[go: up one dir, main page]

WO2012109495A1 - Cibles cellulaires de thiazolidinediones - Google Patents

Cibles cellulaires de thiazolidinediones Download PDF

Info

Publication number
WO2012109495A1
WO2012109495A1 PCT/US2012/024561 US2012024561W WO2012109495A1 WO 2012109495 A1 WO2012109495 A1 WO 2012109495A1 US 2012024561 W US2012024561 W US 2012024561W WO 2012109495 A1 WO2012109495 A1 WO 2012109495A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
mtot
optionally substituted
candidate compound
compound
Prior art date
Application number
PCT/US2012/024561
Other languages
English (en)
Inventor
Gerard R. Colca
Rolf F. Kletzien
William Mcdonald
Steven P. Tanis
Original Assignee
Metabolic Solutions Development Company, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Metabolic Solutions Development Company, Llc filed Critical Metabolic Solutions Development Company, Llc
Publication of WO2012109495A1 publication Critical patent/WO2012109495A1/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to assays for identifying compounds and chemically active agents that possess PPARy-sparing and/or insulin sensitizing activity for the treatment of metabolic diseases, for example, metabolic syndrome, obesity, diabetes mellitus, and neurodegenerative diseases.
  • metabolic diseases for example, metabolic syndrome, obesity, diabetes mellitus, and neurodegenerative diseases.
  • Peroxisome Proliferator Activated Receptors are members of the nuclear hormone receptor super family, which are ligand-activated transcription factors regulating gene expression. PPARs have been implicated in autoimmune diseases and other diseases, i.e. diabetes mellitus, cardiovascular disease, gastrointestinal disease, and Alzheimer's disease.
  • PPARy is the generally accepted site of action for insulin sensitizing thiazolidinedione compounds (TZDs).
  • PPARy is a key regulator of adipocyte differentiation and lipid metabolism. PPARy is also found in other cell types including fibroblasts, myocytes, breast cells, human bone- marrow precursors, and macrophages/monocytes. In addition, PPARy has been shown in macrophage foam cells in atherosclerotic plaques.
  • TZDs developed originally for the treatment of type-2 diabetes, generally exhibit high-affinity as PPARy ligands.
  • thiazolidinediones might mediate their therapeutic effects through direct interactions with PPARy helped to establish the concept that PPARy is a key regulator of glucose and lipid homeostasis.
  • compounds that involve the activation of PPARy also trigger sodium reabsorption and other unpleasant side effects that can negatively influence the health of the patient being administered the compound.
  • TZDs that have reduced binding and/or activation of PPARy ligands, demonstrated beneficial biological properties such as increased insulin sensitivity, reduced blood glucose, reduced blood pressure, increased HDL cholesterol, and preservation of beta cells in the pancreas, without the negative side effects observed with PPARy-activating TZDs.
  • beneficial biological properties such as increased insulin sensitivity, reduced blood glucose, reduced blood pressure, increased HDL cholesterol, and preservation of beta cells in the pancreas.
  • the present invention provides screening assays for the identification of candidate compounds that are capable of binding to a membrane Target of
  • mTOT protein Thiazolidinedione (mTOT protein) protein, or a fusion protein comprising the same.
  • a "mTOT protein" of the present invention includes: a BP44 protein, a BRP44-Like protein, a BP44-BRP44-Like heterodimer and homologs, orthologs, fusions, functional fragments, derivatives and analogs thereof.
  • the method includes: a) combining an mTOT protein, with one or more candidate compounds under conditions to allow specific binding between the mTOT protein and the one or more candidate compounds, and b) detecting specific binding, thereby identifying one or more lead candidate compounds which specifically binds the mTOT protein.
  • the screening method includes verifying that the candidate compound which binds to the mTOT protein is a therapeutic active agent.
  • a screening assay for identifying an insulin sensitizing therapeutic agent effective in treating or preventing an insulin-resistance disease or disorder in a subject comprises: screening one or more candidate compounds in a binding assay, the binding assay comprising the steps: (i) providing an mTOT protein;(ii) contacting the mTOT protein with a candidate compound; and (iii) detecting if the candidate compound specifically binds to the mTOT protein or inhibits the specific binding of a thiazolidinedione compound to the mTOT protein, wherein the candidate compound is identified as a lead candidate compound if the candidate compound specifically binds to the mTOT protein or inhibits the binding of the thiazolidinedione compound to the mTOT protein.
  • the mTOT protein is provided on the surface of a reaction substrate, for example, as used in high-throughput assays wherein samples are spotted or imprinted on the surface of a substantially planar substrate.
  • the mTOT protein can be provided in a reaction receptacle, for example, a tube, a well of a microtiter plate, a pore or sample well of a microfluidic device, or an indentation on the surface of a substrate operable to receive additional reagents or fluids.
  • the mTOT protein is contacted with labeled binding partner in a reaction receptacle, for example, a tube, a well of a microtiter plate, an indentation on the surface of a substrate.
  • the screening method can include repeating steps (i) - (iii) in a high throughput screen.
  • the method further includes assaying the lead candidate compound in an activity assay to determine whether the lead candidate compound is an insulin-sensitizing therapeutic agent.
  • a method for screening a plurality of candidate compounds to identify a lead candidate compound effective against an insulin resistance disease or disorder includes:
  • the method further includes repeating steps (b)-(d) in a high throughput screen.
  • the present invention provides screening assays that are capable of identifying a lead candidate compound.
  • further validation of the lead candidate compound as a therapeutic active agent can be achieved by performing an activity assay that demonstrates the lead candidate compound's ability to mimic the insulin sensitizing activity of a thiazolidinedione compound, for example, a PPARy-sparring thiazolidinedione compound.
  • the activity assay enables the determination whether the lead candidate compound has insulin sensitizing activity or anti-diabetic, anti- obesity, metabolic protective, or neuroprotective activity.
  • Figure 1 represents a chemical structure of mitoglitazone in accordance with the embodiments of the present invention.
  • Figure 2 depicts a photograph of a Western Blot of brown adipose cell lysate after incubation of the cells with varying concentrations of mitoglitazone and probing for BP44 protein.
  • Figures 3 A & 3B depict an alignment of amino acid sequences of BR44 derived from different organisms.
  • Figures 4A& 4B depict consensus sequences between BP44, BRP44-Like protein and homology with a UPF0041 domain sequence.
  • Figure 5 depicts the sequencing results of BP44 protein used in the transfection assays.
  • the optimized BP44-His 6 gene was excised from the DNA2.0 cloning and ligated into the BamHI/NotI sites of the expression vector pcDNA3.1. Correct clones were selected by restriction analysis and the sequence was confirmed by DNA sequence analysis by ACGT, Inc.
  • Figure 6 depicts the sequencing results of BRP44-Like protein used in the transfection assays.
  • the optimized MTOT-Like-His6 gene was excised from the DNA2.0 cloning vector pJ221 with BamHI and Notl and ligated into the BamHI/NotI sites of the expression vector pcDNA3.1. Correct clones were selected by restriction analysis and the sequence was confirmed by DNA sequence analysis by ACGT, Inc.
  • Figure 7 depicts a Western blot indicating the relative sizes of the cloned BP44, BP44-His 6 , BRP44-Like and BRP44-Like His 6 sequences cloned and expressed in HEK293 cells using antibodies to BP44, BRP44-Like and Hex-His.
  • Figure 8 depicts the chemical structure of a thiazolidinedione labeled with a 3H radionuclide for use in various competitive binding assays and a non-thiazolidinedione compound also known to bind to mTOT proteins.
  • FIG. 9 depicts an autoradiography film indicate the specifically labeled native mTOT (-14 kDa) from the wild type HEK293 P2 fraction.
  • the mTOT (-14 kDa) and mTOT 6-His( ⁇ 15.6 kDa) indicated by the two arrows in the P2 fraction from the transiently transfected HEK 293 cells show the increased size of the expressed his-tagged protein is specifically crosslinked.
  • FIG. 9B shows the chemical structure of the photoaffinity crosslinker, 125 I-MSDC-1101.
  • FIG. 9C Autoradiography film showing the crosslinking of rat liver P2 membranes. The arrow indicates the specifically crosslinked mTOT protein in the control DMSO treated membranes (lane 1), and the displacement of the crosslinker with either 25 ⁇ MSDC-160 (lane 2) or 25 ⁇ UK5099 (lane 3).
  • the words "preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.
  • compositional percentages are by weight of the total composition, unless otherwise specified.
  • the word "include” and its variants is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present invention that do not contain those elements or features.
  • the present invention provides assays for screening or identifying candidate compounds including, metals, polypeptides, proteins, lipids, polysaccharides,
  • insulin resistance refers to a state in which a normal amount of insulin produces a subnormal biologic response relative to the biological response in a subject that does not have insulin resistance.
  • Insulin resistance disorder refers to any disease or condition that is caused by or contributed to by insulin resistance.
  • examples ofinsulin resistance disease or disorders include: diabetes mellitus, obesity (obesity can include individuals having a body mass index (BMI) of at least 25 or greater.
  • BMI body mass index
  • Obesity may or may not be associated with insulin resistance), weight gain, metabolic syndrome, insulin- resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy, cholesterol related disorders, such as gallstones, cholescystitis and
  • cholelithiasis gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and bone loss, e.g. osteoporosis.
  • metabolic or inflammation mediated diseases e.g., dyslipidemia, central obesity, rheumatoid arthritis, lupus, myasthenia gravis, vasculitis, Chronic Obstructive Pulmonary Disease (COPD), or inflammatory bowel disease
  • cardiovascular disease e.g. atherosclerosis, arteriosclerosis, angina pectoris, coronary artery disease, congestive heart failure, stroke, or myocardial infarction
  • neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis.
  • Candidate compounds that may be identifiable using the assays provided herein may also be useful for treating or preventing metabolic diseases such as diabetes or obesity.
  • candidate compounds may also be useful in co-therapies directed to the treatment of any of these diseases.
  • a screening method to identify a lead candidate compound of the present invention includes the steps:
  • the new screening methods described herein are predicated, at least in part, on the unexpected discovery that mitoglitazone a PPARy-sparing thiazolidinedione compound, being effective in increasing the sensitivity of insulin, specifically binds to and has an affinity for an mTOT protein, for example, Brain Protein 44 (BP44), Brain R Protein 44-like
  • BRP44-Like protein and a heterodimer of these two proteins (BP44-BRP44-Like).
  • Mitoglitazone is a PPARy-sparing thiazolidinedione shown to effectively stimulate brown adipose tissue ("BAT”) stores, and is useful for treating obesity and other metabolic diseases such as diabetes mellitus.
  • BAT brown adipose tissue
  • a formula for mitoglitazone is shown in Figure 1.
  • an mTOT protein includes any mitochondrial protein that is capable of specifically binding to a thiazolidinedione compound. In some embodiments, an mTOT protein is any mitochondrial protein that is capable of specifically binding to a
  • an mTOT protein is any mitochondrial protein that is capable of specifically binding to PPARy-sparring thiazolidinedione compounds, for example mitoglitazone.
  • an mTOT protein is any mitochondrial protein that is capable of specifically binding to a non-PPARy-sparring thiazolidinedione compound, for example rosiglitazone, troglitazone or pioglitazone.
  • an mTOT protein is any mitochondrial protein that is capable of specifically binding to only PPARy-sparring thiazolidinedione compounds, for example mitoglitazone, but not capable of binding to non- PPARy-sparring thiazolidinedione compounds.
  • mTOT proteins include BP44 protein, BRP44-Like protein and heterodimer proteins comprising both BP44 and BRP44-Like proteins.
  • the mTOT protein is a prokaryotic protein, or a eukaryotic protein, for example a drosophila protein, a yeast protein, a mammalian protein, a human protein.
  • the mTOT protein is a human
  • mitochondrial protein for example, a human BP44 protein, a human BRP44-Like protein or a heterodimer protein comprising both human BP44 and human BRP44-Like proteins.
  • mTOT proteins for use in the screening assays described herein also include orthologs, homologs, functional fragments of mTOT proteins, or fusion proteins comprising the mTOT proteins exemplified herein.
  • mTOT protein or mTOT protein-Like refer to BP44 and BRP44-Like proteins respectively.
  • Brain protein 44 is also known as CGI-129, dJ68L15.3, DKFZp564B167, MGC125752, MGC125753, BRP44, has been previously isolated from brain tissue and other tissue sources.
  • BP44, BRP44, BP44 protein and BP44 peptide are synonyms and are used interchangeably.
  • the term BP44 protein refers to a protein that is a target for the PPARy-sparing thiazolidinediones (e.g., mitoglitazone).
  • BP44 protein can range in size from about 200 amino acids to about 50 amino acids, or from about 150 amino acids to about 50 amino acids or from about 125 amino acids to about 50 amino acids, or from about 200 amino acids to about 75 amino acids, or from about 200 amino acids to about 100 amino acids or from about 200 amino acids to about 125 amino acids.
  • BP44 can include illustrative embodiments wherein BP44 include proteins having 127 or 105 amino acids.
  • the BP44 proteins share a putative conserved domain called the UPF0041 domain as set forth at least in part as shown in SEQ ID NO:41.
  • the theoretical pi of the BP44 protein is 9.67 and has a calculated molecular weight of 12,347 Daltons.
  • accession numbers accompanying examples of BP44 and BRP44- Like proteins are derived from the National Center For Biotechnology Information - NCBI (USA).
  • the BP44 protein (protein and polypeptide both refer to a polymer of amino acids and are used interchangeably herein) used in the various assays of the present invention can include natural or chemically synthesized BP44 protein, full length, substantially full-length, homologs, orthologs, functionally equivalent form of the BP44 protein, or mutant forms of BP44.
  • Exemplary BP44 proteins are provided in Tables 1, 3 and 4 and shown in Figures 3 A, 3B, 4A and 4B.
  • orthologs, homologs, functional fragments, or mutant forms of BP44 refer to proteins having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100%, identity to, or homologous with the BP44 proteins provided in or encoded by any illustrative BP44 proteins or nucleic acids described in Tables 1-4 and shown in Figures 3 A, 3B, 4A and 4B.
  • BP44 protein useful in the present invention is derived from a source, exemplified, but not limited to, those proteins having amino acid sequences provided in Tables 1 , 3 or 4and shown in Figures 3A, 3B, 4A and 4B.
  • the BP44 protein is a human BP44 protein described herein having the amino acid sequence set forth in any one or more of SEQ ID NOs: 1-4, functional fragments or variants of SEQ ID NOs: 1-4, allelic variants of SEQ ID NOs: 1-4, species variants of SEQ ID NOs: 1-4, or homologs of SEQ ID NOs: 1-4.
  • the BP44 protein is a human BP44 protein described herein linked to a His6 tag, having the amino acid sequence set forth in any one or more of SEQ ID NO: 75.
  • BP44 proteins useful in the present invention can include proteins that may be encoded by the polynucleotides of SEQ ID NOs: 5 - 7, and 74 and BP44 proteins encoded by a nucleic acid sequence that hybridizes to the BP44 coding region of a nucleic acid sequence in SEQ ID NOs: 5 - 7 or complementary sequences thereof under "stringent hybridization conditions" as is defined herein and commonly used in the art of molecular biology, for example, in: Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridisation with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
  • a "homolog" of a first gene (for example, a human BRP44 encoding gene as disclosed in SEQ ID NOs: 5 -7) generally refers to a second, different gene that is substantially identical to the first gene, or that encodes a gene product that is substantially identical (i.e. having a % identity greater than 90%) to the gene product encoded by the first gene.
  • An "ortholog" of a first gene refers to a second gene from a different organism that is substantially identical to the first gene, or that encodes a gene product that is substantially identical or substantially identical to the gene product encoded by the first gene.
  • BP44 proteins that find utility in the assays of the present invention can include proteins or polypeptides having at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100%, identity to, or homologous with the BP44 protein having the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 and 8-41, or any full-length BP44 protein described herein.
  • BP44 proteins comprising truncated forms of BP44 having at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% sequence identity or homology to any one of SEQ ID NOs: 1-4, 8-41 and 75 are
  • useful truncated forms comprise functional regions in their amino acid sequence when compared to the full length BP44 protein since they can be functionally identified using the mitochondrial membrane competitive binding crosslinking assay described in Example 1, or can be proven to be functional by directly binding to an unlabeled or labeled mitoglitazone or rosiglitazone compound or analog in vitro or in vivo as exemplified, for example, in the Examples described herein.
  • an mTOT protein can also include a functional region of the BP44 protein.
  • an mTOT protein can include a mitochondrial protein having a conserved domain called the UPF0041 domain.
  • illustrative examples of proteins containing a conserved domain called the UPF0041 domain are provided in a family of proteins called pfam03650, which are part of Super Family Accession No: cl04196.
  • a representative UPF0041 domain is provided in SEQ ID NO: 41 or a protein which contains the consensus sequence of FIG. 4B.
  • Exemplary nucleic acids encoding illustrative BP44 proteins are provided in Table 2 and in Example 3.
  • an illustrative BP44 protein includes an amino acid sequence derived from non-human organisms as described in Table 3 and FIG. 3A & 3B.
  • BP44 proteins include homolog or ortholog proteins of BP44 that belong to the protein family UPF0041 having an identifier: pfam03650illustratively shown in Table 4.
  • illustrative BP44 proteins can include proteins which includes within their amino acid sequence, a domain of pfam03650 having an amino acid sequence comprising:
  • the BP44 protein is a protein having a consensus amino acid sequence:
  • the BP44 protein useful for the assays described herein can be obtained commercially from translatable DNA form from Abnova (BRP44 (Accession No. NP 056230, 1-127 amino acids, full-length recombinant protein with GST tag) Cat. No.
  • human BP44 can be recombinantly produced using Invitrogen's Gene Clone IOH41193 (Invitrogen Corp, Carlsbad, CA USA) using the manufacturer's instructions.
  • the BP44 protein useful for the assays described herein can be obtained commercially from Abnova (BRP44 (Accession No. NP 056230, 1-127 amino acids, full-length recombinant protein with GST tag) Cat. No. H00025874-P01).
  • Antibodies to human and mouse BP44 are commercially available from Novus (Clone (H00025874- M12, Novus, Littleton CO, USA) and Abnova (Clone H00025874-M11, Abnova, Walnut, CA, USA).
  • the BP44 protein for use in the assays described herein can include proteins and polypeptides having protein sequences comprising at least 70 %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 % sequence identity to SEQ ID NOs: 1-4, 8-41 and 75.
  • BRP44-Like proteins can be used as a binding target in the assays described herein alone and/or with BP44, or as part of a heterodimer with BP44.
  • representative BRP44-Like proteins have an amino acid sequence comprising or consisting of those provided in Table 5.
  • Table6 Exemplary nucleotide sequences encoding human BRP44-Like proteins.
  • the BRP44-Like protein used in the various assays of the present invention can include natural or chemically synthesized BRP44-Like protein, full length, substantially full- length, homologs, orthologs, functionally equivalent form of the BRP44-Like protein, mutant forms of BRP44-Like or fusion constructs comprising BRP44-Like protein and a second protein, for example a different mTOT protein or a protein tag, for example His 6 .
  • Exemplary BRP44-Like proteins are provided in Table 5 and in SEQ ID NO: 99.
  • exemplary forms of BRP44-Like proteins refer to proteins having at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99%, identity to, or homologous with the BRP44-Like proteins provided in Table 5, or SEQ ID NO: 99 or encoded by any examples described in Table 6.
  • the mutant BRP44- Like protein can be a truncated polypeptide or a polypeptide with one or more internal deletions.
  • BRP44-Like protein useful in the present fnvention is derived from a human source, exemplified, but not limited to those proteins having amino acid sequences provided in Table 1.
  • the BRP44-Like protein is a human BRP44-Like protein described herein having the amino acid sequence set forth in any one or more of SEQ ID NOs: 42 and 47, functional fragments or variants of SEQ ID NOs: 42 and 47 allelic variants of SEQ ID NOs: 42 and 47, species variants of SEQ ID NOs: 42 and 47, or homologs or orthologs of SEQ ID NOs: 42 and 47, or a fusion construct encoded by the nucleotide sequence of SEQ ID NO: 79.
  • BRP44-Like protein NCBI Accession - NM 016098
  • NCBI Accession - NM 016098 is commercially available from OriGene Catalog. No.
  • TP301461 (OriGene, Rockville, MD, USA).
  • BRP44-Like proteins useful in the present invention can include proteins that may be encoded by the polynucleotides of SEQ ID NOs: 52, 53, and 79 or their complementary sequence thereof and BRP44-Like proteins encoded by a nucleic acid sequence that hybridizes to the BRP44-Like coding region of a nucleic acid sequence in SEQ ID NOs:52, 53, and 79 or complementary sequences thereof under "stringent hybridization conditions" which can include, 50% formamide, 5X SCC and 1% SDS, incubating at 42°C and wash in 0.2X SSC and 0.1% SDS at 65°C.
  • stringent hybridization conditions can include, 50% formamide, 5X SCC and 1% SDS, incubating at 42°C and wash in 0.2X SSC and 0.1% SDS at 65°C.
