cycle and pyruvate dehydrogenase complex deficiency are mitochondrial disorders. Although these disorders may be genetically dissimilar, mitochondrial dysfunction results in an energy deficient state.
  • Mitochondrial diseases should be considered in the differential diagnosis when there are unexplained features, especially when these occur in combination. Mitochondrial disease and disorders can affect multiple organs, resulting in a vast array of symptoms. Symptoms which may affect the brain include, developmental delays, mental retardation, dementia, seizures, neuro-psychiatric disturbances, atypical cerebral palsy, migraines, strokes.
  • Cancer is also one of the most debilitating disease conditions affecting predominantly humans but also a range of animals.
  • the health cost to the world-wide community runs into the billions of dollars, let alone the personal cost to families.
  • Diabetes, mitochondrial disease and cancer are significant conditions requiring expenditure of time and financial resources to develop new methods of treatment, prevention and diagnosis.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NO: correspond numerically to the sequence identifiers ⁇ 400>l (SEQ ID NO:1), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO:1 sequence identifiers ⁇ 400>l
  • SEQ ID NO:2 sequence identifiers identifiers ⁇ 400>2
  • Table 1 A sequence listing is provided after the claims.
  • the subject genes or their expression products are useful as therapeutic or diagnostic agents for conditions such as Type I diabetes, Type II diabetes or other metabolic disorders, abnormal blood pressure, abnormal triglyceride levels, obesity, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance, apoptosis, signal transduction and/or nuclear targeting.
  • the present invention provides, in part, isolated nucleic acid molecules and amino acid sequences which are associated with Type I diabetes, Type II diabetes, abnormal blood presume, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, generic disorders, energy imbalance and/or obesity, as well as in the modulation of apoptosis, signal transduction and/or nuclear targeting.
  • Isolated nucleic acid molecules contemplated by the present invention include the P. obesus sequence designated as CXS-746, which is disclosed in SEQ ID NO:1, and its cognate receptor designated herein as CXS-746R, which is disclosed in SEQ ID NO: 15. Further contemplated are the corresponding human equivalents including the human nucleic acid sequence for CXS-746 disclosed in SEQ ID NO: 18, which encodes the human CXS-746 amino acid sequence disclosed in SEQ ID NO:20. The human CXS-746R nucleic acid and amino acid sequences are disclosed in SEQ ID NOs: 19 and 20, respectively.
  • the present invention contemplates the use of these sequences or mammalian equivalents, including human homologs thereof, or their expression products, in the manufacture of medicaments and diagnostic agents for a range of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity as well as to modulate or detect apoptosis, signal transduction and/or for nuclear targeting.
  • metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity as well as to modulate or detect apoptosis, signal transduction and/or for nuclear targeting.
  • the present invention also provides, therefore, a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof which is secreted or is a cell surface molecule and involved in metabolic signalling in a cell or group of cells.
  • the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NO: 15 or 19 (CXS- 746R) or a nucleotide sequence having at least about 90% similarity to all or part of SEQ ID NO:1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) and/or is capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a complementary form thereof under high stringency conditions.
  • the present invention also provides an isolated expression product or a derivative or homolog thereof which expression product is encoded by a nucleotide sequence the expression product identified as being a secreted or cell surface molecule and involved in metabolic signalling in a cell or group of cells.
  • the present invention is also directed to an isolated expression product or a derivative or homolog thereof wherein the expression product is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ ID NO: 18 (CXS-746) or SEQ ID NO: 15 or SEQ ID NO: 19 (CXS-746R) or a nucleotide sequence having at least 90% similarity to all or part of SEQ ID NO:1 or SEQ ID NO: 18 (CXS-746) or SEQ ID NO: 15 or SEQ ID NO: 19 (CXS-746R) and/or is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO: 18 (CXS-746) or SEQ ID NO: 15 or SEQ ID NO: 19 (CXS-746R) or a complementary form thereof under high stringency conditions.
  • the present invention extends to analogs and mimetics of the subject expression products.
  • homolog includes other mammalian homologs such as from a human.
  • the genetic molecules of the present invention are referred to herein as CXS-746 (SEQ ID NO:1 or 18) or CXS-746R (SEQ ID NO:15 or 19).
  • the expression product may be an RNA (e.g. mRNA) or a protein. Where the expression product is an RNA, the present invention extends to RNA-related molecules associated or related thereto such as RNAi or intron or exon sequences therefrom or short, interfering RNA (si-RNA) or complexes comprising same or hairpin forms thereof or short or long double stranded RNA molecules.
  • compositions comprising CXS- 746 or CXS-746R or its derivatives or homologs or agonists or antagonists of CXS-746 or
  • the present invention is directed to molecules which antagonise CXS- 746 or CXS-746R or enhance the activity of CXS-746 or CXS-746R, or enhance the ability of CXS-746 to bind to its cognate receptor CXS-746R, or which bind to the cognate receptor of CXS-746, thereby mimicking the activity of CXS-746.
  • the present invention is particularly directed to human homologs and orthologs of the genes identified in P. obesus and their use or the use of expression products thereof or homologs, derivatives, analogs or mimetics thereof in therapy and diagnosis.
  • An example of a human CXS-746 mRNA sequence is disclosed in SEQ ID NO: 18, with a corresponding protein sequence disclosed in SEQ ID NO:20.
  • a human mRNA sequence specific for a CXS-746 receptor is disclosed in SEQ ID NO: 19, with an encoded expression product disclosed in SEQ ID NO:21.
  • Another aspect of the present invention contemplates, therefore, a method for treating a subject comprising administering to the subject a treatment effective amount of CXS-746 or CXS-746R or a derivative or homolog thereof or a genetic sequence encoding same or an analog, mimetic, agonist or antagonist of CXS-746 or CXS-746R activity or of CXS- 746 or CXS-746R gene expression for a time and under conditions sufficient to effect treatment.
  • treatments contemplated herein include but are not limited to treatment of metabolic disorders such as diabetes (Types I or II), abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathies, genetic disorders, cancers, energy imbalance and/or obesity as well as modulating apoptosis, signal transduction and/or nuclear targeting.
  • Treatment may be by the administration of a pharmaceutical composition or genetic sequences via gene therapy, antisense therapy or sense or RNAi- or si-RNA-mediated therapy. Treatment is contemplated for human subjects as well as animals such as animals important to livestock industry.
  • expressed sequences are identified as biomarkers CXS-746 and CXS- 746R biomarkers.
  • Nucleotide sequences corresponding to CXS-746 and CXS-746R from P.obesus are disclosed in SEQ ID NOs: 1 and 15, respectively.
  • Human CXS-746 mRNA and amino acid sequences are disclosed in SEQ ID NOs: 18 and 20, respectively, with the human CXS-746R mRNA and amino acid sequences disclosed in SEQ ID NOs: 19 and 21, respectively.
  • the present invention contemplates the use of these sequences in the manufacture of diagnostic agents for metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • the present invention contemplates a method for the diagnosis of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a subject, the method comprising screening for levels of a biomarker including CXS-746 or/and CXS-746R in a biological sample from the subject wherein an altered level of CXS- 746 or CXS-746R compared to a control is indicative of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • a further aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions, the diagnostic agent selected from (a) an antibody to CXS- 746 and/or CXS-746R or their derivatives, homologs, analogs or mimetics; (b) a genetic sequence capable of annealing to a nucleotide strand associated with CXS-746 and/or CXS-746R useful inter alia in PCR, RT-PCR, hybridisation, RFLP analysis and AFLP analysis.
  • metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions
  • the diagnostic agent selected from (a) an antibody to CXS- 746
  • a further aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, genetic disorders, cancers, energy imbalance and/or obesity as well as monitoring apoptosis, signal transduction and/or nuclear targeting, the diagnostic agent selected from an antibody to CXS-746 or CXS-746R or its derivatives, homologs, analogs or mimetics and a genetic sequence comprising or capable of annealing to a nucleotide strand associated with CXS- 746 or CXS-746R useful inter alia in PCR, hybridization, RFLP analysis or AFLP analysis.
  • Figure 1 is a graphical representation of the relative gene expression of chemerin in fractionated mesenteric adipose tissue.
  • Figure 2 is a graphical representation of the association of plasma chemerin levels with BMI and systolic blood pressure. Chemerin levels in human plasma samples from NGT subjects were measured by ELISA. Scatter plots are representative of a) chemerin levels versus BMI and b) chemerin levels versus systolic blood pressure.
  • the subject invention is not limited to specific therapeutic components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
  • a gene includes a single gene or two or more genes
  • an agent includes a single agent, as well as two or more agents
  • reference to “the invention” includes single or multiple aspects of the invention; as well as two or more genes; and so forth.
  • agent should be understood as a reference to any proteinaceous or non-proteinaceous molecule derived from natural, recombinant or synthetic sources.
  • Useful sources include the screening of naturally produced libraries, chemical molecule libraries as well as combinatorial libraries, phage display libraries and in vitro translation- based libraries.
  • Particularly useful agents are those identified by the Signal Sequence Trap (SST) method.
  • the agents may, however, be any proteinaceous molecules such as peptides, polypeptides and proteins or non-proteinaceous molecules such as nucleic acid molecules and small to large natural or synthetically derived organic and inorganic molecules and include antagonists and agonists of the proteins identified by the SST method.
  • the agents may, therefore, also be immunoglobulins such as antibodies or fragments or synthetic or modified forms thereof.
  • An "immunoglobulin” includes an immunoglobulin new antigen receptor (IgNAR) from cartaligenes fish such as sharks (See WO2005/118629)
  • agent means "agent”, “compound”, “active agent”, “pharmacologically active agent”, and
  • “medicament”, “active” and “drug” may be used interchangeably herein to refer to any agent that induces a desired pharmacological and/or physiological effect. Such effects include action on Type I or II diabetes or other metabolic disorders, obesity, mitochondrial dysfunction, myopathies, genetic disorders, energy imbalance, apoptosis, signal transduction and/or nuclear targeting.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • the present invention is predicated in part on the identification of genes associated inter alia with a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, a myopathy, a genetic disorder or a cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, a myopathy, a genetic disorder or a cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, or fragment or homolog or portion thereof wherein the nucleic acid molecule is associated with one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting and is identified as being a secreted or cell surface molecule and involved in metabolic signalling in a cell or group of cells.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting and is identified as being a secreted or cell surface molecule and involved in metabolic signalling in a cell or group of cells.
  • the expression product may be a peptide, polypeptide or protein or mRNA or may be an exon or intron spliced, for example, from an RNA construct.
  • the expression product may also be a hairpin structure which induces or is associated with RNAi.
  • the present invention further provides a nucleic acid molecule composition. Therefore, the present invention provides compositions which comprise a nucleic acid molecule as disclosed in SEQ ID NO:1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R).
  • CXS-746 SEQ ID NO:20
  • CXS-746R SEQ ID NO:21
  • Another aspect of the present invention provides a method for diagnosing a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting by determining the level of expression of a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NO:1 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 19 or a nucleotide sequence having at least about 90% similarity to all or part of SEQ ID NOs: 1 or 15 or 18 or 19 and/or is capable of hybridizing to SEQ ID NOs: 1 or 15 or 18 or 19 or their complementary forms under high stringency conditions at a specified temperature and wherein elevated or reduced levels of expression of one or more of these sequences is indicative of one or more
  • the present invention also provides a method for assessing the presence or absence of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions by determining the level of expression of a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NO:1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% similarity to all or part of SEQ ID NOs: 1 or 18 or SEQ ID NOs: 15 or 19 after alignment and/or is capable of hybridizing to one or more of SEQ ID NOs: 1 or 18 or SEQ ID NOs: 15 or 19 or their complementary forms under high stringency conditions at a specified temperature and
  • Reference herein to similarity is generally at a level of comparison of at least 15 consecutive or substantially consecutive nucleotides such as at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
  • Preferred percentage similarities have at least about 70%, at least about 80%, at least about 90% or above.
  • Examples include 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level,
  • similarity includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
  • references to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”.
  • a “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length, examples include 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25. Because two polynucleotides may each comprise (1) a sequence (i.e.
  • sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity.
  • a “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence.
  • the comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
  • GAP Garnier et al.
  • a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42°C.
  • the temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% and from at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 and 0.9 M for hybridization, and at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 and 0.9 M for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.
  • T m 69.3 + 0.41 (G+C)% (Marmur and Doty J MoI Biol 5: 109, 1962).
  • T m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey Eur J Biochem 46:%3, 1974).
  • Formamide is optional in these hybridization conditions.
  • particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42 0 C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • nucleic acid molecule comprising a sequence of nucleotides the nucleic acid molecule being differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the nucleic acid molecule is a nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% identity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NO: 15 or 19 (CXS- 746R) or its complementary form under high stringency conditions.
  • a further aspect of the present invention provides a nucleic acid molecule or derivative or homolog thereof associated with one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting the nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein the nucleotide sequence is substantially as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a derivative or homolog thereof or having at least about 90% identity to all or part of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R).
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal t
  • the present invention provides a method for the diagnosis of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a subject or the probability of a subject developing metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity, the method comprising screening for levels of CXS-746 protein (SEQ ID NO:20) or mRNA/cDNA (SEQ ID NOs:l and 18) and/or CXS-746R protein (SEQ ID NO:21) or mRNA/cDNA (SEQ ID NOs: 15 and 19) the protein or mRNA/cDNA encoding same or a homolog thereof in a biological sample from the subject, wherein an altered level of CXS- 746 or CXS-746R compared to a normal control is indicative of one
  • CXS-746 or CXS-746R is useful in the treatment, prognosis or diagnosis of abnormal blood pressure.
  • elevated levels of CXS-746 or CXS-746R are indicative of a subject having high blood pressure or a predisposition of developing high blood pressure.
  • decreasing the amount of, or activity of, CXS-746 or CXS- 746R aids in decreasing high blood pressure or diminishing the probability of a subject with a predisposition of having high blood pressure from developing high blood pressure.
  • Decreasing the levels of or activity of CXS-746 or CXS-746R may be by, for example, preventing the expression of CXS-746 or CXS-746R, blocking the activity of CXS-746 or CXS-746R, administering non-functional mimetics of CXS-746 which competitively bind to the CXS-746R or which otherwise antagonize the binding of CXS-746 to CXS-746R, or by any other means whereby the activity or expression of CXS-746 or CXS-746R is diminished.
  • expression include transcriptional, translational and/or post- translational elements of expression of a gene to generate a protein.
  • CXS-746 or CXS-746R are indicative of a subject having low blood pressure or a predisposition for developing low blood pressure. Accordingly, increasing the amount of or activity of CXS-746 or CXS-746R aids in elevating a subject's blood pressure and/or decreasing the probability of a subject with a predisposition of having low blood pressure from developing low blood pressure.
  • Increasing the levels of or activity of CXS-746 or CXS-746R may be by, for example, adding exogenous levels of CXS-746 or CXS-746R or homologs thereof which have similar activity. Alternatively, the expression or level of the CXS-746R could be increased.
  • Other means of increasing the levels or activity of CXS-746 would be known by one of skill in the art and include antagonizing inhibitors of CXS-746 expression or agonizing promoters of CXS-746R expression.
  • CXS-746 or CXS-746R compositions and molecules of the present invention can also be used in combination therapy.
  • antagonists of CXS-746 or CXS-746R could be used in combination with high blood pressure therapies.
  • antagonists of CXS-746 or CXS-746R can be combined with one or more of Angiotensin converting enzyme (ACE) inhibitors: captopril, enalapril, lisinopril, fosinopril, quinapril, ramipril, benazepril, perindopril, trandolapril, moexipril;
  • Angiotensin II receptor antagonists irbesartan, losartan, valsartan, candesartan;
  • Alpha blockers doxazosin, terazosin, prazosin;
  • Beta blockers atenolol, labetalol, metoprolol, propanolol, acebutolo
  • agonists of CXS-746 or CXS-746R could be used in combination with compounds known to increase blood pressure levels.
  • agonists of CXS-746 or CXS-746R can be combined with one or more of Systemic vasoconstrictors: Antihistamines, Adrenaline, Asymmetric dimethylarginine, Adenosine triphosphate, Catecholamines, Cocaine, Decongestants, Endothelin, Ergine, Phenylephrine, Pseudoephedrine, Neuropeptide Y, Norepinephrine, Tetrahydrozoline hydrochloride, Thromboxane, Fludrocortisone and Midodrine.
  • Systemic vasoconstrictors Antihistamines, Adrenaline, Asymmetric dimethylarginine, Adenosine triphosphate, Catecholamines, Cocaine, Decongestants, Endothelin, Ergine, Phenylephrine, Pseu
  • a "control” is defined as a subject having one or more of the following characteristics: lean, young, no genetic predisposition for Type II diabetes (i.e. no family history of Type II diabetes abnormal blood pressure or abnormal triglyceride levels), normoglycemic and/or normoinsulinemic.
  • abnormal blood pressure is defined as blood pressure which is either below or above normal ranges based on a subjects sex and age.
  • abnormal triglyceride levels is defined as triglyceride levels which are either below or above normal ranges based on a subjects sex and age.
  • nucleic acid segment that is complementary to the full length of the mRNA encoding CXS-746 or CXS-746R, or one may use a smaller segment that is complementary to a portion of the RNA.
  • Such smaller segments may be from about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 25, about 30, about 50, about 75, about 100 or even several hundred bases in length and may be contained in larger segments that provide other functions such as promoters, restriction enzyme recognition sites, or other expression or message processing or replication functions.
  • probes are designed to selectively hybridize to a CXS-746 or CXS- 746R or product thereof.
  • a product thereof would include a DNA or RNA strand that is complementary to the mRNA and thus a useful probe would include both the sense and antisense orientations of a particular sequence. Also preferred are the use of probes or primers that are designed to selectively hybridize to a nucleic acid segment having a sequence of SEQ ID NO:1 or SEQ ID NO:15 or SEQ ID NO:18 or SEQ ID NO:19 or the complements thereof.
  • the methods of the present invention may also include determining the amount of hybridized product. Such determination may be by direct detection of a labeled hybridized probe, such as by use of a radioactive, fluorescent or other tag on the probe, or it may be by use of an amplification of a target sequence, and quantification of the amplified product.
  • a preferred method of amplification is a reverse transcriptase polymerase chain reaction (RT-PCR) as described herein. RT-PCR is a preferred method of detection, diagnosis, and/or prognosis of disease or cancer.
  • amplification may comprise contacting the target ribonucleic acids with a pair of amplification primers designed to amplify CXS-746 or CXS-746R mRNA, or even contacting the ribonucleic acids with a pair of amplification primers designed to amplify a nucleic acid segment comprising the nucleic acid sequence or complement of SEQ ID NOs: 1 or 15 or 18 or 19.
  • Diagnostic methods may be based upon the steps of obtaining a biological sample from a subject or patient, contacting the sample nucleic acids from the biological sample with an isolated CXS-746 or CXS-746R nucleic acid segment under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting, and optionally further characterizing, the hybridized complementary nucleic acids thus formed.
  • the methods may involve in situ detection of sample nucleic acids located within the cells of the sample.
  • the sample nucleic acids may also be separated from the cell prior to contact.
  • the sample nucleic acids may be DNA or RNA.
  • the methods may involve the use of isolated CXS-746 or CXS-746R nucleic acid segments that comprises a radio-, enzymatic- or fluorescent-ly detectable label, wherein the hybridized complementary nucleic acids are detected by detecting the label.
  • probes are designed to selectively hybridize to CXS-746 or CXS-746R mRNA or product thereof.
  • a product thereof would include a DNA or RNA strand that is complementary to the mRNA and thus a useful probe would include both the sense and antisense orientations of a particular sequence.
  • probes or primers that are designed to selectively hybridize to a nucleic acid segment having a sequence of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18 or SEQ ID NO:19 or the complements thereof or fragments thereof.
  • some methods may involve detection of expression of a polypeptide product and particularly the expression product encoded by SEQ ID NO:1 or 15 or 18 or 19.
  • detection may be by any means known in the art and may include an immunoassay, an immunoaffinity purification or detection, an ELISA, or an radioimmunoassay, for example.
  • the expression pattern of CXS-746 and/or CXS-746R has been determined, inter alia, to indicate an involvement in the regulation of one or more processes associated with one or more metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • the subject nucleic acid molecules are preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence.
  • a genomic sequence may also comprise exons and introns.
  • a genomic sequence may also include a promoter region or other regulatory regions.
  • the present invention extends, however, to expression products such as mRNA, introns and exons which may also be involved in genetic networking, whether or not they are translated into proteins.
  • the expression products may include complexes comprising RNA or may comprising RNAi or RNAi-type molecules.
  • a homolog is considered to be a CXS-746 or CXS-746R gene from another animal species.
  • the present invention extends to the homologous gene, as determined by nucleotide sequence and/or amino acid sequences and/or function, from primates, including humans, marmosets, orangutans and gorillas, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos).
  • livestock animals e.g. cows, sheep, pigs, horses, donkeys
  • laboratory test animals e.g. mice, rats, guinea pigs, hamsters, rabbits
  • companion animals e.g. cats, dogs
  • captured wild animals e.g. rodents, foxe
  • the present invention also contemplates deimmunized forms of the expression products from one species relative to another species.
  • the deimmunized form of the expression product is a mammalianized form relative to a particular target animal.
  • the target mammal is a human
  • the present invention contemplates use of a humanized form of a non-human expression product.
  • CXS-746 or CXS-746R and their derivatives and homologs may be in isolated or purified form and/or may be ligated to a vector such as an expression vector.
  • Expression may be in a eukaryotic cell line (e.g. mammalian, insect or yeast cells) or in microbial cells (e.g. E. coli) or both.
  • isolated is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means.
  • the nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector.
  • the nucleotide sequence corresponding to CXS-746 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:1 or a derivative or homolog thereof including a nucleotide sequence having similarity to SEQ ID NO:1 or an mRNA sequence as set forth in SEQ ID NO: 18.
  • the nucleotide sequence corresponding to CXS-746R is a cDNA sequence set forth in SEQ ID NO: 15 or an mRNA sequence as set forth in SEQ ID NO: 19.
  • the nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells).
  • the nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions.
  • the nucleic acid molecule may also be part of a vector, such as an expression vector.
  • the derivatives of the nucleic acid molecule of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co- suppression (e.g. RNAi) and fusion nucleic acid molecules.
  • Ribozymes and DNA enzymes are also contemplated by the present invention directed to CXS-746 or CXS-746R or its mRNA. Derivatives and homologs of CXS-746 or CXS-746R are conveniently encompassed by those nucleotide sequences capable of hybridizing to SEQ ID NO:1 or 15 or 18 or 19 or their complementary forms under high stringency conditions.
  • the present invention extends to expression products of CXS-746 or CXS-746R.
  • the expression products can be proteins or mutants, derivatives or homologs thereof as well as a range of RNA molecules.
  • An expression product includes an RNA molecule such as a mRNA transcript as well as a protein.
  • Some genes are non-protein encoding genes and produce mRNA or other RNA type molecules and are involved in regulation by RNA:DNA, RNA:RNA or RNA:protein interaction.
  • the RNA e.g. mRNA
  • the RNA may act directly or via the induction of other molecules such as RNAi or via products mediated from splicing events (e.g. exons or introns).
  • Other genes encode mRNA transcripts which are then translated into proteins.
  • a protein includes a polypeptide.
  • the differentially expressed nucleic acid molecules therefore, may encode mRNAs only or, in addition, proteins. Both mRNAs and proteins are forms of "expression products".
  • Derivatives include fragments, parts, portions, mutants and variants from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of CXS-746 or CXS-746R. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence.
  • substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place.
  • An example of substitutional amino acid variants are conservative amino acid substitutions.
  • Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
  • CXS-746 or CXS-746R should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
  • the derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
  • Another aspect of the present invention provides an isolated protein or other expression product or a derivative or homolog thereof which is associated with one or more of diabetes, mitochondrial disease, myopathy, abnormal blood pressure, abnormal triglyceride levels, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • an isolated protein or derivative or homolog fragment thereof wherein the protein or polypeptide comprises an amino acid sequence disclosed in SEQ ID NO:20 (CXS-746) and/or SEQ ID NO:21 (CXS-746R or an amino acid sequence encoded by SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or an amino acid sequence having at least 90% similarity to all or part thereof and wherein the protein or expression product is associated with one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • CXS-746 or CXS-746R includes reference to isolated or purified naturally occurring CXS-746 or CXS-746R protein or expression product molecules as well as any derivatives or homologs thereof.
  • Derivatives include parts, fragments and portions of CXS-746 or CXS-746R as well as single and multiple amino acid substitutions, deletions and/or additions to CXS-746 or CXS-746R.
  • a derivative of CXS-746 or CXS- 746R is conveniently encompassed by molecules encoded by a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 15 or 18 or 19 under high stringency conditions at a specified temperature.
  • a fragment includes a part, portion, region, domain, N-terminal fragment, a C-terminal fragment, an internal fragments and/or an enzymatically cleaved protein, such as by a membrane cleaving protease.
  • CXS-746 or CXS-746R include chemical analogs.
  • Analogs of CXS- 746 or CXS-746R contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confirmational constraints on the proteinaceous molecule or their analogs.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS);
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 2.
  • Non-conventional Code Non-conventional Code amino acid amino acid
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids, introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
  • the expression product may be an RNA or protein.
  • the term "protein” should be understood to encompass peptides, polypeptides and proteins.
  • the protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • Reference hereinafter to a "protein” includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
  • the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NOs: 1 or 15 or 18 or 19 or a derivative or homolog thereof including a nucleotide sequence having at least about 90% identity to SEQ ID NOs: 1 or 15 or 18 or 19.