  • BRP44-Like proteins that find utility in the assays of the present invention can include proteins or polypeptides having at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 100%, identity to, or homologous with the BRP44-Like protein having the amino acid sequence set forth in any one of SEQ ID NOs: 42 - 51, and 99 or any full-length BRP44-Like protein described herein.
  • BRP44-Like proteins comprising truncated forms of BRP44-Like having at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90% sequence identity or homology to any one of SEQ ID NOs: 42-51 and 99 are contemplated herein.
  • useful truncated forms comprise functional regions in their amino acid sequence when compared to the full length BRP44-Like protein since they can be functionally identified using the mitochondrial membrane competitive binding crosslinking assay described in Example 1 , or can be proven to be functional by directly binding to an unlabeled or labeled mitoglitazone molecule or analog in vitro or in vivo.
  • functional regions of the BRP44-Like protein can include proteins having a conserved domain called the UPF0041 domain which are provided in a family of proteins called pfam03650, which is part of Super Family
  • the sequence of a BP44 protein or a BRP44-Like protein can also be modified by amino acid substitutions, replacements, insertions, deletions, truncations and other modifications. Typically such modifications can be used to prepare mimics of biologically- occurring polypeptides or to generate suitable targets for screening. For example, certain amino acids can be substituted for other amino acids in a polypeptide without appreciable loss of physiological activity (e.g., activities associated with mitoglitazone action intra and inter cellular, for example, anti-diabetic activity, reduction of blood glucose in an animal model and the like). Changes to the amino acid sequence can be conservative changes.
  • the following eight groups each contain amino acids that are regarded conservative substitutions for one another: 1) Alanine (A) and Glycine (G); 2) Aspartic acid (D) and Glutamic acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine ( ); 5) Isoleucine (I), Leucine (L), Methionine (M) and Valine (V); 6) Phenylalanine (F), Tyrosine (Y) and
  • conservative substitution tables providing functionally similar amino acids are well known in the art.
  • one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary
  • substitution ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gin; ile/leu or val; leu/ile or val; lys/arg or gin or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu.
  • An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer- Verlag (1979)).
  • substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered “conservatively modified variations".
  • percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue 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 and multiplying the result by 100 to yield the percentage of sequence identity.
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% percent identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • Sequences are "substantially identical” to each other if they are at least 60%, at least 70%, at least 80% or at least 90% identical. These definitions also refer to the complement of a test sequence. Optionally, the identity exists over a region that is at least about 50 nucleotides in length, or more typically over a region that is 100 to 500 or 1000 or more nucleotides in length.
  • similarity in the context of two or more polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues that are either the same or similar as defined by a conservative amino acid substitutions (i.e., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • a conservative amino acid substitutions i.e., 60% similarity, optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a specified region
  • Sequences are "substantially similar” to each other if they are at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% similar to each other. Optionally, this similarly exists over a region that is at least about 50 amino acids in length, or more typically over a region that is at least about 100 to 500 or 1000 or more amino acids in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities or similarities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2:482 (1970)), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J. Mol. Evol. 35:351-360 (1987)). The method used is similar to the method described by Higgins and Sharp (CABIOS 5:151-153 (1989)). The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package (e.g., version 7.0 (Devereaux et al., Nucl. Acids Res. 12:387-95 (1984)).
  • BLAST and BLAST 2.0 algorithms are described in Altschul et al. (Nuc. Acids Res. 25:3389-402 (1977)), and Altschul et al. (J. Mol. Biol. 215:403-10 (1990)), respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length (W) in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold (Altschul et al., supra).
  • These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., arlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-87).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, typically less than about 0.01, and more typically less than about 0.001.
  • modified BP44 or BRP44-Like proteins or polypeptides for the use in the present assay methods, the hydropathic index of amino acids can be considered. Amino acid substitutions can also be made on the basis of hydrophilicity.
  • the BP44 protein, BRP44-Like protein and BP44-BRP44- Like heterodimer can also be made or expressed as a fusion protein comprising a BP44 protein, RP44-Like protein and BP44-BRP44-Like heterodimer or a ortholog, homolog and functional fragment thereof joined at its N- or C-terminus to a second polypeptide or other molecular entity.
  • the second adjoining polypeptide can be, for example, an epitope, a selectable protein, an enzyme, polyethylene glycol (PEG) and the like.
  • the second polypeptide can be beta-galactosidase, a hexaHis tag, a fluorescence protein tag, for example, a green fluorescent protein (GFP), FLAG, influenza A hemagglutinin, c-Myc, or the like.
  • GFP green fluorescent protein
  • FLAG influenza A hemagglutinin
  • c-Myc c-Myc
  • the BP44 protein,BRP44-Like protein and BP44-BRP44-Like heterodimer can include a C-terminal tag comprising a hexaHis (His 6 -) cleavable sequence.
  • a BP44-BRP44-Like heterodimer refers to a single protein that has fused at least a partial sequence of BP44 as provided in any one of SEQ ID NO: 1-4 and 8-41 with at least a partial sequence of BRP44- Like protein as provided in any one of SEQ ID NO: 42-51.
  • the N- terminal portion of the heterodimer is BP44 and the C-terminal portion is BRP44-Like and vice versa.
  • each portion may represent a full-length sequence or a partial sequence of each of BP44 and BRP44-Like proteins described herein.
  • the mTOT protein BP44-BRP44-Like heterodimer may comprise a linker consisting of one to twenty amino acids, separating the two protein portions.
  • an mTOT protein or fusion proteins thereof for example, an mTOT protein fused with a reporter protein, for example, a 6X His tag, a myc tag, a glutathione enzyme tag, a chloramphenicol
  • acetyltransferase (CAT) protein or a fluorescence protein tag e.g. a green fluorescence protein GFP, Enhanced Green Fluorescent Protein (EGFP), a red fluorescence protein and the like) can be produced as a recombinant protein.
  • a nucleic acid encoding the recombinant mTOT protein or fusion protein thereof is preferably isolated or synthesized.
  • a nucleic acid encoding an mTOT protein can comprise one or more nucleotide sequences provided in one of SEQ ID NOs: 5-7, 52-53, 74, 79 or a complement thereof.
  • nucleic acid encoding the mTOT protein or combination of mTOT proteins is/are isolated using a known method, such as, for example, amplification (e.g., using PCR or splice overlap extension) or isolated from nucleic acid from an organism using one or more restriction enzymes or isolated from a library of nucleic acids.
  • amplification e.g., using PCR or splice overlap extension
  • isolated from nucleic acid from an organism using one or more restriction enzymes or isolated from a library of nucleic acids are known in the art and described, for example, in
  • two non-complementary nucleic acid primer molecules comprising at least about 20 nucleotides in length, and more preferably at least 25 nucleotides in length are hybridized to different strands of a nucleic acid template molecule, and specific nucleic acid molecule copies of the template are amplified enzymatically.
  • the primers hybridize to nucleic acid sequences adjacent to polynucleotides encoding an mTOT protein of the invention, thereby facilitating amplification of the nucleic acid that encodes the mTOT protein.
  • PCR primers for BP44 are commercially available from RealTimePrimers.com, Catalog No. VHPS-894 (RealTimePrimers LLC, Elkins Park, PA USA).
  • the amplified nucleic acid is isolated using a method known in the art and, preferably cloned into a suitable vector.
  • PCR primers can also be selected on the basis of nucleic acid sequences 5' upstream and 3' downstream of the coding sequence of an mTOT protein genes which are obtainable from public gene databases, such as NCBI, and are known to those skilled in the art.
  • cDNA clones of exemplary mTOT proteins are also commercially available and can be transfected into an expression host of interest.
  • Homo sapiens BRP44-Like (NM_016098.1) cDNA clones (available as non-tagged or tagged (Myc-DDK tag or GFP-tagged)) (Catalog No. SC127044, RC201461 and RG201461) OriGene,
  • HEK-293 cell line a eukaryotic cell line
  • HEK-293 cell line a human cell-line
  • a nucleic acid e.g., genomic DNA, cDNA, or RNA that is then reverse transcribed to form cDNA
  • a suitable vector can be then introduced into a suitable organism, for example, a eukaryotic cell or prokaryotic cell, for example, a bacterial cell.
  • a cell comprising the nucleic acid of interest is isolated using methods known in the art and described, for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
  • an mTOT protein-encoding nucleotide sequence for example, SEQ ID NOs: 5-7, 52-53, 74, 79, or a complement thereof, is operably linked to a promoter or other regulatory sequence capable of regulating expression in a cell-free system or cellular system.
  • a nucleic acid comprising a sequence that encodes a BP44 protein is operably linked with a suitable promoter and is expressed in a suitable cell for a time and under conditions sufficient for expression of the BP44 protein to occur.
  • Nucleic acids encoding BP44 proteins, homologs or a functional fragments thereof are readily derived from the publicly available amino acids and
  • nucleic acid sequences set forth in any one of SEQ ID NOs: 1-4, 8-51 and 75.
  • promoter is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers and silencers) that alter expression of a nucleic acid (e.g., a transgene), e.g., in response to a developmental and/or external stimulus, or in a tissue specific manner.
  • a nucleic acid e.g., a transgene
  • promoter is also used to describe a recombinant, synthetic or fusion nucleic acid, or derivative, which confers, activates or enhances the expression of a nucleic acid (e.g., a transgene and/or a selectable marker gene and/or a detectable marker gene) to which it is operably linked.
  • a nucleic acid e.g., a transgene and/or a selectable marker gene and/or a detectable marker gene
  • Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid.
  • the term "in operable connection with” “in connection with” or “operably linked to” means positioning a promoter relative to a nucleic acid (e.g., a transgene) such that expression of the nucleic acid is controlled by the promoter.
  • a promoter is generally positioned 5' (upstream) to the nucleic acid, the expression of which it controls.
  • heterologous promoter/nucleic acid combinations e.g., promoter/transgene and/or promoter/selectable marker gene combinations
  • a suitable promoter can include, but is not limited to, a T3 or a T7 bacteriophage promoter.
  • Typical expression vectors for in vitro expression or cell-free expression are readily known and have been described and include, but are not limited to the TNT T7 and TNT T3 systems (Promega), the pEXPl-DEST and pEXP2-DEST vectors (Invitrogen, Carlsbad, CA USA) and pINVITRO plasmids from (InvivoGen, San Diego, CA USA).
  • Typical promoters suitable for expression of mTOT proteins in bacterial cells include, but are not limited to, the lacZ promoter, the Ipp promoter, temperature-sensitive lamda L or .lamda R promoters, T7 promoter, T3 promoter, SP6 promoter or semi-artificial promoters such as the IPTG-inducible Tac promoter or lacUV5 promoter.
  • a number of other gene construct systems for expressing the nucleic acid fragment of the invention in bacterial cells are well-known in the art and are described for example, in Ausubel et al (In: Current Protocols in Molecular Biology. Wiley Interscience, ISBN 047 150338, 1987), U.S. Pat. No. 5,763,239 (Diversa Corporation) and Sambrook et al (In: Molecular Cloning: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Third Edition 2001).
  • Typical promoters suitable for expression in viruses of eukaryotic cells and eukaryotic cells include the SV40 late promoter, SV40 early promoter and cytomegalovirus (CMV) promoter, CMV IE (cytomegalovirus immediate early) promoter amongst others.
  • CMV cytomegalovirus
  • Preferred vectors for expression in mammalian cells include, but are not limited to, the pcDNA vector suite supplied by Invitrogen, in particular pcDNA 3.1 myc-His-tag comprising the CMV promoter and encoding a C-terminal 6x His and MYC tag; and the retrovirus vector pSRalpha-tkneo.
  • Methods for introducing an isolated nucleic acid molecule or a gene construct comprising same into a cell for expression are well known to those skilled in the art.
  • the technique used for a given organism can depend on known successful techniques suitable for introducing heterogenous nucleic acids for the particular host organism of interest.
  • Means for introducing recombinant DNA into cells include microinjection, transfection mediated by DEAE-dextran, transfection mediated by liposomes such as by using lipofectamine
  • a variety of eukaryotic cell expression systems can be used in the methods according to the present invention.
  • a suitable eukaryotic cell expression system is one in which expression of the BP44 protein in a suitable genetic background causes toxicity.
  • yeast One model eukaryotic organism, yeast, provides a well-established system for genetic and chemical screening. Many genes can be studied in yeast because they are non-essential under certain growth conditions. In addition, homologs and orthologs of yeast genes can be studied in yeast because such homologs and orthologs often have overlapping functions with the yeast genes, allowing deletion or inactivation of the yeast gene.
  • Suitable yeast strains which can be used in the context of the present invention include, for example, Saccharomyces cerevisiae, Saccharomyces uvae, Saccharomyces kl yveri, Schizosaccharomyces pombe, Saccharomyces uvarum, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kl yveri, Yarrowia lipolytica, Candida species such as Candida utilis or Candida cacaoi, Geotrichum species such as Geotrichum fermentans, and the like.
  • the yeast strain can be Saccharomyces cerevisiae.
  • suitable eukaryotic cell expression systems can include, for example, rat, mouse, Drosophila, or C. elegans.
  • the eukaryotic cell expression system can be another non- human, animal, insect or lower model system.
  • a suitable eukaryotic cell expression system also can be human cells or cells isolated from such a non-human eukaryotic organism, such as, for example, a human cell line, rat, mouse, guinea pig, hamster, dog, horse, pig,
  • Drosophila Chinese Hamster Ovary (CHO) or C. elegans cells cultured in vitro.
  • the eukaryotic cell expression system can be genetically engineered to express an mTOT protein and fusion constructs thereof, or may constitutively express these proteins independently without any further manipulation.
  • Drosophila can be genetically engineered to express a BP44 protein that causes over expression in at least some cells in a suitable genetic background.
  • the system can express an endogenous mTOT protein that causes over expression of it in a suitable genetic background.
  • Suitable expression vectors containing mTOT protein DNA/cDNA/RNA for expression of these proteins in vitro are readily available, for example, Homo sapiens BRP44-Like, mRNA (cDNA clone MGC:4871 IMAGE:3452973 complete CDS expressed in pCMV-SPORT6 expression vector in E.coli DH10B), from Invitrogen, (Catalog No. 3452973, Invitrogen, Carlsbad, CA USA).
  • the eukaryotic cell of the expression system can optionally include alleles of, or mutations in, genes that facilitate uptake or increase permeability of a candidate agent(s) when employed in a screening assay.
  • the eukaryotic cell of the cell expression systems e.g., a yeast strain
  • a yeast strain can include mutations in one or more of the yeast genes erg6, pdrl and/or pdr3, which affect membrane efflux pumps and may increase permeability of candidate agents.
  • the genetic background of the eukaryotic cell expression system is one in which expression of the mTOT protein causes some activity that can be measured and at least partially quantified.
  • a cell having a "suitable genetic background" refers to a cell having a genetic makeup in which the mTOT protein is over expressed for the purposes of evaluating the effects of a candidate compound when the cell is used in a screening assay.
  • a useful assay using the discovery outlined herein involves detecting, analyzing or quantifying the effects of compounds on metabolic and/or cellular function in intact cells.
  • mTOT protein i.e.BP44 protein, and/or BRP44-Like protein and/or BP44-BRP44-Like heterodimer
  • fusion constructs comprising an mTOT protein in various cells will result in change in metabolism and/or cell function that can be measured.
  • Candidate compounds may be added to incubations of such cells and those that interfere with the function of the over expressed mTOT protein will then be selected for evaluation of anti-diabetic, anti-obesity, metabolic protective, or neuroprotective activity in one or more activity assays.
  • an mTOT protein or fusion protein comprising same, can be purified using any known methods in the art. Such purification preferably provides an mTOT protein, substantially free of non-specific protein, acids, lipids, nucleic acids, carbohydrates, and the like. Antibodies and other affinity ligands are particularly preferred for producing isolated protein. Antibodies specific for human and mouse BP44 protein are known and described above.
  • the mTOT protein will be in a preparation wherein more than about 80%, 85%, or more than 90% (e.g. 95%, 96%, 97%, 98% or 99%) of the protein in the preparation is an mTOT protein of the present invention or fusion protein comprising same.
  • Standard methods of protein purification can be employed to obtain an isolated mTOT protein of the present invention, including, but not limited to, various high-pressure (or high-performance) liquid chromatography (HPLC) and non-HPLC peptide isolation protocols, such as size exclusion chromatography, ion exchange chromatography, phase separation methods, electrophoretic separations, precipitation methods, salting in/out methods, immunochromatography, and/or other methods.
  • HPLC high-pressure liquid chromatography
  • non-HPLC peptide isolation protocols such as size exclusion chromatography, ion exchange chromatography, phase separation methods, electrophoretic separations, precipitation methods, salting in/out methods, immunochromatography, and/or other methods.
  • a method for isolating an mTOT proteinemploys reversed-phase HPLC using an alkylated silica column such as C 4 -, C 8 - or C 18 -silica is generally used to achieve purification, for example, acetonitrile in an aqueous buffer, usually containing a small amount of trifluoroacetic acid.
  • Ion-exchange chromatography can also be used to separate a peptide based on its charge.
  • affinity purification is useful for isolating a fusion protein comprising a label attached to BP44 protein.
  • Methods for isolating fusion proteins using affinity chromatography are known in the art and described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).
  • an antibody or compound that binds to the label in the case of a polyhistidine tag this may be, for example, nickel-NT A
  • a sample comprising a BP44 fusion protein is then contacted to the immobilized antibody or compound for a time and under conditions sufficient for binding to occur. Following washing to remove any unbound or non-specifically bound protein, the fusion protein is eluted.
  • the degree of purity of the peptide compound may be determined by various methods, including identification of a major large peak on HPLC.
  • a peptide compound that produces a single peak that is at least 95% of the input material on an HPLC column is preferred. Even more preferable is a polypeptide that produces a single peak that is at least 97%, at least 98%, at least 99% or even 99.5% of the input material on an HPLC column.
  • composition analysis of the composition of the mTOT protein can be determined by any of a variety of analytical methods known in the art. Such composition analysis may be conducted using high resolution mass spectrometry to determine the molecular weight of the mTOT protein. Alternatively, hydrolyzing the protein in aqueous acid, and separating, identifying and quantifying the components of the mixture using HPLC, or an amino acid analyzer can confirm the amino acid content of an mTOT protein.
  • Protein sequencers which sequentially degrade the protein and/or peptide and identify the amino acids in order, may also be used to determine the sequence of the mTOT protein. Since some of the mTOT proteins may contain amino and/or carboxy terminal capping groups, it may be necessary to remove the capping group or the capped amino acid residue prior to a sequence analysis. Thin-layer chromatographic methods may also be used to authenticate one or more constituent groups or residues of a desired peptide.
  • the screening assays of the present invention are aimed at identifying compounds that are capable of binding to at least one mTOT protein. .
  • the present invention utilizes the finding that BP44 proteins and in some
  • mTOT proteins are specific targets of PPARy sparring thiazolidinedione compounds, for example, mitoglitazone.
  • Compounds that bind to an mTOT protein are postulated to play a role in insulin sensitization, particularly at sites involving the
  • the present invention provides a method for screening one or more candidate compounds to identify therapeutic agents effective against one or more insulin resistance or metabolic diseases, the method comprises: screening one or more candidate compounds in a binding assay, the binding assay comprising the steps:
  • the mTOT protein or proteins used in the various screening assays can include: mTOT protein having an amino acid sequence as provided in any one of SEQ ID NOs: 1-4, and 8-51, or an mTOT protein having an amino acid sequence as provided in any one of SEQ ID NOs: 1-4, 42, and 47, or an amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-4, 42 or 47, or an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 1-4, 42 or 47, or an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1-4, 42 or 47, or an mTOT protein has an amino acid sequence having at least 96% sequence identity to any one of SEQ ID NOs: 1-4, 42 or 47, or an amino acid sequence having at least 97% sequence identity to any one of SEQ ID NOs: 1-4, 42 or 47, or an amino acid sequence having at least 98% sequence identity to any
  • an illustrative example of an mTOT protein for use in the methods of the present invention includes an mTOT protein encoded by a nucleic acid sequence that specifically hybridizes to the complement of an mTOT nucleic acid sequence of SEQ ID NO: 5-7,or 52-53, under stringent hybridization conditions which are: 50% formamide, 5X SCC and 1% SDS, incubating at 42°C and wash in 0.2X SSC and 0.1% SDS at 65°C and wherein said mTOT protein specifically binds to mitoglitazone.
  • the screening method includes the use of BP44 protein as the mTOT protein. In some embodiments, the screening method includes the use of BRP44-Like protein as the mTOT protein. In some embodiments, the screening method includes the use of BP44-BRP44-Like heterodimer as the mTOT protein, for example as encoded by a ligated nucleotide sequence of SEQ ID Nos: 81 and 86 as described in Example 3.
  • the present invention provides screening assays that are capable of screening candidate compounds that can bind to an mTOT protein.
  • the screening assays of the present invention alsocontemplate assays that are capable of screening candidate compounds that can bind to an mTOT protein.
  • compounds that are capable of mediating metabolic protective effects including, anti-diabetic, anti-obesity, increased insulin sensitivity and reduced inflammation in various cells, including hepatic cells, endothelial cells, epithelial cells, adipocyte cells, muscle cells, neuronal cells and other cells of the brain will also bind to one or more mTOT proteins.
  • mitoglitazone specifically binds to and/or has an affinity to one or more mTOT proteins, for example, BP44 protein and BRP44-Like proteins.
  • mTOT proteins for example, BP44 protein and BRP44-Like proteins.
  • candidate compounds that may bind to an mTOT protein or which actively induce the expression of an mTOT protein in a eukaryotic or bacterial cell, for example, a human cell will also have some physiological effect on other targets of mitoglitazone, by mimicking the activity of mitoglitazone.
  • an optional step in determining whether a candidate compound may exert some physiological effect related to thiazolidinedione activity generally involves determining whether the candidate compound which binds to the mTOT protein is able to exert some measureable insulin-sensitizing activity, anti-diabetic activity, anti-obesity activity, metabolic protective activity, neuroprotective activity, either in vitro and/or in vivo. If such a candidate compound binds to an mTOT protein and is subsequently found to exert some measurable insulin-sensitizing activity, anti-diabetic activity, anti-obesity activity, metabolic protective activity, or neuroprotective activity, then the candidate compound is a therapeutic agent.
  • the present invention provides for at least three broad types of screening assays that can be performed to identify one or more candidate compounds for further testing on the basis that they selectively bind to one or more mTOT proteins.
  • the first assay type generally determines direct binding of an mTOT protein and a candidate compound and then determining whether the candidate compound is biologically active, i.e. whether the candidate compound exerts some measurable insulin-sensitizing activity, anti-diabetic activity, anti-obesity activity, metabolic protective activity, or neuroprotective activity.
  • the second type of screening assays generally involvesprokaryotic or eukaryotic cells that have an over-expressed mTOT protein.
  • the over expression of the mTOT protein in the prokaryotic or eukaryotic cell can be intracellular, for example in the cytosol, nucleus or attached to an organelle membrane or may be confined, embedded in or attached to a cell membrane or both. Such over expression of the mTOT protein will result in some inhibition of the activity caused by such over expression.
  • the term "over-expression" can include expression of the mTOT protein in the treated cell that is greater thanl50% of the expression of the mTOT protein on a percentage weight basis (% wt/wt) of an identical non- treated, normal or wild-type cell.
  • the third general class of screening assays involve spectrophotometric, NMR or some other physical analytical method that can measure the spectral nature of the mTOT protein in the presence and absence of a candidate compound.
  • the screening assay is a fluorescence polarization assay or a FRET assay.
  • a candidate compound can include, but is not limited to: nucleic acids, peptides, proteins, sugars, polysaccharides, glycoproteins, lipids, and small organic molecules.
  • a “candidate compound” is a compound that can be tested in a screening assay of the present invention.
  • small organic molecules typically refers to molecules of a size comparable to those organic molecules generally used in pharmaceuticals. Small organic molecules generally exclude biological polymers (e.g., proteins, nucleic acids, etc.).
  • Preferred small organic molecules range in size up to about 5000 Da. more preferably up to 2000 Da. and most preferably up to about 1000 Da.
  • the candidate compound can be at least one of a metal, a peptide, a protein, a lipid, a polysaccharide, a nucleic acid, a library of small organic molecules, and a drug.
  • determining whether a candidate compound has bound specifically with an mTOT protein requires that at least one of the mTOT protein and candidate compound is labeled with a marker or label.
  • a label can include a fluorescent molecule, a radionuclide, a protein tag, or combinations thereof. Fluorescent molecules can include naturally occurring or synthetic organic and organometallic
  • an illustrative fluorescent molecule can include a compound of Formula (I) or Formula (II) as described herein.
  • an mTOT protein or candidate compound can be labeled with a radionuclide or radioisotope.
  • radionuclide agents useful as labels for use in the screening assays herein can include, but not limited to: 3 H, 14 C, 32 P, or 35 S.
  • the mTOT protein or candidate compound can be labeled with a protein tag.
  • protein tags can include: glutathione-S-transferase, c- myc, Heme-agglutinin (HA), FLAG, avidin, biotin, streptavidin, or a fluorescent protein, in addition to other protein or amino acid based tags that can be used in fusion constructs comprising an mTOT protein or a protein/peptide candidate protein, or linked chemically to an mTOT or candidate compound.
  • a protein tag of the present invention can be readily identified by native fluorescence upon proper excitation and emission wavelength application, by reacting in an enzymic reaction operable to release a detectable signal, or can be detected using a conjugate antibody.
  • the protein tag can include a fluorescent protein or a protein that is capable of fluorescing under appropriate wavelength absorption and emission.
  • the mTOT protein can be recombinantly engineered to be expressed as a fusion construct having a fluorescent protein tag.
  • illustrative fluorescent tags can include: green fluorescent protein, enhanced green fluorescent protein, AcGFPl Fluorescent Protein, AmCyanl Fluorescent Protein, AsRed2 Fluorescent Protein, mBanana Fluorescent Protein, mCherry Fluorescent Protein, Dendra2, Fluorescent Protein, DsRed2 Fluorescent Protein, DsRed-Express Fluorescent Protein, DsRed-Monomer
  • Fluorescent Protein E2-Crimson Fluorescent Protein, GFPuv Fluorescent Protein, HcRedl Fluorescent Protein, mOrange Fluorescent Protein, PAmCherry Fluorescent Protein, mPlum Fluorescent Protein, mRaspberry Fluorescent Protein, mStrawberry Fluorescent, tdTomato Fluorescent Protein, Timer Fluorescent Protein, ZsGreenl Fluorescent Protein, and
  • Zs Yellow 1 Fluorescent Protein All of these fluorescent proteins are commercially available as DNA which can be readily coupled to a gene of interest, i.e. an mTOT gene.
  • the fluorescent proteins can be in monomeric form and can be coupled to either the N-terminus or the C-terminus and are available in vectors suitable for fusion protein construction rom Clontech, (Mountainview, CA USA), for example, bacterial expression vectors cat. No.
  • 632412-pDsRed- Express vector or plasmid or lentiviral vectors operable to clone a fusion construct comprising an mTOT protein fused to a fluorescent protein
  • a fluorescent protein See an exemplary commercially available vector from Clontech pEF-lalpha-DsRed Monomer CI vector Cat. No. 631977.
  • Excitation and emission spectra for all of the exemplified fluorescent proteins are well known and can be found from Clontech product literature or the Clontech website at
  • the screening assay of the present invention includes the screening of one or more candidate compounds in the form of a library of compounds.
  • the library of compounds comprises a combinatorial chemical library as exemplified and described below.
  • Combinatorial chemical libraries can include a plurality of small organic molecules.
  • the combinatorial chemical library can contain at least 1000 candidate compounds.
  • the candidate compound is a small organic molecule.
  • high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate
  • Such "combinatorial chemical libraries” are then screened in one or more assays, as described herein to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity, for example, capable of binding to an mTOT protein, or increase the expression of an mTOT protein in a cell, for example an adipocyte cell or hepatic cell or a eukaryotic cell line.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide (e.g., mutein) library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound).
  • combinatorial mixing of chemical building blocks For example, systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds.
  • combinatorial chemical libraries are well known to those of skill in the art.
  • Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175.
  • Peptide synthesis is by no means the only approach envisioned and intended for use with the present invention.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT).
  • the candidate compound binds to an mTOT protein specifically, or causes increased expression or modulates an activity of the mTOT protein as compared to the expression or activity in the absence of the candidate compound, that candidate compound can be said to be a "lead candidate compound".
  • the lead candidate compound may be validated using an assay capable of demonstrating insulin sensitizing activity, anti-diabetic activity, anti-obesity activity, metabolic protective activity, or neuroprotective activity of the lead candidate compound. If the lead candidate compound demonstrates insulin sensitizing activity, anti-diabetic activity, anti-obesity activity, metabolic protective activity, or neuroprotective activity, the lead candidate compound is a therapeutically active agent.
  • the therapeutically active agent thereby possesses PPARy sparring and/or insulin sensitizing activity suitable for the treatment of a metabolic disease, such as metabolic syndrome, diabetes mellitus, cardiovascular disease, gastrointestinal disease and neurodegenerative diseases.
  • the screening assays described herein are useful for identifying a lead candidate compound from plurality of candidate compounds.
  • subsequent activity assays are employed that measure the insulin-sensitizing activity or anti-diabetic activity, or metabolic protective activity, or neuroprotective activity
  • the lead candidate compound is a therapeutic active agent that putatively modulates a metabolic disease condition in a subject for example a human subject based on its effect on mitochondrial function and/or binding to an mTOT protein.
  • the direct binding screening assay method comprises: (i) screening one or more candidate compounds in a direct binding assay that identifies candidate compounds which bind to an mTOT protein.
  • screening one or more candidate compounds in a binding assay comprising the steps: (i) providing an mTOT protein; (ii) contacting the mTOT protein with a candidate compound; and (iii) detecting if the candidate compound specifically binds to the mTOT protein or inhibits the specific binding of a thiazolidinedione compound to the mTOT protein, wherein the candidate compound is identified as a lead candidate compound if the candidate compound specifically binds to the mTOT protein or inhibits the binding of the
  • ком ⁇ онентs can be designed wherein the candidate compound competes with a known thiazolidinedione compound, for example, any one of mitoglitazone, rosiglitazone, pioglitazone or troglitazone.
  • the competitor compound is any one of mitoglitazone, rosiglitazone, pioglitazone or troglitazone.
  • the competitor compound is any one of mitoglitazone, rosiglitazone, pioglitazone or troglitazone.
  • the competitor compound is
  • the competitor compound is rosiglitazone.
  • Fluorescence Based Assays [0140] In some embodiments, direct binding between a candidate compound and an mTOT protein can be detected and/or measured using fluorescence based assays. Binding of fluorescent candidate compounds can be detected in various ways, including fluorescence energy transfer (FRET), direct spectrophotofluorometric analysis of bound compound, or fluorescence polarization (Rogers, Drug Discovery Today, 1997, 2, 156-160; Hill, Cur.
  • FRET fluorescence energy transfer
  • the mTOT protein can be labeled with a fluorescence molecule, for example, a fluorescence protein tag such as GFP, and then incubated in the presence and absence of a candidate compound under conditions which permit binding of the two agents. The non-bound candidate compound(s) can then be removed from the labeled mTOT protein, and the bound candidate compound can be identified using an appropriate method.
  • the mTOT protein is unlabeled and the candidate compound is labeled.
  • the mTOT protein can be incubated in the presence and absence of a labeled candidate compound and a complex comprising the mTOT protein and labeled candidate compound is subsequently isolated and the candidate compound identified.
  • fluorescence binding assays can involve the use of FRET assays that may be designed for use in screening of compounds with green fluorescent protein (GFP) or some other appropriate fluorescence protein tag.
  • FRET Fluorescence resonance energy transfer
  • candidate compounds can be synthesized which contain a fluorescence probe with the ability to be activated by wavelengths of light that by themselves would not cause a fluorescence fusion tag on an mTOT protein to emit a fluorescence signal.
  • the fluorescence probe would then emit a light at a wavelength that activates the fluorescence fusion tag on the mTOT protein to emit a fluorescence signal when the fluorescence probe is bound to, or in close proximity, to the mTOT protein.
  • the excitation of the fluorescence probe absorbed by the fluorescence fusion tag attached to the mTOT protein.
  • a fluorescence probe of the present invention includes a compound of Formula I:
  • X is -O- or -NR 2 ;
  • Ri is optionally substituted Ci ⁇ straight or branched alkyl or CH 2 C(0)OR 3 ;
  • R 2 is H, optionally substituted Ci -6 straight or branched alkyl, optionally substituted phenyl, optionally substituted 2-pyridyl, optionally substituted 3-pyridyl, optionally substituted 4-pyridyl, optionally substituted 2-pyrimidinyl, optionally substituted 3- pyrimidinyl,or optionally substituted 4-pyrimidinyl;
  • R 3 is H, optionally substituted Ci -6 straight or branched alkyl, or optionally substituted -CH 2 -phenyl;
  • n 2-6.
  • X is -0-.
  • X is -O- and n is 2.
  • X is -O- and n is 3.
  • R ⁇ is optionally substituted C 1-6 straight or branched alkyl.
  • Ri is optionally substituted C 1-6 straight alkyl.
  • Ri is optionally substituted C 3-6 branched alkyl.
  • Ri is methyl, ethyl, propyl, sec-propyl, butyl, t-butyl, pentyl, or 2,2-dimethylbutyl, each of which is optionally substituted.
  • R 2 is optionally substituted C 1-6 straight or branched alkyl.
  • R 2 is optionally substituted C 1-6 straight alkyl.
  • R 2 is optionally substituted C 3-6 branched alkyl.
  • R 2 is methyl, ethyl, propyl, sec-propyl, butyl, t-butyl, pentyl, or 2,2-dimethylbutyl, each of which is optionally substituted.
  • R 2 is H.
  • R 2 is optionally substituted phenyl, optionally substituted 2- pyridyl, optionally substituted 3-pyridyl, optionally substituted 4-pyridyl, optionally substituted 2-pyrimidinyl, optionally substituted 3-pyrimidinyl, or optionally substituted 4- pyrimidinyl.
  • R 3 is H or optionally substituted C 1-6 straight or branched alkyl.
  • R 3 is H.
  • R 3 is optionally substituted Ci_6 straight or branched alkyl.
  • R 3 is optionally substituted Ci-6 straight alkyl.
  • R 3 is optionally substituted C 3-6 branched alkyl.
  • R 3 is methyl, ethyl, propyl, sec-propyl, butyl, t-butyl, pentyl, or 2,2-dimethylbutyl, each of which is optionally substituted.
  • R 3 is optionally substituted -CH 2 - phenyl.
  • X is -OH, -OCH 3 , -N(R 2 ) 2 ;
  • Ri is H, optionally substituted C 1-6 straight or branched alkyl, optionally substituted phenyl, optionally substituted -CH 2 -phenyl, optionally substituted 2-pyridyl, optionally substituted 3-pyridyl, or optionally substituted 4-pyridyl;
  • Each R 2 is independently H, optionally substituted Ci-6 straight or branched alkyl, optionally substituted phenyl, optionally substituted 2-pyridyl, optionally substituted 3- pyridyl, optionally substituted 4-pyridyl, optionally substituted 2-pyrimidinyl, optionally substituted 3-pyrimidinyl, or optionally substituted 4-pyrimidinyl, or
  • n 2-6;
  • n 2-6.
  • X is -OH. In other embodiments, X is -OCH. In other instances, X is -N(R 2 ) 2 .
  • Ri is H.
  • Ri is optionally substituted Ci -6 straight or branched alkyl.
  • Ri is optionally substituted C 1-6 straight alkyl.
  • R ⁇ is optionally substituted C 3-6 branched alkyl.
  • Ri is methyl, ethyl, propyl, .seopropyl, butyl, t-butyl, pentyl, or 2,2-dimethylbutyl, each of which is optionally substituted.
  • R 2 is optionally substituted Ci.6 straight or branched alkyl.
  • R 2 is optionally substituted C 1-6 straight alkyl.
  • R 2 is optionally substituted C 3- 6 branched alkyl.
  • R 2 is methyl, ethyl, propyl, sec-propyl, butyl, t-butyl, pentyl, or 2,2-dimethylbutyl, each of which is optionally substituted.
  • R 2 is H.
  • R 2 is optionally substituted phenyl, optionally substituted 2- pyridyl, optionally substituted 3-pyridyl, optionally substituted 4-pyridyl, optionally substituted 2-pyrimidinyl, optionally substituted 3-pyrimidinyl, or optionally substituted 4- pyrimidinyl.
  • two R 2 substituents and the nitrogen atom to which they are attached form an optionally substituted 3-7 membered heterocyclic ring.
  • two R 2 substituents and the nitrogen atom to which they are attached form a rin selected from:
  • each of Z l5 Z 2 , Z 3 , Z4, and Z 5 are independently selected from -NH-, -0-, -S-, or -CH 2 -.
  • two R 2 substituents and the nitrogen atom to which they are attached form a pyrrolidine-yl, piperidine-yl, piperazine-yl, morpholine-yl, or
  • the compound of Formula I is Compound A:
  • the compound of Formula II is Compound B:
  • Exemplary methods for making probes of Formulae I or II include synthetic pathways such as those illustrated in Schemes 1 and 2 below.
  • the alcohol functionality can be converted to a leaving group (e.g., tosyl, mesyl, chloro, bromo, or the like) to generate intermediate (4a) using an appropriate reagent (e.g. p-toluenesulfonyl chloride,
  • One exemplary experimental procedure used to generate compound A of Formula I includes: heating a mixture of 7-hydroxy-4-methylcoumarin (1.76 g, 10 mmol), potassium carbonate (2.07 g, 15 mmol), 2-chloroethanol (1.34 mg, 20.0 mmol), and potassium iodided (80 mg, 0.5 mmol) in N,N-dimethylformamide (50 ml, 600 mmol) at 110 °C for about 5 hours, cool to room temperature, and stir at room temperature.
  • the starting material (lb) undergoes protection of its secondary amine group using any suitable protecting group (e.g., a BOC protecting group) followed by conversion of the primary alcohol functionality to a leaving group (e.g., a mesyl or tosyl group) to generate intermediate (2b).
  • a suitable protecting group e.g., a BOC protecting group
  • a leaving group e.g., a mesyl or tosyl group
  • Starting material (3b) is treated with a strong base (e.g., NaH or the like) and reacted with intermediate (2b) to generate the BOC-protected intermediate (4b), which is deprotected with a strong acid (e.g., trifluoroacetic acid (TFA)) and reacted with an optionally substituted 2-(2-oxo-2H-chromen-4-yl)acetic acid in the presence of a coupling reagent(s), (e.g., EDC and HOBt) to generate exemplary compound B of Formula II.
  • a strong base e.g., NaH or the like
  • a strong acid e.g., trifluoroacetic acid (TFA)
  • TFA trifluoroacetic acid
  • candidate compounds that block the interaction of a compound labeled with an exemplified probe of formula I or formula II and which is known to specifically and/or directly bind to a fluorescently tagged mTOT protein will result in a decrease in fluorescence.
  • Such blocking candidate compounds are potentially active agents with respect to metabolic diseases and can be validated using assays described in Examples 1 and 2.
  • the fluorescence based assays described herein may use intact cells or membranes isolated from cells that express the fluorescence tagged mTOT protein.
  • the fluorescence tagged mTOT protein can include any one of GFP labeled mTOT protein.
  • Other assays may be used to identify candidate compounds capable of exerting some metabolic disease therapeutic effect, including assays that identify candidate compounds operable to bind to the mTOT protein through measuring direct binding of candidate compounds to the mTOT protein.
  • Other exemplary screening methods employ direct binding between an mTOT protein and a TZD that is known to bind to the mTOT protein of interest, for example, BP44 and mitoglitazone.
  • candidate compounds are added to the binding complex one at a time and the dissociation of the mTOT protein from the TZD, for example, mitoglitazone can be detected using affinity ultrafiltration and ion spray mass spectroscopy/HPLC.or other physical and analytical methods.
  • candidate compounds that inhibit the specific binding of known compounds that bind to mTOT are then identified as lead candidate compounds.
  • the binding of an mTOT protein with one or more candidate compounds can be evaluated indirectly using the yeast two hybrid system described in Fields et al., Nature, 340:245-246 (1989), and Fields et al., Trends in Genetics, 10:286-292 (1994), both of which are incorporated herein by reference in its entirety.
  • the two-hybrid system is a genetic assay for detecting interactions between two proteins or polypeptides. It can be used to identify proteins that bind to a known protein of interest, or to delineate domains or residues critical for an interaction. Variations on this methodology have been developed to clone genes that encode DNA binding proteins, to identify peptides that bind to a protein, and to screen for drugs.
  • the two-hybrid system exploits the ability of a pair of interacting proteins to bring a transcription activation domain into close proximity with a DNA binding domain that binds to an upstream activation sequence (UAS) of a reporter gene, and is generally performed in yeast.
  • UAS upstream activation sequence
  • the assay requires the construction of two hybrid genes encoding (1) a DNA- binding domain that is fused to a first protein and (2) an activation domain fused to a second protein.
  • the DNA-binding domain targets the first hybrid protein to the UAS of the reporter gene; however, because most proteins lack an activation domain, this DNA-binding hybrid protein does not activate transcription of the reporter gene.
  • the second hybrid protein which contains the activation domain, cannot by itself activate expression of the reporter gene because it does not bind the UAS. However, when both hybrid proteins are present, the non- covalent interaction of the first and second proteins tethers the activation domain to the UAS, activating transcription of the reporter gene.
  • this assay can be used to detect agents that interfere with the binding interaction.
  • Expression of the reporter gene is monitored as different candidate compounds are added to the system.
  • the presence of an inhibitory agent for example a competitive binding compound results in lack of a reporter signal.
  • the yeast two-hybrid screening assay of the present invention can also be used to identify proteins that bind to the gene product.