  • Another aspect of the present invention is directed to an isolated expression product that is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the protein is selected from the list consisting of:
  • SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a derivative or homolog thereof or a sequence encoding an amino acid sequence having at least about 90% similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of the protein;
  • the protein of the present invention is preferably in isolated form.
  • isolated is meant a protein having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject protein, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
  • sequence similarity and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g.
  • sequence identity will be understood to mean the "match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
  • the nucleotide sequence or amino acid sequence of the present invention may correspond to exactly the same sequence of the naturally occurring gene (or corresponding cDNA) or protein or may carry one or more nucleotide or amino acid substitutions, additions and/or deletions.
  • the nucleotide sequences set forth in SEQ ID NOs: 1 or 18 correspond to the genes referred to herein as CXS-746 and SEQ ID NO: 15 and 19 correspond to CXS-746R.
  • the corresponding expression products are CXS-746 (SEQ ID NO:20) and CXS-746R (SEQ ID NO:21).
  • Reference herein to CXS-746 or CXS-746R includes, where appropriate, reference to the genomic gene or cDNA as well as any naturally occurring or induced derivatives.
  • the present invention further encompasses mutants, fragments, parts and portions of the nucleotide sequence corresponding to CXS-746 or CXS-746R.
  • CXS-746 or CXS-746R permits the generation of a range of therapeutic molecules capable of modulating expression of CXS-746 or CXS-746R or modulating the activity of CXS-746 or CXS-746R.
  • Modulators contemplated by the present invention includes agonists and antagonists of CXS-746 or CXS-746R expression.
  • Antagonists of CXS-746 or CXS-746R expression include antisense molecules, ribozymes and co-suppression molecules.
  • Agonists include molecules which increase promoter activity or which interfere with negative regulatory mechanisms.
  • Antagonists of CXS-746 or CXS-746R include antibodies and inhibitor peptide fragments. All such molecules may first need to be modified to enable such molecules to penetrate cell membranes. Alternatively, viral agents may be employed to introduce genetic elements to modulate expression of CXS-746 or CXS-746R.
  • the present invention contemplates, therefore, a method for modulating expression of
  • CXS-746 or CXS-746R in a mammal, the method comprising contacting the CXS-746 or CXS-746R gene with an effective amount of a modulator of CXS-746 or CXS-746R expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate the expression of CXS-746 or CXS-746R.
  • a nucleic acid molecule encoding CXS-746 or CXS-746R or a derivative or homolog thereof may be introduced into a cell to enhance the ability of that cell to produce CXS-746 or CXS-746R, conversely, CXS-746 or CXS-746R antisense sequences such as oligonucleotides may be introduced to decrease the availability of CXS-746 or CXS-746R molecules.
  • Another aspect of the present invention contemplates a method of modulating activity of CXS-746 or CXS-746R in a mammal, the method comprising administering to the mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease CXS-746 or CXS-746R activity.
  • the molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of CXS-746 or CXS-746R or their ligands.
  • Modulating levels of CXS-746 or CXS-746R expression is important in the treatment of a range of conditions including metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the present invention has application in the treatment of humans as well as in the veterinary and animal husbandry industries. Accordingly, subjects contemplated for treatment in accordance with the present invention includes, but is not limited to humans, primates, livestock animals (e.g. pigs, sheep, cows, horses, donkeys), laboratory test animals (e.g.
  • mice mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. dogs, cats) and captured wild animals (e.g. foxes, kangaroos, deer).
  • a particularly preferred host is a human, primate or livestock animal.
  • the present invention contemplates therapeutic and prophylactic uses of CXS-746 or CXS-746R amino acid and nucleic acid molecules in addition to CXS-746 or CXS-746R agonistic and antagonistic agents.
  • the present invention contemplates, therefore, a method of modulating expression of CXS- 746 or CXS-746R in a mammal, the method comprising contacting the CXS-746 or CXS- 746R genes with an effective amount of an agent for a time and under conditions sufficient to up-regulate, down-regulate or otherwise modulate expression of CXS-746 or CXS- 746R.
  • an agent for a time and under conditions sufficient to up-regulate, down-regulate or otherwise modulate expression of CXS-746 or CXS- 746R.
  • antisense sequences such as oligonucleotides may be utilized.
  • nucleic acid molecules encoding CXS-746 or CXS-746R or derivatives thereof may be introduced to up-regulate one or more specific functional activities.
  • Another aspect of the present invention contemplates a method of modulating activity of CXS-746 or CXS-746R in a subject, the method comprising administering to the subject a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease CXS-746 or CXS-746R activity.
  • Modulation of activity by the administration of an agent to a mammal can be achieved by one of several techniques, including but in no way limited to introducing into the mammal a proteinaceous or non-proteinaceous molecule which:
  • nucleic acid molecule comprising a sequence of nucleotides the nucleic acid molecule which has is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the isolated molecule is encoded by a nucleic acid molecule selected from the list consisting of a nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% identity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS -746R) or its complementary form under high stringency conditions in the manufacture of a medicament for the treatment of one or more diseases and/or conditions.
  • a nucleic acid molecule selected from the list consisting of a nucleic acid molecule comprises a nucleotide sequence
  • the proteinaceous molecule may be derived from natural or recombinant sources including fusion proteins or following, for example, natural product screening or the screening of chemical libraries.
  • the non-proteinaceous molecule may be, for example, a nucleic acid molecule or may be derived from natural sources, such as for example natural product screening or may be chemically synthesized.
  • the present invention contemplates chemical analogs of CXS-746 or CXS-746R or small molecules capable of acting as agonists or antagonists.
  • Chemical agonists may not necessarily be derived from CXS-746 or CXS- 746R but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties.
  • Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing CXS-746 or CXS-746R from carrying out their normal biological functions.
  • Antagonists include monoclonal antibodies, antisense and sense nucleic acids which prevent transcription or translation of CXS-746 or CXS-746R genes or mRNA in mammalian cells. Modulation of expression may also be achieved utilizing antigens, RNA, RNAi, ribosomes, DNAzymes, RNA aptamers or antibodies.
  • the proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the expression of CXS-746 or CXS-746R or the activity of CXS-746 or CXS- 746R, the molecule acts directly if it associates with CXS-746 or CXS-746R to modulate expression or activity, the molecule acts indirectly if it associates with a molecule other than CXS-746 or CXS-746R which other molecule either directly or indirectly modulates the expression or activity of CXS-746 or CXS-746R. Accordingly, the method of the present invention encompasses the regulation of CXS-746 or CXS-746R expression or activity via the induction of a cascade of regulatory steps.
  • the molecules which may be administered to a mammal in accordance with the present invention may also be linked to a targeting means such as a monoclonal or polyclonal antibody, which provides specific delivery of these molecules to the target cells.
  • a targeting means such as a monoclonal or polyclonal antibody, which provides specific delivery of these molecules to the target cells.
  • a further aspect of the present invention relates to the use of the invention in relation to mammalian disease conditions.
  • the present invention is particularly useful but in no way limited to use in a therapeutic or prophylactic treatment of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • another aspect of the present invention relates to a method of treating a mammal suffering from a condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, the method comprising administering to the mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of CXS-746 or CXS-746R or sufficient to modulate the activity of CXS-746 or CXS-746R.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting
  • the present invention relates to a method of treating a mammal suffering from a disease condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, the method comprising administering to the mammal an effective amount of CXS-746 or CXS-746R.
  • a disease condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting
  • muscle refers to any abnormal conditions or disease of the muscle tissues, which include the muscles over our bones (skeletal muscle) and the heart (cardiac muscle).
  • Mitochondrial dysfunction relates to abnormalities in mitochondria.
  • Mitochondria are part of the cell (organelle) that is responsible for energy production.
  • the organelle consists of two sets of membranes, a smooth continuous outer coat and an inner membrane arranged in tubules or in folds that form plate-like double membranes (cristae).
  • Mitochondria are the principal energy source of the cell, containing the cytochrome enzymes of terminal electron transport and the enzymes of the citric acid cycle, fatty acid oxidation, and oxidative phosphorylation. They are responsible for converting nutrients into energy as well as many other specialized tasks.
  • Mitochondria are complex organelles located in virtually all cells of the body. A large degree of their complexity is due to the fact that over 1000 proteins are located in the mitochondria. Thirteen of these proteins are encoded by the mitochondrial DNA (mtDNA), while the remainder are nuclear-encoded, and imported into the mitochondria.
  • Symptoms of mitochondrial dysfunction include weakness (which may be intermittent), neuropathic pain, absent reflexes, gastrointestinal problem (gastroesophogeal reflux, delayed gastric emptying, constipation, pseudo-obstruction), fainting, absent or excessive sweating resulting in temperature regulation problems, hypotonia, cramping and muscle pain, proximal renal tubular wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes, cardiac conduction defects (heart blocks) and cardiomyopathy, hypoglycemia (low blood sugar) and liver failure, visual loss and blindness, hearing loss and deafness, and diabetes and exocrine pancreatic failure (inability to make digestive enzymes).
  • mitochondrial dysfunction There may also be systemic problems associated with mitochondrial dysfunction, including failure to gain weight, short stature, fatigue, respiratory problems.
  • Mitochondrial defects have been linked to Alzheimer's, Parkinson's, diabetes, autism, and the aging process.
  • Other disease associated with mitochondrial dysfunction include, LIC
  • LCAD LCHAD, Leigh Disease, LHON, Luft Disease, MAD, MCA, MELAS, MERRP, mitochondrial DNA depletion, Mitochondrial Encephalopathy, MNGIE, NARP, Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency, SCAD, SCHAD and VLCAD.
  • Alpers Disease or Progressive Infantile Poliodystrophy, includes symptoms such as seizures, dementia, spasticity, blindness, liver dysfunction, and cerebral degeneration. (Luft Proceedings of the National Academy of Sciences of the United States of America 91(19):873 ⁇ -8, 1994).
  • Barth syndrome or LIC Lethal Infantile Cardiomyopathy
  • LIC Lethal Infantile Cardiomyopathy
  • Carnitine- Acyl-Carnitine Deficiency is an autosomal recessive disorder, the symptoms of which are seizures, apnea, bradycardia, vomiting, lethargy, coma, enlarged liver, limb weakness, myoglobin in the urine, Reye-like symptoms triggered by fasting.
  • Carnitine Deficiency is an autosomal recessive disease, the symptoms of which include Cardiomyopathy, failure to thrive, and altered consciousness or coma, sometimes hypotonia.
  • Co-Enzyme QlO Deficiency is most likely an autosomal recessive disease, the symptoms of which include Encephalomyopathy, mental retardation, exercise intolerance, ragged-red fibers, and recurrent myoglobin in the urine.
  • NADH-CoQ reductase deficiency is an autosomal disease, the symptoms of which are classified by three major forms: (1) fatal infantile multisystem disorder, characterized by developmental delay, muscle weakness, heart disease, congenital lactic acidosis, and respiratory failure; (2) myopathy beginning in childhood or in adult life, manifesting as exercise intolerance or weakness. Elevated lactic acid common; and (3) mitochondrial encephalomyopathy (including MELAS), which may begin in childhood or adult life and consists of variable combinations of symptoms and signs, including ophthalmoplegia, seizures, dementia, ataxia, hearing loss, pigmentary retinopathy, sensory neuropathy, and uncontrollable movements. In addition, this disorder may cause Leigh Syndrome.
  • encephalomyopathy which is typically normal for the first 6 to 12 months of life and then show developmental regression, ataxia, lactic acidosis, optic atrophy, ophthalmoplegia, nystagmus, dystonia, pyramidal signs, respiratory problems and frequent seizures; and
  • myopathy Two main variants: (a) Fatal infantile myopathy: may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged-red fibers, respiratory failure, and kidney problems: and (b) Benign infantile myopathy: may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged- red fibers, respiratory problems, but (if the child survives) followed by spontaneous improvement.
  • Complex V Deficiency or ATP synthase deficiency includes symptoms such as slow, progressive myopathy.
  • CPEO or Chronic Progressive External Ophthalmoplegia Syndrome includes symptoms such as visual myopathy, retinitis pigmentosa, dysfunction of the central nervous system. It is caused by single mitochondrial DNA deletions, with Mitochondrial DNA point mutation, A3243G being the most common (Luft 1994 Supra).
  • CPT I Deficiency is an autosomal recessive disease and includes symptoms such as enlarged liver and recurrent Reye-like episodes triggered by fasting or illnesses.
  • CPT II Deficiency is an autosomal recessive disease, the symptoms of which include exercise intolerance, fasting intolerance, muscle pain, muscle stiffness, and myoglobin in the urine and in infants, Reye-like syndrome, enlarged liver, hypoglycemia, enlarged heart and cardiac arrhythmia.
  • KSS KSS or Kearns-Sayre Syndrome
  • Symptoms associated with KSS include progressive external ophthalmoplegia, pigmentary retinopathy, heart block, and high cerebrospinal protein.
  • Lactic Acidosis is associated with the accumulation of lactic acid due to its production exceeding its use. Chronic lactic acidosis is a common symptom of mitochondrial disease.
  • LCAD or Long-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, which causes a fatal syndrome, in infants, typified by failure to thrive, enlarged liver, enlarged heart, metabolic encephalopathy and hypotonia.
  • LCHAD is an autosomal recessive disorder, characterized by symptoms such as encephalopathy, liver dysfunction, cardiomyopathy, and myopathy. Also pigmentary retinopathy and peripheral neuropathy.
  • Leigh Disease or Syndrome or Subacute Necrotizing Encephalomyelopathy is characterized by symptoms such as Seizures, hypotonia, fatigue, nystagmus, poor reflexes, eating and swallowing difficulties, breathing problems and poor motor function.
  • LHON or Leber Hereditary Optic Neuropathy is caused by mitochondrial DNA point mutations, including G14459A, among others. Symptoms associated with LHON include primarily blindness in young men. Less common symptoms include mild dementia, ataxia, spasticity, peripheral neuropathy and heart conduction defects.
  • MAD or Glutaric Aciduria Type II or multiple Acyl-CoA Dehydrogenase Deficiency is caused by defects of the flavoproteins responsible for transferring electrons (ETF or ETF- dehydrogenase) therefore affecting the function of all six ETF-funneling acyl-CoA dehydrogenases
  • MCAD or Medium-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, which afflicts infants or young children with episodes of encephalopathy, enlarged and fatty degeneration of the liver, and low carnitine in the blood.
  • MELAS or Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes is caused by mitochondrial DNA point mutations, the most common of which is A3243G. It is characterized by symptoms: Short stature, seizures, stroke-like episodes with focused neurological deficits, recurrent headaches, cognitive regression, disease progression ragged-red fibers (Koo et al. Annals of Neurology 34(l):25-32, 1993). MERRF or Myoclonic Epilepsy and Ragged-Red Fiber Disease is caused by the mitochondrial DNA point mutations A8344G and T8356C. Its symptoms include myoclonus, epilepsy, progressive ataxia, muscle weakness and degeneration, deafness and dementia (Luft 1994 Supra).
  • mitochondrial DNA Depletion There are three forms of mitochondrial DNA Depletion. These include: (1) congenital myopathy: Neonatal weakness, hypotonia requiring assisted ventilation, possible renal dysfunction. Severe lactic acidosis. Prominent ragged-red fibers. Death due to respiratory failure usually occurs prior to one year of age; (2) infantile myopathy: Following normal early development until one year old, weakness appears and worsens rapidly, causing respiratory failure and death typically within a few years; and (3) hepatopathy, enlarged liver and intractable liver failure, myopathy, severe lactic acidosis. Death is typical within the first year.
  • Mitochondrial Encephalopathy also includes Encephalomyopathy and Encephalomyelopathy.
  • MNGIE Myoneurogastrointestinal Disorder and Encephalopathy
  • symptoms such as progressive external ophthalmoplegia, limb weakness, peripheral neuropathy, digestive tract disorders, leukodystrophy, lactic acidosis and ragged red fibers.
  • NARP or Neuropathy, Ataxia, and Retinitis Pigmentosa is caused by mitochondrial DNA point mutations in genes associated with Complex V, including T8993G, (also T8993C by some researchers). Leigh Syndrome may result if the percentage of mutation is high enough.
  • Pearson Syndrome is characterized by symptoms associated with bone marrow and pancreas dysfunction. It is caused by single mitochondrial DNA deletions. Inheritance is usually sporadic. Those who survive infancy usually develop Kearns-Sayre Syndrome. Pyruvate Carboxylase Deficiency is an autosomal recessive disorder, the symptoms of which include lactic acidosis, hypoglycemia, severe retardation, failure to thrive, in addition to seizures and spasticity.
  • Pyruvate Dehydrogenase Deficiency is characterized by symptoms such as lactic acidosis, ataxia, pyruvic acidosis, spinal and cerebellar degeneration. Less common symptoms include agenesis of the corpus callosum and lesions in the basal ganglia, cerebellum, and brain stem, growth delay, hypotonia, seizures and polyneuropathy.
  • SCAD Short-Chain Acyl-CoA Dehydrogenase Deficiency
  • SCAD Short-Chain Acyl-CoA Dehydrogenase Deficiency
  • SCHAD is an autosomal recessive disorder, characterized by encephalopathy and possibly liver disease or cardiomyopathy.
  • VLCAD or Very Long-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, characterized by various manifestations, ranging from fatal infantile encephalopathy to recurrent myoglobin in the urine, similar to the myopathic form of CPT II deficiency.
  • an “effective amount” means an amount necessary at least partly to attain the desired physiological effect or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • CXS-746 or CXS-746R or agents capable of modulating the expression or activity of the molecules may be co-administered with one or more other compounds or other molecules.
  • co-administered is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes.
  • sequential administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
  • the present invention relates to the use of an agent capable of modulating the expression of CXS-746 or CXS-746R or a derivative or homolog thereof in the manufacture of a medicament for the treatment of a condition characterized by a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer.
  • the present invention relates to the use of an agent capable of modulating the activity of CXS-746 or CXS-746R or a derivative or homolog thereof in the manufacture of a medicament for the treatment of a condition characterized by diabetes, mitochondrial dysfunction, myopathy, genetic disorder, abnormal blood pressure, abnormal triglyceride levels, or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a further aspect of the present invention relates to the use of CXS-746 or CXS-746R or derivative or homolog thereof in the manufacture of a medicament for the treatment of a condition characterized by a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • Still yet another aspect of the present invention relates to agents for use in modulating the expression of CXS-746 or CXS-746R or a derivative or homolog thereof.
  • a further aspect relates to agents for use in modulating CXS-746 or CXS-746R a derivative or homolog thereof activity.
  • Still another aspect of the present invention relates to CXS-746 or CXS-746R or derivative or homolog thereof for use in treating a condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • the mammal undergoing treatment may be a human or an animal in need of therapeutic or prophylactic treatment.
  • treating and “treatment” as used herein refer to a reduction in the severity and/or frequency of one ore more symptoms associated with inter alia diabetes, mitochondrial dysfunction, myopathy, genetic disorder, abnormal blood pressure, abnormal triglyceride levels, or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
  • Treating" a subject may involve prevention of the disorder or disease condition or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting a disease or disorder as well as decreasing the severity of one or more symptoms associated with the disease or disorder.
  • such conditions involve, weakness (which may be intermittent), neuropathic pain, absent reflexes, gastrointestinal problem (gastroesophogeal reflux, delayed gastric emptying, constipation, pseudo-obstruction), fainting, absent or excessive sweating resulting in temperature regulation problems weakness, hypotonia, cramping, muscle pain, proximal renal tubular wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes, cardiac conduction defects (heart blocks) and cardiomyopathy, hypoglycemia (low blood sugar) and liver failure, visual loss and blindness, hearing loss and deafness, diabetes and exocrine pancreatic failure (inability to make digestive enzymes), mitochondrial dysfunction, including failure to gain weight, short statue, fatigue and respiratory problems.
  • the present invention contemplates in one embodiment a composition comprising a modulator of CXS-746 or CXS-746R expression or CXS-746 or CXS-746R activity and one or more pharmaceutically acceptable carriers and/or diluents.
  • the composition comprises CXS-746 or CXS-746R or a derivative or homolog thereof and one or more pharmaceutically acceptable carriers and/or diluents.
  • active components all such components of such a composition are referred to as "active components”.
  • compositions of active components in a form suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • sterile aqueous solutions where water soluble
  • sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active components in the required amount in the appropriate solvent with optionally other ingredients, as required, followed by sterilization by, for example, filter sterilization, irradiation or other convenient means. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • CXS-746 or CXS-746R themselves are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound.
  • compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
  • the amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained.
  • Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ⁇ g and 2000 mg of active compound.
  • the tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
  • the principal active component may be compounded for convenient and effective administration in sufficient amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • a unit dosage form can, for example, contain the principal active component in amounts ranging from 0.5 ⁇ g to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 ⁇ g to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the the ingredients.
  • CXS-746 or CXS-746R will range from 0.01 ng/kg/body weight to above 10,000 mg/kg/body weight.
  • Alternative amounts range from
  • CXS-746 or CXS-746R may be administered per minute, hour, day, week, month or year depending on the condition being treated.
  • the route of administration may vary and includes intravenous, intraperitoneal, sub-cutaneous, intramuscular, intranasal, via suppository, via infusion, via drip, orally or via other convenient means.
  • the pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating CXS-746 or CXS-746R expression or CXS-746 or CXS-746R activity.
  • the vector may, for example, be a viral vector.
  • Still another aspect of the present invention is directed to antibodies specific for CXS-746 or CXS-746R and derivatives and homologs of CXS-746 and CXS-746R.
  • Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to CXS-746 or CXS-746R or may be specifically raised to CXS-746 or CXS-746R or derivatives or homologs thereof.
  • CXS-746 or CXS-746R or their derivatives or homologs may first need to be associated with a carrier molecule.
  • the antibodies and/or recombinant CXS-746 or CXS-746R or their derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
  • CXS-746 or CXS-746R and derivatives or homologs thereof can be used to screen for naturally occurring antibodies to CXS-746 or CXS-746R which may occur in certain autoimmune diseases or where cell death is occurring. These may occur, for example in some autoimmune diseases.
  • specific antibodies can be used to screen for CXS-746 or CXS-746R.
  • Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.
  • Antibodies to CXS-746 or CXS-746R of the present invention may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the CXS-746 or CXS-746R or may be specifically raised to the CXS-746 or CXS-746R or their derivatives. In the case of the latter, the CXS-746 or CXS-746R protein may need first to be associated with a carrier molecule. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool or as a means for purifying CXS-746 or CXS-746R.
  • CXS-746 or CXS-746R proteins can be used to screen for CXS-746 or CXS-746R proteins.
  • the latter would be important, for example, as a means for screening for levels of CXS-746 or CXS-746R in a cell extract or other biological fluid or purifying CXS-746 or CXS-746R made by recombinant means from culture supernatant fluid.
  • Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
  • Immunoassays in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
  • antibodies binding to the encoded proteins of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the disease marker antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen may be detected. Detection is generally achieved by the addition of a second antibody specific for the target protein, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the disease marker antigen are immobilized onto the well surface and then contacted with the antibodies of the invention. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen is detected. Where the initial antibodies are linked to a detectable label, the immunocomplexes may be detected directly. Again, the immunocomplexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
  • Another ELISA in which the proteins or peptides are immobilized involves the use of antibody competition in the detection.
  • labelled antibodies are added to the wells, allowed to bind to the disease marker protein, and detected by means of their label.
  • the amount of marker antigen in an unknown sample is then determined by mixing the sample with the labelled antibodies before or during incubation with coated wells.
  • the presence of marker antigen in the sample acts to reduce the amount of antibody available for binding to the well and thus reduces the ultimate signal. This is appropriate for detecting antibodies in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
  • ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunocomplexes. These are described as follows:
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the control sample and/or clinical or biological sample to be tested under conditions effective to allow immunecomplex (antigen/antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
  • Under conditions effective to allow immunecomplex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • suitable conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° to 27° C, or may be overnight at about 4°C or so.
  • the contacted surface is washed so as to remove non-complexed material.
  • a preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immunocomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunocomplexes may be determined.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for 2 hours at room temperature in a PB S -containing solution such as PBS-Tween).
  • the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-3-ethyl-benzthiazoline-6- sulfonic acid [ABTS] and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-3-ethyl-benzthiazoline-6- sulfonic acid [ABTS] and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
  • the antibodies of this invention will be used to quantify and localize the expression of the encoded marker proteins.
  • the antibody for example, will be labeled by any one of a variety of methods and used to visualize the localized concentration of the cells producing the encoded protein.
  • the invention also relates to an in vivo method of imaging metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions using the above described monoclonal antibodies.
  • this method involves administering to a subject an imaging- effective amount of a detectably-labeled disease-specific monoclonal antibody or fragment thereof and a pharmaceutically effective carrier and detecting the binding of the labeled monoclonal antibody to the diseased, or in the case of down regulated marker genes, healthy tissue.
  • in vivo imaging refers to any method which permits the detection of a labeled monoclonal antibody of the present invention or fragment thereof that specifically binds to a diseased tissue located in the subject's body.
  • a "subject” is a mammal, preferably a human.
  • An “imaging effective amount” means that the amount of the detectably-labeled monoclonal antibody, or fragment thereof, administered is sufficient to enable detection of binding of the monoclonal antibody or fragment thereof to the diseased tissue, or the binding of the monoclonal antibody or fragment thereof in greater proportion to healthy tissue relative to diseased tissue.
  • a factor to consider in selecting a radionuclides for in vivo diagnosis is that the half-life of a nuclide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host, as well as background, is minimized.
  • a radionuclides used for in vivo imaging will lack a particulate emission, but produce a large number of photons in a 140-2000 keV range, which may be readily detected by conventional gamma cameras.