  • a fusion polynucleotide encoding both an mTOT protein (or fragment) and a UAS binding domain i.e., a first protein
  • a large number of hybrid genes each encoding a different second protein fused to an activation domain are produced and screened in the assay.
  • the second protein is encoded by one or more members of a total cDNA or genomic DNA fusion library, with each second protein-coding region being fused to the activation domain.
  • This system is applicable to a wide variety of proteins, and it is not even necessary to know the identity or function of the second binding protein.
  • the system is highly sensitive and can detect interactions not revealed by other methods; even transient interactions may trigger transcription to produce a stable mR A that can be repeatedly translated to yield the reporter protein.
  • the quantitative measurement of bound candidate compound to the mTOT protein can be determined by any method that distinguishes between the folded and unfolded states of the mTOT protein, for example, by adding an antibody that recognizes the folded state but not the unfolded state and vice versa. Then the amount of bound antibody can be measured and quantified using standard immunological procedures, for example Enzyme Linked Immunosorbent Assay (ELISA).
  • ELISA Enzyme Linked Immunosorbent Assay
  • Antibodies to the mTOT proteins for example, BP44 and BRP44-Like are known and commercially available.
  • the function of the mTOT protein need not necessarily be known in order for this assay to be performed. Virtually any agent can be assessed by this method as a candidate compound, including, but not limited to, metals, polypeptides, proteins, lipids, polysaccharides, polynucleotides and small organic molecules.
  • the binding assays of the present invention involve measuring the binding of a candidate compound in the presence or absence of a competitive compound that is known to bind to the mTOT protein used in the assay.
  • the competitive binding assay to identify a lead candidate compound effective against an insulin resistance disease or disorder includes the steps: (a) providing a candidate compound and at least one mTOT protein; (b) incubating the at least one mTOT protein with a competitor compound in the presence of the candidate compound to produce a test combination; (c) incubating the at least one mTOT protein with said competitor compound in the absence of the candidate compound to produce a corresponding control combination; (d) measuring the amount of the competitor compound bound to the mTOT protein in the test combination and in the control combination; and (e) selecting as a lead candidate compound any candidate compound that causes a measurable decrease in the amount of competitor compound bound to the mTOT protein measured in step (d) in the test combination relative to the control combination.
  • the magnitude in reduction of competitor compound bound to the mTOT protein in the test combination relative to the control combination reflects the affinity of the candidate compound for the at least one mTOT protein.
  • the method further includes repeating steps (b)-(d) in a high throughput screen.
  • providing the candidate compound and at least one mTOT protein can include affixing the mTOT protein to a solid substrate, for example, a glass slide, a silicon chip, or other solid substrates used in high-throughput applications in which 1 to 1,000 reaction spots are printed or spotted in an array format.
  • providing the candidate compound and at least one mTOT protein is carried out in solution in a reaction vessel or some containment receptacle.
  • providing the candidate compound and at least one mTOT protein can include admixing the mTOT protein and candidate compound in solution contained within a reaction vessel or receptacle.
  • a reaction vessel or receptacle can include a well in a microtiter plate, or other multi-well plate, or plastic or other solid polymeric vial or glass vial or tubes, eppendorf tubes or such containers or liquid handling devices conventionally used in chemistry and biological syntheses and reactions.
  • the mTOT protein can include an isolated mTOT protein or a fusion construct thereof. In some embodiments, the mTOT protein is unlabeled. In some embodiments, the mTOT protein is a fusion of an mTOT protein and a tag or label which facilitates specific binding or isolation from a mixture using the tag or label, for example a GST, c-myc, heme-agglutinin (HA), avidin, steptavidin, biotin, FLAG, hexa-Histidine (His6), a fluorphore or fluorescent protein (e.g. GFP, EGFP, DsRed and other fluorescent proteins described herein) and the like.
  • a tag or label which facilitates specific binding or isolation from a mixture using the tag or label, for example a GST, c-myc, heme-agglutinin (HA), avidin, steptavidin, biotin, FLAG, hexa-Histidine (His6), a flu
  • the mTOT protein can include at least one of: a BP44 protein, a BRP44-Like protein or a heterodimer of BP44-BRP44Like proteins.
  • the mTOT protein is a human BP44, BRP44-Like or a heterodimer of BP44-BRP44Like proteins.
  • the mTOT protein is a protein provided in tables 1,3,4 and 5 and Figures 3A-4B.
  • the competitor compound can be a TZD which is known to bind to a target compound as described herein.
  • the competitor compound can be any one of mitoglitazone, rosiglitazone, pioglitazone, MSDC-160, U 5099, or troglitazone.
  • the competitor compound can be labeled with a radionuclide, protein tag or a fluorescent molecule or the competitor compound is unlabelled, but competes with a labeled photoaffinity crosslinker, such as 125 I MSDC -1101 as shown in FIG. 9B.
  • Competitive assays useful in the present screening methods are described in the examples below, for example, with reference to FIG. 9C.
  • direct binding screening methods can comprise using competitive screening assays in which a neutralizing antibodies capable of binding to an mTOT protein, specifically compete with a candidate compound for binding to the mTOT protein.
  • the competitor compound is a radiolabelled antibody capable of specifically binding to an mTOT protein.
  • Such antibodies have been described herein.
  • Other labels such as radiolabeled competitive binding studies are described in A. H. Lin et al., Antimicrobial Agents and Chemotherapy, 1997, vol. 41, no. 10. pp. 2127-2131, the disclosure of which is incorporated herein by reference in its entirety.
  • the competitor compound is labeled with, for example, a radioactive isotope, for example, 3 H, 32 P, 35 S or 14 C.
  • the competitor compound is a TZD compound labeled with a radioisotope.
  • the competitor compound is radiolabelled mitoglitazone or radiolabelled rosiglitazone.
  • the competitor compound is a TZD compound labeled with 3 H.
  • the competitor compound is 3 H mitoglitazone or 3 H rosiglitazone.
  • the competitor compound is 3 H mitoglitazone.
  • the competitor compound is 3 H rosiglitazone.
  • measuring the amount of the competitor compound bound to the mTOT protein in the test combination and in the control combination can include determining the amount of competitor compound bound to the mTOT protein.
  • measuring the amount of the competitor compound bound to the mTOT protein in the test combination and in the control combination can include determining the amount of candidate compound bound to the competitor compound and by logical operation, the more candidate compound bound to the mTOT protein indicates that less competitor compound is bound to the mTOT protein.
  • the competitive assays of the present invention can include the step of measuring the amount of labeled competitor compound bound to an mTOT protein by measuring the amount of labeled competitor compound remaining in solution or attached to a solid substrate after incubation with a non-labeled candidate compound.
  • measuring the amount of the competitor compound bound to the mTOT protein in the test combination and in the control combination includes measuring the amount of labeled competitor compound remaining bound to the mTOT protein after removal of unbound candidate compound and unbound labeled competitor compound. If the candidate compound reduces the amount of labeled competitor compound bound to the mTOT protein in the test combination relative to the control combination, then the candidate compound is a lead candidate compound.
  • an optional step can include a subsequent activity assay to identify whether the bound candidate compound is a therapeutic active agent.
  • a candidate compound Once a candidate compound has been identified as a compound that specifically binds to an mTOT protein it is a lead candidate compound.
  • Lead candidate compounds can be confirmed as being a therapeutic active agent by further evaluating its activity in activity assays used to confirm the insulin sensitizing or anti-diabetic activity of thiazolidinediones, such as mitoglitazone.
  • Representative activity assays include the mitochondrial membrane competitive binding crosslinking assay, the induction of BP44 protein expression in brown adipose tissue assay, and the Drosophila melanogaster model of diet-induced insulin resistance assay. Representative activity assays are further described in the examples section.
  • an illustrative high-throughput screening method for identifying candidate compounds capable of directly binding to an mTOT protein is described in Wieboldt et ah, Anal. Chem., 69:1683-1691 (1997), incorporated herein by reference in its entirety.
  • This assay method is capable of screening in high-throughput fashion combinatorial libraries of 20-30 agents at a time in solution phase for binding to the mTOT protein.
  • candidate compounds that bind to the mTOT protein can be separated from other library components by simple membrane washing.
  • the bound candidate compound(s) that are retained on the filter can be subsequently liberated from the mTOT protein, and analyzed by HPLC and electrospray (ion spray) ionization mass spectroscopy. This procedure selects candidate compounds originally present in the library with the greatest affinity for the mTOT protein, and is particularly useful for small molecule libraries.
  • binding between a candidate compound and an mTOT protein can be measured by over expressing the mTOT protein on the surface of a cell or over expressing the mTOT protein within a cell as described above. As a result of such over expression, some measureable effect occurring within the cell as a result can be made.
  • HE 293 cells can be cultured in vitro to express the mTOT protein in either a transient or stable fashion using the methods herein for expressing an mTOT protein in prokaryotic or eukaryotic cells recombinantly.
  • mTOT protein either on a membrane surface or expressed intracellularly (nuclear, organelle or cytoplasmic, for example, expressed on the surface of a mitochondrial membrane) may be employed in the present assays. While not wishing to be bound by any particular theory, it is believed that the mTOT protein over expression can result in changes in cell function relative to control cells (non-transfected or cells not over- expressing the mTOT protein). In some embodiments, changes in intracellular calcium will occur due to over expression of the mTOT protein in cell culture, for example, HEK 293 cells. Such calcium levels in the cell can be determined using a calcium-sensitive dye.
  • Candidate compound is added to a tissue culture medium (at concentrations ranging from 0.01-50 micromolar) harboring the cells over expressing the mTOT protein. Specific binding between candidate compound and mTOT protein can be detected as reversal of the calcium fluctuations and return them to levels of intracellular calcium shown in control cells without overexpression of the mTOT protein.
  • An illustrative example of such an assay is as follows. Cells over expressing the mTOT protein fail to grow, but the additions of a positive modulator, for example mitoglitazone, known to have positive metabolic activity return the cells to normal growth.
  • a positive modulator for example mitoglitazone
  • This system can then be used in vitro or in vivo to select other candidate compounds that can bind to the mTOT protein and in a similar fashion to the positive modulator mitoglitazone, return the cells to normal growth.
  • candidate compounds can then be evaluated and validated using other activity assays for anti-diabetic, anti-obesity, metabolic protective, or neuroprotective actions as described herein.
  • Determining whether a test compound binds to an mTOT protein can also be accomplished by measuring the intrinsic fluorescence of the mTOT protein and determining whether the intrinsic fluorescence is modulated in the presence of a candidate compound.
  • the intrinsic fluorescence of the mTOT protein is measured as a function of the tryptophan residue(s) of the mTOT protein.
  • fluorescence of the mTOT protein is measured and compared to the fluorescence intensity of the mTOT protein in the presence of the candidate compound, wherein a decrease in fluorescence intensity indicates binding of the test compound to an mTOT protein.
  • a method for performing the intrinsic fluorescence measurement is set forth in "Principles of Fluorescence Spectroscopy" by Joseph R. Lakowicz, New York, Plenum Press, 1983 (ISBN 0306412853) and
  • an optional step can include a subsequent activity assay to identify whether the bound candidate compound is a therapeutic active agent.
  • a candidate compound Once a candidate compound has been identified as a compound that specifically binds to an mTOT protein it is a lead candidate compound. Lead candidate compounds can be confirmed as being a therapeutic active agent by further evaluating it's activity in standard known activity assays used to confirm the activity of thiazolidinediones, such as
  • Representative activity assays include the mitochondrial membrane competitive binding crosslinking assay, the induction of BP44 protein expression in brown adipose tissue assay, and the Drosophila melanogaster model of diet-induced insulin resistance assay.
  • the present invention is also drawn to the discovery of a previously unrecognized target of the insulin sensitizers TZDs called mTOT protein.
  • mTOT proteins and mTOT protein are used interchangeably. Because mTOT proteins of the present invention are well conserved phylogenetically, the present inventors evaluated whether not a fly mTOT protein could bind and respond to insulin sensitizers. Indeed, mitochondrial fractions from wild type flies contained a protein of about 18-19 kDA that specifically interacted with the TZD probe.
  • the inventors have conducted preliminary studies which indicate that the knock down of either the mTOT protein BP44 or BRP44-Like gene products, produces reduced hemolymph glucose levels and extends life span as do the active drugs, suggesting that the TZDs may be attenuating the activity of the recently identified mitochondrial mTOT protein complex.
  • the present invention provides compositions and methods for modulating mTOT protein gene product activity involved in insulin sensitivity.
  • the present invention is directed to compounds, particularly functional nucleic acids, for example, antisense oligonucleotides, which are targeted to a nucleic acid encoding an mTOT protein as provided herein.
  • Pharmaceutical and other compositions comprising the compounds of the invention are also provided.
  • methods of modulating the expression of an mTOT protein in cells, tissues or in an organism comprises contacting said cells, tissues or organism with one or more functional nucleic acid compounds or compositions of the present invention to inhibit, reduce or prevent the expression and/or activity of an mTOT protein in the cell, tissue or organism.
  • an animal particularly a mammal, and more particularly a human, having, or suspected of having, or being prone to a disease or condition associated with expression of an mTOT protein, for example, diabetes mellitus, cardiovascular disease, gastrointestinal disease, or Alzheimer's disease, by administering a therapeutically or prophylactically effective amount of one or more of the functional nucleic acid compounds or compositions of the invention.
  • an mTOT protein for example, diabetes mellitus, cardiovascular disease, gastrointestinal disease, or Alzheimer's disease
  • a functional nucleic acid for example, an antisense oligonucleotide which modulates the expression of an mTOT protein gene product is administered in a liposome formulation.
  • the liposome formulation is an amphoteric liposome formulation.
  • the amphoteric liposome formulation comprises one or more amphoteric lipids.
  • the amphoteric liposome is formed from a lipid phase comprising a mixture of lipid components with amphoteric properties, which may, for example, be selected from the group consisting of (i) a stable cationic lipid and a chargeable anionic lipid, (ii) a chargeable cationic lipid and chargeable anionic lipid and (iii) a stable anionic lipid and a chargeable cationic lipid.
  • the liposome may further comprise neutral lipids.
  • the amphoteric liposomes comprise DOPE, POPC, CHEMS and MoChol.
  • the molar ratio of POPC/DOPE/MoChol/CHEMS is about 6/24/47/23.
  • the present invention provides co-therapies comprising a functional nucleic acid compound that hybridizes to at least a portion of oligonucletide sequence selected from SEQ ID NOs: 5-7,52-73 or the complement thereof, and another TZD therapy agent, wherein the TZD therapy agent is selected from a therapeutically effective amount of mitoglitazone, pioglitazone, rosiglitazone or combinations thereof.
  • the functional nucleic acid compound includes an additional oligonucleotide or oligonucleoside.
  • the additional functional nucleic acid can include any one of SEQ ID NOs: 54-73 or the complementary nucleotide sequence thereof.
  • the oligonucleotides are between 15 and 35 base pairs in length. In still another embodiment, the oligonucleotides have a phosphorothiolate backbone.
  • the method of treating a subject with an insulin insensitivity disorder for example, diabetes mellitus, metabolic syndrome, cardiovascular disease, gastrointestinal disease or Alzheimer's disease, where the daily dose of functional nucleic acid compound is from 0.1 mg/m 2 to 300 mg/m 2 oligomer of body surface area of a patient.
  • an insulin insensitivity disorder for example, diabetes mellitus, metabolic syndrome, cardiovascular disease, gastrointestinal disease or Alzheimer's disease
  • the daily dose of functional nucleic acid compound is from 0.1 mg/m 2 to 300 mg/m 2 oligomer of body surface area of a patient.
  • the oligonucleotide is administered intravenously to a diabetes type II patient.
  • the present invention utilizes the finding that mTOT proteins, for example, BP44 proteins, BRP44 Like proteins, or BP44-BRP44-Like heterodimers among others exemplified herein, are specific targets of thiazolidinedione compounds, for example, rosiglitazone and PPARy sparring mitoglitazone.
  • thiazolidinedione compounds for example, rosiglitazone and PPARy sparring mitoglitazone.
  • Compounds that bind to mTOT proteins are postulated to play a role in insulin sensitization, particularly at sites involving the mitochondria.
  • an inhibitor of mTOT protein gene expression can be a functional nucleic acid.
  • the category of "functional nucleic acids" is the category of "functional nucleic acids"
  • siRNA molecules encompasses siRNA molecules, shRNA molecules, miRNA molecules, and antisense nucleic acid molecules.
  • siRNA refers to small inhibitory RNA duplexes that induce the RNA interference (RNAi) pathway. These molecules can vary in length (generally 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNA have unpaired overhanging bases on the 5' or 3' end of the sense strand and/or the antisense strand.
  • siRNA includes duplexes of two separate strands, as well as single strands that can form hairpin structures comprising a duplex region.
  • an oligonucleotide having a nucleotide sequence encoding a gene and “polynucleotide having a nucleotide sequence encoding a gene,” means a nucleic acid sequence comprising the coding region of a gene, or in other words the nucleic acid sequence that encodes a gene product.
  • the coding region may be present in a cDNA, genomic DNA or R A form.
  • the oligonucleotide or polynucleotide may be single-stranded (i.e., the sense strand) or double-stranded.
  • oligonucleotide refers to a short length of single-stranded polynucleotide chain. Oligonucleotides are typically less than 200 residues long (e.g., between 8 and 100), however, as used herein, the term is also intended to encompass longer polynucleotide chains (e.g., as large as 5000 residues). Oligonucleotides are often referred to by their length. For example a 24 residue oligonucleotide is referred to as a "24-mer”.
  • Oligonucleotides can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides.
  • Such structures can include, but are not limited to, duplexes, hairpins, cruciforms, bends, and triplexes.
  • the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, for the sequence “A-G-T”, is complementary to the sequence "T-C-A”.
  • Complementarity may be “partial”, in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
  • the term "completely complementary,” for example when used in reference to a functional nucleic acid compound of the present invention refers to an functional nucleic acid, for example an antisense oligonucleotide where all of the nucleotides are complementary to a target sequence (e.g., a gene).
  • Exemplary partially complementary oligonucleotides are those that can still hybridize to the target sequence under physiological conditions.
  • the term “partially complementary” refers to oligonucleotides that have regions of one or more non-complementary nucleotides both internal to the oligonucleotide or at either end. Oligonucleotides with mismatches at the ends may still hybridize to the target sequence.
  • the term "homology” refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity).
  • a partially complementary sequence is a nucleic acid molecule that at least partially inhibits a completely complementary nucleic acid molecule from hybridizing to a target nucleic acid is "substantially homologous”. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution
  • a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous nucleic acid molecule to a target under conditions of low stringency.
  • conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target that is substantially non-complementary (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non-complementary target.
  • substantially homologous refers to any functional nucleic acid that can hybridize to either or both strands of the double-stranded nucleic acid sequence under conditions of low stringency as described above.
  • substantially homologous refers to any functional nucleic acid that can hybridize (i.e., it is the complement of) the single-stranded nucleic acid sequence under conditions of low stringency as described above.
  • hybridization is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, the Tm of the formed hybrid, and the G:C ratio within the nucleic acids. A single molecule that contains pairing of complementary nucleic acids within its structure is said to be “self- hybridized”.
  • Tm is used in reference to the "melting temperature".
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands.
  • stringency is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted.
  • low stringency conditions a nucleic acid sequence of interest will hybridize to its exact complement, sequences with single base mismatches, closely related sequences (e.g., sequences with 90% or greater homology), and sequences having only partial homology (e.g., sequences with 50-90% homology).
  • intermediate stringency conditions a nucleic acid sequence of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely related sequences (e.g., 90% or greater homology).
  • a nucleic acid sequence of interest will hybridize only to its exact complement, and (depending on conditions such a temperature) sequences with single base mismatches. In other words, under conditions of high stringency the temperature can be raised so as to exclude hybridization to sequences with single base mismatches.
  • High stringency conditions when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 0.1X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • hybridization comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5X Denhardt's reagent and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • SDS 5X Denhardt's reagent
  • 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 1.0X SSPE, 1.0% SDS at 42°C when a probe of about 500 nucle
  • Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5X SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5X Denhardt's reagent [50X Denhardt's contains per 500 ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and 100 ⁇ g/ml denatured salmon sperm DNA followed by washing in a solution comprising 5X SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length is employed.
  • 5X SSPE 43.8 g/1 NaCl, 6.9 g/1 NaH 2 P0 4 H 2 0 and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH
  • 5X Denhardt's reagent 50X Denhardt's
  • the present invention is not limited to the hybridization of probes of about 500 nucleotides in length.
  • the present invention contemplates the use of probes between approximately 8 nucleotides up to several thousand (e.g., at least 5,000) nucleotides in length, for example, 8 to 5,000, or 8 to 2,000, or 8 to 1,000, or 8 to 500, or 8 to 250, or 8 to 200, or 8 to 100, or 8 to 75, or 8 to 50, or 8 to 30 nucleotides in length.
  • stringency conditions may be altered for probes of other sizes (See e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization
  • physiological conditions refers to specific stringency conditions that approximate or are conditions inside an animal (e.g., a human).
  • exemplary physiological conditions for use in vitro include, but are not limited to, 37°C, 95% air, 5% C0 2 , commercial medium for culture of mammalian cells (e.g., DMEM media available from Gibco, MD), 5-10% serum (e.g., calf serum or horse serum), additional buffers, and optionally hormone (e.g., insulin and epidermal growth factor).
  • isolated when used in relation to a nucleic acid, as in “an isolated oligonucleotide” or “isolated polynucleotide” refers to a nucleic acid sequence that is identified and separated from at least one component or contaminant with which it is ordinarily associated in its natural source. Isolated nucleic acid is such present in a form or setting that is different from that in which it is found in nature. In contrast, non-isolated nucleic acids as nucleic acids such as DNA and RNA found in the state they exist in nature.
  • a given DNA sequence e.g., a gene
  • RNA sequences such as a specific mRNA sequence encoding a specific protein
  • isolated nucleic acid encoding a given protein includes, by way of example, such nucleic acid in cells ordinarily expressing the given protein where the nucleic acid is in a chromosomal location different from that of natural cells, or is otherwise flanked by a different nucleic acid sequence than that found in nature.
  • the isolated nucleic acid, oligonucleotide, or polynucleotide may be present in single- stranded or double-stranded form.
  • the oligonucleotide or polynucleotide when an isolated nucleic acid, oligonucleotide or polynucleotide is to be utilized to inhibit the translation of an mTOT protein, the oligonucleotide or polynucleotide will contain at a minimum the non-coding strand or the antisense strand that binds to the sense mRNA encoding an mTOT protein (i.e., the oligonucleotide or polynucleotide may be single-stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide or polynucleotide may be double- stranded).
  • purified or “to purify” refers to the removal of
  • recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by the removal of host cell proteins; the percent of recombinant polypeptides is thereby increased in the sample.
  • the functional nucleic acid compounds of the present invention target the mTOT protein gene or a messenger RNA transcript or cDNA thereof.
  • exemplary functional nucleic acids hybridize to a coding sequence mRNA copy of the cDNA shown in SEQ ID NOs: 5-7 and 52-53 or a complementary nucleotide sequence thereof.
  • the exemplary functional nucleic acids stringently hybridize to a mRNA copy of the cDNA shown in SEQ ID NOs: 5-7 and 52-53.
  • mTOT protein cDNA sequences of SEQ ID NOs: 5-7 and 52-53 and mRNA sequences or complementary sequences thereof are merely exemplary mTOT protein mRNA sequences, other variant sequences (including splicing variants) which encode other isoforms of mTOT protein gene products, for example, human BP44 or BP44-Like polypeptides of Tables 1-5 and shown in Figures 3A, 3B, 4A and 4B are also contemplated herein.
  • the rational design process can involve the use of a computer program to evaluate the criteria for every sequence of 18-30 base pairs or only sequences of a fixed length, e.g., 19 base pairs.
  • the computer program is designed such that it provides a report ranking of all of the potential siRNAs 18-30 base pairs, ranked according to which sequences generate the highest value. A higher value refers to a more efficient siRNA for a particular target gene.
  • the computer program that may be used may be developed in any computer language that is known to be useful for scoring nucleotide sequences. Additionally, rather than run every sequence through one and/or another formula, one may compare a subset of the sequences, which may be desirable if for example only a subset are available.
  • BLAST Basic Local Alignment Search Tool
  • scan the sequence and to identify regions of moderate GC context then perform relevant calculations using one of the above-described formulas on these regions. These calculations can be done manually or with the aid of a computer.
  • inhibition of mTOT protein genes includes inhibition of human BP44, and/or human BRP44-Like genes, i.e. genomic DNA, cDNA, mRNA, mutant or alternative splice variants thereof, or complementary nucleotide sequences thereof.
  • target is used in a variety of different forms throughout this disclosure and is defined by the context in which it is used.
  • target mRNA refers to a messenger RNA to which a given siRNA can be directed against.
  • target sequence and “target site” refer to a sequence within the mRNA to which the sense strand of an siRNA shows varying degrees of homology and the antisense strand exhibits varying degrees of complementarity.
  • siRNA target can refer to the gene, mRNA, cDNA or protein against which an siRNA is directed.
  • target silencing can refer to the state of a gene, or the corresponding mRNA or protein.
  • the siRNA can comprise partially purified RNA, substantially pure RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally- occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides.
  • Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, including modifications that make the siRNA resistant to nuclease digestion.
  • the siRNA can be targeted to any stretch of approximately 18-30 contiguous nucleotides in any of the target mRNA sequences, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially complementary to, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) complementary to, e.g., having for example 3, 2, 1, or 0 mismatched nucleotide(s), a target mRNA sequence.
  • Techniques for selecting target sequences for siR A are provided, for example, in Tuschl, T. et al., "The siRNA User Guide", revised Oct.
  • the sense strand of the present siRNA comprises a nucleotide sequence identical to any contiguous stretch of about 18 to about 30 nucleotides in the mTOT protein mRNA.
  • specific siRNAs for downregulating the activity and/or expression of an mTOT proteins can include: 5 ' -GCTG ATGCTGCCCG AG AA ATT-3 ' (SEQ ID NO:54) starting at position 217 of SEQ ID NOs: 5 or 5'-
  • one or both strands of the siRNA can also comprise a 3' overhang.
  • a "3' overhang” refers to at least one unpaired nucleotide extending from the 3 '-end of a duplexed RNA strand.
  • the siRNA comprises at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxynucleotides) in length, preferably from 1 to about 5 nucleotides in length, more preferably from 1 to about 4 nucleotides in length, and particularly preferably from about 2 to about 4 nucleotides in length.
  • the length of the overhangs can be the same or different for each strand.
  • the 3' overhang is present on both strands of the siRNA, and is 2 nucleotides in length.
  • each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
  • one or both strands of the siRNA can also comprise a hairpin insert.
  • a hairpin insert refers to at least one nucleotide insert ranging from about 3 to about 10 nucleotides, preferably from 3 to 6 nucleotides in length positioned within the siRNA sequence, for example approximating the center of the sequence.
  • exemplary siRNA sequences useful in the treatment, inhibition or prevention methods of the present invention can comprise or consist of: 5'-
  • siRNAs can be synthesized and may be commercially available from Dharmacon or
  • siRNAs, shRNAs and lentiviral vectors capable of expressing siRNA or shRNA sequences useful in inhibiting mTOT protein expression and/or activity are commercially available under Catalog Nos.: sc-141751, and sc-95332 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
  • siRNA useful in inhibiting the expression of mTOT protein proteins in vitro and in vivo can be obtained using a number of techniques known to those of skill in the art.
  • the siRNA can be chemically synthesized or recombinantly produced using methods known in the art, such as the Drosophila in vitro system described in U.S. Patent Application Publication No. 2002/0086356, filed March 30, 2001 to Tuschl et al, the entire disclosure of which is herein incorporated by reference.
  • the siRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer.
  • the siRNA can be synthesized as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • Commercial suppliers of synthetic RNA molecules or synthesis reagents include Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo. USA), Pierce Chemical (part of Perbio Science, Rockford, 111. USA), Glen Research (Sterling, Va. USA), ChemGenes (Ashland, Mass. USA) and Cruachem (Glasgow, UK).
  • siRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter.
  • suitable promoters for expressing siRNA from a plasmid include, for example, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant plasmids of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
  • siRNA expressed from recombinant plasmids can either be isolated from cultured cell expression systems by standard techniques, or can be expressed intracellularly at or near the area of neovascularization in vivo.
  • the use of recombinant plasmids to deliver siRNA to cells in vivo is discussed in more detail below.
  • siRNA can be expressed from a recombinant plasmid either as two separate, complementary RN A molecules, or as a single RNA molecule with two complementary regions.
  • a plasmid comprising nucleic acid sequences for expressing an siRNA can comprise a sense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter, and an antisense RNA strand coding sequence in operable connection with a polyT termination sequence under the control of a human U6 RNA promoter.
  • the plasmid is ultimately intended for use in producing a recombinant adeno-associated viral vector or retroviral vector comprising the same nucleic acid sequences for expressing an siRNA in vivo or in vitro upon appropriate transfection.
  • An exemplary selective siRNA with hairpin insert pairs operable to be used in a plasmid for expressing the shRNA can comprise or consist of the pair:
  • exemplary selective siRNA with hairpin insert
  • oligonucleotide pairs operable to be used in a plasmid for expressing the shRNA to inhibit an mTOT protein mRNA can comprise or consist of the pairs:
  • in operable connection with a polyT termination sequence means that the nucleic acid sequences encoding the sense or antisense strands are immediately adjacent to the polyT termination signal in the 5' direction. During transcription of the sense or antisense sequences from the plasmid, the polyT termination signals act to terminate transcription.
  • promoter under the control of a promoter means that the nucleic acid sequences encoding the sense or antisense strands are located 3' of the promoter, so that the promoter can initiate transcription of the sense or antisense coding sequences.
  • the siRNA can also be expressed from recombinant viral vectors intracellularly at or near the area of the mTOT protein gene expressed in vitro or in vivo.
  • the recombinant viral vectors of the invention comprise sequences encoding the siRNA and any suitable promoter for expressing the siRNA sequences. Suitable promoters include, in addition to others described specifically herein, the U6 or HI RNA pol III promoter sequences and the cytomegalovirus promoter. Selection of other suitable promoters is within the skill in the art.
  • the recombinant viral vectors of the invention can also comprise inducible or regulatable promoters for expression of the siRNA in a particular tissue or in a particular intracellular environment.
  • the siRNA can be expressed from a recombinant viral vector either as two separate, complementary RNA molecules, or as a single RNA molecule with two complementary regions.
  • molecule(s) to be expressed can be used, for example vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., Antiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like.
  • the tropism of the viral vectors can also be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses.
  • an AAV vector of the invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like.
  • Preferred viral vectors are those derived from AV and AAV.
  • the siRNA is expressed as two separate, complementary single- stranded RNA molecules from a recombinant AAV vector comprising, for example, either the U6 or HI RNA promoters, or the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • a suitable AV vector for expressing the siRNA, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
  • Suitable AAV vectors for expressing the siRNA, methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61 : 3096-3101; Fisher J et al. (1996), J. Virol., 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No.
  • siRNA containing a given target sequence can be evaluated using standard techniques for measuring the levels of RNA or protein in cells.
  • siRNA can be delivered to cultured cells, and the levels of target mRNA can be measured by Northern blot or dot blotting techniques, or by quantitative RT-PCR.
  • the levels of mTOT protein in the infected cells can be measured by ELISA or Western blot.
  • a shRNA nucleic acid molecule refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • One portion or segment of a duplex stem of the shRNA structure is anti-sense strand or complementary, e.g., fully complementary, to a section of about 18 to about 40 or more nucleotides of the mRNA of the target gene, for example, mTOT protein BP44 and/or BRP44-Like.
  • shRNAs mimic the natural precursors of micro RNAs (miRNAs). miRNAs are noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level during plant and animal development.
  • miRNAs are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre- miRNA, probably by Dicer, an RNase IH-type enzyme, or a homolog thereof.
  • pre- miRNA Naturally- occurring miRNA precursors
  • Pre-miRNA have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem.
  • the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide "loop" in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other.
  • Short hairpin RNAs, or engineered RNA precursors, of the invention are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNAi agents (e.g., siRNAs of the invention).
  • the shRNA molecules of the invention are designed to produce any of the siRNAs described above when processed in a cell e.g., by Dicer present within the cell.
  • the requisite elements of a shRNA molecule include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or double-stranded stem portion. The two portions need not be fully or perfectly
  • the first and second “stem” portions are connected by a portion having a sequence that, has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a "loop" portion in the shRNA molecule.
  • the shRNA molecules are processed to generate siRNAs.
  • shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide "loop" in a portion of the stem, for example a one-, two- or three-nucleotide loop.
  • the stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides.
  • the overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.
  • Us uracils
  • Ts thymidines
  • One strand of the stem portion of the shRNA is further sufficiently complementary (e.g., antisense) to a target RNA of mTOT protein (e.g., mRNA of BP44 or BRP44-Like, as provided in SEQ ID NO:5-7 & 52-53, or any mRNA encoding any of the mTOT proteins of Tables 1, 3, 4 and 5) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi).
  • RNAi RNA interference
  • the antisense portion can be on the 5' or 3' end of the stem.
  • the stem portions of a shRNA are preferably about 15 to about 50 nucleotides in length.
  • the two stem portions are about 18 or 19 to about 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length.
  • the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway.
  • the stem can be longer than 30 nucleotides.
  • a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).
  • the two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem.
  • the two portions can be, but need not be, fully or perfectly complementary.
  • the loop in the shRNAs or engineered RNA precursors may differ from natural pre- miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences.
  • the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.
  • a preferred loop consists of or comprises a "tetraloop" sequences. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.
  • shRNAs of the invention include the sequences of a desired siRNA molecule described above.
  • the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target RNA (e.g., mTOT protein mRNA), for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation.
  • the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5' UTR (untranslated region), coding sequence, or 3* UTR, provided said portion is distant from the site of the gain-of-function mutation.
  • This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides.
  • the last two nucleotides of the nucleotide sequence can be selected to be UU.
  • This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA.
  • This sequence can replace a stem portion of a wild-type pre- miRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized.
  • DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA.
  • the efficacy of the functional nucleic acids useful herein can be increased when the functional nucleic acids, for example, siRNAs are mTOT protein targeted, delivered systemically, repeatedly and safely.
  • Low transfection efficiency, nuclease degeneration, poor tissue penetration and non-specific immune degradation can be overcome when the functional nucleic acids are incorporated into protective and functional vehicles, for example, viral vectors, liposomes complexed with polyethyleneimine (PEI), linked with vascular endothelial growth factor (VEGF) receptor- 2 and PEI that was PEGylated with an RGD peptide ligand at the distal end, protamine-antibody fusion protein, and tumor-targeting immunoliposome complexes.
  • PI polyethyleneimine
  • VEGF vascular endothelial growth factor
  • the functional nucleic acid is administered to the subject either as naked siRNA, in conjunction with a delivery reagent, or as a recombinant plasmid or viral vector which expresses the siRNA.
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as
  • oligonucleotides having non-naturally-occurring portions which function similarly.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
  • target nucleic acid and “nucleic acid encoding mTOT protein” encompass DNA encoding mTOT protein, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA.
  • RNA including pre-mRNA and mRNA
  • cDNA derived from such RNA.
  • the specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This minhibition of function of a target nucleic acid by compounds that specifically hybridize to it is generally referred to as "antisense".
  • the functions of DNA to be interfered with include replication and transcription.
  • RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.
  • an “antisense” functional nucleic acid can include a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double- stranded cDNA molecule, or complementary to an mRNA sequence, for example, as provided in any one or more of SEQ ID NOs: 5-7, 52 or 53.
  • the antisense nucleic acid can be complementary to an entire mTOT protein coding strand, or to only a portion thereof (e.g., coding region of a human mTOT protein nucleotide sequence).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" of the coding strand of a nucleotide sequence encoding an mTOT protein protein (e.g., the 5' or 3' untranslated regions).
  • an antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used (see, e.g., Protocols for Oligonucleotide Conjugates.
  • the antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., R A transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Antisense nucleic acids can also be produced from synthetic methods such as phosphoramidite methods, H-phosphonate methodology, and phosphite trimester methods.
  • Antisense nucleic acids can also be produced by PCR methods. Such methods produce cDNA and cRNA sequences complementary to the mRNA.
  • antisense molecules can be modified or unmodified RNA, DN A, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis, for example, inhibition in the expression of one or more mTOT protein proteins.
  • Antisense molecules can be modified or unmodified RNA, DNA, or mixed polymer oligonucleotides and primarily function by specifically binding to matching sequences resulting in inhibition of peptide synthesis, for example, inhibition in the expression of mTOT protein proteins.
  • oligonucleotide binds to target RNA by Watson Crick base-pairing and blocks gene expression by preventing ribosomal translation of the bound sequences either by steric blocking or by activating RNase H enzyme.
  • Antisense molecules can also alter protein synthesis by interfering with RNA processing or transport from the nucleus into the cytoplasm.
  • An antisense nucleic acid can be an a-anomeric nucleic acid molecule.
  • An a- anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other.
  • the antisense nucleic acid molecule can also comprise a 2'-0-methylribonucleotideor a chimeric RNA-DNA analog, and can have mixed internucleoside linkages (see, e.g., Protocols for Oligonucleotide Conjugates. Totowa N.J.: Humana Press, 1994).
  • methods for treating diabetes mellitus, metabolic syndrome, cardiovascular disease, gastrointestinal disease or Alzheimer's disease with one or more functional nucleic acid comprises administering a functional nucleic acid to a subject in need thereof.
  • antisense nucleic acid molecules are typically administered to a subject (systemically, e.g., by intravenous administration, or locally, e.g.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For systemic
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies that bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein.
  • vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are also known in the art (e.g., Wacheck et al.
  • mTOT protein gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the mTOT protein (e.g., the mTOT protein promoter and/or enhancers) to form triple helical structures that prevent transcription of an mTOT protein gene in target cells (see generally, Hurst, H.C., Breast Cancer Res 2001, 3:395-398; Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14:807-15), these references are incorporated herein by reference in their entireties.
  • the potential sequences that can be targeted for triple helix formation can be increased by creating a so-called
  • Switchback nucleic acid molecule is synthesized in an alternating 5'-3', 3'-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizeable stretch of either purines or pyrimidines to be present on one strand of a duplex.
  • the present invention employs functional nucleic acids, particularly antisense oligonucleotides, for use in inhibiting, reducing or preventing the expression of one or more mTOT proteins, ultimately inhibiting the amount of mTOT protein produced.
  • Antisense technology is emerging as an effective means for reducing the expression of specific gene products and is uniquely useful in a number of therapeutic, diagnostic, and research applications for the minhibition of mTOT protein.
  • the overall effect of such interference with target nucleic acid function is inhibition of the expression of an mTOT protein as described herein.
  • inhibition is the preferred form of gene expression and mRNA is a preferred target.
  • the functional nucleic acids of the present invention inhibit expression of mTOT protein in vitro or in vivo, by at least 10% as measured in a suitable assay, such as those described in the examples below.
  • the compounds of the present invention inhibit expression of mTOT protein in vitro or in vivo, by at least 25%.
  • the compounds of the present invention inhibit expression of mTOT protein in vitro or in vivo, by at least 40%.
  • the compounds of the present invention inhibit expression of mTOT protein in vitro or in vivo, by at least 50%. In a further embodiment of this invention, the compounds of the present invention inhibit expression of mTOT protein in vitro or in vivo, by at least 60%. In another embodiment of this invention, the compounds of the present invention inhibit expression of mTOT protein in vitro or in vivo, by at least 70% or at least 80% or higher.
  • Targeting an antisense oligonucleotide to a particular nucleic acid is a multistep process. In some embodiments, the process begins with the identification of a nucleic acid sequence encoding an mTOT protein of interest for which the function is to be inhibited. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., detection or inhibition of expression of the protein, results.
  • An antisense nucleic acid can be designed such that it is complementary to the entire coding region of mTOT protein mRNA, but in general, is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of mTOT protein mRNA.
  • an intragenic site for the mTOT protein is the region encompassing the translation initiation or termination codon of the open reading frame (ORF) of the gene.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of mTOT protein mRNA, e.g., between the -10 and +10 regions of the target gene nucleotide sequence of interest.
  • the translation initiation codon is typically 5'-AUG (in transcribed mRNA molecules; 5 -ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the "AUG codon", the “start codon” or the “AUG start codon”.
  • a minority of genes have a translation initiation codon having the RNA sequence 5'- GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5*-ACG and 5"-CUG have been shown to function in vivo.
  • the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically
  • start codon and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding mTOT protein, regardless of the sequence(s) of such codons.
  • a translation termination codon (or "stop codon”) of a gene may have one of three sequences, i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are 5 -TAA, 5'-TAG and 5'-TGA, respectively).
  • start codon region and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation initiation codon.
  • stop codon region and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5' or 3') from a translation termination codon.
  • An antisense oligonucleotide can be, e.g., about 5- 100 or from about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more nucleotides in length.
  • the 5' untranslated region (5'UTR) of mTOT protein known in the art to refer to the portion of the mRNA in the 5' direction from the translation initiation codon, and thus including nucleotides between the 5' cap site and the translation initiation codon of the mRNA or corresponding nucleotides on the gene, is the target region.
  • the 3' untranslated region (3'UTR) of mTOT protein known in the art to refer to the portion of an mRNA in the 3' direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3' end of an mRNA or corresponding nucleotides on the gene is the target region.
  • a further target region includes the 5' cap of the mRNA for mTOT protein that comprises an N7-methylated guanosine residue joined to the 5'-most residue of the mRNA via a 5'-5' triphosphate linkage.
  • the 5' cap region of the mRNA is considered to include the 5' cap structure itself as well as the first 50 nucleotides adjacent to the cap.
  • the 5' cap region itself is also a target region according to this invention.
  • mRNA splice sites are target regions of the gene encoding mTOT protein, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease.