  • a radionuclides may be bound to an antibody either directly or indirectly by using an intermediary functional group.
  • Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylene diaminetetracetic acid
  • metallic ions suitable for use in this invention are 99m Tc, 123 I, 131 I, 11 1 In, 131 1, 97 Ru, 67 Cu, 67 Ga, 125 1, 68 Ga, 72 As, 89 Zr, and 201 Tl.
  • the monoclonal antibody or fragment thereof may be labeled by any of several techniques known to the art.
  • the methods of the present invention may also use paramagnetic isotopes for purposes of in vivo detection.
  • Elements particularly useful in Magnetic Resonance Imaging (“MRI") include 157 Gd, 55 Mn, 162 Dy,
  • Administration of the labeled antibody may be local or systemic and accomplished intravenously, intraarterially, via the spinal fluid or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has lapsed, for example 30 minutes to 48 hours, for the monoclonal antibody or fragment thereof to bind with the target tissue, either diseased and/or healthy tissue, the area of the subject under investigation is examined by routine imaging techniques such as MRI, SPECT, planar scintillation imaging and emerging imaging techniques, as well.
  • routine imaging techniques such as MRI, SPECT, planar scintillation imaging and emerging imaging techniques, as well.
  • the exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan.
  • the distribution of the bound radioactive isotope and its increase or decrease with time is then monitored and recorded. By comparing the results with data obtained from studies of clinically normal individuals, the presence and extent of the diseased tissue may be determined.
  • the present invention provides methods for the in vivo diagnosis of disease in a patient. Such methods generally comprise administering to a patient an effective amount of a disease specific antibody, which antibody is conjugated to a marker, such as a radioactive isotope or a spin-labeled molecule, that is detectable by non-invasive methods.
  • a disease specific antibody which antibody is conjugated to a marker, such as a radioactive isotope or a spin-labeled molecule, that is detectable by non-invasive methods.
  • the antibody- marker conjugate is allowed sufficient time to come into contact with reactive antigens that be present within the tissues of the patient, and the patient is then exposed to a detection device to identify the detectable marker.
  • any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available antiimmunoglobulin antibody.
  • An antibody as contemplated herein includes any antibody specific to any region of CXS-746 or CXS-746R. Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art.
  • Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of CXS-746 or CXS-746R, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
  • the use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product.
  • the preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein Nature 256:495-499, 1975; Kohler and Milstein European Journal of Immunology 6:511-519, 1976).
  • Another aspect of the present invention contemplates a method of detecting CXS-746 or CXS-746R or a derivative or homolog thereof in a biological sample from a subject, the method comprising contacting the biological sample with an antibody specific CXS-746 or CXS-746R or their antigenic derivatives or homologs for a time and under conditions sufficient for a complex to form, and then detecting the complex.
  • the presence of the complex is indicative of the presence CXS-746 or CXS-746R.
  • This assay may be quantitated or semi-quantitated to determine a propensity to develop mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting or to monitor a therapeutic regimen.
  • CXS-746 or CXS-746R may be accomplished in a number of ways such as by Western blotting and ELISA procedures.
  • a wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target.
  • Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule.
  • a second antibody specific to the CXS-746 or CXS-746R, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-CXS-746-labeled or CXS-746R-labeled antibody. Any unreacted material is washed away, and the presence of CXS-746 or CXS- 746R is determined by observation of a signal produced by the reporter molecule.
  • the results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten.
  • Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent.
  • the sample is one which might contain CXS-746 or CXS-746R including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid.
  • the sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
  • the solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay.
  • the binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g.
  • the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of CXS- 746 or CXS-746R.
  • the second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to CXS-746 or CXS-746R.
  • An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
  • reporter molecule as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative.
  • the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
  • an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate.
  • glutaraldehyde or periodate As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan.
  • Commonly used enzymes include horseradish peroxidase, glucose oxidase, ⁇ -galactosidase and alkaline phosphatase, amongst others.
  • the substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase.
  • fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above.
  • the enzyme-labeled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample.
  • a "reporter molecule” also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
  • fluorescent compounds such as fiuorecein and rhodamine
  • fluorescent compounds may be chemically coupled to antibodies without altering their binding capacity.
  • the fluorochrome-labeled antibody When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
  • the fluorescent labeled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.
  • Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method.
  • reporter molecules such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
  • the present invention also contemplates genetic assays such as involving PCR analysis to detect CXS-746 or CXS-746R or its derivatives.
  • the assays of the present invention may also extend to measuring CXS-746 or CXS-746R in association with another gene or molecule.
  • the present invention may also be described in certain embodiments as a kit for use in detecting or diagnosing metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • a representative kit may comprise one or more nucleic acid segments as described above that selectively hybridize to CXS-746 or CXS-746R and a container for each of the one or more nucleic acid segments.
  • the nucleic acid segments may be combined in a single tube.
  • the nucleic acid segments would be designed to selectively hybridize to a nucleic acid segment that includes the sequence or complement of SEQ ID NOs: 1 or 15 or 18 or 19.
  • the nucleic acid segments may also include a pair of primers for amplifying the target mRNA.
  • kits may also include any buffers, solutions, solvents, enzymes, nucleotides, or other components for hybridization, amplification or detection reactions.
  • Preferred kit components include reagents for RT-PCR, in situ hybridization, Northern analysis and/or RPA.
  • the kit for use in detecting metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions may comprise an antibody which immunoreacts with a CXS-746 or CXS-746R polypeptide and a container for the antibody.
  • an antibody may be a polyclonal or a monoclonal antibody and may be included in a kit with reagents, secondary antibodies, labeling means, or other components for polypeptide detection including, but not limited to an ELISA kit.
  • the present invention further comprises the prognosis and/or diagnosis of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions by measuring the amounts of nucleic acid amplification products formed as above.
  • the amounts of nucleic amplification products identified in an individual patient may be compared with groups of normal individuals or individuals with an identified disease state. Diagnosis may be accomplished by finding that the patient's levels of CXS-746 and CXS-746R fall within the normal range, or within the range observed in individuals with the disease state. Further comparison with groups of individuals of varying disease state progression may provide a prognosis for the individual patient.
  • the present invention further broadly comprises kits for performing the above-mentioned procedures, containing amplification primers and/or hybridization probes.
  • Another aspect of the present invention comprises the detection and diagnosis of diabetes or a condition associated therewith or the likelihood of developing diabetes or obesity or a predisposition of developing diabetes by combining measurement of levels of markers of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • markers of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
  • An embodiment of the invention comprises combining measurement of SEQ ID NOs: 1 or 15 or 18 or 19 with other markers associated with metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions, such as BMI, weight, waste to hip ratio, fasting glucose, fasting insulin and percent body fat.
  • kits for detection and measurement of the levels of two or more disease state markers in biological samples are kits for detection and measurement of the levels of two or more disease state markers in biological samples.
  • kits may incorporate a variety of methodologies for detection and measurement of disease state markers, including but not limited to oligonucleotide probes, primers for nucleic acid amplification, antibodies which bind specifically to protein products of disease state marker genes, and other proteins or peptides which bind specifically to disease state marker gene products.
  • the present invention concerns immunodetection kits for use with the immunodetection methods described above.
  • the encoded marker proteins or peptides may be employed to detect antibodies and the corresponding antibodies may be employed to detect encoded proteins or peptides, either or both of such components may be provided in the kit.
  • the immunodetection kits thus comprise, in suitable container means, an encoded protein or peptide and/or a first antibody that binds to an encoded protein or peptide, and an immunodetection reagent.
  • the encoded protein or peptide, or the first antibody that binds to the encoded protein or peptide may be bound to a solid support, such as a column matrix or well of a microtiter plate.
  • the immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody or antigen, and detectable labels that are associated with or attached to a secondary binding ligand.
  • Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody or antigen, and secondary antibodies that have binding affinity for a human antibody.
  • suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody or antigen, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.
  • kits may further comprise a suitably aliquoted composition of the encoded protein or polypeptide antigen, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
  • kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody or antigen may be placed, and preferably, suitably aliquoted. Where a second or third binding ligand or additional component is provided, the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed.
  • the kits of the present invention will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • a Psammomys obesus colony is maintained at Deakin University, Waurn Ponds, Geelong, Victoria, Australia with the breeding pairs fed ad libitum a diet of lucerne and standard laboratory chow. Animals are weaned at four weeks of age and sustained on a diet of standard laboratory chow from which 12% of energy was derived from fat, 63% from carbohydrate and 25% from protein (Barastoc, Pakenham, Australia). Animals are housed in a humidity and temperature controlled room (22 ⁇ 1°C) with a 12- 12-hour light-dark cycle.
  • Group A animals are lean, normoglycemic and normoinsulinemic (NGT), group B animals are obese, normoglycemic and hyperinsulinemic (IGT), and group C animals are obese, hyperglycemic and hyperinsulinemia (Type II diabetic).
  • Plasma insulin concentrations were determined using a double antibody solid phase radioimmunoassay (Phadeseph, Kabi Pharmacia Diagnostics, Sweden).
  • the tissue samples were homogenised in Trizol (Invitrogen Life Technologies) and l/5 th volume chloroform was added to the homogenate, which was then mixed and incubated at room temperature for 3 min.
  • the homogenates were then separated by centrifugation at 13000xg for 15 min (4 0 C). Following centrifugation the aqueous supernatant was removed and added to an equal volume of 70% ethanol.
  • This system utilises capillary electrophoresis to separate and detect nucleic acid fragments by size through the interconnected micro channels on a Nano chip (Agilent Technologies).
  • Good quality RNA was signified by an electropherogram displaying a marker peak, and two ribosomal peaks of which the 18s band is at an approximate ratio of 1 :2 to the 28s band.
  • Phenotypic parameters of human normal and type 2 diabetic subjects were compared using a Students t-test or a Mann- Whitney U test for data that was not normally distributed. Associations between circulating CXS-746 levels and phenotypic measures were determined using Pearson correlation (for normally distributed data) or Spearman correlation (for non-normally distributed data) in SPSS. Multivariate linear regression was used to determine if associations were independent of other variables. EXAMPLE 5 Signal Sequence Trap
  • RNA was extracted as described in Example 3 from liver of lean, normoglycaemic, normoinsulinaemic (NGT), obese normoglycaemic, hyperinsulinaemic (IGT) and obese Type II diabetic (T2D) P. obesus in the fed and fasted states. Equal amounts of RNA from each group were pooled and the mRNA extracted using an oligo(dT) cellulose column.
  • the mRNA was reverse transcribed using random 9mer primers to enrich for the 5' end of mRNA.
  • the random 9mer primers were engineered to contain a Not I restriction site (underlined) which was used for cloning:
  • the cDNA was run on a 1.5% w/v TBE agarose gel.
  • the 300 to 800 base pair products were cut out of the gel, purified and quantitated using standard methodologies.
  • the Not I-Sal I digested skeletal muscle library was ligated into the retrovirus plasmid vector pLNCX2, 5' to a murine interleukin 3 (mIL-3) gene that was engineered to lack a signal sequence. Transformation conditions were according to standard methodologies. Transformation of bacterial cells generated approximately 200,000 transformants. The plasmid library obtained from the 200,000 transformants was transfected into a retrovirus packaging cell line (293 Plat E).
  • the retrovirus library that was produced was used to infect the mIL-3 dependent cell line FDCPl.
  • the retrovirus library and cells were placed in 6 well dishes and centrifuged for 1 hour at 1000xg, to increase the frequency of infection.
  • the cells were then incubtated at 37 0 C for 24 hrs in media containing mIL-3.
  • the infected cells were washed four times to eradicate IL-3 from the media then plated into 96-well plates.
  • Cells infected with retroviruses that contain an in-frame signal sequence and secreted mIL-3 were evidenced by live cells growing in each well after several days. After 2-3 weeks, genomic DNA was extracted from positive clones using standard methodologies.
  • First round PCR primers were as follows: Forward 5'-CTGGTTTAGTGAACCGTCAGATC (SEQ ID NO:3) and Reverse 5'-CTCCTTGACAATAGAGCTGCAA (SEQ ID NO:4).
  • the DNA was denatured for 2 min at 94 0 C, and amplified by 35 cycles of 94°C for 30 sec, 56 0 C for 30 sec and 72 0 C for 1 min, followed by a final extension of 7 min at 72 0 C.
  • the first round PCR product was diluted 1 :100 and 1 ⁇ l used as template for a second round of amplification to reduce the levels of genomic DNA in the final PCR product.
  • Second round PCR primers were designed immediately adjacent to the plasmid insert site so the amplified cDNA product contains very little mIL-3 or vector sequence which may potentially interfere with hybridisation to the microarray chip.
  • the primer sequences were as follows: Forward 5'-TAGCGCTACCGGACTCAGAT (SEQ ID NO:5) and Reverse 5'- CGGCCACTGATTGAAGCTT (SEQ ID NO:6).
  • PCR conditions were the same as for first round PCR. Products were visualized by TBE agarose gel (1.% w/v) electrophoresis at 6V/cm for 60 min to ensure successful amplification had taken place.
  • PCR products were purified using the Arraylt vacuum manifold system (TeleChem International, Sunnyvale, CA) and resuspended in 20 ⁇ L of Ix spotting solution (TeleChem) at a concentration of approximately 0.5 mg/ml in 384 well plate format. 5 ⁇ L of the resuspended purified cDNA solution was transferred to 384 well uniplates (Whatman Inc, Clifton, USA). This cDNA was arrayed onto Super Amine Microarray Substrates (TeleChem) using a Chip Writer Pro robotic arrayer (BioRad) fitted with 32 Stealth SMP-03 quill tipped microarray pins (Telechem). The distance between adjacent cDNA spots was 200 ⁇ M.
  • Each pin drew 0.25 ⁇ L of cDNA and deposited approximately 0.6nL on each slide. Humidity was maintained between 55-65% during printing. Spotted DNAs were allowed to dry overnight, after which the slides were washed and blocked as recommended by the manufacturer (TeleChem).
  • Fluorescently labeled cDNA was prepared from 20 ⁇ g of total RNA using an indirect labelling method (Superscript Indirect cDNA Labelling System, Invitrogen) as per the manufacturers instructions. cDNA synthesis was performed in a 40 ⁇ L reaction containing 5 ⁇ g oligo-dT primer, 400U Superscript IH (Invitrogen), Ix first strand buffer, 0.0 IM DTT, 0.5mM of each dATP, dCTP and dGTP, 0.15OmM dTTP (Amersham, Buckinghamshire, UK) and 0.2mM aminoallyl-dUTP (Sigma, St. Louis, MO).
  • Superscript Indirect cDNA Labelling System Invitrogen
  • Synthesis was conducted in a GeneAmp PCR System 9700 (PE Applied Systems) at 46°C for 2 hours. The reaction was stopped by addition of 5 ⁇ l of 0.5M EDTA and RNA was hydrolysed by addition of 20 ⁇ l of IM NaOH at 70°C for 20 minutes. The reaction was neutralized with 25 ⁇ l of IM HCl and the cDNA was purified using SNAP purification kits according to manufacturer's instructions (Invitrogen) and eluted in nuclease-free water. The cDNA was concentrated by ethanol precipitation and the cDNA pellet was resuspended in 5 ⁇ l coupling buffer. Cy3 or Cy5 monofunctional NHS ester reactive dyes (Amersham) were dissolved in 5 ⁇ l of DMSO and added to the cDNA. The coupling reaction was conducted in the dark for 1 hour.
  • Dye-coupled cDNA was purified using SNAP DNA purification columns (Invitrogen), combined and added to lO ⁇ g of mouse Cotl DNA (Invitrogen). The cDNAs were again concentrated with Micron 30 spin columns (Millipore). The cDNA was hybridised in a 50 ⁇ L volume containing the labeled cDNA, 50% v/v formamide, 5x SSC, 8 ⁇ g PolydA, 2.5x Denhardt's solution, 4 ⁇ g yeast tRNA and 0.1% SDS. The cDNA was then denatured at 98 0 C for 2 min and maintained at 6O 0 C until required.
  • the hybridisation solution was mounted onto an array slide under a Lifterslip (Erie Glass) and hybridisation was conducted in a humid hybridisation chamber, in a hybridisation oven, at 42 0 C for 16 hours. Following hybridisation the array slides were removed from their chamber and washed for 2 min in each of a 0.5X SSC and 0.1% w/v SDS, 0.5x SSC and 0.01% w/v SDS, 0.6x SSC and 0.06% w/v SDS solution. The array slides were dried in a centrifuge for 1 min at 500xg.
  • Fluorescent images of the microarrays were acquired using a GenePix 4000B scanner (Axon Instruments, Union City CA, USA) and the images were analysed using GenePix Pro 5.1 (Axon Instruments). Slides were scanned for both Cy3 and Cy5 signal at a lO ⁇ M pixel resolution. Laser intensity and amplification of the photomultiplier tubes were adjusted to ensure approximately equal overall signal intensity for both Cy3 and Cy5. False colour images were generated for each dye and combined to provide a representation of the relative Cy3 and Cy5 intensities. Individual cDNA spots were flagged if spot size was too small, if the overall signal intensity was too low, or if the Cy3 and Cy5 signal intensities within the spots were not linearly related. GenePix Pro allows for the 'flagging' of bad elements (defined by present GenePix Pro parameters as feature signal intensity; feature background; element morphology; elements size and the percentage of pixels greater than feature background) that were then excluded from further analysis.
  • Signal intensities for each feature were corrected for local background and features that failed to meet quality criteria (e.g. low expression values, poor feature morphology; small feature size or small percentage of pixels greater than feature background) were omitted.
  • quality criteria e.g. low expression values, poor feature morphology; small feature size or small percentage of pixels greater than feature background
  • Signal intensity was normalised to the mean intensity of all respective signal intensities, providing a relative measure of gene expression for each element on the microarray slide.
  • genes encoding SST positive cDNAs in liver tissue of lean, normoglycaemic, normoinsulinaemic (NGT), obese normoglycaemic, hyperinsulinaemic (IGT) or obese Type II diabetic (T2D) P. obesus obesity and Type II diabetes was determined using microarray analysis. Differential gene expression as measured by microarray was assessed using the independent samples t-test algorithm in the Acuity software. Genes were considered to be differentially expressed if the significance of the t- test was p ⁇ 0.05. A number of differentially expressed genes were identified and their expression was confirmed by real time PCR. RNA was reverse transcribed using Superscript Hi RT system (Invitrogen).
  • Oligonucleotide primers for were designed using the Primer Express 2.0 software program (Applied Biosystems), and real time PCR was performed using SYBR Green PCR master mix and an ABI PRISM 7700 sequence detector (Applied Biosystems).
  • the primer sequences for P. obesus CXS-746 real time gene expression were as follows: forward 5'- TGGGCCTTCCGAGAGATG (SEQ ID NO: 7) and reverse 5'- AATTCCAACCGCACAAAGGT (SEQ ID NO:8) or reverse 5 1 - AGACGACCACACAGGTCACGTA (SEQ ID NO:24).
  • obesus CXS-746 receptor real time gene expression were as follows: forward 5'- AGCTTTGACCGCTGCATCTC (SEQ ID NO:9) and reverse 5'- GGAACTCAAGAAGAAAGCCAAGAG (SEQ ID NO: 10).
  • the primer sequences for murine CXS-746 real time gene expression were as follows: forward 5'- CCAACTGCCCCAAGAAGGA (SEQ ID NO: 11) and reverse 5'- CGCCTTCTCCCGTTTGGT (SEQ ID NO: 12).
  • the primer sequences for murine CXS- 746 receptor real time gene expression were as follows: forward 5'- TGGCCGACTTCCTGTTCAAC (SEQ ID NO: 13) and reverse 5'- CCCGAACACCCAGTGGTAGT (SEQ ID NO: 14).
  • LGiLGNGLVi IIATFKMKKT VNMVWFLNLA VADFLFNVFL PIHITYAAMD YHWVFGTAMC KISNFLLIHN MFTSVFLLTI ISSDRCISVL LPVWSQNHRS VRLAYMACMV IWVLAFFLSS PSLVFRDTAN LHGKISCFNN FSLSTPGSSS WPTHSQMDPV GYSRHMVVTV TRFLCGFLVP VLIITACYLT IVCKLQRNRL AKTKKPFKII VTIIITFFLC WCPYHTLNLL ELHHTAMPGS VFSLGLPLAT ALAIANSCMN PILYVFMGQD FKKFKVALFS RLVNALSEDT GHSSYPSHRS FTKMSSMNER TSMNERETGM
  • CXS-746 was sequence identified as a positive clone from a SST using P. obesus liver
  • RNA Comparison of the P. obesus CXS-746 sequence to GenBank revealed it was homologous to the chemerin gene. Alternate names for this gene are tazarotene-induced gene 2 (TIG2); HP 10433, retinoic acid receptor responder (tazarotene induced) 2
  • CMKLRl chemokine-like receptor 1
  • Chemerin was initially identified as a novel transcript that was upregulated in psoriasis lesions treated with the retinoic acid receptor agonist tazarotene (Nagapal et al. Journal of Investative Dermatolatology 109:91-5, 1997).
  • the human chemerin gene has been to chromosome 7q36.1 and encodes a 7340bp mRNA.
  • Chemerin mRNA expression is induced by RAR agonists (such as tazarotene) and not by RXR or Vitamin D Receptor specific agonists (Nagapal et al. 1997 supra).
  • the chemerin protein is secreted as prochemerin and is proteolytically cleaved at the C-terminus by extracellular proteases to yield a 15kDa mature polypeptide.
  • Chemerin is expressed by many tissues including spleen, lymph nodes, adrenal gland, liver, pancreas, small intestine and mature chemerin protein has been isolated from human ascetic fluid that was secondary to ovarian cancer, hemof ⁇ ltrate and serum (Zabel et al. Journal of Biological Chemistry 280: 34661-6, 2005).
  • CXS-746 has been identified as a natural ligand of chemokine-like receptor 1 (CMKLRl), a previously orphan protein G-coupled receptor (Samson et al. European Journal of Immunology 28: 1689-700, 1998).
  • CMKLRl chemokine-like receptor 1
  • CMKLRl is expressed by macrophages and immature dendritic cells and is found in lymph node, parathyroid glands and developing osteogenic and cartilaginous tissue (Meder et al. FEBS Letters 555:495-9, 2003).
  • CXS-746 binding to the CMKLRl receptor induces intracellular calcium release and MAPK pathway activation (Vermi et al. Journal of Experimental Medicine 201:509-15, 2005).
  • CXS-746 is a potent chemoattractant for macrophages and dendritic cells expressing the G-protein coupled receptor CMKLRl.
  • CXS-746 and its receptor CMKLRl may play a pivotal role as a link between innate and adaptive immunity where tissue infiltration by neutrophils promote the recruitment of APC at inflammatory sites (Vermi et al. 2005 supra).
  • CMKLRl is used as a co-receptor by the immunodeficiency viruses HIV-I and SIV (Samson et al. 199S supra).
  • CXS-746 gene was found to be expressed in all tissues examined in P. obesus, but was highest in the liver, subscapular fat, intramuscular fat, epididymal fat, mesenteric fat, perirenal fat and the kidney.
  • CXS-746 receptor CXS-746R was also expressed in all tissues examined in P. obesus, where highest expression was observed in adipose tissue and the lung. This is the first description of CXS-746 and CXS-746 receptor expression in adipose tissue depots. The results are shown in Table 3. TABLE 3 CXS-746 and CXS-746 Receptor Tissue Distribution ;Relatiye[GeneiExpression
  • CXS-746 gene expression in mesenteric fat of NGT, IGT and T2D P obesus, in the fed and fasted state was analysed by real time PCR.
  • CXS-746 receptor gene expression in the mesenteric fat of NGT, IGT and T2D P. obesus, in the fed and fasted state was also analysed by real time PCR.
  • CXS-746 and CXS-746 receptor gene expression as measured by SYBR Green Real Time PCR in P. obesus visceral and subcutaneous adipose tissue
  • CXS-746 receptor gene expression was also measured in visceral and subcutaneous adipose tissue from fed NGT, IGT and T2D P. obesus by real time PCR.
  • 3T3-L1 adipocytes were cultured in high glucose DMEM (25mM), supplemented with 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin Two days after the cells reached confluence, differentiation of fibroblasts into adipocytes was initiated by the addition of high glucose DMEM. 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin, 0.5mM l-,ethyl-3-isobutulxanthine (IBMX), 2.5uM dexamethasone and 0.166U/ml insulin for three days.
  • IBMX 0.5mM l-,ethyl-3-isobutulxanthine
  • the medium was then aspirated and replaced with glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin and 0.166U/ml of insulin for two days. Following these treatments, the cells were cultured in glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin.
  • Real time PCR showed that, compared to undifferentiated fibroblasts, CXS-746 gene expression was markedly upregulated during differentiation (day 3, 4, 5 and 6, pO.OOl) and was approximately 20 fold higher in fully differentiated adipocytes (day 7,8 and 9, pO.OOl).
  • 3T3-L1 adipocytes were cultured in high glucose DMEM (25mM), supplemented with 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin. Two days after the cells reached confluence, differentiation of fibroblasts into adipocytes was initiated by the addition of high glucose DMEM. 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin, 0.5mM l-,ethyl-3-isobutulxanthine (IBMX), 2.5uM dexamethasone and 0.166U/ml insulin for three days.
  • IBMX 0.5mM l-,ethyl-3-isobutulxanthine
  • the medium was then aspirated and replaced with high glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin and 0.166U/ml of insulin for two days. Following these treatments, the cells were cultured in high glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin.
  • Real time PCR showed that, compared to undifferentiated fibroblasts, CXS-746 receptor gene expression was markedly downregulated during differentiation (day 1, 2, 3 p ⁇ 0.001) and was approximately 10 fold lower in fully differentiated adipocytes (day 7, 8 and 9, p ⁇ 0.001).