  • aberrant fusion junctions due to
  • mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as "fusion transcripts". Introns can be effective target regions for antisense oligonucleotides targeted, for example, to DNA or pre-mRNA of mTOT protein.
  • RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as
  • pre-mRNA variants are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and extronic regions.
  • pre-mRNA variants Upon excision of one or more exon or intron regions or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants". Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.
  • Variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more than one start codon or stop codon.
  • Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA.
  • Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mR A or mRNA.
  • One specific type of alternative stop variant is the "polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the "poly A stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.
  • oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or R A molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other.
  • oligonucleotide and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense oligonucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • An antisense oligonucleotide is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of expression and/or activity of an mTOT protein, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense oligonucleotide to non-target sequences under conditions in which specific binding is desired.
  • Such conditions include, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.
  • the antisense oligonucleotides of the present invention comprise at least 80% sequence complementarity to a target region within the target nucleic acid of mTOT protein to which they are targeted. In another embodiment, the antisense oligonucleotides of the present invention comprise at least 90% sequence complementarity to a target region within the target nucleic acid of mTOT protein to which they are targeted. In still another embodiment of this invention, the antisense oligonucleotides of the present invention comprise at least 95% sequence complementarity to a target region within the target nucleic acid of mTOT protein to which they are targeted.
  • an antisense oligonucleotide in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an antisense
  • oligonucleotide with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • BLAST programs Basic local alignment search tools
  • Antisense oligonucleotides and other functional nucleic acids of the invention which hybridize to the target mTOT encoding DNA or RNA and inhibit expression of the target, are identified as taught herein. Representative sequences of these compounds are hereinbelow identified as embodiments of the invention.
  • the sites to which these representative antisense oligonucleotides are specifically hybridizable are hereinbelow referred to as "illustrative target regions" and are therefore sites for targeting.
  • the term "illustrative target region” is defined as at least an 8-nucleobase portion of a target region of mTOT protein, to which an active antisense oligonucleotide is targeted.
  • an illustrative target region is at least 15 nucleobases. In still another embodiment an illustrative target region is at least 20 nucleobases. In another embodiment an illustrative target region is at 30 nucleobases. In yet another embodiment an illustrative target region is at least 40 nucleobases. In still another embodiment an illustrative target region is at least 50
  • an illustrative target region is at 60 nucleobases. In still another embodiment an illustrative target region is at least 70 nucleobases. In another embodiment an illustrative target region is at least 80 nucleobases or more. In other embodiments, the illustrative target region consists of consecutive nucleobases. Thus, target regions 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative target regions are considered to be suitable target regions as well. While not wishing to be bound by theory, it is presently believed that these illustrative target regions represent regions of the target nucleic acid that are accessible for hybridization.
  • Exemplary additional target regions include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5 '-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases).
  • additional target regions are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3'-terminus of one of the illustrative target regions (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases).
  • the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the target region and continuing until the DNA or RNA contains about 8 to about 80 nucleobases.
  • Antisense oligonucleotides are commonly used as research reagents and diagnostics. For example, antisense oligonucleotides, which are able to inhibit gene expression with seventeen specificity, are often used by those of ordinary skill to elucidate the function of particular genes. Antisense oligonucleotides are also used, for example, to distinguish between functions of various members of a biological pathway. Antisense minhibition has, therefore, been harnessed for research use.
  • the antisense oligonucleotides of the present invention can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of mTOT protein genes expressed within cells and tissues.
  • Expression patterns within cells or tissues treated with one or more antisense oligonucleotides are compared to control cells or tissues not treated with antisense oligonucleotides and the patterns produced are analyzed for differential levels of mTOT protein gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the mTOT protein genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds that affect expression patterns.
  • Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression) (Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.
  • Antisense oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals, particularly mammals, and including humans.
  • Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that oligonucleotides can be useful therapeutic modalities that can be configured to be useful in treatment regimes for treatment of cells, tissues and animals, especially humans. Specific routes of administration and dosages can be ascertained using routine experimentation, to which several clinical trials and experimental protocols for delivering functional nucleic acids such as siRNA, miRNA and antisense oligonucleotides are well known.
  • antisense oligonucleotides are a particular form of functional nucleic acids
  • the present invention comprehends other functional nucleic acids with the broad category of antisense oligonucleotides, including but not limited to oligonucleotide mimetics such as are described below.
  • the antisense oligonucleotides in accordance with this invention preferably comprise compounds at least about 8 nucleobases in length (i.e. linked nucleosides).
  • antisense oligonucleotides of this invention are antisense oligonucleotides of at least about 12 nucleobases in length.
  • antisense oligonucleotides of this invention comprise about 20 nucleobases in length. In still another embodiment, antisense oligonucleotides of this invention comprise about 30 nucleobases in length. In yet another embodiment, antisense oligonucleotides of this invention comprise about 40 nucleobases in length. In still another embodiment, antisense oligonucleotides of this invention comprise about 50 nucleobases in length. In another embodiment, antisense oligonucleotides of this invention comprise about 60 nucleobases in length. In still another embodiment, antisense oligonucleotides of this invention comprise about 70 nucleobases in length.
  • antisense oligonucleotides of this invention comprise about 80 nucleobases in length.
  • Antisense oligonucleotides include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides that hybridize to the target nucleic acid encoding mTOT protein and modulate its expression.
  • GCS external guide sequence
  • Antisense oligonucleotides spanning from 8 to 80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense oligonucleotides described herein are considered to be suitable antisense oligonucleotides as well.
  • exemplary antisense oligonucleotides include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5'-terminus of one of the illustrative antisense oligonucleotides (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5'-terminus of the antisense oligonucleotide which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases).
  • such antisense oligonucleotides include at least 12 consecutive nucleobases from the S'-terminus of one of the illustrative antisense oligonucleotides.
  • the antisense oligonucleotide includes at least 20 consecutive nucleobases from the 5'-terminus of one of the illustrative antisense oligonucleotides.
  • the antisense oligonucleotide includes at least 30 consecutive nucleobases from the 5'-terminus of one of the illustrative antisense oligonucleotides.
  • the antisense oligonucleotide includes at least 50 consecutive nucleobases from the 5'-terminus of one of the illustrative antisense oligonucleotides. In still another embodiment, the antisense oligonucleotide includes at least 60 or more consecutive nucleobases from the 5'-terminus of one of the illustrative antisense oligonucleotides.
  • antisense oligonucleotides are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3'- terminus of one of the illustrative antisense oligonucleotides (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3'-terminus of the antisense oligonucleotide which is specifically hybridizable to the target nucleic acid and continuing until the DNA or RNA contains about 8 to about 80 nucleobases).
  • such antisense oligonucleotides include at least 12 consecutive nucleobases from the 3'-terminus of one of the illustrative antisense
  • the antisense oligonucleotide includes at least 20 consecutive nucleobases from the 3'-terminus of one of the illustrative antisense oligonucleotides. In a further embodiment, the antisense oligonucleotide includes at least 30 consecutive nucleobases from the 3'-terminus of one of the illustrative antisense
  • the antisense oligonucleotide includes at least 50 consecutive nucleobases from the 3'-terminus of one of the illustrative antisense oligonucleotides. In still another embodiment, the antisense oligonucleotide includes at least 60 or more consecutive nucleobases from the 3'-terminus of one of the illustrative antisense oligonucleotides.
  • nucleoside is a base-sugar combination.
  • the base portion of the nucleoside is normally a heterocyclic base.
  • the two most common classes of such heterocyclic bases are the purines and the pyrimidines.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.
  • the phosphate group can be linked to either the 2', 3* or 5' hydroxyl moiety of the sugar.
  • the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound.
  • linear structures can be further joined to form a circular structure, however, open linear structures are generally preferred.
  • linear structures may also have internal nucleobase complementarity and may therefore fold in a manner as to produce a double stranded structure.
  • the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide.
  • the normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.
  • antisense oligonucleotides useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages.
  • oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.
  • modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
  • Preferred modified oligonucleotide backbones include, for example,
  • phosphorothioates chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates,
  • phosphoramidates including 3 -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
  • oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage i.e. a single inverted nucleoside residue that may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
  • Various salts, mixed salts and free acid forms are also included.
  • Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991 , 254, 1497-1500.
  • Most preferred embodiments of the invention are oligonucleotides with
  • phosphorothioate backbones and oligonucleosides with heteroatom backbones and in particular ⁇ CH 2 -NH-0-CH 2 -, -CH 2 -N(CH 3 )-0-CH 2 - (known as a methylene (methylimino) or MMI backbone], -CH 2 -0-N(CH 3 )-CH 2 ⁇ , -CH 2 -N(CH 3 )- N(CH 3 )-CH 2 - and -0-N(CH 3 )- CH 2 -CH 2 - [wherein the native phosphodiester backbone is represented as ⁇ 0-P-0-CH 2 -) of the above referenced U.S. Pat. No.
  • Modified oligonucleotides may also contain one or more substituted sugar moieties.
  • Preferred oligonucleotides comprise one of the following at the position: OH; F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Q to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Particularly preferred are 0[(CH 2 ) n O] m CH 3 , 0(CH 2 ) n OCH 3 , 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 , 0(CH 2 ) crampONH 2 , and 0(CH 2 ) n ON[(CH 2 ) n CH 3 ] 2 , where n and m are from 1 to about 10.
  • oligonucleotides comprise one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, CI, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, poly-alkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN
  • a preferred modification includes 2'-methoxyethoxy (2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2"-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486- 504) i.e., an alkoxyalkoxy group.
  • a further preferred modification includes
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE, as described in examples hereinbelow
  • 2'-dimethylamino-ethoxyethoxy also known in the art as 2'-0-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE
  • 2'-0-CH 2 -0-CH2-N(CH 3 )2 also described in examples hereinbelow.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • a further preferred modification includes Locked Nucleic Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety.
  • the linkage is preferably a methylene (-CH 2 -) discipline group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1 or 2.
  • LNAs and preparation thereof are described in International Published Patent Application Nos. WO 98/39352 and WO
  • Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, "unmodified” or “natural”
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and
  • nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(lH-pyrimido[5,4-b][l,4]benzoxazi- n-2(3H)-one), phenothiazine cytidine (lH-pyrimido[5,4-b] [l,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b]
  • nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications.
  • the present invention also includes antisense oligonucleotides that are chimeric compounds.
  • "Chimeric” antisense oligonucleotides or “chimeras”, in the context of this invention, are antisense oligonucleotides, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.
  • oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid.
  • An additional region of the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid.
  • oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression.
  • the cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as interferon-induced RNAseL which cleaves both cellular and viral RNA.
  • Chimeric antisense oligonucleotides of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described herein. Such compounds have also been referred to in the art as hybrids or gapmers.
  • the antisense oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • the functional nucleic acid compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
  • Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos.:
  • prodrug indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
  • prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in International Published Patent Application No. WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in International Published Patent Application No. WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.
  • pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like.
  • Suitable amines are ⁇ , ⁇ ' dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., "Pharmaceutical Salts", J. of Pharma Sci. , 1977, 66, 1-19).
  • the base addition salts of said acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner.
  • the free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner.
  • a "pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates.
  • Suitable pharmaceutically acceptable salts include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phosphp acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonico
  • Suitable pharmaceutically acceptable cation are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
  • the present invention also includes pharmaceutical compositions and formulations that include the antisense oligonucleotides of the invention.
  • the pharmaceutical compositions and formulations that include the antisense oligonucleotides of the invention.
  • compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • Oligonucleotides with at least one 2'-0-methoxyethyl modification are believed to be particularly useful for oral administration.
  • compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • Coated condoms, gloves and the like may also be useful.
  • Preferred topical formulations include those in which the
  • oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g.
  • Oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids.
  • Preferred fatty acids and esters include but are not limited arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, Iinoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1 -monocaprate, l-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C.sub.1-10 alkyl ester (e.g. isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.
  • arachidonic acid oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, Iinoleic acid, lin
  • compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators.
  • Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof.
  • Preferred bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid,
  • Preferred fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl l-monocaprate, l-dodecylazacycloheptan-2-one, an acylcamitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g.
  • penetration enhancers for example, fatty acids/salts in combination with bile acids/salts.
  • a particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA.
  • Further penetration enhancers include polyoxyethylene- 9-lauryl ether, polyoxyethylene-20-cetyl ether.
  • Antisense oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles.
  • Oligonucleotide complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches.
  • Particularly preferred complexing agents include chitosan, N-trimethylchitosan,
  • poly-L-lysine polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
  • polymethylacrylate polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG).
  • compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions that may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions may be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.
  • compositions of the present invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions may be formulated and used as foams.
  • Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.
  • the preparation of such compositions and formulations is generally known to those skilled in the pharmaceutical and formulation arts and may be applied to the formulation of the compositions of the present invention.
  • compositions of the present invention may be prepared and formulated as emulsions.
  • Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 ⁇ in diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.
  • Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other.
  • emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
  • a water-in-oil (w/o) emulsion When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion.
  • an oily phase when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion.
  • Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase.
  • Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed.
  • compositions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.
  • Such complex formulations often provide certain advantages that simple binary emulsions do not.
  • Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion.
  • a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.
  • Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids.
  • Synthetic surfactants also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988:, Marcel Dekker, Inc., New York, N.Y., volume 1 , p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
  • Surfactants are typically amphiphilic and comprise a hydrophilic and a
  • hydrophobic portion The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations.
  • HLB hydrophile/lipophile balance
  • surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 88, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).
  • Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia.
  • Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid
  • polar inorganic solids such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.
  • non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume l, p. 199).
  • Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxy vinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.
  • polysaccharides for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth
  • cellulose derivatives for example, carboxymethylcellulose and carboxypropylcellulose
  • synthetic polymers for example, carbomers, cellulose ethers, and carboxy
  • emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives.
  • preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid.
  • Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation.
  • Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite
  • antioxidant synergists such as citric acid, tartaric acid, and lecithin.
  • Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
  • compositions of antisense oligonucleotides and functional nucleic acids are formulated as microemulsions.
  • a microemulsion may be defined as a system of water, oil and amphiphile, which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume I, p. 245).
  • microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte.
  • microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).
  • Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants.
  • ionic surfactants non-ionic surfactants
  • Brij 96 polyoxyethylene oleyl ethers
  • polyglycerol fatty acid esters tetraglycerol monolaurate (ML310),
  • the cosurfactant usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
  • Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art.
  • the aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol.
  • the oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.
  • Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs.
  • Lipid based microemulsions both o/w and w/o have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin.
  • microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile drugs, peptides or oligonucleotides. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications.
  • microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of oligonucleotides and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of oligonucleotides and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.
  • Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the oligonucleotides and nucleic acids of the present invention.
  • Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories including surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.
  • oligonucleotides are sequestered in lipids (e.g., liposomes or micelles) to aid in delivery (See e.g., U.S. Patents 6,458,382, 6,429,200; U.S. Patents 6,458,382, 6,429,200; U.S. Patents 6,458,382, 6,429,200; U.S. Patents 6,458,382, 6,429,200; U.S. Patent
  • liposome refers to one or more lipids forming a complex, usually surrounded by an aqueous solution. Liposomes are generally spherical structures comprising lipids fatty acids, lipid bilayer type structures, unilamellar vesicles and amorphous lipid vesicles. Generally, liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. The liposomes may be unilamellar vesicles (possessing a single bilayer membrane) or multilamellar (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer).
  • Liposomes of the present invention may also include a DNAi oligonucleotide as defined below, either bound to the liposomes or sequestered in or on the liposomes.
  • the molecules include, but are not limited to, DNAi oligonucleotides and/or other agents used to treat diseases such as cancer.
  • encapsulation incorporation, or association of a drug, molecule, compound, including a DNAi oligonucleotide, with the lipids of a liposome.
  • the molecule may be associated with the lipid bilayer or present in the aqueous interior of the liposome or both.
  • “Sequestered” includes encapsulation in the aqueous core of the liposome.
  • part or all of the molecule is located in the aqueous core of the liposome and part outside of the liposome in the aqueous phase of the liposomal suspension, where part of the molecule is located in the aqueous core of the liposome and part in the lipid portion of the liposome, or part sticking out of the liposomal exterior, where molecules are partially or totally embedded in the lipid portion of the liposome, and includes molecules associated with the liposomes, with all or part of the molecule associated with the exterior of the liposome.
  • the oligonucleotide and/or other agents must be stably sequestered in the liposomes until eventual uptake in the target tissue or cells. Accordingly, the guidelines for liposomal formulations of the FDA regulate specific preclinical tests for liposomal drugs (http://www.fda.gov/cder/guidance/2191dft.pdf). After injection of liposomes into the blood stream, serum components interact with the liposomes, which can lead to permeabilization of the liposomes. However, release of a drug or molecule that is encapsulated in a liposome depends on molecular dimensions of the drug or molecule.
  • a plasmid of thousands of base pairs is released much more slowly than smaller oligonucleotides or other small molecules.
  • liposomes used for delivery may be amphoteric liposomes, such as those described in US 2009/0220584, incorporated herein by reference.
  • Amphoteric liposomes are a class of liposomes having anionic or neutral charge at about pH 7.5 and cationic charge at pH 4.
  • Lipid components of amphoteric liposomes may be themselves amphoteric, and/or may consist of a mixture of anionic, cationic, and in some cases, neutral species, such that the liposome is amphoteric.
  • an "amphoteric liposome” is a liposome with an amphoteric character, as defined below.
  • sequestered, sequestering, or sequester refers to encapsulation, incorporation, or association of a drug, molecule, compound, including a functional nucleic acid compound of the present invention, with the lipids of a liposome.
  • the compound may be associated with the lipid bilayer or present in the aqueous interior of the liposome or both.
  • Sequestered includes encapsulation in the aqueous core of the liposome. It also
  • part or all of the functional nucleic acid is located in the aqueous core of the liposome and part outside of the liposome in the aqueous phase of the liposomal suspension, where part of the molecule is located in the aqueous core of the liposome and part in the lipid portion of the liposome, or part sticking out of the liposomal exterior, where molecules are partially or totally embedded in the lipid portion of the liposome, and includes molecules associated with the liposomes, with all or part of the molecule associated with the exterior of the liposome.
  • polydispersity index is a measure of the heterogeneity of the particle dispersion (heterogeneity of the diameter of liposomes in a mixture) of the liposomes.
  • a polydispersity index can range from 0.0 (homogeneous) to 1.0 (heterogeneous) for the size distribution of liposomal formulations.
  • the amphoteric liposomes include one or more amphoteric lipids or alternatively a mix of lipid components with amphoteric properties. Suitable amphoteric lipids are disclosed in PCT International Publication Number WO02/066489 as well as in PCT International Publication Number WO03/070735, the contents of both of which are incorporated herein by reference. Alternatively, the lipid phase may be formulated using pH-responsive anionic and/or cationic components, as disclosed in PCT International Publication Number
  • WO02/066012 the contents of which are incorporated by reference herein.
  • Cationic lipids sensitive to pH are disclosed in PCT International Publication Numbers WO02/066489 and WO03/070220, in Budker, et al. 1996, Nat. Biotechnol., 14(6):760-4, and in US Patent Number 6,258,792 the contents of which are incorporated by reference herein, and can be used in combination with constitutively charged anionic lipids or with anionic lipids that are sensitive to pH.
  • the cationic charge may also be introduced from constitutively charged lipids that are known to those skilled in the art in combination with a pH sensitive anionic lipid. (See also PCT International Publication Numbers WO05/094783,
  • Amphoteric liposomes of the present invention include (1) amphoteric lipids or a mixture of lipid components with amphoteric properties, (2) neutral lipids, (3) one or more functional nucleic acid compounds, (4) a cryoprotectant and/or lyoprotectant, (5) or spray- drying protectant.
  • the inhibitory mTOT protein functional nucleic acid compounds-liposomes have a defined size distribution and polydispersity index.
  • amphoter or “amphoteric” character refers to a structure, being a single substance (e.g., a compound) or a mixture of substances (e.g., a mixture of two or more compounds) or a supramolecular complex (e.g., a liposome) comprising charged groups of both anionic and cationic character wherein
  • Amphoter I Lipid Pairs refers to lipid pairs containing a stable cation and a chargeable anion. Examples include without limitation DDAB/CHEMS, DOTAP/CHEMS and DOTAP/DOPS. In some aspects, the ratio of the percent of cationic lipids to anionic lipids is lower than 1.
  • Amphoter II Lipid Pairs refers to lipid pairs containing a chargeable cation and a chargeable anion. Examples include without limitation Mo- Chol/CHEMS, DPIM/CHEMS or DPIM/DG-Succ. In some aspects, the ratio of the percent of cationic lipids to anionic lipids is between about 5 and 0.2.
  • Amphoter HI Lipid Pairs refers to lipid pairs containing a chargeable cation and stable anion. Examples include without limitation Mo-Chol/DOPG or Mo-Chol/Chol-S04. In one embodiment, the ratio of the percent of cationic lipids to anionic lipids is higher than 1.