  • 3T3-L1 adipocytes were fully differentiated into adipocytes for 8 days (as described above) were stimulated with murine TNF- ⁇ (3ng/ml) for 72 hours.
  • RNA was extracted and the expression of CXS-746 and CXS-746 receptor was determined by real time PCR. Expression of CXS-746 was significantly upregulated by TNF- ⁇ treatment in mature 3T3 adipocytes (p ⁇ 0.001). In contrast, TNF- ⁇ treatment did not significantly affect CXS-746 receptor expression (Table 8).
  • 3T3-L1 adipocytes were fully differentiated into adipocytes for 8 days (as described above) and were stimulated with lO ⁇ M troglitazone or 5mM aspirin for 24 hours. Unstimulated cells were used as a control. RNA was extracted and the expression of CXS- 746 and CXS-746 receptor was determined by real time PCR (Table 9). Expression of CXS-746 was significantly downregulated by troglitazone treatment in mature 3T3 adipocytes (p ⁇ 0.001). In contrast, aspirin treatment did not significantly affect expression of CXS-746 receptor.
  • KRP Krebs Ringer Phosphate
  • the digested samples were filtered through a nylon mesh into 25ml fresh KRP buffer and centrifuged 300g, 10 min at room temperature The floating adipocyte layer was collected, and the supernatant aspirated to reveal the pellet fraction. Both fractions were snap frozen in liquid nitrogen and stored -8O 0 C for subsequent RNA extraction.
  • CXS-746 and CXS-746 receptor are predominantly expressed by adipocytes in adipose tissue
  • mesenteric adipose tissue from NGT and IGT P obesus was fractionated into cellular populations comprised of adipocytes and stromal-vascular cells.
  • CXS-746 receptor gene expression was not significantly different in adipocyte and stromal-vascular cells from adipose tissue of P obesus.
  • An ELISA was developed using commercially available unlabeled and biotinylated polyclonal antihuman chemerin antibodies (R&D Systems, USA). Primary unlabelled antibody was diluted to 1 ⁇ g/ml in PBS and coated onto Maxisorp ELISA plates (Nunc, USA) in lOO ⁇ l at 4°C overnight. The plates were washed with PBS 0.05% tween 20 (PBST) and blocked using 200 ⁇ l blocking buffer (3% BSA in PBST) for 1 hour. The blocking solution was removed and plasma samples (diluted 1 :60 with blocking buffer; lOO ⁇ l per sample in duplicate) were added to the plate.
  • PBST PBS 0.05% tween 20
  • the assays were measured using a BioRad microtiter plate reader (Model 550) at 450nm with a reference of 630nm. Interassay coefficient of variation was less than 10% and the within assay coefficient of variation was less than 5%.
  • the sensitivity of the ELISA assay was 1-10 ng/ml and the mid range of the assay was 5ng/ml. The least detectable concentration of human chemerin was 0.5ng/ml.
  • Serum CXS-746 levels are correlated with metabolic syndrome related phenotypes
  • CXS-746 levels were significantly associated with measures of body fat (BMI, fat mass, weight and WHR) and metabolic syndrome related phenotypes (fasting glucose, fasting insulin, plasma triglycerides and blood pressure; Table 12,) in NGT subjects.
  • BMI body fat
  • metabolic syndrome related phenotypes fasting glucose, fasting insulin, plasma triglycerides and blood pressure; Table 12,
  • Homa b quantitative estimate of ⁇ -cell function
  • homa_s quantitative estimate of insulin sensitivity
  • BMI body mass index
  • WHR waist hip ratio
  • HDL high dense lipoprotein
  • TG triglycerides
  • BP blood pressure TABLE 12
  • CXS-746 is associated with metabolic syndrome related phenotypes
  • Plasma samples were obtained as part of the San Antonio Family Heart Study (SAFHS), a cohort which was first assessed in 1991 and was designed to primarily investigate the genetics of cardiovascular disease and its risk factors in Mexican Americans.
  • SAFHS San Antonio Family Heart Study
  • the SAFHS includes 1,431 individuals in 42 extended families at baseline. Samples were ascertained by way of a single adult Mexican American proband selected at random, without regard to presence or absence of disease and almost exclusively from Mexican American census tracts in San Antonio. To ensure large, multigenerational pedigrees, probands had to have at least 6 age-eligible offspring and/or siblings living in San Antonio. All first, second, and third degree relatives of the proband and of the proband's spouse, aged 16 years or above, were eligible to participate in the study. All protocols were approved by the Institutional Review Board of the University of Texas Health Science Centre at San Antonio (San Antonio, TX).
  • Serum CXS-746 levels are correlated with metabolic syndrome related phenotypes in
  • Statistical analyses were performed using linear regression analysis and bivariate correlations to determine whether CXS-746 was associated with adiposity and/or metabolic syndrome related phenotypes.
  • CXS-746 was significantly associated with a number of phenotypes tested including BMI, fasting glucose and fasting serum insulin (Table 14).
  • CXS-746 is associated with obesity and metabolic syndrome related phenotypes in NGT subjects from the SAFHS cohort

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Abstract

The present invention provides in part isolated nucleic acid molecules and amino acid sequences comprising a nucleotide sequence encoding or complementary to sequences encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequences derived from Psammonys obesus which are SEQ DD NOS: 1 and 15 and the corresponding human equivalents which are SEQ ED NO.18 and 19. It is also directed to the use of these sequences in the manufacture of medicament and diagnostic agents for a range of metabolic conditions.

Description

THERAPEUTIC AGENTS, TARGETS AND DIAGNOSTICS
FIELD
The present invention relates generally to the field of therapeutic agents and targets. More particularly, the present invention is directed to the identification ' of molecules or antagonists or agonists thereof for use in medical treatment and/or diagnostic protocols. The present invention further contemplates a screening protocol for potential therapeutic targets.
BACKGROUND
Bibliographic details of references provided in the subject specification are listed at the end of the specification.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
The increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the veterinary and allied human and animal health fields. This is particularly the case in the investigation of the genetic bases involved in the etiology of certain disease conditions. Diseases of particular concern include disorders associated with diabetes and mitochondrial dysfunction as well as myopathies, genetic disorders and cancers and in modulating apoptosis, signal transduction and/or nuclear targeting.
Diabetes represents a significant and debilitating disease. The incidence of diabetes is increasing rapidly. It has been estimated that there were about 700,000 persons with diabetes in Australia in 1995 while in the US, the prevalence of diabetes increased from 4.9% in 1990 to 6.9% in 1999 (Mokdad Diabetes Care 24(2):A\2, 2001). There are two main types of diabetes referred to as Type I and Type II diabetes.
Type I diabetes, also known as insulin-dependent diabetes mellitus (IDDM), results from an inability to produce insulin. It can develop at any age, although it usually develops in children and young adults and is also referred to as juvenile-onset diabetes. Once it has developed, Type I diabetes is a life-long condition.
Type II diabetes occurs later in life and is sometimes known as late-onset diabetes or non- insulin-dependent diabetes mellitus (NIDDM), because insulin treatment is not always needed. Type II diabetes develops when the body becomes resistant to insulin. This happens when the body's tissues, such as muscle, do not respond fully to the actions of insulin, so cannot make use of glucose in the blood. The pancreas responds by producing more insulin. In addition, the liver, where glucose is stored, releases more glucose to try to increase the amount of glucose available. Eventually, the pancreas becomes less able to produce enough insulin and the tissues become more resistant to insulin. As a result, blood glucose levels slowly start to rise.
Mitochondrial dysfunction refers to any illness resulting from a deficiency of any mitochondrial-located protein which is involved in energy metabolism. Therefore, deficiencies of the respiratory (electron transport) chain, either resulting from a deficiency in one or more of the mitochondrial or nuclear-encoded proteins, are mitochondrial disorders. Also, by definition, disorders of the fatty acid (beta) oxidation, Krebs cycle and pyruvate dehydrogenase complex deficiency are mitochondrial disorders. Although these disorders may be genetically dissimilar, mitochondrial dysfunction results in an energy deficient state.
There is no one identifying feature of mitochondrial disease. Subjects can have combinations of problems whose onset may occur from before birth to late adult life. Mitochondrial diseases should be considered in the differential diagnosis when there are unexplained features, especially when these occur in combination. Mitochondrial disease and disorders can affect multiple organs, resulting in a vast array of symptoms. Symptoms which may affect the brain include, developmental delays, mental retardation, dementia, seizures, neuro-psychiatric disturbances, atypical cerebral palsy, migraines, strokes.
Cancer is also one of the most debilitating disease conditions affecting predominantly humans but also a range of animals. The health cost to the world-wide community runs into the billions of dollars, let alone the personal cost to families.
Diabetes, mitochondrial disease and cancer, therefore, are significant conditions requiring expenditure of time and financial resources to develop new methods of treatment, prevention and diagnosis.
There is a need to identify exported or cell surface molecules involved in cell signalling. Such molecules or genes encoding same are useful targets for therapeutic and diagnostic agents.
SUMMARY
Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NO: correspond numerically to the sequence identifiers <400>l (SEQ ID NO:1), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.
It is proposed herein that the subject genes or their expression products are useful as therapeutic or diagnostic agents for conditions such as Type I diabetes, Type II diabetes or other metabolic disorders, abnormal blood pressure, abnormal triglyceride levels, obesity, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance, apoptosis, signal transduction and/or nuclear targeting.
The present invention provides, in part, isolated nucleic acid molecules and amino acid sequences which are associated with Type I diabetes, Type II diabetes, abnormal blood presume, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, generic disorders, energy imbalance and/or obesity, as well as in the modulation of apoptosis, signal transduction and/or nuclear targeting.
Isolated nucleic acid molecules contemplated by the present invention include the P. obesus sequence designated as CXS-746, which is disclosed in SEQ ID NO:1, and its cognate receptor designated herein as CXS-746R, which is disclosed in SEQ ID NO: 15. Further contemplated are the corresponding human equivalents including the human nucleic acid sequence for CXS-746 disclosed in SEQ ID NO: 18, which encodes the human CXS-746 amino acid sequence disclosed in SEQ ID NO:20. The human CXS-746R nucleic acid and amino acid sequences are disclosed in SEQ ID NOs: 19 and 20, respectively.
The present invention contemplates the use of these sequences or mammalian equivalents, including human homologs thereof, or their expression products, in the manufacture of medicaments and diagnostic agents for a range of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity as well as to modulate or detect apoptosis, signal transduction and/or for nuclear targeting.
The present invention also provides, therefore, a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof which is secreted or is a cell surface molecule and involved in metabolic signalling in a cell or group of cells.
More particularly, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NO: 15 or 19 (CXS- 746R) or a nucleotide sequence having at least about 90% similarity to all or part of SEQ ID NO:1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) and/or is capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a complementary form thereof under high stringency conditions.
The present invention also provides an isolated expression product or a derivative or homolog thereof which expression product is encoded by a nucleotide sequence the expression product identified as being a secreted or cell surface molecule and involved in metabolic signalling in a cell or group of cells.
The present invention is also directed to an isolated expression product or a derivative or homolog thereof wherein the expression product is encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ ID NO: 18 (CXS-746) or SEQ ID NO: 15 or SEQ ID NO: 19 (CXS-746R) or a nucleotide sequence having at least 90% similarity to all or part of SEQ ID NO:1 or SEQ ID NO: 18 (CXS-746) or SEQ ID NO: 15 or SEQ ID NO: 19 (CXS-746R) and/or is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO: 18 (CXS-746) or SEQ ID NO: 15 or SEQ ID NO: 19 (CXS-746R) or a complementary form thereof under high stringency conditions.
The present invention extends to analogs and mimetics of the subject expression products.
Reference to "homolog" includes other mammalian homologs such as from a human.
The genetic molecules of the present invention are referred to herein as CXS-746 (SEQ ID NO:1 or 18) or CXS-746R (SEQ ID NO:15 or 19). The expression product may be an RNA (e.g. mRNA) or a protein. Where the expression product is an RNA, the present invention extends to RNA-related molecules associated or related thereto such as RNAi or intron or exon sequences therefrom or short, interfering RNA (si-RNA) or complexes comprising same or hairpin forms thereof or short or long double stranded RNA molecules.
Even yet another aspect of the present invention relates to a composition comprising CXS- 746 or CXS-746R or its derivatives or homologs or agonists or antagonists of CXS-746 or
CXS-746R together with one or more pharmaceutically acceptable carriers and/or diluents.
In another aspect, the present invention is directed to molecules which antagonise CXS- 746 or CXS-746R or enhance the activity of CXS-746 or CXS-746R, or enhance the ability of CXS-746 to bind to its cognate receptor CXS-746R, or which bind to the cognate receptor of CXS-746, thereby mimicking the activity of CXS-746.
The present invention is particularly directed to human homologs and orthologs of the genes identified in P. obesus and their use or the use of expression products thereof or homologs, derivatives, analogs or mimetics thereof in therapy and diagnosis. An example of a human CXS-746 mRNA sequence is disclosed in SEQ ID NO: 18, with a corresponding protein sequence disclosed in SEQ ID NO:20. A human mRNA sequence specific for a CXS-746 receptor is disclosed in SEQ ID NO: 19, with an encoded expression product disclosed in SEQ ID NO:21.
Another aspect of the present invention contemplates, therefore, a method for treating a subject comprising administering to the subject a treatment effective amount of CXS-746 or CXS-746R or a derivative or homolog thereof or a genetic sequence encoding same or an analog, mimetic, agonist or antagonist of CXS-746 or CXS-746R activity or of CXS- 746 or CXS-746R gene expression for a time and under conditions sufficient to effect treatment.
In accordance with this and other aspects of the present invention, treatments contemplated herein include but are not limited to treatment of metabolic disorders such as diabetes (Types I or II), abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathies, genetic disorders, cancers, energy imbalance and/or obesity as well as modulating apoptosis, signal transduction and/or nuclear targeting. Treatment may be by the administration of a pharmaceutical composition or genetic sequences via gene therapy, antisense therapy or sense or RNAi- or si-RNA-mediated therapy. Treatment is contemplated for human subjects as well as animals such as animals important to livestock industry.
In one embodiment, expressed sequences are identified as biomarkers CXS-746 and CXS- 746R biomarkers. Nucleotide sequences corresponding to CXS-746 and CXS-746R from P.obesus are disclosed in SEQ ID NOs: 1 and 15, respectively. Human CXS-746 mRNA and amino acid sequences are disclosed in SEQ ID NOs: 18 and 20, respectively, with the human CXS-746R mRNA and amino acid sequences disclosed in SEQ ID NOs: 19 and 21, respectively.
The present invention contemplates the use of these sequences in the manufacture of diagnostic agents for metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
Accordingly, of the present invention contemplates a method for the diagnosis of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a subject, the method comprising screening for levels of a biomarker including CXS-746 or/and CXS-746R in a biological sample from the subject wherein an altered level of CXS- 746 or CXS-746R compared to a control is indicative of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
A further aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions, the diagnostic agent selected from (a) an antibody to CXS- 746 and/or CXS-746R or their derivatives, homologs, analogs or mimetics; (b) a genetic sequence capable of annealing to a nucleotide strand associated with CXS-746 and/or CXS-746R useful inter alia in PCR, RT-PCR, hybridisation, RFLP analysis and AFLP analysis.
A further aspect of the present invention is directed to a diagnostic agent for use in monitoring or diagnosing conditions such as but not limited to diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, genetic disorders, cancers, energy imbalance and/or obesity as well as monitoring apoptosis, signal transduction and/or nuclear targeting, the diagnostic agent selected from an antibody to CXS-746 or CXS-746R or its derivatives, homologs, analogs or mimetics and a genetic sequence comprising or capable of annealing to a nucleotide strand associated with CXS- 746 or CXS-746R useful inter alia in PCR, hybridization, RFLP analysis or AFLP analysis.
TABLE 1
Summary of Sequence Identifiers
Figure imgf000010_0001
24 CXS-746 P.obesus Reverse
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a graphical representation of the relative gene expression of chemerin in fractionated mesenteric adipose tissue.
Figure 2 is a graphical representation of the association of plasma chemerin levels with BMI and systolic blood pressure. Chemerin levels in human plasma samples from NGT subjects were measured by ELISA. Scatter plots are representative of a) chemerin levels versus BMI and b) chemerin levels versus systolic blood pressure.
DETAILED DESCRIPTION
Unless otherwise indicated, the subject invention is not limited to specific therapeutic components, manufacturing methods, dosage regimens, or the like, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in the subject specification, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" includes a single gene or two or more genes; "an agent" includes a single agent, as well as two or more agents; reference to "the invention" includes single or multiple aspects of the invention; as well as two or more genes; and so forth.
Reference herein to an "agent" should be understood as a reference to any proteinaceous or non-proteinaceous molecule derived from natural, recombinant or synthetic sources. Useful sources include the screening of naturally produced libraries, chemical molecule libraries as well as combinatorial libraries, phage display libraries and in vitro translation- based libraries. Particularly useful agents are those identified by the Signal Sequence Trap (SST) method. The agents may, however, be any proteinaceous molecules such as peptides, polypeptides and proteins or non-proteinaceous molecules such as nucleic acid molecules and small to large natural or synthetically derived organic and inorganic molecules and include antagonists and agonists of the proteins identified by the SST method. The agents may, therefore, also be immunoglobulins such as antibodies or fragments or synthetic or modified forms thereof. An "immunoglobulin" includes an immunoglobulin new antigen receptor (IgNAR) from cartaligenes fish such as sharks (See WO2005/118629)
The terms "agent", "compound", "active agent", "pharmacologically active agent",
"medicament", "active" and "drug" may be used interchangeably herein to refer to any agent that induces a desired pharmacological and/or physiological effect. Such effects include action on Type I or II diabetes or other metabolic disorders, obesity, mitochondrial dysfunction, myopathies, genetic disorders, energy imbalance, apoptosis, signal transduction and/or nuclear targeting. The terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "agent", "compound", "active agent", "pharmacologically active agent", "medicament", "active" and "drug" are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
The present invention is predicated in part on the identification of genes associated inter alia with a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, a myopathy, a genetic disorder or a cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an expression product or a derivative, or fragment or homolog or portion thereof wherein the nucleic acid molecule is associated with one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathies, genetic disorders or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting and is identified as being a secreted or cell surface molecule and involved in metabolic signalling in a cell or group of cells.
The expression product may be a peptide, polypeptide or protein or mRNA or may be an exon or intron spliced, for example, from an RNA construct. The expression product may also be a hairpin structure which induces or is associated with RNAi.
The present invention further provides a nucleic acid molecule composition. Therefore, the present invention provides compositions which comprise a nucleic acid molecule as disclosed in SEQ ID NO:1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R). The corresponding human expression product is referred to as CXS-746 (SEQ ID NO:20) or CXS-746R (SEQ ID NO:21).
Another aspect of the present invention provides a method for diagnosing a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting by determining the level of expression of a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NO:1 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 19 or a nucleotide sequence having at least about 90% similarity to all or part of SEQ ID NOs: 1 or 15 or 18 or 19 and/or is capable of hybridizing to SEQ ID NOs: 1 or 15 or 18 or 19 or their complementary forms under high stringency conditions at a specified temperature and wherein elevated or reduced levels of expression of one or more of these sequences is indicative of one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or or in modulating apoptosis, signal transduction and/or nuclear targeting.
The present invention also provides a method for assessing the presence or absence of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions by determining the level of expression of a nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding an expression product or a derivative or homolog thereof wherein the nucleotide sequence is as substantially set forth in SEQ ID NO:1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% similarity to all or part of SEQ ID NOs: 1 or 18 or SEQ ID NOs: 15 or 19 after alignment and/or is capable of hybridizing to one or more of SEQ ID NOs: 1 or 18 or SEQ ID NOs: 15 or 19 or their complementary forms under high stringency conditions at a specified temperature and wherein elevated or reduced levels of expression of one or more of these sequences is indicative of one or more of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
Reference herein to similarity is generally at a level of comparison of at least 15 consecutive or substantially consecutive nucleotides such as at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399 or 400 consecutive or substantially consecutive nucleotides. Preferred percentage similarities have at least about 70%, at least about 80%, at least about 90% or above. Examples include 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level,
"similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length, examples include 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (Nucl Acids Res 25:3389, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, Chapter 15, 1994-1998).
Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% and from at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 and 0.9 M for hybridization, and at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8 and 0.9 M for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide, such as 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15 M for hybridization, and at least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15 M for washing conditions. In general, washing is carried out Tm = 69.3 + 0.41 (G+C)% (Marmur and Doty J MoI Biol 5: 109, 1962). However, the Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey Eur J Biochem 46:%3, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-420C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides the nucleic acid molecule being differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the nucleic acid molecule is a nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% identity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NO: 15 or 19 (CXS- 746R) or its complementary form under high stringency conditions.
A further aspect of the present invention provides a nucleic acid molecule or derivative or homolog thereof associated with one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting the nucleic acid molecule comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a sequence encoding an expression product wherein the nucleotide sequence is substantially as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a derivative or homolog thereof or having at least about 90% identity to all or part of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R).
In another embodiment, the present invention provides a method for the diagnosis of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a subject or the probability of a subject developing metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity, the method comprising screening for levels of CXS-746 protein (SEQ ID NO:20) or mRNA/cDNA (SEQ ID NOs:l and 18) and/or CXS-746R protein (SEQ ID NO:21) or mRNA/cDNA (SEQ ID NOs: 15 and 19) the protein or mRNA/cDNA encoding same or a homolog thereof in a biological sample from the subject, wherein an altered level of CXS- 746 or CXS-746R compared to a normal control is indicative of one or more metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions or the probability of developing these conditions.
In another aspect, CXS-746 or CXS-746R is useful in the treatment, prognosis or diagnosis of abnormal blood pressure. For example, elevated levels of CXS-746 or CXS-746R are indicative of a subject having high blood pressure or a predisposition of developing high blood pressure. Accordingly, decreasing the amount of, or activity of, CXS-746 or CXS- 746R aids in decreasing high blood pressure or diminishing the probability of a subject with a predisposition of having high blood pressure from developing high blood pressure. Decreasing the levels of or activity of CXS-746 or CXS-746R may be by, for example, preventing the expression of CXS-746 or CXS-746R, blocking the activity of CXS-746 or CXS-746R, administering non-functional mimetics of CXS-746 which competitively bind to the CXS-746R or which otherwise antagonize the binding of CXS-746 to CXS-746R, or by any other means whereby the activity or expression of CXS-746 or CXS-746R is diminished. The term "expression" include transcriptional, translational and/or post- translational elements of expression of a gene to generate a protein.
Conversely, low levels of CXS-746 or CXS-746R are indicative of a subject having low blood pressure or a predisposition for developing low blood pressure. Accordingly, increasing the amount of or activity of CXS-746 or CXS-746R aids in elevating a subject's blood pressure and/or decreasing the probability of a subject with a predisposition of having low blood pressure from developing low blood pressure. Increasing the levels of or activity of CXS-746 or CXS-746R may be by, for example, adding exogenous levels of CXS-746 or CXS-746R or homologs thereof which have similar activity. Alternatively, the expression or level of the CXS-746R could be increased. Other means of increasing the levels or activity of CXS-746 would be known by one of skill in the art and include antagonizing inhibitors of CXS-746 expression or agonizing promoters of CXS-746R expression.
The CXS-746 or CXS-746R compositions and molecules of the present invention can also be used in combination therapy. For example, antagonists of CXS-746 or CXS-746R could be used in combination with high blood pressure therapies. For example, antagonists of CXS-746 or CXS-746R can be combined with one or more of Angiotensin converting enzyme (ACE) inhibitors: captopril, enalapril, lisinopril, fosinopril, quinapril, ramipril, benazepril, perindopril, trandolapril, moexipril; Angiotensin II receptor antagonists: irbesartan, losartan, valsartan, candesartan; Alpha blockers: doxazosin, terazosin, prazosin; Beta blockers: atenolol, labetalol, metoprolol, propanolol, acebutolol, betaxolol, bisoprolol, carvedilol, nadolol, penbutolol, sotalol, timolol; Calcium channel blockers: amlodipine, diltiazem, verapamil, felodipine, isradipine, nicardipine, nifedipine; Diuretics: hydrochlorothiazide (HCTZ), bendroflumethiazide, chlortalidone, furosemide, bumetanide, torsemide, metolazone, spironolactone and combination products (which usually contain HCTZ and one other drug)
Conversely, agonists of CXS-746 or CXS-746R could be used in combination with compounds known to increase blood pressure levels. For example, agonists of CXS-746 or CXS-746R can be combined with one or more of Systemic vasoconstrictors: Antihistamines, Adrenaline, Asymmetric dimethylarginine, Adenosine triphosphate, Catecholamines, Cocaine, Decongestants, Endothelin, Ergine, Phenylephrine, Pseudoephedrine, Neuropeptide Y, Norepinephrine, Tetrahydrozoline hydrochloride, Thromboxane, Fludrocortisone and Midodrine.
As used herein, a "control" is defined as a subject having one or more of the following characteristics: lean, young, no genetic predisposition for Type II diabetes (i.e. no family history of Type II diabetes abnormal blood pressure or abnormal triglyceride levels), normoglycemic and/or normoinsulinemic. As used herein, "abnormal blood pressure" is defined as blood pressure which is either below or above normal ranges based on a subjects sex and age.
As used herein, "abnormal triglyceride levels" is defined as triglyceride levels which are either below or above normal ranges based on a subjects sex and age.
In the practice of this embodiment, one may use a nucleic acid segment that is complementary to the full length of the mRNA encoding CXS-746 or CXS-746R, or one may use a smaller segment that is complementary to a portion of the RNA. Such smaller segments may be from about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 25, about 30, about 50, about 75, about 100 or even several hundred bases in length and may be contained in larger segments that provide other functions such as promoters, restriction enzyme recognition sites, or other expression or message processing or replication functions. In related embodiments such probes are designed to selectively hybridize to a CXS-746 or CXS- 746R or product thereof. A product thereof would include a DNA or RNA strand that is complementary to the mRNA and thus a useful probe would include both the sense and antisense orientations of a particular sequence. Also preferred are the use of probes or primers that are designed to selectively hybridize to a nucleic acid segment having a sequence of SEQ ID NO:1 or SEQ ID NO:15 or SEQ ID NO:18 or SEQ ID NO:19 or the complements thereof.
The methods of the present invention may also include determining the amount of hybridized product. Such determination may be by direct detection of a labeled hybridized probe, such as by use of a radioactive, fluorescent or other tag on the probe, or it may be by use of an amplification of a target sequence, and quantification of the amplified product. A preferred method of amplification is a reverse transcriptase polymerase chain reaction (RT-PCR) as described herein. RT-PCR is a preferred method of detection, diagnosis, and/or prognosis of disease or cancer. In the practice of such a method, amplification may comprise contacting the target ribonucleic acids with a pair of amplification primers designed to amplify CXS-746 or CXS-746R mRNA, or even contacting the ribonucleic acids with a pair of amplification primers designed to amplify a nucleic acid segment comprising the nucleic acid sequence or complement of SEQ ID NOs: 1 or 15 or 18 or 19.
Diagnostic methods may be based upon the steps of obtaining a biological sample from a subject or patient, contacting the sample nucleic acids from the biological sample with an isolated CXS-746 or CXS-746R nucleic acid segment under conditions effective to allow hybridization of substantially complementary nucleic acids, and detecting, and optionally further characterizing, the hybridized complementary nucleic acids thus formed.
The methods may involve in situ detection of sample nucleic acids located within the cells of the sample. The sample nucleic acids may also be separated from the cell prior to contact. The sample nucleic acids may be DNA or RNA.
The methods may involve the use of isolated CXS-746 or CXS-746R nucleic acid segments that comprises a radio-, enzymatic- or fluorescent-ly detectable label, wherein the hybridized complementary nucleic acids are detected by detecting the label. In related embodiments such probes are designed to selectively hybridize to CXS-746 or CXS-746R mRNA or product thereof. A product thereof would include a DNA or RNA strand that is complementary to the mRNA and thus a useful probe would include both the sense and antisense orientations of a particular sequence. Also preferred are the use of probes or primers that are designed to selectively hybridize to a nucleic acid segment having a sequence of SEQ ID NO:1, SEQ ID NO:15, SEQ ID NO:18 or SEQ ID NO:19 or the complements thereof or fragments thereof.
In the practice of the subject invention, some methods may involve detection of expression of a polypeptide product and particularly the expression product encoded by SEQ ID NO:1 or 15 or 18 or 19. Such detection may be by any means known in the art and may include an immunoassay, an immunoaffinity purification or detection, an ELISA, or an radioimmunoassay, for example. The expression pattern of CXS-746 and/or CXS-746R has been determined, inter alia, to indicate an involvement in the regulation of one or more processes associated with one or more metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions. The subject nucleic acid molecules are preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence. A genomic sequence may also comprise exons and introns. A genomic sequence may also include a promoter region or other regulatory regions. The present invention extends, however, to expression products such as mRNA, introns and exons which may also be involved in genetic networking, whether or not they are translated into proteins. Furthermore, the expression products may include complexes comprising RNA or may comprising RNAi or RNAi-type molecules.
A homolog is considered to be a CXS-746 or CXS-746R gene from another animal species. The present invention extends to the homologous gene, as determined by nucleotide sequence and/or amino acid sequences and/or function, from primates, including humans, marmosets, orangutans and gorillas, livestock animals (e.g. cows, sheep, pigs, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. cats, dogs) and captured wild animals (e.g. rodents, foxes, deer, kangaroos). The present invention also contemplates deimmunized forms of the expression products from one species relative to another species. In one preferred embodiment, the deimmunized form of the expression product is a mammalianized form relative to a particular target animal. In a most preferred embodiment where the target mammal is a human, the present invention contemplates use of a humanized form of a non-human expression product.
CXS-746 or CXS-746R and their derivatives and homologs may be in isolated or purified form and/or may be ligated to a vector such as an expression vector. Expression may be in a eukaryotic cell line (e.g. mammalian, insect or yeast cells) or in microbial cells (e.g. E. coli) or both.
By "isolated" is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means. The nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure. The nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells). The nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions. The nucleic acid molecule may also be part of a vector, such as an expression vector.
In one embodiment, the nucleotide sequence corresponding to CXS-746 is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:1 or a derivative or homolog thereof including a nucleotide sequence having similarity to SEQ ID NO:1 or an mRNA sequence as set forth in SEQ ID NO: 18. In a related embodiment, the nucleotide sequence corresponding to CXS-746R is a cDNA sequence set forth in SEQ ID NO: 15 or an mRNA sequence as set forth in SEQ ID NO: 19.
The nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E. coli) or a eukaryotic cell (e.g. yeast cells, fungal cells, insect cells, mammalian cells or plant cells). The nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions. The nucleic acid molecule may also be part of a vector, such as an expression vector.
The derivatives of the nucleic acid molecule of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co- suppression (e.g. RNAi) and fusion nucleic acid molecules. Ribozymes and DNA enzymes are also contemplated by the present invention directed to CXS-746 or CXS-746R or its mRNA. Derivatives and homologs of CXS-746 or CXS-746R are conveniently encompassed by those nucleotide sequences capable of hybridizing to SEQ ID NO:1 or 15 or 18 or 19 or their complementary forms under high stringency conditions.
The present invention extends to expression products of CXS-746 or CXS-746R. The expression products can be proteins or mutants, derivatives or homologs thereof as well as a range of RNA molecules.
An expression product includes an RNA molecule such as a mRNA transcript as well as a protein. Some genes are non-protein encoding genes and produce mRNA or other RNA type molecules and are involved in regulation by RNA:DNA, RNA:RNA or RNA:protein interaction. The RNA (e.g. mRNA) may act directly or via the induction of other molecules such as RNAi or via products mediated from splicing events (e.g. exons or introns). Other genes encode mRNA transcripts which are then translated into proteins. A protein includes a polypeptide. The differentially expressed nucleic acid molecules, therefore, may encode mRNAs only or, in addition, proteins. Both mRNAs and proteins are forms of "expression products".
Derivatives include fragments, parts, portions, mutants and variants from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of CXS-746 or CXS-746R. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.
Chemical and functional equivalents of CXS-746 or CXS-746R should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.
The derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
Another aspect of the present invention provides an isolated protein or other expression product or a derivative or homolog thereof which is associated with one or more of diabetes, mitochondrial disease, myopathy, abnormal blood pressure, abnormal triglyceride levels, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
In one aspect of the present invention, there is provided an isolated protein or derivative or homolog fragment thereof wherein the protein or polypeptide comprises an amino acid sequence disclosed in SEQ ID NO:20 (CXS-746) and/or SEQ ID NO:21 (CXS-746R or an amino acid sequence encoded by SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or an amino acid sequence having at least 90% similarity to all or part thereof and wherein the protein or expression product is associated with one or more of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, a genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
Reference herein to CXS-746 or CXS-746R includes reference to isolated or purified naturally occurring CXS-746 or CXS-746R protein or expression product molecules as well as any derivatives or homologs thereof. Derivatives include parts, fragments and portions of CXS-746 or CXS-746R as well as single and multiple amino acid substitutions, deletions and/or additions to CXS-746 or CXS-746R. A derivative of CXS-746 or CXS- 746R is conveniently encompassed by molecules encoded by a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 15 or 18 or 19 under high stringency conditions at a specified temperature.
As used herein a fragment includes a part, portion, region, domain, N-terminal fragment, a C-terminal fragment, an internal fragments and/or an enzymatically cleaved protein, such as by a membrane cleaving protease.
Other derivatives of CXS-746 or CXS-746R include chemical analogs. Analogs of CXS- 746 or CXS-746R contemplated herein include, but are not limited to, modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confirmational constraints on the proteinaceous molecule or their analogs.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 2.
TABLE 2 Codes for non-conventional amino acids
Non-conventional Code Non-conventional Code amino acid amino acid
α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyc lopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile
D-alanine Dal L-N-methylleucine Nmleu
D-arginine Darg L-N-methyllysine Nmlys
D-aspartic acid Dasp L-N-methylmethionine Nmmet
D-cysteine Dcys L-N-methylnorleucine Nmnle
D-glutamine DgIn L-N-methylnorvaline Nmnva
D-glutamic acid DgIu L-N-methylornithine Nmorn
D-histidine Dhis L-N-methylphenylalanine Nmphe
D-isoleucine DiIe L-N-methylproline Nmpro
D-leucine Dleu L-N-methylserine Nmser
D-lysine Dlys L-N-methylthreonine Nmthr
D-methionine Dmet L-N-methyltryptophan Nmtrp
D-ornithine Dorn L-N-methyltyrosine Nmtyr
D-phenylalanine Dphe L-N-methylvaline Nmval
D-proline Dpro L-N-methylethylglycine Nmetg
D-serine Dser L-N-methyl-t-butylglycine Nmtbug
D-threonine Dthr L-norleucine NIe Non-conventional Code Non-conventional Code amino acid amino acid
D-tryptophan Dtrp L-norvaline Nva
D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib
D-valine Dval α-methyl-γ-aminobutyrate Mgabu
D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa
D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen
D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap
D-α-methylaspartate Dmasp α-methylpenicillamine Mpen
D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine NgIu
D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg
D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn
D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu
D-α-methylleucine Dmleu α-napthylalanine Anap
D-α-methyllysine Dmlys N-benzylglycine Nphe
D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine NgIn
D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn
D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu
D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp
D-α-methylserine Dmser N-cyclobutylglycine Ncbut
D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep
D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex
D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec
D-α-methylvaline Dmval N-cylcododecylglycine Ncdod
D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct
D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro
D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund
D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm
D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe Non-conventional Code Non-conventional Code amino acid amino acid
D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
D-N-methylglutamate Dnmglu N-(I -hydroxy ethyl)glycine Nthr
D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser
D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis
D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp
D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu
N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet
D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen
N-methylglycine NaIa D-N-methylphenylalanine Dnmphe
N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro
N-(I -methylpropyl)glycine Nile D-N-methylserine Dnmser
N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr
D-N-methyltryptophan Dnmtrp N-(I -methylethyl)glycine Nval
D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr
L-?-butylglycine Tbug N-(thiomethyl)glycine Ncys
L-ethylglycine Etg penicillamine Pen
L-homophenylalanine Hphe L-α-methylalanine Mala
L-α-methylarginine Marg L-α-methylasparagine Masn
L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug
L-α-methylcysteine Mcys L-methylethylglycine Metg
L-α-methylglutamine MgIn L-α-methylglutamate MgIu
L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe
L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet
L-α-methylleucine Mleu L-α-methyllysine Mlys
L-α-methylmethionine Mmet L-α-methylnorleucine MnIe Non-conventional Code Non-conventional Code amino acid amino acid
L-α-methylnorvaline Mnva L-α-methylornithine Morn
L-α-methylphenylalanine Mphe L-α-methylproline Mpro
L-α-raethylserine Mser L-α-methylthreonine Mthr
L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr
L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe
N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine
1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Cα and N α-methylamino acids, introduction of double bonds between Cα and Cβ atoms of amino acids and the formation of cyclic peptides or analogs by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
All such modifications may also be useful in stabilizing the CXS-746 or CXS-746R molecules for use in in vivo administration protocols or for diagnostic purposes.
As stated above, the expression product may be an RNA or protein. The term "protein" should be understood to encompass peptides, polypeptides and proteins. The protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Reference hereinafter to a "protein" includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
In one embodiment, the expression product is encoded by a sequence of nucleotides as set forth in SEQ ID NOs: 1 or 15 or 18 or 19 or a derivative or homolog thereof including a nucleotide sequence having at least about 90% identity to SEQ ID NOs: 1 or 15 or 18 or 19.
Higher similarities are also contemplated by the present invention such as greater than 80% or 90% or 95% or 96% or 97% or 98% or 99% or above. Further examples include 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100%.
Another aspect of the present invention is directed to an isolated expression product that is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the protein is selected from the list consisting of:
(i) an expression product encoded by a nucleotide sequence substantially as set forth in
SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a derivative or homolog thereof or a sequence encoding an amino acid sequence having at least about 90% similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of the protein;
(ii) an expression product encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a derivative or homolog thereof; and (iii) an expression product as set forth in SEQ ID NO:20 (CXS-746) or SEQ ID NO:21 (CXS-746R).
The protein of the present invention is preferably in isolated form. By "isolated" is meant a protein having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject protein, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100% of subject protein relative to other components as determined by molecular weight, amino acid sequence or other convenient means. The protein of the present invention may also be considered, in a preferred embodiment, to be biologically pure.
The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by- nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, GIy, VaI, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, GIu, Asn, GIn, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
The nucleotide sequence or amino acid sequence of the present invention may correspond to exactly the same sequence of the naturally occurring gene (or corresponding cDNA) or protein or may carry one or more nucleotide or amino acid substitutions, additions and/or deletions. The nucleotide sequences set forth in SEQ ID NOs: 1 or 18 correspond to the genes referred to herein as CXS-746 and SEQ ID NO: 15 and 19 correspond to CXS-746R. The corresponding expression products are CXS-746 (SEQ ID NO:20) and CXS-746R (SEQ ID NO:21). Reference herein to CXS-746 or CXS-746R includes, where appropriate, reference to the genomic gene or cDNA as well as any naturally occurring or induced derivatives. Apart from the substitutions, deletions and/or additions to the nucleotide sequence, the present invention further encompasses mutants, fragments, parts and portions of the nucleotide sequence corresponding to CXS-746 or CXS-746R.
The identification of CXS-746 or CXS-746R permits the generation of a range of therapeutic molecules capable of modulating expression of CXS-746 or CXS-746R or modulating the activity of CXS-746 or CXS-746R. Modulators contemplated by the present invention includes agonists and antagonists of CXS-746 or CXS-746R expression. Antagonists of CXS-746 or CXS-746R expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter activity or which interfere with negative regulatory mechanisms. Antagonists of CXS-746 or CXS-746R include antibodies and inhibitor peptide fragments. All such molecules may first need to be modified to enable such molecules to penetrate cell membranes. Alternatively, viral agents may be employed to introduce genetic elements to modulate expression of CXS-746 or CXS-746R.
The present invention contemplates, therefore, a method for modulating expression of
CXS-746 or CXS-746R in a mammal, the method comprising contacting the CXS-746 or CXS-746R gene with an effective amount of a modulator of CXS-746 or CXS-746R expression for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate the expression of CXS-746 or CXS-746R. For example, a nucleic acid molecule encoding CXS-746 or CXS-746R or a derivative or homolog thereof may be introduced into a cell to enhance the ability of that cell to produce CXS-746 or CXS-746R, conversely, CXS-746 or CXS-746R antisense sequences such as oligonucleotides may be introduced to decrease the availability of CXS-746 or CXS-746R molecules.
Another aspect of the present invention contemplates a method of modulating activity of CXS-746 or CXS-746R in a mammal, the method comprising administering to the mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease CXS-746 or CXS-746R activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of CXS-746 or CXS-746R or their ligands.
Modulating levels of CXS-746 or CXS-746R expression is important in the treatment of a range of conditions including metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting. The present invention has application in the treatment of humans as well as in the veterinary and animal husbandry industries. Accordingly, subjects contemplated for treatment in accordance with the present invention includes, but is not limited to humans, primates, livestock animals (e.g. pigs, sheep, cows, horses, donkeys), laboratory test animals (e.g. mice, rats, guinea pigs, hamsters, rabbits), companion animals (e.g. dogs, cats) and captured wild animals (e.g. foxes, kangaroos, deer). A particularly preferred host is a human, primate or livestock animal.
Accordingly, the present invention contemplates therapeutic and prophylactic uses of CXS-746 or CXS-746R amino acid and nucleic acid molecules in addition to CXS-746 or CXS-746R agonistic and antagonistic agents.
The present invention contemplates, therefore, a method of modulating expression of CXS- 746 or CXS-746R in a mammal, the method comprising contacting the CXS-746 or CXS- 746R genes with an effective amount of an agent for a time and under conditions sufficient to up-regulate, down-regulate or otherwise modulate expression of CXS-746 or CXS- 746R. For example, antisense sequences such as oligonucleotides may be utilized.
Conversely, nucleic acid molecules encoding CXS-746 or CXS-746R or derivatives thereof may be introduced to up-regulate one or more specific functional activities.
Another aspect of the present invention contemplates a method of modulating activity of CXS-746 or CXS-746R in a subject, the method comprising administering to the subject a modulating effective amount of an agent for a time and under conditions sufficient to increase or decrease CXS-746 or CXS-746R activity.
Modulation of activity by the administration of an agent to a mammal can be achieved by one of several techniques, including but in no way limited to introducing into the mammal a proteinaceous or non-proteinaceous molecule which:
(i) modulates expression of CXS-746 or CXS-746R;
(ii) functions as an antagonist of CXS-746 or CXS-746R; and
(iii) functions as an agonist of CXS-746 or CXS-746R.
Yet another aspect of the present invention contemplates the use an isolated nucleic acid molecule comprising a sequence of nucleotides the nucleic acid molecule which has is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the isolated molecule is encoded by a nucleic acid molecule selected from the list consisting of a nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% identity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS -746R) or its complementary form under high stringency conditions in the manufacture of a medicament for the treatment of one or more diseases and/or conditions.
The proteinaceous molecule may be derived from natural or recombinant sources including fusion proteins or following, for example, natural product screening or the screening of chemical libraries. The non-proteinaceous molecule may be, for example, a nucleic acid molecule or may be derived from natural sources, such as for example natural product screening or may be chemically synthesized. The present invention contemplates chemical analogs of CXS-746 or CXS-746R or small molecules capable of acting as agonists or antagonists. Chemical agonists may not necessarily be derived from CXS-746 or CXS- 746R but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing CXS-746 or CXS-746R from carrying out their normal biological functions. Antagonists include monoclonal antibodies, antisense and sense nucleic acids which prevent transcription or translation of CXS-746 or CXS-746R genes or mRNA in mammalian cells. Modulation of expression may also be achieved utilizing antigens, RNA, RNAi, ribosomes, DNAzymes, RNA aptamers or antibodies.
The proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the expression of CXS-746 or CXS-746R or the activity of CXS-746 or CXS- 746R, the molecule acts directly if it associates with CXS-746 or CXS-746R to modulate expression or activity, the molecule acts indirectly if it associates with a molecule other than CXS-746 or CXS-746R which other molecule either directly or indirectly modulates the expression or activity of CXS-746 or CXS-746R. Accordingly, the method of the present invention encompasses the regulation of CXS-746 or CXS-746R expression or activity via the induction of a cascade of regulatory steps.
The molecules which may be administered to a mammal in accordance with the present invention may also be linked to a targeting means such as a monoclonal or polyclonal antibody, which provides specific delivery of these molecules to the target cells. A further aspect of the present invention relates to the use of the invention in relation to mammalian disease conditions. For example, the present invention is particularly useful but in no way limited to use in a therapeutic or prophylactic treatment of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
Accordingly, another aspect of the present invention relates to a method of treating a mammal suffering from a condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, the method comprising administering to the mammal an effective amount of an agent for a time and under conditions sufficient to modulate the expression of CXS-746 or CXS-746R or sufficient to modulate the activity of CXS-746 or CXS-746R.
In another aspect, the present invention relates to a method of treating a mammal suffering from a disease condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting, the method comprising administering to the mammal an effective amount of CXS-746 or CXS-746R.
As used herein "myopathy" refers to any abnormal conditions or disease of the muscle tissues, which include the muscles over our bones (skeletal muscle) and the heart (cardiac muscle).
Mitochondrial dysfunction relates to abnormalities in mitochondria. Mitochondria are part of the cell (organelle) that is responsible for energy production. The organelle consists of two sets of membranes, a smooth continuous outer coat and an inner membrane arranged in tubules or in folds that form plate-like double membranes (cristae). Mitochondria are the principal energy source of the cell, containing the cytochrome enzymes of terminal electron transport and the enzymes of the citric acid cycle, fatty acid oxidation, and oxidative phosphorylation. They are responsible for converting nutrients into energy as well as many other specialized tasks. Mitochondria are complex organelles located in virtually all cells of the body. A large degree of their complexity is due to the fact that over 1000 proteins are located in the mitochondria. Thirteen of these proteins are encoded by the mitochondrial DNA (mtDNA), while the remainder are nuclear-encoded, and imported into the mitochondria.
Symptoms of mitochondrial dysfunction include weakness (which may be intermittent), neuropathic pain, absent reflexes, gastrointestinal problem (gastroesophogeal reflux, delayed gastric emptying, constipation, pseudo-obstruction), fainting, absent or excessive sweating resulting in temperature regulation problems, hypotonia, cramping and muscle pain, proximal renal tubular wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes, cardiac conduction defects (heart blocks) and cardiomyopathy, hypoglycemia (low blood sugar) and liver failure, visual loss and blindness, hearing loss and deafness, and diabetes and exocrine pancreatic failure (inability to make digestive enzymes).
There may also be systemic problems associated with mitochondrial dysfunction, including failure to gain weight, short stature, fatigue, respiratory problems.
Mitochondrial defects have been linked to Alzheimer's, Parkinson's, diabetes, autism, and the aging process. Other disease associated with mitochondrial dysfunction include, LIC
(Lethal Infantile Cardiomyopathy), Beta-oxidation Defects, COX Deficiency,
Mitochondrial Cytopathy, Alpers Disease, Barth syndrome, Carnitine-Acyl-Carnitine
Deficiency, Carnitine Deficiency, Co-Enzyme QlO Deficiency, Complex I Deficiency,
Complex II Deficiency, Complex III Deficiency, Complex IV Deficiency, Complex V Deficiency, CPEO, CPT I Deficiency, Glutaric Aciduria Type II, KSS, lactic acidosis,
LCAD, LCHAD, Leigh Disease, LHON, Luft Disease, MAD, MCA, MELAS, MERRP, mitochondrial DNA depletion, Mitochondrial Encephalopathy, MNGIE, NARP, Pearson Syndrome, Pyruvate Carboxylase Deficiency, Pyruvate Dehydrogenase Deficiency, SCAD, SCHAD and VLCAD.
Alpers Disease, or Progressive Infantile Poliodystrophy, includes symptoms such as seizures, dementia, spasticity, blindness, liver dysfunction, and cerebral degeneration. (Luft Proceedings of the National Academy of Sciences of the United States of America 91(19):873\-8, 1994).
Barth syndrome or LIC (Lethal Infantile Cardiomyopathy) is an X-linked recessive disorder the symptoms of which include skeletal myopathy, cardiomyopathy, short stature, and neutropenia. (Christodoulou American Journal of Medical Genetics 50(3):255-64, 1994).
Carnitine- Acyl-Carnitine Deficiency is an autosomal recessive disorder, the symptoms of which are seizures, apnea, bradycardia, vomiting, lethargy, coma, enlarged liver, limb weakness, myoglobin in the urine, Reye-like symptoms triggered by fasting.
Carnitine Deficiency is an autosomal recessive disease, the symptoms of which include Cardiomyopathy, failure to thrive, and altered consciousness or coma, sometimes hypotonia.
Co-Enzyme QlO Deficiency is most likely an autosomal recessive disease, the symptoms of which include Encephalomyopathy, mental retardation, exercise intolerance, ragged-red fibers, and recurrent myoglobin in the urine.
Complex I Deficiency or NADH dehydrogenase (NADH-CoQ reductase) deficiency is an autosomal disease, the symptoms of which are classified by three major forms: (1) fatal infantile multisystem disorder, characterized by developmental delay, muscle weakness, heart disease, congenital lactic acidosis, and respiratory failure; (2) myopathy beginning in childhood or in adult life, manifesting as exercise intolerance or weakness. Elevated lactic acid common; and (3) mitochondrial encephalomyopathy (including MELAS), which may begin in childhood or adult life and consists of variable combinations of symptoms and signs, including ophthalmoplegia, seizures, dementia, ataxia, hearing loss, pigmentary retinopathy, sensory neuropathy, and uncontrollable movements. In addition, this disorder may cause Leigh Syndrome.
Complex II Deficiency or Succinate dehydrogenase deficiency, the symptoms of which include encephalomyopathy and various manifestations, including failure to thrive, developmental delay, hyoptonia, lethargy, respiratory failure, ataxia, myoclonus and lactic acidosis. May also cause Leigh Syndrome.
Complex III Deficiency or Ubiquinone-cytochrome c oxidoreductase deficiency, symptoms of which are categorized in four major forms: (1) fatal infantile encephalomyopathy, congenital lactic acidosis, hypotonia, dystrophic posturing, seizures, and coma. Ragged-red fibers common; (2) encephalomyopathies of later onset (childhood to adult life): various combinations of weakness, short stature, ataxia, dementia, hearing loss, sensory neuropathy, pigmentary retinopathy, and pyramidal signs. Ragged-red fibers common. Possible lactic acidosis; (3) myopathy, with exercise intolerance evolving into fixed weakness. Ragged-red fibers common. Possible lactic acidosis; and (4) infantile histiocytoid cardiomyopathy.