  • DOPS Dioleoylphosphatidylserine [0369] POPS Palmitoyl-oleoylphosphatidylserine
  • DOTMA 1 ,2-dioleyloxypropy l)-N,N,N-trimethylammoniumchloride
  • DOTAP (1 ,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt
  • DPTAP (1 ,2-dipalmitoyloxypropyl)-N,N,N-trirnethylarnmonium salt
  • DOTMA (1 ,2-dioleyloxypropy l)-N,N,N-trimethylammonium chloride
  • HistDG 1,2 Dipalmitoylglycerol-hemisuccinat-N_-Histidinyl- hemisuccinate, & Distearoyl, Dimyristoyl, Dioleoyl or palmitoyl-oleoylderivatives
  • DGSucc 1,2 Dipalmitoyglycerol-3-hemisuccinate & Distearoyl-, dimyristoyl- Dioleoyl or palmitoyl-oleoylderivatives
  • DOSC (l,2-dioleoyl-3-succinyl-sn-glyceryl choline ester
  • DOGSDO (1 ,2-dioleoyl-sn-glycero-3-succinyl-2-hydroxy ethyl disulfide ornithine
  • lipids that are suitable for use in the compositions in accordance with the present invention.
  • the membrane anchors of the lipids are shown exemplarily and serve only to illustrate the lipids of the invention and are not intended to limit the same.
  • the overall molecule assumes its pH-dependent charge characteristics by the simultaneous presence of cationic and anionic groups in the "amphoteric substance" molecule portion. More specifically, an amphoteric substance is characterized by the fact that the sum of its charge components will be precisely zero at a particular pH value. This point is referred to as isoelectric point (IP). Above the IP the compound has a negative charge, and below the IP it is to be regarded as a positive cation, the IP of the amphoteric lipids according to the invention ranging between 4.5 and 8.5. [0416] The overall charge of the molecule at a particular pH value of the medium can be calculated as follows:
  • ni number of such groups in the molecule.
  • a compound is formed by coupling the amino group of histidine to cholesterol hemisuccinate.
  • the product has a negative charge because the carboxyl function which is present therein is in its fully dissociated form, and the imidazole function only has low charge.
  • an acid pH value of about 4 the situation is reversed: the carboxyl function now is largely discharged, while the imidazole group is essentially fully protonated, and the overall charge of the molecule therefore is positive.
  • the amphoteric lipid is selected from the group consisting of HistChol, HistDG, isoHistSuccDG, Acylcamosine and HCChol. In another embodiment, the amphoteric lipid is HistChol.
  • Amphoteric lipids can include, without limitation, derivatives of cationic lipids which include an anionic substituent.
  • Amphoteric lipids include, without limitation, the compounds having the structure of the formula:
  • Z is a sterol or an aliphatic
  • Sterol is selected from the group consisting of cholesterol, sitosterol, campesterol, desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol, sigmasterol, 22-hydroxycholesterol, 25 hydroxycholesterol, lanosterol, 7-dehydrocholesteril, dihydrocholesterol,
  • Each Wl is independently an unsubstituted aliphatic
  • Each W2 is independently an aliphatic optionally substituted with HO(0)C-aliphatic- amino or carboxy;
  • HET is an amino, an optionally substituted heterocycloaliphatic or an optionally substituted heteroaryl.
  • the HET is an optionally substituted heterocycloaliphatic including at least one nitrogen ring atom, or an optionally substituted heteroaryl including at least one nitrogen ring atom.
  • the HET is morpholinyl, piperidinyl, piperazinlyl, pyrimidinyl, or pyridinyl.
  • the cationic lipid has the structure
  • the sterol is cholesterol.
  • amphoteric lipids include, without limitation, the compounds having the structure of the formula:
  • Z is a structure according to the general formula
  • Sterol is selected from the group consisting of cholesterol, sitosterol, campesterol, desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol, sigmasterol, 22-hydroxycholesterol, 25 hydroxycholesterol, lanosterol, 7-dehydrocholesterol, dihydrocholesterol,
  • Each Wl is independently an unsubstituted aliphatic with up to 8 carbon atoms
  • Each W2 is independently an aliphatic , carboxylic acid with up to 8 carbon atoms and 0, 1, or 2 ethyleneically unsaturated bonds;
  • HET is an amino, an optionally substituted heterocycloaliphatic or an optionally substituted heteroaryl.
  • the HET is an optionally substituted heterocycloaliphatic including at least one nitrogen ring atom, or an optionally substituted heteroaryl including at least one nitrogen ring atom.
  • the HET is morpholinyl, piperidinyl, piperazinlyl, pyrimidinyl, or pyridinyl.
  • the cationic lipid has the structure Sterol-X-spacerl-Y-spacer2-morpholinyl or Sterol-X-spacerl-Y-spacer2-imidazolyl.
  • the sterol is cholesterol.
  • the lipid phase can be formulated using pH-responsive anionic and/or cationic components, as disclosed in PCT International Publication Number WO02/066012, the contents of which are incorporated by reference herein.
  • Cationic lipids sensitive to pH are disclosed in PCT International Publication Numbers WO02/066489 and WO03/070220, in Budker, et al. (1996), Nat Biotechnol. 14(6):760-4, and in US Patent Number 6,258,792, the contents of all of which are incorporated by reference herein.
  • the cationic charge may be introduced from constitutively charged lipids known to those skilled in the art in combination with a pH sensitive anionic lipid.
  • the mixture of lipid components may comprise (i) a stable cationic lipid and a chargeable anionic lipid, (ii) a chargeable cationic lipid and chargeable anionic lipid or (iii) a stable anionic lipid and a chargeable cationic lipid.
  • the charged groups can be divided into the following 4 groups.
  • Nitrogen bases with preferred pKa values are also formed by substituting nitrogen atoms one or more times with low molecular weight alkane hydroxyls, such as hydroxymethyl or hydroxyethyl groups.
  • alkane hydroxyls such as hydroxymethyl or hydroxyethyl groups.
  • amphoteric liposomes contain variable amounts of such membrane-forming or membrane-based amphiphilic materials, so that they have an amphoteric character. This means that the liposomes can change the sign of the charge completely.
  • the amount of charge carrier of a liposome, present at a given pH of the medium can be calculated using the following formula:
  • ni is the number of these groups in the liposome.
  • cationic components include DPIM, CHIM, DORIE, DDAB, DAC-Chol, TC-Chol, DOTMA, DOGS, (C18) 2 Gly + ⁇ , ⁇ -dioctadecylamido-glycine, CTAB, CPyC, DODAP DMTAP, DPTAP, DOTAP, DC-Choi, MoChol, HisChol and DOEPC.
  • cationic lipids include DMTAP, DPTAP, DOTAP, DC-Choi, MoChol and HisChol.
  • the cationic lipids can be compounds having the structure of the formula
  • L is a sterol or [aliphatic(C(0)0)-] 2 -alkyl-;
  • Sterol is selected from the group consisting of cholesterol, sitosterol, campesterol, desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol, sigmasterol, 22-hydroxycholesterol,
  • Each spacer 1 and spacer 2 is independently an unsubstituted aliphatic;
  • HET is an amino, an optionally substituted heterocycloaliphatic or an optionally substituted heteroaryl.
  • the HET is an optionally substituted heterocycloaliphatic including at least one nitrogen ring atom, or an optionally substituted heteroaryl including at least one nitrogen ring atom.
  • the HET is morpholinyl, piperidinyl, piperazinlyl, pyrimidinyl or pyridinyl.
  • the cationic lipid has the structure
  • the sterol is cholesterol.
  • pH sensitive cationic lipids can be compounds having the structure of the formula
  • L is a structure according to the general formula
  • Sterol is selected from the group consisting of cholesterol, sitosterol, campesterol, desmosterol, fucosterol, 22-ketosterol, 20-hydroxysterol, sigmasterol, 22-hydroxycholesterol, 25 hydroxycholesterol, lanosterol, 7-dehydrocholesterol, dihydrocholesterol,
  • Each spacer 1 and spacer 2 is independently an unsubstituted aliphatic with 1-8 carbon atoms;
  • HET is an amino, an optionally substituted heterocycloaliphatic or an optionally substituted heteroaryl.
  • the HET is an optionally substituted heterocycloaliphatic including at least one nitrogen ring atom, or an optionally substituted heteroaryl including at least one nitrogen ring atom.
  • the HET is morpholinyl, piperidinyl, piperazinlyl, pyrimidinyl or pyridinyl.
  • the cationic lipid has the structure
  • the sterol is cholesterol.
  • the amphoteric mixtures further comprise anionic lipids, either constitutively or conditionally charged in response to pH, and such lipids are also known to those skilled in the art.
  • lipids for use with the invention include DOGSucc, POGSucc, DMGSucc, DPGSucc, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG, DMPA, DPP A, DOPA, POPA, CHEMS and CetylP.
  • anionic lipids include DOGSucc, DMGSucc, DMPG, DPPG, DOPG, POPG, DMPA, DPP A, DOPA, POPA, CHEMS and CetylP.
  • Neutral lipids include any lipid that remains neutrally charged at a pH between about 4 and 9.
  • Neutral lipids include, without limitation, cholesterol, other sterols and derivatives thereof, phospholipids, and combinations thereof.
  • the phospholipids include any one phospholipid or combination of phospholipids capable of forming liposomes. They include phosphatidylcholines, phosphatidylethanolamines, lecithin and fractions thereof, phosphatidic acids, phosphatidylglycerols, phosphatidylinolitols, phosphatidylserines, plasmalogens and sphingomyelins.
  • the phosphatidylcholines include, without limitation, those obtained from egg, soy beans or other plant sources or those that are partially or wholly synthetic or of variable lipid chain length and unsaturation, POPC, OPPC, natural or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC, DSPC, DOPC and derivatives thereof.
  • phosphatidylcholines are POPC, non-hydrogenated soy bean PC and non-hydrogenated egg PC.
  • Phosphatidylethanolamines include, without limitation, DOPE, DMPE and DPPE and derivatives thereof.
  • Phosphatidylglycerols include, without limitation, DMPG, DLPG, DPPG, and DSPG.
  • Phosphatidic acids include, without limitation, DSPA, DMPA, DLPA and DPPA.
  • Sterols include cholesterol derivatives such as 3-hydroxy-5.6-cholestene and related analogs, such as 3-amino-5.6-cholestene and 5,6-cholestene, cholestane, cholestanol and related analogs, such as 3-hydroxy-cholestane; and charged cholesterol derivatives such as cholesteryl-beta-alanine and cholesterol hemisuccinate.
  • neutral lipids include but are not limited to DOPE, POPC, soy bean PC or egg PC and cholesterol.
  • the invention provides a mixture comprising amphoteric liposomes and a functional nucleic acid compound.
  • the amphoteric liposomes have an isoelectric point of between 4 and 8.
  • the amphoteric liposomes are negatively charged or neutral at pH 7.4 and positively charged at pH 4.
  • the amphoteric liposomes include amphoteric lipids.
  • the amphoteric lipids can be HistChol, HistDG, isoHistSucc DG, Acylcarnosine, HCChol or combinations thereof.
  • the amphoteric liposomes include a mixture of one or more cationic lipids and one or more anionic lipids.
  • the cationic lipids can be DMTAP, DPTAP, DOTAP, DC-Choi, MoChol or HisChol, or combinations thereof
  • the anionic lipids can be CHEMS, DGSucc, Cet-P, DMGSucc, DOGSucc, POGSucc, DPGSucc, DG Succ, DMPS, DPPS, DOPS, POPS, DMPG, DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA or combinations thereof.
  • the liposomes also include neutral lipids.
  • the neutral lipids include sterols and derivatives thereof.
  • the sterols comprise cholesterol and derivatives thereof.
  • the neutral lipids may also include neutral phospholipids.
  • the phospholipids include phosphatidylcholines or phosphatidylcholines and phosphoethanolamines.
  • the phosphatidylcholines are POPC, OPPC, natural or hydrogenated soy bean PC, natural or hydrogenated egg PC, DMPC, DPPC or DOPC and derivatives thereof and the phosphatidylethanolamines are DOPE, DMPE, DPPE or derivatives and combinations thereof.
  • the phosphatidylcholine is POPC, OPPC, soy bean PC or egg PC and the phosphatidylethanolamines is DOPE.
  • the lipids of the amphoteric liposomes include DOPE, POPC, CHEMS and MoChol; POPC, Choi, CHEMS and DOTAP; POPC, Choi, Cet-P and MoChol, or POPC, DOPE, MoChol and DMGSucc.
  • the amphoteric liposomes of the mixture of the invention can be formed from a lipid phase comprising a mixture of lipid components with amphoteric properties, wherein the total amount of charged lipids in the liposome can vary from 5 mole% to 70 mole%, the total amount of neutral lipids may vary from 20 mole% to 70 mole%, and a functional nucleic acid compound.
  • the amphoteric liposomes include 3 to 20 mole% of POPC, 10 to 60 mole% of DOPE, 10 to 60 mole% of MoChol and 10 to 50 mole% of CHEMS.
  • the liposomes can include POPC, DOPE, MoChol and CHEMS in the molar ratios of POPC/DOPE/MoChol/CHEMS of about 6/24/47/23 or 15/45/20/20.
  • the liposomes include 3 to 20 mole% of POPC, 10 to 40 mole% of DOPE, 15 to 60 mole% of MoChol and 15 to 60 mole% of DMGSucc.
  • the liposomes include POPC, DOPE, DMGSucc and MoChol in the molar ratios of
  • the liposomes include 10 to 50 mole% of POPC, 20 to 60 mole% of Choi, 10 to 40 mole% of CHEMS and 5 to 20 mole% of DOTAP.
  • the liposomes include POPC, Choi, CHEMS and DOTAP in the molar ratio of
  • the liposomes include 10 to 40 mole% of POPC, 20 to 50 mole% of Choi, 5 to 30 mole% of Cet-P and 10 to 40 mole% of MoChol.
  • POPC/Chol/Cet-P MoChol is about 35/35/10/20.
  • the functional nucleic acid compound contained in the amphoteric liposomal mixture comprises one or more functional nucleic acids that hybridize to SEQ ID NOs: 5-7, 52-53 or to a nucleic acid (DNA or RNA) that encode any one mTOT proteins of Tables 1, 3,4, or 5,and shown in Figures 3 A, 3B, 4A and 4B, and complementary nucleotides sequences thereof or portions thereof.
  • the functional nucleic acid can be one or more of SEQ ID NOs: 54-73 or the complement thereof.
  • the functional nucleic acid compounds contained in the liposomal mixture are between 15 and 35 base pairs in length.
  • amphoteric liposome-functional nucleic acid compound mixture includes at least one functional nucleic acid compounds as set forth in SEQ ID NO: 54-73 and amphoteric liposomes comprising POPC, DOPE, MoChol and CHEMS in the molar ratio of POPC/DOPE/MoChol/CHEMS of about 6/24/47/23.
  • the amphoteric liposomes of the mixture can include a size between 50 and 500 ⁇ . In one embodiment, the size is between 80 and 300 nm and in another embodiment the size is between 90 and 200 nm.
  • the amphoteric liposomes may have an isoelectric point between 4 and 8.
  • the amphoteric liposomes may be negatively charged or neutral at pH 7.4 and positively charged at pH 4.
  • the amphoteric liposomes have a functional nucleic acid compound concentration of at least about 2 mg/ml at a lipid concentration of 10 to 100 mM or less.
  • the invention provides a method of preparing amphoteric liposomes containing a functional nucleic acid compound.
  • the method includes using an active loading procedure and in another, a passive loading procedure.
  • the method produces liposomes using manual extrusion, machine extrusion, homogenization, microfluidization or ethanol injection.
  • a functional nucleic acid compound concentration of at least about 2 mg/ml at a lipid concentration of 10 to 100 mM or less.
  • the method has an encapsulation efficiency of at least 35%.
  • amphoteric liposomes formulations may comprise POPC/ DOPE/ MoChol/ CHEMS at molar ratios of 6/24/47/23, respectively.
  • Such liposomes are cholesterol-rich and negatively-charged. This is unique among lipid delivery systems and contributes to cellular uptake.
  • HLB hydrophile/lipophile balance
  • Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure.
  • Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters.
  • Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class.
  • the polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.
  • Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.
  • the most important members of the anionic surfactant class are the alkyl sulfates and the soaps.
  • Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
  • amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
  • formulation and therapeutically effective compositions may optionally contain a penetration enhancer.
  • a penetration enhancer can include a fatty acid.
  • Various exemplary fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, l-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C MO alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.
  • Bile salts The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935).
  • the term "bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives.
  • the bile salts of the invention include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences,
  • Chelating agents as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of oligonucleotides through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
  • Chelating agents of the invention include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14: 43-51).
  • EDTA disodium ethylenediaminetetraacetate
  • citric acid e.g., citric acid
  • salicylates e.g., sodium salicylate, 5-methoxysalicylate and homovanilate
  • N-acyl derivatives of collagen e.g., laureth-9 and N-amino acyl derivative
  • Non-chelating non-surfactants As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate
  • This class of penetration enhancers include, for example, unsaturated cyclic ureas, 1 -alky 1- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39: 621-626).
  • Agents that enhance uptake of functional nucleic acid compounds at the cellular level may also be added to the pharmaceutical and other compositions of the present invention.
  • cationic lipids such as lipofectin (Junichi et al, U.S. Pat. No.
  • cationic glycerol derivatives include cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., International Published Patent Application No. WO 97/30731), are also known to enhance the cellular uptake of oligonucleotides.
  • nucleic acids include glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.
  • glycols such as ethylene glycol and propylene glycol
  • pyrrols such as 2-pyrrol
  • azones such as 2-pyrrol
  • terpenes such as limonene and menthone.
  • compositions of the present invention also incorporate carrier compounds in the formulation.
  • carrier compound or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation.
  • the coadministration of a nucleic acid and a carrier compound typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the
  • phosphorothioate oligonucleotide in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4'isothiocyano- stilbene-2,2'-disulfonic acid (Miyao et al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
  • a “pharmaceutical carrier” or “excipient” is a “pharmaceutical carrier” or “excipient”
  • Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars,
  • lubricants e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.
  • disintegrants e.g., starch, sodium starch glycolate, etc.
  • wetting agents e.
  • compositions of the present invention can also be used to formulate the compositions of the present invention.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin,
  • Formulations for topical administration of nucleic acids may include sterile and non- sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases.
  • the solutions may also contain buffers, diluents and other suitable additives.
  • Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration that do not deleteriously react with nucleic acids can be used.
  • Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.
  • compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels.
  • the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • additional materials useful in physically formulating various dosage forms of the compositions of the present invention such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers.
  • such materials when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention.
  • the formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.
  • Aqueous suspensions may contain substances that increase the viscosity of the suspension including, for example, sodium carboxvmethylcellulose, sorbitol and or dextran.
  • the suspension may also contain stabilizers.
  • compositions of the invention may contain one or more antisense oligonucleotides, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense oligonucleotides targeted to a second nucleic acid target.
  • antisense oligonucleotides particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense oligonucleotides targeted to a second nucleic acid target.
  • Two or more combined compounds may be used together or sequentially.
  • the invention provides a method of introducing the functional nucleic acid compound-amphoteric liposome mixture to cells or an animal.
  • the functional nucleic acid compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits for a variety of conditions related to the expression of mTOT protein. Among such conditions are inhibition of mTOT protein expression, i.e., the reduction or inhibition of the activity of mTOT protein. Such treatment is useful for the treatment, prophylaxis and management of an insulin resistance disorder.
  • an "insulin resistance disorder” as discussed herein refers to any disease, disorder or condition that is caused by or contributed to by insulin resistance.
  • insulin resistance disease or disorders include: diabetes mellitus, obesity (obesity can include individuals having a body mass index (BMI) of at least 25 or greater, obesity may or may not be associated with insulin resistance), weight gain, metabolic syndrome, insulin-resistance syndromes, syndrome X, complications associated with insulin resistance, for example: high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including cognitive functions in dementia, retinopathy, peripheral neuropathy, nephropathy, glomerulonephritis, glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis, some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such
  • osteoporosis e.g., dyslipidemia, central obesity, rheumatoid arthritis, lupus, myasthenia gravis, vasculitis, Chronic Obstructive Pulmonary Disease (COPD), or inflammatory bowel disease
  • metabolic or inflammation mediated diseases e.g., dyslipidemia, central obesity, rheumatoid arthritis, lupus, myasthenia gravis, vasculitis, Chronic Obstructive Pulmonary Disease (COPD), or inflammatory bowel disease
  • cardiovascular disease e.g. atherosclerosis, arteriosclerosis, angina pectoris, coronary artery disease, congestive heart failure, stroke, or myocardial infarction
  • neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis.
  • an animal preferably a human, suspected of having a disease or disorder that can be treated by inhibiting the expression of mTOT protein, is treated by administering one or more functional nucleic acid compounds in accordance with this invention.
  • the compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of an antisense oligonucleotide to a suitable pharmaceutically acceptable diluent or carrier.
  • Administration of the functional nucleic acid compounds and methods of the invention may also be useful prophylactically, e.g., to prevent or delay insulin insensitivity, enhance glucose utilization and improve mitochondrial function, for example.