Complex IV Deficiency or Cytochrome c oxidase deficiency is caused by a defect in Complex IV of the respiratory chain, the symptoms of which can be categorized in two major forms: (1) encephalomyopathy, which is typically normal for the first 6 to 12 months of life and then show developmental regression, ataxia, lactic acidosis, optic atrophy, ophthalmoplegia, nystagmus, dystonia, pyramidal signs, respiratory problems and frequent seizures; and (2) myopathy: Two main variants: (a) Fatal infantile myopathy: may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged-red fibers, respiratory failure, and kidney problems: and (b) Benign infantile myopathy: may begin soon after birth and accompanied by hypotonia, weakness, lactic acidosis, ragged- red fibers, respiratory problems, but (if the child survives) followed by spontaneous improvement.
Complex V Deficiency or ATP synthase deficiency includes symptoms such as slow, progressive myopathy.
CPEO or Chronic Progressive External Ophthalmoplegia Syndrome includes symptoms such as visual myopathy, retinitis pigmentosa, dysfunction of the central nervous system. It is caused by single mitochondrial DNA deletions, with Mitochondrial DNA point mutation, A3243G being the most common (Luft 1994 Supra).
CPT I Deficiency is an autosomal recessive disease and includes symptoms such as enlarged liver and recurrent Reye-like episodes triggered by fasting or illnesses.
CPT II Deficiency is an autosomal recessive disease, the symptoms of which include exercise intolerance, fasting intolerance, muscle pain, muscle stiffness, and myoglobin in the urine and in infants, Reye-like syndrome, enlarged liver, hypoglycemia, enlarged heart and cardiac arrhythmia.
KSS or Kearns-Sayre Syndrome, in most cases is caused by large mitochondria DNA deletions. Symptoms associated with KSS include progressive external ophthalmoplegia, pigmentary retinopathy, heart block, and high cerebrospinal protein.
Lactic Acidosis is associated with the accumulation of lactic acid due to its production exceeding its use. Chronic lactic acidosis is a common symptom of mitochondrial disease.
LCAD or Long-Chain Acyl-CoA Dehydrogenase Deficiency, is an autosomal recessive disorder, which causes a fatal syndrome, in infants, typified by failure to thrive, enlarged liver, enlarged heart, metabolic encephalopathy and hypotonia. LCHAD is an autosomal recessive disorder, characterized by symptoms such as encephalopathy, liver dysfunction, cardiomyopathy, and myopathy. Also pigmentary retinopathy and peripheral neuropathy.
Leigh Disease or Syndrome or Subacute Necrotizing Encephalomyelopathy is characterized by symptoms such as Seizures, hypotonia, fatigue, nystagmus, poor reflexes, eating and swallowing difficulties, breathing problems and poor motor function.
LHON or Leber Hereditary Optic Neuropathy is caused by mitochondrial DNA point mutations, including G14459A, among others. Symptoms associated with LHON include primarily blindness in young men. Less common symptoms include mild dementia, ataxia, spasticity, peripheral neuropathy and heart conduction defects.
Luft Disease is characterized by symptoms such as hypermetabolism, with fever, heat intolerance, profuse perspiration, polyphagia, polydipsia, ragged-red fibers, and resting tachycardia. In addition to exercise intolerance with mild weakness.
MAD or Glutaric Aciduria Type II or multiple Acyl-CoA Dehydrogenase Deficiency is caused by defects of the flavoproteins responsible for transferring electrons (ETF or ETF- dehydrogenase) therefore affecting the function of all six ETF-funneling acyl-CoA dehydrogenases
MCAD or Medium-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, which afflicts infants or young children with episodes of encephalopathy, enlarged and fatty degeneration of the liver, and low carnitine in the blood.
MELAS or Mitochondrial Encephalomyopathy Lactic Acidosis and Strokelike Episodes is caused by mitochondrial DNA point mutations, the most common of which is A3243G. It is characterized by symptoms: Short stature, seizures, stroke-like episodes with focused neurological deficits, recurrent headaches, cognitive regression, disease progression ragged-red fibers (Koo et al. Annals of Neurology 34(l):25-32, 1993). MERRF or Myoclonic Epilepsy and Ragged-Red Fiber Disease is caused by the mitochondrial DNA point mutations A8344G and T8356C. Its symptoms include myoclonus, epilepsy, progressive ataxia, muscle weakness and degeneration, deafness and dementia (Luft 1994 Supra).
There are three forms of mitochondrial DNA Depletion. These include: (1) congenital myopathy: Neonatal weakness, hypotonia requiring assisted ventilation, possible renal dysfunction. Severe lactic acidosis. Prominent ragged-red fibers. Death due to respiratory failure usually occurs prior to one year of age; (2) infantile myopathy: Following normal early development until one year old, weakness appears and worsens rapidly, causing respiratory failure and death typically within a few years; and (3) hepatopathy, enlarged liver and intractable liver failure, myopathy, severe lactic acidosis. Death is typical within the first year.
Mitochondrial Encephalopathy also includes Encephalomyopathy and Encephalomyelopathy.
MNGIE or Myoneurogastrointestinal Disorder and Encephalopathy, include symptoms such as progressive external ophthalmoplegia, limb weakness, peripheral neuropathy, digestive tract disorders, leukodystrophy, lactic acidosis and ragged red fibers.
NARP or Neuropathy, Ataxia, and Retinitis Pigmentosa is caused by mitochondrial DNA point mutations in genes associated with Complex V, including T8993G, (also T8993C by some researchers). Leigh Syndrome may result if the percentage of mutation is high enough.
Pearson Syndrome is characterized by symptoms associated with bone marrow and pancreas dysfunction. It is caused by single mitochondrial DNA deletions. Inheritance is usually sporadic. Those who survive infancy usually develop Kearns-Sayre Syndrome. Pyruvate Carboxylase Deficiency is an autosomal recessive disorder, the symptoms of which include lactic acidosis, hypoglycemia, severe retardation, failure to thrive, in addition to seizures and spasticity.
Pyruvate Dehydrogenase Deficiency is characterized by symptoms such as lactic acidosis, ataxia, pyruvic acidosis, spinal and cerebellar degeneration. Less common symptoms include agenesis of the corpus callosum and lesions in the basal ganglia, cerebellum, and brain stem, growth delay, hypotonia, seizures and polyneuropathy.
SCAD or Short-Chain Acyl-CoA Dehydrogenase Deficiency, is an autosomal recessive disorder characterized by symptoms such as failure to thrive, developmental delay and hypoglycemia.
SCHAD is an autosomal recessive disorder, characterized by encephalopathy and possibly liver disease or cardiomyopathy.
VLCAD or Very Long-Chain Acyl-CoA Dehydrogenase Deficiency is an autosomal recessive disorder, characterized by various manifestations, ranging from fatal infantile encephalopathy to recurrent myoglobin in the urine, similar to the myopathic form of CPT II deficiency.
An "effective amount" means an amount necessary at least partly to attain the desired physiological effect or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition of the individual to be treated, the taxonomic group of the individual to be treated, the degree of protection desired, the formulation of the vaccine, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
In accordance with these methods, CXS-746 or CXS-746R or agents capable of modulating the expression or activity of the molecules may be co-administered with one or more other compounds or other molecules. By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.
In yet another aspect, the present invention relates to the use of an agent capable of modulating the expression of CXS-746 or CXS-746R or a derivative or homolog thereof in the manufacture of a medicament for the treatment of a condition characterized by a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer.
In still yet another aspect, the present invention relates to the use of an agent capable of modulating the activity of CXS-746 or CXS-746R or a derivative or homolog thereof in the manufacture of a medicament for the treatment of a condition characterized by diabetes, mitochondrial dysfunction, myopathy, genetic disorder, abnormal blood pressure, abnormal triglyceride levels, or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
A further aspect of the present invention relates to the use of CXS-746 or CXS-746R or derivative or homolog thereof in the manufacture of a medicament for the treatment of a condition characterized by a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
Still yet another aspect of the present invention relates to agents for use in modulating the expression of CXS-746 or CXS-746R or a derivative or homolog thereof. A further aspect relates to agents for use in modulating CXS-746 or CXS-746R a derivative or homolog thereof activity.
Still another aspect of the present invention relates to CXS-746 or CXS-746R or derivative or homolog thereof for use in treating a condition characterized by one or more symptoms of a metabolic condition such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
In a related aspect of the present invention, the mammal undergoing treatment may be a human or an animal in need of therapeutic or prophylactic treatment.
The terms "treating" and "treatment" as used herein refer to a reduction in the severity and/or frequency of one ore more symptoms associated with inter alia diabetes, mitochondrial dysfunction, myopathy, genetic disorder, abnormal blood pressure, abnormal triglyceride levels, or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting.
"Treating" a subject may involve prevention of the disorder or disease condition or adverse physiological event in a susceptible individual as well as treatment of a clinically symptomatic individual by inhibiting a disease or disorder as well as decreasing the severity of one or more symptoms associated with the disease or disorder. Generally, such conditions involve, weakness (which may be intermittent), neuropathic pain, absent reflexes, gastrointestinal problem (gastroesophogeal reflux, delayed gastric emptying, constipation, pseudo-obstruction), fainting, absent or excessive sweating resulting in temperature regulation problems weakness, hypotonia, cramping, muscle pain, proximal renal tubular wasting resulting in loss of protein, magnesium, phosphorous, calcium and other electrolytes, cardiac conduction defects (heart blocks) and cardiomyopathy, hypoglycemia (low blood sugar) and liver failure, visual loss and blindness, hearing loss and deafness, diabetes and exocrine pancreatic failure (inability to make digestive enzymes), mitochondrial dysfunction, including failure to gain weight, short statue, fatigue and respiratory problems.
Accordingly, the present invention contemplates in one embodiment a composition comprising a modulator of CXS-746 or CXS-746R expression or CXS-746 or CXS-746R activity and one or more pharmaceutically acceptable carriers and/or diluents. In another embodiment, the composition comprises CXS-746 or CXS-746R or a derivative or homolog thereof and one or more pharmaceutically acceptable carriers and/or diluents.
For brevity, all such components of such a composition are referred to as "active components".
The compositions of active components in a form suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or other medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active components in the required amount in the appropriate solvent with optionally other ingredients, as required, followed by sterilization by, for example, filter sterilization, irradiation or other convenient means. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When CXS-746 or CXS-746R themselves are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.
The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.
Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active component may be compounded for convenient and effective administration in sufficient amounts with a suitable pharmaceutically acceptable carrier in dosage unit form. A unit dosage form can, for example, contain the principal active component in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the the ingredients.
In general terms, effective amounts of CXS-746 or CXS-746R will range from 0.01 ng/kg/body weight to above 10,000 mg/kg/body weight. Alternative amounts range from
0.1 ng/kg/body weight is above 1000 mg/kg/body weight. CXS-746 or CXS-746R may be administered per minute, hour, day, week, month or year depending on the condition being treated. The route of administration may vary and includes intravenous, intraperitoneal, sub-cutaneous, intramuscular, intranasal, via suppository, via infusion, via drip, orally or via other convenient means.
The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating CXS-746 or CXS-746R expression or CXS-746 or CXS-746R activity. The vector may, for example, be a viral vector.
Still another aspect of the present invention is directed to antibodies specific for CXS-746 or CXS-746R and derivatives and homologs of CXS-746 and CXS-746R. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to CXS-746 or CXS-746R or may be specifically raised to CXS-746 or CXS-746R or derivatives or homologs thereof. In the case of the latter, CXS-746 or CXS-746R or their derivatives or homologs may first need to be associated with a carrier molecule. The antibodies and/or recombinant CXS-746 or CXS-746R or their derivatives of the present invention are particularly useful as therapeutic or diagnostic agents.
For example, CXS-746 or CXS-746R and derivatives or homologs thereof can be used to screen for naturally occurring antibodies to CXS-746 or CXS-746R which may occur in certain autoimmune diseases or where cell death is occurring. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for CXS-746 or CXS-746R. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Antibodies to CXS-746 or CXS-746R of the present invention may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the CXS-746 or CXS-746R or may be specifically raised to the CXS-746 or CXS-746R or their derivatives. In the case of the latter, the CXS-746 or CXS-746R protein may need first to be associated with a carrier molecule. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool or as a means for purifying CXS-746 or CXS-746R.
For example, specific antibodies can be used to screen for CXS-746 or CXS-746R proteins. The latter would be important, for example, as a means for screening for levels of CXS-746 or CXS-746R in a cell extract or other biological fluid or purifying CXS-746 or CXS-746R made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
Immunoassays, in their most simple and direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like may also be used.
In one exemplary ELISA, antibodies binding to the encoded proteins of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the disease marker antigen, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen may be detected. Detection is generally achieved by the addition of a second antibody specific for the target protein, that is linked to a detectable label. This type of ELISA is a simple "sandwich ELISA". Detection may also be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
In another exemplary ELISA, the samples suspected of containing the disease marker antigen are immobilized onto the well surface and then contacted with the antibodies of the invention. After binding and washing to remove non-specifically bound immunocomplexes, the bound antigen is detected. Where the initial antibodies are linked to a detectable label, the immunocomplexes may be detected directly. Again, the immunocomplexes may be detected using a second antibody that has binding affinity for the first antibody, with the second antibody being linked to a detectable label.
Another ELISA in which the proteins or peptides are immobilized, involves the use of antibody competition in the detection. In this ELISA, labelled antibodies are added to the wells, allowed to bind to the disease marker protein, and detected by means of their label. The amount of marker antigen in an unknown sample is then determined by mixing the sample with the labelled antibodies before or during incubation with coated wells. The presence of marker antigen in the sample acts to reduce the amount of antibody available for binding to the well and thus reduces the ultimate signal. This is appropriate for detecting antibodies in an unknown sample, where the unlabeled antibodies bind to the antigen-coated wells and also reduces the amount of antigen available to bind the labeled antibodies.
Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immunocomplexes. These are described as follows:
In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
In ELISAs, it is probably more customary to use a secondary or tertiary detection means rather than a direct procedure. Thus, after binding of a protein or antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the control sample and/or clinical or biological sample to be tested under conditions effective to allow immunecomplex (antigen/antibody) formation. Detection of the immunecomplex then requires a labeled secondary binding ligand or antibody, or a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or third binding ligand.
"Under conditions effective to allow immunecomplex (antigen/antibody) formation" means that the conditions preferably include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
The "suitable" conditions also mean that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours, at temperatures preferably on the order of 25° to 27° C, or may be overnight at about 4°C or so.
Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immunocomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immunocomplexes may be determined.
To provide a detecting means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate. Thus, for example, one will desire to contact and incubate the first or second immunecomplex with a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immunecomplex formation (e.g., incubation for 2 hours at room temperature in a PB S -containing solution such as PBS-Tween).
After incubation with the labeled antibody, and subsequent to washing to remove unbound material, the amount of label is quantified, e.g., by incubation with a chromogenic substrate such as urea and bromocresol purple or 2,2'-azido-di-3-ethyl-benzthiazoline-6- sulfonic acid [ABTS] and H2O2, in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectra spectrophotometer.
The antibodies of this invention will be used to quantify and localize the expression of the encoded marker proteins. The antibody, for example, will be labeled by any one of a variety of methods and used to visualize the localized concentration of the cells producing the encoded protein.
The invention also relates to an in vivo method of imaging metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions using the above described monoclonal antibodies. Specifically, this method involves administering to a subject an imaging- effective amount of a detectably-labeled disease-specific monoclonal antibody or fragment thereof and a pharmaceutically effective carrier and detecting the binding of the labeled monoclonal antibody to the diseased, or in the case of down regulated marker genes, healthy tissue. The term "in vivo imaging" refers to any method which permits the detection of a labeled monoclonal antibody of the present invention or fragment thereof that specifically binds to a diseased tissue located in the subject's body. A "subject" is a mammal, preferably a human. An "imaging effective amount" means that the amount of the detectably-labeled monoclonal antibody, or fragment thereof, administered is sufficient to enable detection of binding of the monoclonal antibody or fragment thereof to the diseased tissue, or the binding of the monoclonal antibody or fragment thereof in greater proportion to healthy tissue relative to diseased tissue.
A factor to consider in selecting a radionuclides for in vivo diagnosis is that the half-life of a nuclide be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation upon the host, as well as background, is minimized. Ideally, a radionuclides used for in vivo imaging will lack a particulate emission, but produce a large number of photons in a 140-2000 keV range, which may be readily detected by conventional gamma cameras.
A radionuclides may be bound to an antibody either directly or indirectly by using an intermediary functional group. Intermediary functional groups which are often used to bind radioisotopes which exist as metallic ions to antibody are diethylenetriaminepentaacetic acid (DTPA) and ethylene diaminetetracetic acid (EDTA). Examples of metallic ions suitable for use in this invention are 99mTc, 123I, 131I, 11 1In, 1311, 97Ru, 67Cu, 67Ga, 1251, 68Ga, 72As, 89Zr, and 201Tl.
In accordance with the present invention, the monoclonal antibody or fragment thereof may be labeled by any of several techniques known to the art. The methods of the present invention may also use paramagnetic isotopes for purposes of in vivo detection. Elements particularly useful in Magnetic Resonance Imaging ("MRI") include 157Gd, 55Mn, 162Dy,
52Cr, and 56Fe. Administration of the labeled antibody may be local or systemic and accomplished intravenously, intraarterially, via the spinal fluid or the like. Administration may also be intradermal or intracavitary, depending upon the body site under examination. After a sufficient time has lapsed, for example 30 minutes to 48 hours, for the monoclonal antibody or fragment thereof to bind with the target tissue, either diseased and/or healthy tissue, the area of the subject under investigation is examined by routine imaging techniques such as MRI, SPECT, planar scintillation imaging and emerging imaging techniques, as well. The exact protocol will necessarily vary depending upon factors specific to the patient, as noted above, and depending upon the body site under examination, method of administration and type of label used; the determination of specific procedures would be routine to the skilled artisan. The distribution of the bound radioactive isotope and its increase or decrease with time is then monitored and recorded. By comparing the results with data obtained from studies of clinically normal individuals, the presence and extent of the diseased tissue may be determined.
It will be apparent to those of skill in the art that a similar approach may be used to radio- image the production of the encoded disease marker proteins in human patients. The present invention provides methods for the in vivo diagnosis of disease in a patient. Such methods generally comprise administering to a patient an effective amount of a disease specific antibody, which antibody is conjugated to a marker, such as a radioactive isotope or a spin-labeled molecule, that is detectable by non-invasive methods. The antibody- marker conjugate is allowed sufficient time to come into contact with reactive antigens that be present within the tissues of the patient, and the patient is then exposed to a detection device to identify the detectable marker.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available antiimmunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of CXS-746 or CXS-746R. Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of CXS-746 or CXS-746R, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler and Milstein Nature 256:495-499, 1975; Kohler and Milstein European Journal of Immunology 6:511-519, 1976).
Another aspect of the present invention contemplates a method of detecting CXS-746 or CXS-746R or a derivative or homolog thereof in a biological sample from a subject, the method comprising contacting the biological sample with an antibody specific CXS-746 or CXS-746R or their antigenic derivatives or homologs for a time and under conditions sufficient for a complex to form, and then detecting the complex. The presence of the complex is indicative of the presence CXS-746 or CXS-746R. This assay may be quantitated or semi-quantitated to determine a propensity to develop mitochondrial dysfunction, myopathy, genetic disorder or cancer or in modulating apoptosis, signal transduction and/or nuclear targeting or to monitor a therapeutic regimen. The presence of CXS-746 or CXS-746R may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target.
Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-CXS-746 or antibody-CXS-746R complex, a second antibody specific to the CXS-746 or CXS-746R, labeled with a reporter molecule capable of producing a detectable signal, is then added and incubated, allowing time sufficient for the formation of another complex of antibody-CXS-746-labeled or CXS-746R-labeled antibody. Any unreacted material is washed away, and the presence of CXS-746 or CXS- 746R is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which might contain CXS-746 or CXS-746R including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from room temperature to about 37°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of CXS- 746 or CXS-746R. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to CXS-746 or CXS-746R.
An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, β-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen- antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. A "reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
Alternately, fluorescent compounds, such as fiuorecein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.
As in the EIA, the fluorescent labeled antibody is allowed to bind to the first antibody- hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method.
However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
The present invention also contemplates genetic assays such as involving PCR analysis to detect CXS-746 or CXS-746R or its derivatives.
The assays of the present invention may also extend to measuring CXS-746 or CXS-746R in association with another gene or molecule. The present invention may also be described in certain embodiments as a kit for use in detecting or diagnosing metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions. A representative kit may comprise one or more nucleic acid segments as described above that selectively hybridize to CXS-746 or CXS-746R and a container for each of the one or more nucleic acid segments. In certain embodiments the nucleic acid segments may be combined in a single tube. In certain embodiments the nucleic acid segments would be designed to selectively hybridize to a nucleic acid segment that includes the sequence or complement of SEQ ID NOs: 1 or 15 or 18 or 19. In further embodiments, the nucleic acid segments may also include a pair of primers for amplifying the target mRNA. Such kits may also include any buffers, solutions, solvents, enzymes, nucleotides, or other components for hybridization, amplification or detection reactions. Preferred kit components include reagents for RT-PCR, in situ hybridization, Northern analysis and/or RPA.
In certain embodiments the kit for use in detecting metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions may comprise an antibody which immunoreacts with a CXS-746 or CXS-746R polypeptide and a container for the antibody. Such an antibody may be a polyclonal or a monoclonal antibody and may be included in a kit with reagents, secondary antibodies, labeling means, or other components for polypeptide detection including, but not limited to an ELISA kit.
The present invention further comprises the prognosis and/or diagnosis of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions by measuring the amounts of nucleic acid amplification products formed as above. The amounts of nucleic amplification products identified in an individual patient may be compared with groups of normal individuals or individuals with an identified disease state. Diagnosis may be accomplished by finding that the patient's levels of CXS-746 and CXS-746R fall within the normal range, or within the range observed in individuals with the disease state. Further comparison with groups of individuals of varying disease state progression may provide a prognosis for the individual patient. The present invention further broadly comprises kits for performing the above-mentioned procedures, containing amplification primers and/or hybridization probes.
Another aspect of the present invention comprises the detection and diagnosis of diabetes or a condition associated therewith or the likelihood of developing diabetes or obesity or a predisposition of developing diabetes by combining measurement of levels of markers of metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions. An embodiment of the invention comprises combining measurement of SEQ ID NOs: 1 or 15 or 18 or 19 with other markers associated with metabolic conditions such as Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions, such as BMI, weight, waste to hip ratio, fasting glucose, fasting insulin and percent body fat. Yet another aspect of the present invention comprises kits for detection and measurement of the levels of two or more disease state markers in biological samples. The skilled practitioner will realize that such kits may incorporate a variety of methodologies for detection and measurement of disease state markers, including but not limited to oligonucleotide probes, primers for nucleic acid amplification, antibodies which bind specifically to protein products of disease state marker genes, and other proteins or peptides which bind specifically to disease state marker gene products.
In still further embodiments, the present invention concerns immunodetection kits for use with the immunodetection methods described above. As the encoded marker proteins or peptides may be employed to detect antibodies and the corresponding antibodies may be employed to detect encoded proteins or peptides, either or both of such components may be provided in the kit. The immunodetection kits thus comprise, in suitable container means, an encoded protein or peptide and/or a first antibody that binds to an encoded protein or peptide, and an immunodetection reagent.
In certain embodiments, the encoded protein or peptide, or the first antibody that binds to the encoded protein or peptide, may be bound to a solid support, such as a column matrix or well of a microtiter plate.
The immunodetection reagents of the kit may take any one of a variety of forms, including those detectable labels that are associated with or linked to the given antibody or antigen, and detectable labels that are associated with or attached to a secondary binding ligand. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody or antigen, and secondary antibodies that have binding affinity for a human antibody.
Further suitable immunodetection reagents for use in the present kits include the two- component reagent that comprises a secondary antibody that has binding affinity for the first antibody or antigen, along with a third antibody that has binding affinity for the second antibody, the third antibody being linked to a detectable label.
The kits may further comprise a suitably aliquoted composition of the encoded protein or polypeptide antigen, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
The kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit. The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the antibody or antigen may be placed, and preferably, suitably aliquoted. Where a second or third binding ligand or additional component is provided, the kit will also generally contain a second, third or other additional container into which this ligand or component may be placed. The kits of the present invention will also typically include a means for containing the antibody, antigen, and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
The present invention is further described by the following non-limiting Examples.
EXAMPLE 1
Psammomys obesus colony
A Psammomys obesus colony is maintained at Deakin University, Waurn Ponds, Geelong, Victoria, Australia with the breeding pairs fed ad libitum a diet of lucerne and standard laboratory chow. Animals are weaned at four weeks of age and sustained on a diet of standard laboratory chow from which 12% of energy was derived from fat, 63% from carbohydrate and 25% from protein (Barastoc, Pakenham, Australia). Animals are housed in a humidity and temperature controlled room (22 ± 1°C) with a 12- 12-hour light-dark cycle.
Group A animals are lean, normoglycemic and normoinsulinemic (NGT), group B animals are obese, normoglycemic and hyperinsulinemic (IGT), and group C animals are obese, hyperglycemic and hyperinsulinemia (Type II diabetic).
EXAMPLE 2 Analytical methods
Whole blood glucose was measured immediately using an enzymatic glucose analyser (Model 27, Yellow Springs Instruments, OH). Plasma insulin concentrations were determined using a double antibody solid phase radioimmunoassay (Phadeseph, Kabi Pharmacia Diagnostics, Sweden).
EXAMPLE 3 RNA extraction
Total RNA was extracted from tissue and 3T3-L1 adipocytes in a two-step process utilising Trizol (Invitrogen Life Technologies, Carlsbad, USA) and RNeasy (Qiagen, Hilden, Germany) protocols. The tissue samples were homogenised in Trizol (Invitrogen Life Technologies) and l/5th volume chloroform was added to the homogenate, which was then mixed and incubated at room temperature for 3 min. The homogenates were then separated by centrifugation at 13000xg for 15 min (40C). Following centrifugation the aqueous supernatant was removed and added to an equal volume of 70% ethanol. The solution was mixed and the RNA extracted using RNeasy kit spin columns (Qiagen) according to manufacturer's instructions. The RNA was eluted using RNAase free water and total RNA integrity, quantity and concentration was assessed using the RNA 6000 Nano Assay (Agilent Technologies, Palo Alto, USA) with the Agilent 2100 Bioanalyser (Agilent Technologies) as per the manufacturer's instructions. This system utilises capillary electrophoresis to separate and detect nucleic acid fragments by size through the interconnected micro channels on a Nano chip (Agilent Technologies). Good quality RNA was signified by an electropherogram displaying a marker peak, and two ribosomal peaks of which the 18s band is at an approximate ratio of 1 :2 to the 28s band.
EXAMPLE 4
Statistical Analysis
Statistical analyses were performed using SPSS for Windows version 14.0 (SPSS Inc.). Differences between groups were compared using a one-way analysis of variance (ANOVA). Levene's test for homogeneity of variance was used to determine if variance between six animal groups (NGT fed, NGT fasted, IGT fed, IGT fasted, T2D fed, T2D fasted) was equal or not. If homogeneity was not equal, the Games-Howell post hoc analysis was used, and if equal, LSD was used to determine if there were significant differences between groups or correlations with phenotypic characteristics. Differences and correlations were considered significant if p<0.05.
Phenotypic parameters of human normal and type 2 diabetic subjects were compared using a Students t-test or a Mann- Whitney U test for data that was not normally distributed. Associations between circulating CXS-746 levels and phenotypic measures were determined using Pearson correlation (for normally distributed data) or Spearman correlation (for non-normally distributed data) in SPSS. Multivariate linear regression was used to determine if associations were independent of other variables. EXAMPLE 5 Signal Sequence Trap
Total RNA was extracted as described in Example 3 from liver of lean, normoglycaemic, normoinsulinaemic (NGT), obese normoglycaemic, hyperinsulinaemic (IGT) and obese Type II diabetic (T2D) P. obesus in the fed and fasted states. Equal amounts of RNA from each group were pooled and the mRNA extracted using an oligo(dT) cellulose column.
The mRNA was reverse transcribed using random 9mer primers to enrich for the 5' end of mRNA. The random 9mer primers were engineered to contain a Not I restriction site (underlined) which was used for cloning:
pTCTAGATCGCGAGCGGCCGCCCNNNNNNNNN (SEQ ID NO:2)
After second strand synthesis, Sal I adapter addition and Not I digestion, the cDNA was run on a 1.5% w/v TBE agarose gel. The 300 to 800 base pair products were cut out of the gel, purified and quantitated using standard methodologies.
The Not I-Sal I digested skeletal muscle library was ligated into the retrovirus plasmid vector pLNCX2, 5' to a murine interleukin 3 (mIL-3) gene that was engineered to lack a signal sequence. Transformation conditions were according to standard methodologies. Transformation of bacterial cells generated approximately 200,000 transformants. The plasmid library obtained from the 200,000 transformants was transfected into a retrovirus packaging cell line (293 Plat E).
The retrovirus library that was produced was used to infect the mIL-3 dependent cell line FDCPl. The retrovirus library and cells were placed in 6 well dishes and centrifuged for 1 hour at 1000xg, to increase the frequency of infection. The cells were then incubtated at 370C for 24 hrs in media containing mIL-3. The infected cells were washed four times to eradicate IL-3 from the media then plated into 96-well plates. Cells infected with retroviruses that contain an in-frame signal sequence and secreted mIL-3 were evidenced by live cells growing in each well after several days. After 2-3 weeks, genomic DNA was extracted from positive clones using standard methodologies.
EXAMPLE 6 cDNA microarray production
To amplify cDNAs using genomic DNA a nested PCR protocol was used. First round PCR primers were as follows: Forward 5'-CTGGTTTAGTGAACCGTCAGATC (SEQ ID NO:3) and Reverse 5'-CTCCTTGACAATAGAGCTGCAA (SEQ ID NO:4). The DNA was denatured for 2 min at 940C, and amplified by 35 cycles of 94°C for 30 sec, 560C for 30 sec and 720C for 1 min, followed by a final extension of 7 min at 720C. The first round PCR product was diluted 1 :100 and 1 μl used as template for a second round of amplification to reduce the levels of genomic DNA in the final PCR product. Second round PCR primers were designed immediately adjacent to the plasmid insert site so the amplified cDNA product contains very little mIL-3 or vector sequence which may potentially interfere with hybridisation to the microarray chip. The primer sequences were as follows: Forward 5'-TAGCGCTACCGGACTCAGAT (SEQ ID NO:5) and Reverse 5'- CGGCCACTGATTGAAGCTT (SEQ ID NO:6). PCR conditions were the same as for first round PCR. Products were visualized by TBE agarose gel (1.% w/v) electrophoresis at 6V/cm for 60 min to ensure successful amplification had taken place.
PCR products were purified using the Arraylt vacuum manifold system (TeleChem International, Sunnyvale, CA) and resuspended in 20μL of Ix spotting solution (TeleChem) at a concentration of approximately 0.5 mg/ml in 384 well plate format. 5μL of the resuspended purified cDNA solution was transferred to 384 well uniplates (Whatman Inc, Clifton, USA). This cDNA was arrayed onto Super Amine Microarray Substrates (TeleChem) using a Chip Writer Pro robotic arrayer (BioRad) fitted with 32 Stealth SMP-03 quill tipped microarray pins (Telechem). The distance between adjacent cDNA spots was 200μM. Each pin drew 0.25 μL of cDNA and deposited approximately 0.6nL on each slide. Humidity was maintained between 55-65% during printing. Spotted DNAs were allowed to dry overnight, after which the slides were washed and blocked as recommended by the manufacturer (TeleChem).
EXAMPLE 7 cDNA microarray hybridization
Fluorescently labeled cDNA was prepared from 20μg of total RNA using an indirect labelling method (Superscript Indirect cDNA Labelling System, Invitrogen) as per the manufacturers instructions. cDNA synthesis was performed in a 40μL reaction containing 5μg oligo-dT primer, 400U Superscript IH (Invitrogen), Ix first strand buffer, 0.0 IM DTT, 0.5mM of each dATP, dCTP and dGTP, 0.15OmM dTTP (Amersham, Buckinghamshire, UK) and 0.2mM aminoallyl-dUTP (Sigma, St. Louis, MO). Synthesis was conducted in a GeneAmp PCR System 9700 (PE Applied Systems) at 46°C for 2 hours. The reaction was stopped by addition of 5μl of 0.5M EDTA and RNA was hydrolysed by addition of 20μl of IM NaOH at 70°C for 20 minutes. The reaction was neutralized with 25μl of IM HCl and the cDNA was purified using SNAP purification kits according to manufacturer's instructions (Invitrogen) and eluted in nuclease-free water. The cDNA was concentrated by ethanol precipitation and the cDNA pellet was resuspended in 5μl coupling buffer. Cy3 or Cy5 monofunctional NHS ester reactive dyes (Amersham) were dissolved in 5μl of DMSO and added to the cDNA. The coupling reaction was conducted in the dark for 1 hour.
Dye-coupled cDNA was purified using SNAP DNA purification columns (Invitrogen), combined and added to lOμg of mouse Cotl DNA (Invitrogen). The cDNAs were again concentrated with Micron 30 spin columns (Millipore). The cDNA was hybridised in a 50μL volume containing the labeled cDNA, 50% v/v formamide, 5x SSC, 8μg PolydA, 2.5x Denhardt's solution, 4μg yeast tRNA and 0.1% SDS. The cDNA was then denatured at 980C for 2 min and maintained at 6O0C until required. The hybridisation solution was mounted onto an array slide under a Lifterslip (Erie Glass) and hybridisation was conducted in a humid hybridisation chamber, in a hybridisation oven, at 420C for 16 hours. Following hybridisation the array slides were removed from their chamber and washed for 2 min in each of a 0.5X SSC and 0.1% w/v SDS, 0.5x SSC and 0.01% w/v SDS, 0.6x SSC and 0.06% w/v SDS solution. The array slides were dried in a centrifuge for 1 min at 500xg.
Fluorescent images of the microarrays were acquired using a GenePix 4000B scanner (Axon Instruments, Union City CA, USA) and the images were analysed using GenePix Pro 5.1 (Axon Instruments). Slides were scanned for both Cy3 and Cy5 signal at a lOμM pixel resolution. Laser intensity and amplification of the photomultiplier tubes were adjusted to ensure approximately equal overall signal intensity for both Cy3 and Cy5. False colour images were generated for each dye and combined to provide a representation of the relative Cy3 and Cy5 intensities. Individual cDNA spots were flagged if spot size was too small, if the overall signal intensity was too low, or if the Cy3 and Cy5 signal intensities within the spots were not linearly related. GenePix Pro allows for the 'flagging' of bad elements (defined by present GenePix Pro parameters as feature signal intensity; feature background; element morphology; elements size and the percentage of pixels greater than feature background) that were then excluded from further analysis.
Median Cy3 and Cy5 signal intensities for each cDNA spot were imported from Genepix
Pro files and data transformation conducted using Acuity (Version 4.0, Axon Instruments).
Signal intensities for each feature were corrected for local background and features that failed to meet quality criteria (e.g. low expression values, poor feature morphology; small feature size or small percentage of pixels greater than feature background) were omitted.
The ratio of Cy3 to Cy 5 was calculated and the data logarithmically transformed (base2).
Signal intensity was normalised to the mean intensity of all respective signal intensities, providing a relative measure of gene expression for each element on the microarray slide.
The expression of genes encoding SST positive cDNAs in liver tissue of lean, normoglycaemic, normoinsulinaemic (NGT), obese normoglycaemic, hyperinsulinaemic (IGT) or obese Type II diabetic (T2D) P. obesus obesity and Type II diabetes was determined using microarray analysis. Differential gene expression as measured by microarray was assessed using the independent samples t-test algorithm in the Acuity software. Genes were considered to be differentially expressed if the significance of the t- test was p<0.05. A number of differentially expressed genes were identified and their expression was confirmed by real time PCR. RNA was reverse transcribed using Superscript Hi RT system (Invitrogen). Oligonucleotide primers for were designed using the Primer Express 2.0 software program (Applied Biosystems), and real time PCR was performed using SYBR Green PCR master mix and an ABI PRISM 7700 sequence detector (Applied Biosystems). The primer sequences for P. obesus CXS-746 real time gene expression were as follows: forward 5'- TGGGCCTTCCGAGAGATG (SEQ ID NO: 7) and reverse 5'- AATTCCAACCGCACAAAGGT (SEQ ID NO:8) or reverse 51- AGACGACCACACAGGTCACGTA (SEQ ID NO:24). The primer sequences for P. obesus CXS-746 receptor real time gene expression were as follows: forward 5'- AGCTTTGACCGCTGCATCTC (SEQ ID NO:9) and reverse 5'- GGAACTCAAGAAGAAAGCCAAGAG (SEQ ID NO: 10). The primer sequences for murine CXS-746 real time gene expression were as follows: forward 5'- CCAACTGCCCCAAGAAGGA (SEQ ID NO: 11) and reverse 5'- CGCCTTCTCCCGTTTGGT (SEQ ID NO: 12). The primer sequences for murine CXS- 746 receptor real time gene expression were as follows: forward 5'- TGGCCGACTTCCTGTTCAAC (SEQ ID NO: 13) and reverse 5'- CCCGAACACCCAGTGGTAGT (SEQ ID NO: 14).
EXAMPLE 8
CXS-746
Sequence of Psammomys obesus of CXS-746
5 - -
Figure imgf000075_0001
ATCAAACCAAATGGGAAAAAGCGGCAATGCCTTGCCTGTATCAAACTGGACCCCAAGG- 3 '
(SEQIDNO:!). Sequence of human CXS-746 (mRNA)
5' -
GCGGGACGGT CAGGGGAGAC CTCCAGGCGC AGGGAAGGAC GGCCAGGGTG ACACGGAAGC ATGCGACGGC TGCTGATCCC TCTGGCCCTG TGGCTGGGTG CGGTGGGCGT GGGCGTCGCC GAGCTCACGG AAGCCCAGCG CCGGGGCCTG CAGGTGGCCC TGGAGGAATT TCACAAGCAC CCGCCCGTGC AGTGGGCCTT CCAGGAGACC AGTGTGGAGA GCGCCGTGGA CACGCCCTTC CCAGCTGGAA TATTTGTGAG GCTGGAATTT AAGCTGCAGC AGACAAGCTG CCGGAAGAGG GACTGGAAGA AACCCGAGTG CAAAGTCAGG CCCAATGGGA GGAAACGGAA ATGCCTGGCC TGCATCAAAC TGGGCTCTGA GGACAAAGTT CTGGGCCGGT TGGTCCACTG CCCCATAGAG ACCCAAGTTC TGCGGGAGGC TGAGGAGCAC CAGGAGACCC AGTGCCTCAG GGTGCAGCGG GCTGGTGAGG ACCCCCACAG CTTCTACTTC CCTGGACAGT TCGCCTTCTC CAAGGCCCTG CCCCGCAGCT AAGCCAGCAC TGAGCTGCGT GGTGCCTCCA GGACCGCTGC GGGTGGTAAC CAGTGGAAGA CCCCAGCCCC CAGGGAGAGG AACCCGTTCT ATCCCCAGCC ATGATAATAA AGCTGCTCTC CCAAAAAAAA- 3 ' (SEQ ID NO: 18)
Sequence of human CXS-746 (Amino Acid)
MRRLLIPLAL WLGAVGVGVA ELTEAQRRGL QVALEEFHKH PPVQWAFQET SVESAVDTPF PAGIFVRLEF KLQQTSCRKR DWKKPECKVR PNGRKRKCLA CIKLGSEDKV LGRLVHCPIE TQVLREAEEH QETQCLRVQR AGEDPHSFYF PGQFAFSKAL PRS (SEQ ID NO:20)
Sequence ofPsammomys obesus CXS-746 R
5'
Figure imgf000076_0001
GCCGGACTCTGTCTTCAGCCTGGGGCTACCAAA- 3' (SEQ ID NO: 15)
Sequence of human CXS-746R (mRNA)
5' -
GAATTCGGCA CGAGTCAGGG AAGCAGCCCC GGCGGCCAGC AGGGAGCTCA GGACAGAGCA
GGCTCCCTGG GAAGCCTCCG GGTGATAGGG GTGTTCCAGC TGCGGCGCTC TGGGGGTTCA
GAGGGGGATC TTGAATGAAC AAATGAATGA ACTGCTTTCT GGGCAAACAG CCACAGCCAG AGGAGCCTGT GATTGGCAGA AAGAAGCCAG GGTGTGCAAG TCTCCCCAAC AGCCTCGAGT GGCCTGCAGT CACAGGGAAC CCTCAGGAAG ACCTTCCGGG CAGAGACCAG AGGGAAGCCC ATCTCTCCAG CAGAACTGCT TGGATTTTTC TACCAGGAGG CTCAGGGCTC TGCAACAATG ATAGCAGAAG CTGATGGCAT CTAGAGATCT AGGCTGGGAC TAGCACAGCA TCACTTCTAC CACTTTCTGT TGGTCACAGC AACTCACCAT GCCAGTGCAG ATTCAAGGGG AGGAGAAATA GAGTCCACTT CTTGATGGGA GGCGTGACAT AGAATGGAGG ATGAAGATTA CAACACTTCC ATCAGTTACG GTGATGAATA CCCTGATTAT TTAGACTCCA TTGTGGTTTT GGAGGACTTA TCCCCCTTGG AAGCCAGGGT GACCAGGATC TTCCTGGTGG TGGTCTACAG CATCGTCTGC TTCCTCGGGA TTCTGGGCAA TGGTCTGGTG ATCATCATTG CCACCTTCAA GATGAAGAAG ACAGTGAACA TGGTCTGGTT CCTCAACCTG GCAGTGGCAG ATTTCCTGTT CAACGTCTTC CTCCCAATCC ATATCACCTA TGCCGCCATG GACTACCACT GGGTTTTCGG GACAGCCATG TGCAAGATCA GCAACTTCCT TCTCATCCAC AACATGTTCA CCAGCGTCTT CCTGCTGACC ATCATCAGCT CTGACCGCTG CATCTCTGTG CTCCTCCCTG TCTGGTCCCA GAACCACCGC AGCGTTCGCC TGGCTTACAT GGCCTGCATG GTCATCTGGG TCCTGGCTTT CTTCTTGAGT TCCCCATCTC TCGTCTTCCG GGACACAGCC AACCTGCATG GGAAAATATC CTGCTTCAAC AACTTCAGCC TGTCCACACC TGGGTCTTCC TCGTGGCCCA CTCACTCCCA AATGGACCCT GTGGGGTATA GCCGGCACAT GGTGGTGACT GTCACCCGCT TCCTCTGTGG CTTCCTGGTC CCAGTCCTCA TCATCACAGC TTGCTACCTC ACCATCGTGT GCAAACTGCA GCGCAACCGC CTGGCCAAGA CCAAGAAGCC CTTCAAGATT ATTGTGACCA TCATCATTAC CTTCTTCCTC TGCTGGTGCC CCTACCACAC ACTCAACCTC CTAGAGCTCC ACCACACTGC CATGCCTGGC TCTGTCTTCA GCCTGGGTTT GCCCCTGGCC ACTGCCCTTG CCATTGCCAA CAGCTGCATG AACCCCATTC TGTATGTTTT CATGGGTCAG GACTTCAAGA AGTTCAAGGT GGCCCTCTTC TCTCGCCTGG TCAATGCTCT AAGTGAAGAT ACAGGCCACT CTTCCTACCC CAGCCATAGA AGCTTTACCA AGATGTCATC AATGAATGAG AGGACTTCTA TGAATGAGAG GGAGACCGGC ATGCTTTGAT CCTCACTGTG GAACCCCTCA ATGGACTCTC TCAACCCAGG GACACCCAAG GATATGTCTT CTGAAGATCA AGGCAAGAAC CTCTTTAGCA TCCACCAATT TTCACTGCAT TTTGCATGGG ATGAACAGTG TTTTATGCTG GGAATCTAGG GCCTGGAACC CCTTTCTTCT AGTGGACAGA ACATGCTGTG TTCCATACAG CCTTGGACTA GCAATTTATG CTTCTTGGGA GGCCAGCCTT GACTGACTCA AAGCAAAAAA GGAAGAATTC -3 ' (SEQ ID NO: 19)
Sequence of human CXS-746R (Amino Acid)
MEDEDYNTSI SYGDEYPDYL DSIVVLEDLS PLEARVTRIF LVVVYSIVCF
LGiLGNGLVi IIATFKMKKT VNMVWFLNLA VADFLFNVFL PIHITYAAMD YHWVFGTAMC KISNFLLIHN MFTSVFLLTI ISSDRCISVL LPVWSQNHRS VRLAYMACMV IWVLAFFLSS PSLVFRDTAN LHGKISCFNN FSLSTPGSSS WPTHSQMDPV GYSRHMVVTV TRFLCGFLVP VLIITACYLT IVCKLQRNRL AKTKKPFKII VTIIITFFLC WCPYHTLNLL ELHHTAMPGS VFSLGLPLAT ALAIANSCMN PILYVFMGQD FKKFKVALFS RLVNALSEDT GHSSYPSHRS FTKMSSMNER TSMNERETGM
(SEQ ID NO:21) CXS-746 identification and sequence homology
CXS-746 was sequence identified as a positive clone from a SST using P. obesus liver
RNA. Comparison of the P. obesus CXS-746 sequence to GenBank revealed it was homologous to the chemerin gene. Alternate names for this gene are tazarotene-induced gene 2 (TIG2); HP 10433, retinoic acid receptor responder (tazarotene induced) 2
(RARRES2). Analysis of gene expression found CXS-746 gene expression to be unaltered in the liver of IGT or T2D animals compared to the expression of CXS-746 in the liver of nGT animals. Subsequent investigation of CXS-746 mRNA expression in a large number of P. obesus tissues demonstrated that CXS-746 was highly expressed in adipose tissue and was further characterised.
CXS-746 receptor identification and sequence homology
Human and mouse orthologues of CXS-746 have been shown to be the natural ligand for a G-protein coupled receptor, CMKLRl. Highly conserved sequences from mouse, rat and human orthologues of this gene were identified and used to design PCR primers homologous to these sequences. The primer sequences for cloning P. obesus CXS-746 receptor cDNA were as follows: Forward 5'- GCC ATGTGC AAGATC AGC AA (SEQ ID NO: 16) and Reverse 5'- GGTAGCCCCAGGCTGAAGA (SEQ ID NO: 17). These primers were used to successfully amplify P. obesus CXS-746 receptor cDNA. Comparison of the P. obesus CXS-746 receptor sequence to GenBank revealed it was homologous to the chemokine-like receptor 1 (CMKLRl) gene. Alternate names for this gene are DEZ, ChemR23, MGC126105 and MGC126106.
CXS-746 bioinformatics Chemerin was initially identified as a novel transcript that was upregulated in psoriasis lesions treated with the retinoic acid receptor agonist tazarotene (Nagapal et al. Journal of Investative Dermatolatology 109:91-5, 1997). The human chemerin gene has been to chromosome 7q36.1 and encodes a 7340bp mRNA. Chemerin mRNA expression is induced by RAR agonists (such as tazarotene) and not by RXR or Vitamin D Receptor specific agonists (Nagapal et al. 1997 supra). The chemerin protein is secreted as prochemerin and is proteolytically cleaved at the C-terminus by extracellular proteases to yield a 15kDa mature polypeptide. Chemerin is expressed by many tissues including spleen, lymph nodes, adrenal gland, liver, pancreas, small intestine and mature chemerin protein has been isolated from human ascetic fluid that was secondary to ovarian cancer, hemofϊltrate and serum (Zabel et al. Journal of Biological Chemistry 280: 34661-6, 2005).
CXS-746 has been identified as a natural ligand of chemokine-like receptor 1 (CMKLRl), a previously orphan protein G-coupled receptor (Samson et al. European Journal of Immunology 28: 1689-700, 1998). CMKLRl is expressed by macrophages and immature dendritic cells and is found in lymph node, parathyroid glands and developing osteogenic and cartilaginous tissue (Meder et al. FEBS Letters 555:495-9, 2003). CXS-746 binding to the CMKLRl receptor induces intracellular calcium release and MAPK pathway activation (Vermi et al. Journal of Experimental Medicine 201:509-15, 2005). CXS-746 is a potent chemoattractant for macrophages and dendritic cells expressing the G-protein coupled receptor CMKLRl. CXS-746 and its receptor CMKLRl may play a pivotal role as a link between innate and adaptive immunity where tissue infiltration by neutrophils promote the recruitment of APC at inflammatory sites (Vermi et al. 2005 supra). In addition, CMKLRl is used as a co-receptor by the immunodeficiency viruses HIV-I and SIV (Samson et al. 199S supra).
CXS-746 and CXS-746 receptor gene expression in different tissues as measured by SYBR Green Real Time PCR
The CXS-746 gene was found to be expressed in all tissues examined in P. obesus, but was highest in the liver, subscapular fat, intramuscular fat, epididymal fat, mesenteric fat, perirenal fat and the kidney. Similarly CXS-746 receptor (CXS-746R) was also expressed in all tissues examined in P. obesus, where highest expression was observed in adipose tissue and the lung. This is the first description of CXS-746 and CXS-746 receptor expression in adipose tissue depots. The results are shown in Table 3. TABLE 3 CXS-746 and CXS-746 Receptor Tissue Distribution
Figure imgf000080_0001
;Relatiye[GeneiExpression
Testes 1.93 0.45
Ovary 2.58 1.47
CXS-746 and CXS-746 Receptor gene expression in P. obesus mesenteric fat as measured by SYBR Green Real Time PCR
CXS-746 gene expression in mesenteric fat of NGT, IGT and T2D P obesus, in the fed and fasted state, was analysed by real time PCR. CXS-746 gene expression was significantly higher in IGT and T2D than NGT animals in the fasted state (p=0.004 and p^O.Ol respectively; Table 4). CXS-746 receptor gene expression in the mesenteric fat of NGT, IGT and T2D P. obesus, in the fed and fasted state, was also analysed by real time PCR. CXS-746 receptor gene expression was significantly higher in IGT and T2D than NGT animals in the fasted state (p=0.009 and p=0.05 respectively). The results are shown in Table 4. These data show that elevated expression of CXS-746 and CXS-746 receptor mRNA in adipose tissue was associated with obesity and Type II diabetes.
TABLE 4
CXS-746 gene expression in P. obesus mesenteric fat
Figure imgf000081_0001
CXS-746 and CXS-746 receptor gene expression as measured by SYBR Green Real Time PCR in P. obesus visceral and subcutaneous adipose tissue
CXS-746 gene expression in fed NGT, IGT and T2D P. obesus visceral and subcutaneous adipose tissue was analysed by real time PCR. CXS-746 gene expression was significantly higher in IGT and T2D subcutaneous tissue compared to NGT subcutaneous adipose tissue
(p=0.006 and p=0.0001 respectively). Gene expression in visceral adipose tissue was also higher in IGT and T2D compared to NGT, however, expression was significant higher only T2D visceral adipose tissue compared with NGT visceral adipose tissue (p=0.01).
CXS-746 gene expression was significantly higher in subcutaneous compared to visceral adipose tissue in NGT (p= 0.03). CXS-746 gene expression was not significantly different in subcutaneous compared to visceral adipose tissue in IGT or T2D animals.
CXS-746 receptor gene expression was also measured in visceral and subcutaneous adipose tissue from fed NGT, IGT and T2D P. obesus by real time PCR. CXS-746 receptor gene expression was significantly higher in IGT subcutaneous tissue compared to NGT subcutaneous adipose tissue (p=0.03) and showed a trend toward being higher in T2D animals however this difference was not significant (p=0.07). Expression of CXS- 746 receptor mRNA in visceral adipose tissue was higher in IGT (p=0.02) and T2D (p=0.03) compared to expression in visceral adipose tissue of NGT animals. CXS-746 receptor gene expression was significantly higher in subcutaneous compared to visceral adipose tissue in T2D animals (p= 0.03). CXS-746 receptor gene expression was not significantly different in subcutaneous compared to visceral adipose tissue in IGT or T2D animals.
TABLE 5 CXS-746 gene expression in P. obesus visceral and subcutaneous adipose tissue
Figure imgf000083_0001
CXS-746 gene expression in 3T3-L1 adipocytes during differentiation as measured by SYBR Green Real Time PCR
3T3-L1 adipocytes were cultured in high glucose DMEM (25mM), supplemented with 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin Two days after the cells reached confluence, differentiation of fibroblasts into adipocytes was initiated by the addition of high glucose DMEM. 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin, 0.5mM l-,ethyl-3-isobutulxanthine (IBMX), 2.5uM dexamethasone and 0.166U/ml insulin for three days. The medium was then aspirated and replaced with glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin and 0.166U/ml of insulin for two days. Following these treatments, the cells were cultured in glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin. Real time PCR showed that, compared to undifferentiated fibroblasts, CXS-746 gene expression was markedly upregulated during differentiation (day 3, 4, 5 and 6, pO.OOl) and was approximately 20 fold higher in fully differentiated adipocytes (day 7,8 and 9, pO.OOl). These data clearly shows that mature adipocytes express high levels of CXS-746 and that the expression of CXS-746 observed in adipose tissue is predominantly within adipocytes. TABLE 6
CXS-746 gene expression in 3T3-L1 adipocytes undergoing differentiation
Figure imgf000084_0001
EXAMPLE 9
CXS-746 receptor gene expression in 3T3-L1 adipocytes during differentiation as measured by SYBR Green Real Time PCR
3T3-L1 adipocytes were cultured in high glucose DMEM (25mM), supplemented with 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin. Two days after the cells reached confluence, differentiation of fibroblasts into adipocytes was initiated by the addition of high glucose DMEM. 10% fetal bovine serum, 50units/ml of penicillin and 50ug/ml of streptomycin, 0.5mM l-,ethyl-3-isobutulxanthine (IBMX), 2.5uM dexamethasone and 0.166U/ml insulin for three days. The medium was then aspirated and replaced with high glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin and 0.166U/ml of insulin for two days. Following these treatments, the cells were cultured in high glucose DMEM, 10% fetal bovine serum, 50U/ml of penicillin and 50ug/ml of streptomycin. Real time PCR showed that, compared to undifferentiated fibroblasts, CXS-746 receptor gene expression was markedly downregulated during differentiation (day 1, 2, 3 p<0.001) and was approximately 10 fold lower in fully differentiated adipocytes (day 7, 8 and 9, p<0.001). These data clearly shows that mature adipocytes express reduced levels of CXS-746 receptor compared with fibroblasts. Moreover these data suggests that mature adipocytes are likely to respond poorly to CXS-746 stimulation.
TABLE 7 CXS-746 receptor gene expression in 3T3-L1 adipocytes undergoing differentiation
Figure imgf000085_0001
Figure imgf000086_0001
CXS-746 and CXS-746 receptor gene expression in 3T3-L1 adipocytes following TN F- a stimulation as measured by SYBR Green Real Time PCR
3T3-L1 adipocytes were fully differentiated into adipocytes for 8 days (as described above) were stimulated with murine TNF-α (3ng/ml) for 72 hours. RNA was extracted and the expression of CXS-746 and CXS-746 receptor was determined by real time PCR. Expression of CXS-746 was significantly upregulated by TNF-α treatment in mature 3T3 adipocytes (p<0.001). In contrast, TNF-α treatment did not significantly affect CXS-746 receptor expression (Table 8). TABLE 8
CXS-746 and CXS-746 receptor gene expression in 3T3-L1 adipocytes following TNF-a stimulation
Figure imgf000087_0001
CXS-746 and CXS-746 receptor gene expression in 3T3-L1 adipocytes following troglitazone or aspirin stimulation as measured by SYBR Green Real Time PCR
3T3-L1 adipocytes were fully differentiated into adipocytes for 8 days (as described above) and were stimulated with lOμM troglitazone or 5mM aspirin for 24 hours. Unstimulated cells were used as a control. RNA was extracted and the expression of CXS- 746 and CXS-746 receptor was determined by real time PCR (Table 9). Expression of CXS-746 was significantly downregulated by troglitazone treatment in mature 3T3 adipocytes (p<0.001). In contrast, aspirin treatment did not significantly affect expression of CXS-746 receptor. Treatment of mature 3T3-L1 adipocytes with troglitazone induced a marked 8 fold downregulation of CXS-746 receptor expression which was highly significant (pO.OOl). Treatment of mature 3T3-L1 adipocytes with aspirin also induced a markedly significant downregulation of CXS-746 receptor expression (p<0.001; Table 9) Both troglitazone and aspirin have been shown to improve insulin sensitivity and ameliorate Type II diabetes. These data, therefore, suggest that downregulation of CXS- 746 signalling via suppression of CXS-746 and/or CXS-746 receptor expression may improve insulin resistance and ameliorate Type II diabetes. TABLE 9
CXS-746 and CXS-746 receptor gene expression in 3T3-L1 adipocytes following treatment with troglitazone or aspirin
Figure imgf000088_0001
EXAMPLE 10
Fractionation of adipose tissue
Approximately 2 grams of mesenteric adipose tissue was minced and washed several times with Krebs Ringer Phosphate (KRP) buffer (12.5mM HEPES, 120Mm NaCl, 1.2mM MgSO4, ImM CaCl2, 0.4mM NaH2PO4, 0.6mM Na2HPO4, 6mM KCI, 5mM glucose, 3% BSA fraction V, pH 7.4) to remove any dead cells, connective tissue or blood cells. The tissues were digested in 0.75mg/ml of collagenase Type I (Worthington biochemical) in a shaking water bath (37°C) for -20 minutes. The digested samples were filtered through a nylon mesh into 25ml fresh KRP buffer and centrifuged 300g, 10 min at room temperature The floating adipocyte layer was collected, and the supernatant aspirated to reveal the pellet fraction. Both fractions were snap frozen in liquid nitrogen and stored -8O0C for subsequent RNA extraction.
EXAMPLE 11
CXS-746 and CXS-746 receptor are predominantly expressed by adipocytes in adipose tissue
To determine which cells within adipose tissue express CXS-746 and CXS-746, mesenteric adipose tissue from NGT and IGT P obesus was fractionated into cellular populations comprised of adipocytes and stromal-vascular cells. CXS-746 expression was significantly higher in adipocytes compared to stromal-vascular cells in both NGT and IGT animals (p=0.0001), and CXS-746 expression was significantly higher in adipocytes of IGT compared to the adipocytes of NGT animals (p=0.01; table 10). In contrast, CXS-746 receptor gene expression was not significantly different in adipocyte and stromal-vascular cells from adipose tissue of P obesus. These results show that in adipose tissue, CXS-746 is predominantly expressed within adipocytes while CXS-746 receptor is expressed by both adipocytes and stromal-vascular cells.
TABLE 10 The relative gene expression of CXS-746, CXS-746 receptor, Leptin and CD68 in fractionated mesenteric adipose tissue
Figure imgf000089_0001
EXAMPLE 12 Human Samples
All plasma samples were obtained during the course of large scale epidemiological studies of obesity, diabetes and metabolic syndrome from Mauritius. Mauritius is a subtropical island located in the south western Indian Ocean with a population of about 1.3 million An estimated 70% of the population are of Asian Indian origin (54% Hindu and 16% Muslim), 2% are of Chinese origin, and 28% are of the "general" population, which mainly comprises people with mixed African and Malagasy ancestry with some European and Indian admixture (Creoles). The three ethnic groups each demonstrate a high prevalence of obesity and diabetes (12, 13). The samples consisted of 256 individuals, where 142 had normal glucose tolerance (NGT) and 114 had type 2 diabetes (T2D). Samples were randomly selected between individuals who had an age between 35 and 65 years. All samples were obtained with informed consent and all protocols were approved by the Inner Eastern Health Care Network Institution review board.
EXAMPLE 13 ELISA
An ELISA was developed using commercially available unlabeled and biotinylated polyclonal antihuman chemerin antibodies (R&D Systems, USA). Primary unlabelled antibody was diluted to 1 μg/ml in PBS and coated onto Maxisorp ELISA plates (Nunc, USA) in lOOμl at 4°C overnight. The plates were washed with PBS 0.05% tween 20 (PBST) and blocked using 200μl blocking buffer (3% BSA in PBST) for 1 hour. The blocking solution was removed and plasma samples (diluted 1 :60 with blocking buffer; lOOμl per sample in duplicate) were added to the plate. After 2 hours at room temperature the plate was washed with PBST and biotionylated antichemerin antibody (1 μg/ml) was added to each well. After 2 hours the plate was washed and strepavidin horseradish peroxidase (100ng/ml, Sigma) was added to the plate. After 1 hour at room temperature the plate was washed with PBST and the assay was developed using lOOμl/well 3,3', 5,5'- tetramethylbenzidine (0.1mg/ml) dissolved in citrate buffer (5OmM Na2HPO4, 25mM citric acid, pH 5.0). The reaction was stopped after 10 minutes by addition of 50 μl IM H3PO4. The assays were measured using a BioRad microtiter plate reader (Model 550) at 450nm with a reference of 630nm. Interassay coefficient of variation was less than 10% and the within assay coefficient of variation was less than 5%. The sensitivity of the ELISA assay was 1-10 ng/ml and the mid range of the assay was 5ng/ml. The least detectable concentration of human chemerin was 0.5ng/ml. EXAMPLE 14
Serum CXS-746 levels are correlated with metabolic syndrome related phenotypes
To determine whether circulating levels of CXS-746 were associated with Type II diabetes and/or measures of obesity, plasma levels of CXS-746 were measured by ELISA in human plasma samples from 256 individuals aged 35-65 years, of whom 142 were NGT and 114 had T2D. The phenotypic characteristics of each group are presented in Table 11. Plasma CXS-746 levels were not significantly different between the NGT and T2D groups. It was also tested whether there was a difference in plasma CXS-746 levels between NGT subjects with BMI >30 kg/m2 (n=18) and those with a BMI <25 kg/m2 (n=75). Plasma CXS-746 levels were significantly higher in subjects with BMI >30 kg/m2 compared with those with a BMI<25 kg/m2 (296.5 ± 61.2 vs 222.7 ± 67.1, p=6.2xlθ"5). Linear regression analysis was performed to determine whether plasma CXS-746 levels were associated with adiposity and/or metabolic syndrome related phenotypes. Strong correlations with age and gender were observed due to higher CXS-746 levels in females compared to males and older individuals compared to younger individuals. After adjusting for age and gender, CXS-746 levels were significantly associated with measures of body fat (BMI, fat mass, weight and WHR) and metabolic syndrome related phenotypes (fasting glucose, fasting insulin, plasma triglycerides and blood pressure; Table 12,) in NGT subjects. After further adjustment for BMI, plasma CXS-746 levels were still independently associated with metabolic syndrome related phenotypes including systolic blood pressure (p=0.001) and plasma triglycerides (p=0.009) but not measures of insulin sensitivity or glucose homeostasis. When the data was adjusted for age, gender, BMI and triglycerides, CXS-746 levels were only associated with systolic and diastolic blood pressure (p=0.002 and p=0.004, respectively). These results clearly demonstrate that circulating CXS-746 levels are associated with key characteristics of metabolic syndrome including BMI, plasma triglyceride levels and blood pressure. TABLE 11
Physical and metabolic characteristics of study subjects
Figure imgf000092_0001
Homa b, quantitative estimate of β-cell function; homa_s, quantitative estimate of insulin sensitivity; BMI, body mass index; WHR, waist hip ratio; HDL, high dense lipoprotein, TG, triglycerides; BP, blood pressure TABLE 12 CXS-746 is associated with metabolic syndrome related phenotypes
Figure imgf000093_0001
Associations were performed using multivariate linear regression. The data was adjusted for age, gender, BMI and plasma triglycerides. BMI, body mass index; BP, blood pressure; homa s, quantitative estimate of insulin sensitivity; TG, plasma triglyceride levels *denotes values were obtained using Spearmeans correlation, all other data were obtained using Pearsons correlations. EXAMPLE 15 Human Samples
Plasma samples were obtained as part of the San Antonio Family Heart Study (SAFHS), a cohort which was first assessed in 1991 and was designed to primarily investigate the genetics of cardiovascular disease and its risk factors in Mexican Americans. The SAFHS includes 1,431 individuals in 42 extended families at baseline. Samples were ascertained by way of a single adult Mexican American proband selected at random, without regard to presence or absence of disease and almost exclusively from Mexican American census tracts in San Antonio. To ensure large, multigenerational pedigrees, probands had to have at least 6 age-eligible offspring and/or siblings living in San Antonio. All first, second, and third degree relatives of the proband and of the proband's spouse, aged 16 years or above, were eligible to participate in the study. All protocols were approved by the Institutional Review Board of the University of Texas Health Science Centre at San Antonio (San Antonio, TX).
EXAMPLE 16
Serum CXS-746 levels are correlated with metabolic syndrome related phenotypes in
SAFHS samples
Subjects who were normal NGT subjects between the Mauritius and SAFHS populations were compared. To define NGT subjects, all samples with a fasting glucose value of <6.1mmol/l were selected. The statistical analysis hereon was performed as outlined in the next paragraph.
As shown in Example 14, circulating CXS-746 levels were significantly associated with metabolic syndrome related phenotypes in the Mauritius population. This is also shown in a separate population, the SAFHS population. Circulating CXS-746 levels were measured in 1306 individuals aged between 15-92, of whom 1114 were NGT and 192 had T2D. The phenotypic characteristics of these samples are presented in Table 13. It was also tested whether there was a difference in plasma CXS-746 levels between NGT subjects with a BMK25kg/m2 (n=289) and those with a BMI>30 kg/m2 (n=349). Plasma CXS-746 levels were significantly higher in subjects with a BMI>30 kg/m2 compared with those with a BMI <25 kg/m2 (209 ± 4.98 vs 165 ± 4 16 respectively, p=5.1 xlO'9). Statistical analyses were performed using linear regression analysis and bivariate correlations to determine whether CXS-746 was associated with adiposity and/or metabolic syndrome related phenotypes. CXS-746 was significantly associated with a number of phenotypes tested including BMI, fasting glucose and fasting serum insulin (Table 14). Strong correlations with age and gender were also observed due to higher CXS-746 levels in females compared to males (188 ± 65 and 168 ± 55 respectively) and older individuals compared to younger individuals. After adjusting for age and gender, circulating CXS-746 levels were still significant in metabolic syndrome related parameters tested including BMI, fasting serum insulin, blood glucose (Table 14). This clearly demonstrates that CXS-746 is significantly associated with the development of the metabolic syndrome independent of age and gender. When the data were adjusted further for age, gender and BMI using linear regression analysis, significant associations were observed with HDL cholesterol, total serum cholesterol and triglycerides. Similar trends were observed in a separate population, Mauritius, where circulating CXS-746 levels were significantly associated with BMI, triglycerides and blood pressure independent of age and gender. This clearly demonstrates that CXS-746 is associated with metabolic syndrome related phenotypes independent of age and gender and could be a predictor for the development of the metabolic syndrome
TABLE 13
Physical and metabolic characteristics of SAFHS subjects
Figure imgf000095_0001
Figure imgf000096_0001
TABLE 14
CXS-746 is associated with obesity and metabolic syndrome related phenotypes in NGT subjects from the SAFHS cohort
Figure imgf000096_0002
Figure imgf000097_0001
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
BIBLIOGRAPHY
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Kohler and Milstein Nature 256:495-499, 1975
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Vermi et al. Journal of Experimental Medicine 201:509-15, 2005
WO 02/062994 Zabel et al Journal of Biological Chemistry 280: 34661-6, 2005

Claims

CLAIMS:
1. An isolated nucleic acid molecule comprising a sequence of nucleotides said nucleic acid molecule differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the nucleic acid molecule is selected from the list consisting of a nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% identity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or its complementary form under high stringency conditions.
2. A pharmaceutical composition comprising at least one isolated nucleic acid molecule according to Claim 1.
3. Use of an isolated nucleic acid molecule comprising a sequence of nucleotides said nucleic acid molecule which is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the isolated molecule is encoded by a nucleic acid molecule selected from the list consisting of a nucleic acid molecule comprises a nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a nucleotide sequence having at least about 90% identity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or its complementary form in the manufacture of a medicament for the treatment of one or more diseases and/or conditions.
4. An isolated expression product that is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the protein is selected from the list consisting of:
(i) an expression product encoded by a nucleotide sequence substantially as set forth in SEQ ID NOs:l or 18 (CXS-746) or SEQ ID NOs:15 or 19 (CXS-746R) or a derivative, homolog or analog thereof or a sequence encoding an amino acid sequence having at least about 90% similarity to this sequence or a derivative, homolog, analog, chemical equivalent or mimetic of said protein; and
(ii) an expression product encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) or a derivative, homolog or analog thereof.
5. A pharmaceutical composition comprising at least one an expression product according to Claim 4.
6. Use of an isolated expression product that is differentially expressed in cells from a subject having one or more diseases and/or conditions wherein the expression product is selected from the listing consisting of:
(i) an expression product encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:1 (CXS-746) or a derivative or homolog thereof or a sequence encoding an amino acid sequence having at least about 80% similarity to this sequence or a derivative or homolog of said protein;
(ii) an expression product encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:1 (CXS-746) or a derivative, homolog or analog thereof in the manufacture of a medicament for the treatment of one or more diseases and/or conditions; and
(iii) an expression product as disclosed in SEQ ID NO:20 (CXS-746) or SEQ ID NO:21 (CXS-7746R).
7. A method of modulating expression of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) in a mammal, said method comprising contacting SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) with an effective amount of an agent capable of modulating SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) expression for a time and under conditions sufficient to up-regulate or down- regulate or otherwise modulate expression SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs:15 or l9 (CXS-746R).
8. A method of modulating activity of the expression product of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) in a mammal, said method comprising administering to said mammal an effective amount of an agent capable of modulating the activity of the expression product of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) for a time and under conditions sufficient to increase or decrease or otherwise modulate the activity of the expression product of SEQ ID NOs: 1 or 18 (CXS- 746) or SEQ ID NOs: 15 or 19 (CXS-746R).
9. A method of treating a mammal suffering from a disease and/or condition, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to up-regulate or down-regulate or otherwise modulate expression SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R).
10. A method of treating a mammal suffering from a disease and/or condition, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to increase or decrease or otherwise modulate the activity of the expression product of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or l9 (CXS-746R).
11. The method according to any one of Claims 9 and 10 wherein the disease and/or condition is selected from Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity.
12. Use of an agent capable of modulating the expression of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) in the manufacture of a medicament for the treatment of a disease and/or condition.
13. Use of an agent capable of modulating the activity of an expression product of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R) in the manufacture of a medicament for the treatment of a disease and/or condition.
14. A method of treating a mammal suffering from a diseases and/or condition, said method comprising administering to said mammal an effective amount of one or more of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R).
15. A method of treating a mammal suffering from a diseases and/or condition, said method comprising administering to said mammal an effective amount of the expression product of one or more of SEQ ID NOs: 1 or 18 (CXS-746) or SEQ ID NOs: 15 or 19 (CXS-746R).
16. The method according to any one of Claims 14 and 15 wherein the disease and/or condition is selected from Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity.
17. A method of diagnosing Type I diabetes Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a subject, comprising the steps of: (a) obtaining one or more biological samples from said subject; and (b) measuring levels of a biomarker, wherein a difference of in the level of said specific biomarker in said biological sample(s) compared to a control sample is indicative of said subject having Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
18. The method of Claim 17, wherein an elevated level of said biomarker in said subject compared to a control is indicative of said subject having Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
19. The method of Claim 17 or 18 in which said biomarker is CXS-746.
20. The method of Claim 17 or 18 in which said biomarker is CXS-746R.
21. The method of Claim 19, wherein said decorin specific nucleic acid sequence is disclosed in SEQ ID NOs: 1 or 18 or its complement.
22. The method of Claim 20, wherein said CXS-746R nucleic acid sequence is disclosed in SEQ ID NOs: 15 or 19 or its complement.
23. The method of Claim 17, wherein the biological sample is selected from whole blood, blood plasma, serum, mucus, urine, semen, respiratory fluid, lymph fluid and saliva.
24. The method of Claim 17, wherein said biomarker is a ribonucleic acid.
25. The method of Claim 24 wherein said detecting is further defined as contacting said ribonucleic acids with an oligonucleotide probe that hybridizes thereto under high stringency conditions and then detecting hybridization.
26. The method of Claim 25, wherein said hybridization is detected by Northern hybridization or in situ hybridization.
27. The method of Claim 25, further comprising determining the amount of ribonucleic acid to which the probe hybridizes.
28. The method of Claim 25 in which the sequence of said probe is selected to bind specifically to a CXS-746 mRNA or a fragment thereof or a cDNA thereof.
29. The method of Claim 25, wherein said sequence of said probe is selected to bind specifically to a CXS-746R mRNA or a fragment thereof or a cDNA thereof.
30. The method of Claim 28, wherein said oligonucleotide probe is selected to bind specifically to an isolated nucleic acid having a sequence or its complement as disclosed in SEQ ID NOs:l or l8.
31. The method of Claim 29 wherein said oligonucleotide probe is selected to bind specifically to an isolated nucleic acid having a sequence or its complement as disclosed in SEQ ID NOs: 15 or 19.
32. The method of Claim 25, wherein said ribonucleic acids are amplified to form nucleic acid amplification products.
33. The method of Claim 32, wherein said amplification is by RT-PCR.
34. The method of Claim 33, wherein said amplification comprises contacting said ribonucleic acids with a pair of amplification primers designed to amplify a CXS-746 or CXS-746R mRNA.
35. The method of Claim 34, wherein said amplification comprises contacting said ribonucleic acids with a pair of amplification primers designed to amplify a nucleic acid segment comprising a detectable segment of a nucleic acid having the sequence or complement of SEQ ID NO:1 or SEQ ID NO:15 or SEQ ID NO:18 or SEQ ID NO:19.
36. The method of Claim 35, wherein said detectable segment is from about 100 bases in length up to about the length of the coding sequences of SEQ ID NO:1 or SEQ ID NO: 15 or SEQ ID NO: 18 or SEQ ID NO: 19.
37. The method of Claim 17, further defined as detecting difference in quantity of expression of a biomarker polypeptide.
38. The method of Claim 37, wherein the biomarker is CXS-746.
39. The method of Claim 37, wherein the biomarker is CXS-746R.
40. The method of Claim 37, wherein said detection is by immunoassay.
41. The method of Claim 40, wherein said immunoassay is an ELISA.
42. The method of Claim 40, wherein said immunoassay is a radioimmunoassay.
43. The method of Claim 37, wherein the CXS-746 specific amino acid sequence is disclosed in SEQ ID NO:20.
44. The method of Claim 38, wherein the CXS-7746R specific amino acid sequence is disclosed in SEQ ID NO:21.
45. The method of Claim 37, wherein said CXS-746 polypeptide is encoded by SEQ ID NOs:l or l8.
46. The method of Claim 39, wherein said CXS-746R polypeptide is encoded by SEQ ID NOs: 15 or 19.
47. A method of diagnosing Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing said conditions in a subject comprising:
(a) obtaining a serum sample from said subject; (b) contacting said serum sample with an antibody immunoreactive with CXS- 746 or CXS-746R to form an immunocomplex;
(c) detecting said immunocomplex; and
(d) comparing the quantity of said immunocomplex to the quantity of immunocomplex formed under identical conditions with the same antibody and a control serum from one or more controls, wherein an increase in quantity of said immunocomplex in serum from said subject relative to said control serum is indicative of Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions.
48. The method of Claim 47, wherein said immunocomplex is detected in a Western blot assay.
49. The method of Claim 47, wherein said immunocomplex is detected in an ELISA.
50. A kit for use in diagnosing Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a biological sample, comprising: (a) one or more nucleic acid segments that selectively hybridize to a CXS-746 or CXS-746R mRNA; and (b) a container for each of said one or more nucleic acid segments.
51. The kit of Claim 50, wherein said one or more nucleic acid segments selectively hybridize to a nucleic acid segment that includes the sequence or complement of SEQ ID NOs:l or l5 or l8 or l9
52. The kit of Claim 51, wherein said one or more nucleic acid segments is a pair of primers for amplifying said mRNA.
53. A kit for use in diagnosing diabetes or a complication arising from Type I diabetes, Type II diabetes, abnormal blood pressure, abnormal triglyceride levels, mitochondrial dysfunction, myopathy, genetic disorders, energy imbalance and/or obesity or a predisposition for developing these conditions in a biological sample, comprising: (a) one or more antibodies which immunoreact with a CXS-746 or CXS-746R protein or peptide; and (b) a container for said antibodies.
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