  • the functional nucleic acid compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding mTOT protein, enabling sandwich and other assays to easily be constructed to exploit this fact.
  • Hybridization of the functional nucleic acid compounds of the invention with a nucleic acid encoding mTOT protein can be detected by means known in the art. Such means may include conjugation of an enzyme to the functional nucleic acid compounds, radiolabelling of the functional nucleic acid compounds or any other suitable detection means. Kits using such detection means for detecting the level of mTOT protein in a sample may also be prepared.
  • the administered mixtures can reduce or stop aberrant glucose metabolism, insulin insensitivity, improved glucose utilization and improved mitochondrial function in mammals.
  • the introduction of the mixture results in a reduction hyperglycemia.
  • the mixture is administered to a cell, a non-human animal or a human to treat or prophylactically treat insulin insensitivity disease or disorder.
  • the mixture is introduced to an animal at a dosage of between 0.001 ⁇ g per kg of body weight to 100 mg per kg of body weight. Precise amounts of the functional nucleic acid to be administered typically will be guided by judgment of a medical practitioner, typically titrating from a high dose to a tolerable lower dose having comparable insulin sensitizing activity.
  • a unit dose will generally depend on the route of administration and be in the range of 10 ng/kg body weight to 100 mg/kg body weight per day, more typically in the range of 100 ng/kg body weight to about 10 mg/kg body weight per day or most preferably, in the range of about 0.1 mg/kg body weight to about 10 mg/kg body weight.
  • the method provides administration of a daily dose of one or more functional nucleic acid or antisense oligonucleotides (absent any vehicle) in an amount ranging from about 0.01 mg/m 2 to 300 mg/m 2 functional nucleic acid per body surface area of a patient.
  • the oligonucleotide is administered intravenously to a patient.
  • the dose is administered daily for up to 5 days of a three to ten week treatment cycle.
  • continuous or intermittent intravenous infusions may be made sufficient to maintain concentrations of at least from about 10 nanomolar to about 100 micromolar of the functional nucleic acid compound in the blood or plasma of the subject.
  • the mixture is introduced to the animal one or more times per day or continuously.
  • the mixture is introduced to the animal via topical, pulmonary or parenteral administration or via a medical device.
  • the mixture administered to the animal or cells further includes a TZD agent, for example, a therapeutically effective dose of mitoglitazone or pioglitazone.
  • compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual functional nucleic acid, and can generally be estimated based on EC50 found to be effective in vitro and in vivo animal models.
  • dosage is from 0.001 ⁇ g to 100 mg per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the functional nucleic acid is administered in maintenance doses, ranging from 0.001 ⁇ g to 100 g per kg of body weight, once or more daily, to once every 20 years.
  • prodrug refers to compounds that are transformed (typically rapidly) in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety. Examples of
  • esters of the compounds of this invention include, but are not limited to, alkyl esters (for example with between about one and about six carbons) the alkyl group is a straight or branched chain. Acceptable esters also include cycloalkyl esters and arylalkyl esters such as, but not limited to benzyl. Examples of pharmaceutically acceptable amides of the compounds of this invention include, but are not limited to, primary amides, and secondary and tertiary alkyl amides (for example with between about one and about six carbons). Amides and esters of the compounds of the present invention may be prepared according to conventional methods. A thorough discussion of prodrugs is provided in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems", Vol. 14 of the A.C.S.
  • the term "therapeutically effective amount” is an amount of a functional nucleic acid operable to inhibit mTOT protein expression in a cell, tissue or organism, for example, an animal patient, preferably a mammal, such as a human, that when administered to a subject or patient, ameliorates a symptom of the disease.
  • the amount of a functional nucleic acid of the invention which constitutes a “therapeutically effective amount” will vary depending on the functional nucleic acid, the presence of a TZD co-administered agent, the disease state and its severity, the bioavailability characteristics of the compound and/or inhibitor, the age of the patient to be treated, and the like.
  • the therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their knowledge and to this disclosure.
  • the dosage or dosages comprising the therapeutically effective amounts are not toxic and perform to accepted medical practices commensurate with an appropriate risk/benefit ratio.
  • Treating" or "treatment” of a disease, disorder, or syndrome includes any one or more of: (i) preventing the disease, disorder, or syndrome from occurring in a human, i.e. causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome and (iv) alleviate one or more symptoms of the disease, disorder , or syndrome.
  • adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine
  • Co-administration or “combined administration” or the like as utilized herein are meant to include modes of administration of the selected active, therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.
  • Coadministration can also include delivery of the active ingredients in a "fixed combination", e.g. a functional nucleic acid compound and a TZD are both administered to a patient simultaneously in the form of a single entity or dosage.
  • the term “non-fixed combination” means that the active ingredients, e.g. a functional nucleic acid compound and a TZD, are both administered to a patient as separate entities, either simultaneously, concurrently or sequentially with no specific time limits, such that the administration provides therapeutically effective levels of the combination of active agents in the body of the patient.
  • the invention is directed to a method of treating a disease or disorder associated, caused or linked with insulin insensitivity, for example, diabetes mellitus, cardiovascular disease and gastrointestinal disease, and Alzheimer's disease, which method comprises administering to a patient a therapeutically effective amount of a functional nucleic acid compound, as defined in the present disclosure, where mitochondrial function or insulin sensitivity is enhanced, at least in part, by the inhibition of mTOT protein; optionally in combination with one or more TZDs.
  • the mTOT protein inhibitor can be a functional nucleic acid compound.
  • the present invention provides a method for treating a disease or disorder associated with insulin insensitivity in a patient in need thereof, the method comprising administering to said patient a therapeutically effective dose of a functional nucleic acid compound that inhibits the expression of mTOT protein in said patient.
  • the administration includes administering one or more functional nucleic acid compounds operable to inhibit the expression of an mTOT gene or RNA, for example, the functional nucleic acid can include the nucleotide sequence of any one of SEQ ID NOs: 54-73, or a complement thereof.
  • the administration comprises administering a therapeutically effective amount of one or more functional nucleic acids having the nucleotide sequence of any one of SEQ ID NOs: 54-73or a complement thereof in a pharmaceutical composition. In a further embodiment, the administration comprises administering a therapeutically effective amount of one or more functional nucleic acid compounds having the nucleotide sequence of any one of SEQ ID NOs: 54-73 or a complement thereof in a pharmaceutical composition comprising a liposome. In a further embodiment, the administration comprises administering a
  • the administration comprises administering a combination of a therapeutically effective amount of one or more functional nucleic acid compounds having the nucleotide sequence of any one of SEQ ID NOs: 54-73 or a complement thereof in a pharmaceutical composition formulated for systemic administration via intravenous administration and a TZD, for example, mitoglitazone or pioglitazone.
  • a TZD for example, mitoglitazone or pioglitazone.
  • the method of the invention comprises administering to a patient a therapeutically effective amount of a functional nucleic acid compound. In another embodiment, the method of the invention comprises administering to a patient a
  • a functional nucleic acid compound in combination with a therapeutically effective dose of a TZD, for example, mitoglitazone.
  • a photoaffinity crosslinker can be synthesized by coupling a carboxylic acid analog of pioglitazone to a p-azido-benzyl group containing ethylamine as described in Amer. J. Physiol 256:E252-E260. (See FIG 9B).
  • the crosslinker can be iodinated carrier free, using a modification of the Iodogen (Pierce) procedure and purified using open column
  • crosslinking is defined as labeling that is prevented by the presence of competing drug.
  • Competitive binding assays are conducted in 50 mM Tris, pH8.0. All crosslinking reactions are conducted in triplicate using 8 concentrations of competitor ranging from 0-25 ⁇ . (See Figure 9 C, compounds 2 and 3.
  • Each crosslinking reaction tube contains 20 ⁇ g of crude mitochondrial enriched rat liver membranes, 0.1 ⁇ of 125 I-MSDC-1 101 , and -/+ competitor drug with a final concentration of 1% DMSO.
  • the binding assay reaction is performed at room temperature in the dark for 20 minutes and stopped by exposure to 180,000 ⁇ , ⁇ .
  • the membranes are pelleted at 20,000 x g for 5 minutes, the pellet is resuspended in Laemmli sample buffer containing 1% BME and run on 10-20% Tricine gels. Following electrophoresis the gels are dried under vacuum and exposed to Kodak BioMax MS film at -80°C The density of the resulting specifically labeled autoradiography bands are quantitated using ImageJ software (NIH) and IC50 values determined by non-linear analysis using GraphPad PrismTM . Selected compounds in this assay demonstrated an IC 50 of less than 20 ⁇ , less than 5 ⁇ or less than 1 ⁇ .
  • the crosslinking to this protein band is emblematic of the ability of the ability of the PPAR-sparing compounds to bind to the mitochondria, the key organelle responsible for the effectiveness of these compounds for the therapeutic effects sought to be determined.
  • the mitochondrial membrane cross-linking assay can also be performed using transfected mTOT cells.
  • a comparison of HEK293 wild type cells with HEK293 cells transiently transfected with pcDNA 3.1+ (human BP44 c- terminal 6-his-tagged) for 48 hours was made.
  • the cells were fractionated by Dounce homogenization in 50 mM Tris-HCl (pH 8.0), 250 mM sucrose containing a protease inhibitor cocktail (Roche Complete). The homogenates were subjected to differential centrifugation resulting in fractions PI (800 x g nuclear pellet), P2 (20,000 x g mitochondrial pellet) and S2 (20,000 x g cytosolic supernatant).
  • PI 800 x g nuclear pellet
  • P2 20,000 x g mitochondrial pellet
  • S2 20,000 x g cytosolic supernatant
  • crosslinking reaction tube contains 20 ⁇ g of HEK293 fractions, 0.1 of 125I-MSDC-1101, and -/+ 25 ⁇ MSDC-0160 in a final concentration of 1% DMSO.
  • the binding assay reaction is performed at room temperature in the dark for 20 minutes and stopped by exposure to UV light (180,000 ⁇ -ioules).
  • the membranes are pelleted at 20,000 x g for 5 minutes, the pellet is resuspended in Laemmli sample buffer containing 1% BME and 10 ⁇ g total protein is run on 10-20% Tricine gels. Following electrophoresis the gels are dried under vacuum and exposed to Kodak BioMax MS film at -80°C.
  • FIG.9B shows the chemical structure of the photoaffinity crosslinker, 125 I-MSDC-1101.
  • PPARy-sparing thiazolidinedione like activity can be measured by assaying the effect of a candidate compound's ability to induce expression of BP44 in an adipocyte precursor cell.
  • Brown adipose precursor cells can be isolated from mouse interscapular brown fat pads and inoculated into 35 mm culture dishes. Cultures can be maintained in Dulbecco's modified-Eagle's medium containing 10% fetal calf serum. The medium is changed the following day and every second day thereafter.
  • the precursor cells are confluent and the appropriate control vehicle, candidate compounds, or a prototype PPARy-sparing thiazolidinedione such as MSDC-0160 (a positive control PPARy-sparing TZD) are added to the medium of each culture dish.
  • the medium containing the candidate compound is replaced every other day.
  • Each treatment group is typically assayed in triplicate.
  • the cultures are treated with KHM buffer (20 mM Hepes, pH 7.2, containing 10 mM potassium acetate, 2 mM magnesium acetate) also containing 1% NP-40 detergent plus a protease inhibitor cocktail, transferred to microfuge tubes and pelleted at 8,000 x g for 5 minutes.
  • KHM buffer (20 mM Hepes, pH 7.2, containing 10 mM potassium acetate, 2 mM magnesium acetate) also containing 1% NP-40 detergent plus a protease inhibitor cocktail
  • the post-nuclear lysates contained in the supernatant are collected and stored at - 80°C.
  • the lysate protein concentration is equalized to 1 ⁇ g/ul in SDS sample buffer and 20 ⁇ g/sample is separated by SDS-PAGE under reducing conditions.
  • Western blot analysis is carried out on the separated proteins.
  • the blot is subjected to incubation with a specific rabbit polyclonal antibody that recognizes only BRP44 and is followed by incubation with a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase.
  • the ⁇ 14 kDa BP44 protein is detected using an enhanced chemiluminesence reagent followed by exposure to detection film.
  • Expression levels of the BP44 protein are quantified by densitometry using ImageJ software (U.S. National Institutes of Health).
  • the candidate compound's ability to induce expression of BP44 is compared to the positive and negative control samples.
  • a candidate compound that induces the expression of BP44 in Brown adipose precursor cells relative to negative controls suggests that the candidate compound possesses PPARy-sparing thiazolidinedione like activity.
  • Results shown in Figure 2 illustrate the effect of mitoglitazone (a PPARy-sparring thiazolidinedione) on the expression of BP44.
  • mitoglitazone a PPARy-sparring thiazolidinedione
  • the expression of BP44 in brown adipose cells is proportional to the activity of a PPARy-sparring thiazolidinedionemitoglitazone.
  • This validation activity assay can be used to confirm the identity of therapeutic active compounds of those selected lead candidate compounds after screening.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Endocrinology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé d'identification d'un composé candidat tête de série qui est efficace dans le traitement ou la prévention d'une maladie ou d'un trouble d'insulino-résistance chez un sujet, comprenant : le criblage d'un ou plusieurs composés candidats dans un essai de liaison, l'essai de liaison comprenant les étapes de : (i) apport d'une protéine mTOT ; (ii) mise en contact de la protéine mTOT avec un composé candidat ; et (iii) détection si le composé candidat se lie spécifiquement à la protéine mTOT ou inhibe la liaison spécifique d'un composé thiazolidinedione à la protéine mTOT. Le composé candidat est identifié en tant que composé candidat tête de série si le composé candidat se lie spécifiquement à la protéine mTOT ou inhibe la liaison du composé thiazolidinedione à la protéine mTOT.
PCT/US2012/024561 2011-02-09 2012-02-09 Cibles cellulaires de thiazolidinediones WO2012109495A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201161441260P 2011-02-09 2011-02-09
US61/441,260 2011-02-09
US201261583996P 2012-01-06 2012-01-06
US61/583,996 2012-01-06

Publications (1)

Publication Number Publication Date
WO2012109495A1 true WO2012109495A1 (fr) 2012-08-16

Family

ID=45852696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/024561 WO2012109495A1 (fr) 2011-02-09 2012-02-09 Cibles cellulaires de thiazolidinediones

Country Status (1)

Country Link
WO (1) WO2012109495A1 (fr)

Citations (74)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4426330A (en) 1981-07-20 1984-01-17 Lipid Specialties, Inc. Synthetic phospholipid compounds
US4534899A (en) 1981-07-20 1985-08-13 Lipid Specialties, Inc. Synthetic phospholipid compounds
US5010175A (en) 1988-05-02 1991-04-23 The Regents Of The University Of California General method for producing and selecting peptides with specific properties
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
WO1991019735A1 (fr) 1990-06-14 1991-12-26 Bartlett Paul A Banques de peptides modifies resistant a la protease
WO1992000091A1 (fr) 1990-07-02 1992-01-09 Bioligand, Inc. Banque de bio-oligomeres aleatoires, son procede de synthese et son mode d'emploi
US5108921A (en) 1989-04-03 1992-04-28 Purdue Research Foundation Method for enhanced transmembrane transport of exogenous molecules
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US5227170A (en) 1989-06-22 1993-07-13 Vestar, Inc. Encapsulation process
US5252479A (en) 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
WO1993020242A1 (fr) 1992-03-30 1993-10-14 The Scripps Research Institute Bibliotheques chimiques combinatoires codees
US5264221A (en) 1991-05-23 1993-11-23 Mitsubishi Kasei Corporation Drug-containing protein-bonded liposome
WO1993024641A2 (fr) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Virus adeno-associe a sequences terminales inversees utilisees comme promoteur
WO1993024510A1 (fr) 1992-05-25 1993-12-09 Centre National De La Recherche Scientifique (Cnrs) Composes biologiquement actifs de type phosphotriesters
US5288514A (en) 1992-09-14 1994-02-22 The Regents Of The University Of California Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support
WO1994013788A1 (fr) 1992-12-04 1994-06-23 University Of Pittsburgh Systeme porteur viral de recombinaison
US5354844A (en) 1989-03-16 1994-10-11 Boehringer Ingelheim International Gmbh Protein-polycation conjugates
US5356633A (en) 1989-10-20 1994-10-18 Liposome Technology, Inc. Method of treatment of inflamed tissues
WO1994026764A1 (fr) 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
US5395619A (en) 1993-03-03 1995-03-07 Liposome Technology, Inc. Lipid-polymer conjugates and liposomes
US5417978A (en) 1993-07-29 1995-05-23 Board Of Regents, The University Of Texas System Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use
US5459127A (en) 1990-04-19 1995-10-17 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
US5462854A (en) 1993-04-19 1995-10-31 Beckman Instruments, Inc. Inverse linkage oligonucleotides for chemical and enzymatic processes
US5469854A (en) 1989-12-22 1995-11-28 Imarx Pharmaceutical Corp. Methods of preparing gas-filled liposomes
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5506337A (en) 1985-03-15 1996-04-09 Antivirals Inc. Morpholino-subunit combinatorial library and method
US5512295A (en) 1994-11-10 1996-04-30 The Board Of Trustees Of The Leland Stanford Junior University Synthetic liposomes for enhanced uptake and delivery
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5521291A (en) 1991-09-30 1996-05-28 Boehringer Ingelheim International, Gmbh Conjugates for introducing nucleic acid into higher eucaryotic cells
US5525735A (en) 1994-06-22 1996-06-11 Affymax Technologies Nv Methods for synthesizing diverse collections of pyrrolidine compounds
US5527528A (en) 1989-10-20 1996-06-18 Sequus Pharmaceuticals, Inc. Solid-tumor treatment method
US5534259A (en) 1993-07-08 1996-07-09 Liposome Technology, Inc. Polymer compound and coated particle composition
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5539083A (en) 1994-02-23 1996-07-23 Isis Pharmaceuticals, Inc. Peptide nucleic acid combinatorial libraries and improved methods of synthesis
US5543152A (en) 1994-06-20 1996-08-06 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US5543158A (en) 1993-07-23 1996-08-06 Massachusetts Institute Of Technology Biodegradable injectable nanoparticles
US5547932A (en) 1991-09-30 1996-08-20 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
US5549974A (en) 1994-06-23 1996-08-27 Affymax Technologies Nv Methods for the solid phase synthesis of thiazolidinones, metathiazanones, and derivatives thereof
US5556948A (en) 1993-01-22 1996-09-17 Mitsubishi Chemical Corporation Phospholipid derivatized with PEG bifunctional linker and liposome containing it
US5569588A (en) 1995-08-09 1996-10-29 The Regents Of The University Of California Methods for drug screening
US5580575A (en) 1989-12-22 1996-12-03 Imarx Pharmaceutical Corp. Therapeutic drug delivery systems
US5583020A (en) 1992-11-24 1996-12-10 Ribozyme Pharmaceuticals, Inc. Permeability enhancers for negatively charged polynucleotides
US5591721A (en) 1994-10-25 1997-01-07 Hybridon, Inc. Method of down-regulating gene expression
US5593853A (en) 1994-02-09 1997-01-14 Martek Corporation Generation and screening of synthetic drug libraries
US5595756A (en) 1993-12-22 1997-01-21 Inex Pharmaceuticals Corporation Liposomal compositions for enhanced retention of bioactive agents
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
WO1997030731A2 (fr) 1996-02-21 1997-08-28 The Immune Response Corporation Procede de preparation de complexes porteurs de polynucleotides pour administration a des cellules
US5705188A (en) 1993-02-19 1998-01-06 Nippon Shinyaku Company, Ltd. Drug composition containing nucleic acid copolymer
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5763239A (en) 1996-06-18 1998-06-09 Diversa Corporation Production and use of normalized DNA libraries
WO1998039352A1 (fr) 1997-03-07 1998-09-11 Takeshi Imanishi Nouveaux analogues de bicyclonucleoside et d'oligonucleotide
WO1999014226A2 (fr) 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
US6258792B1 (en) 1996-04-12 2001-07-10 University Of Pittsburgh Cationic cholesteryl derivatives containing cyclic polar groups
US20020086356A1 (en) 2000-03-30 2002-07-04 Whitehead Institute For Biomedical Research RNA sequence-specific mediators of RNA interference
US6429200B1 (en) 1998-07-17 2002-08-06 Mirus Corporation Reverse micelles for delivery of nucleic acids
WO2002066012A2 (fr) 2001-02-21 2002-08-29 Novosom Ag Liposomes amphoteres et leur utilisation
WO2002066489A2 (fr) 2001-02-21 2002-08-29 Novosom Ag Sterols amphoteres et leur utilisation
US6458382B1 (en) 1999-11-12 2002-10-01 Mirus Corporation Nucleic acid transfer complexes
WO2003070735A2 (fr) 2002-02-19 2003-08-28 Novosom Ag Elements permettant la preparation de liposomes amphoteres
WO2003070220A1 (fr) 2002-02-19 2003-08-28 Novosom Ag Lipides cationiques sensibles au ph, liposomes et nanocapsules qui les contiennent
WO2004000928A2 (fr) 2002-06-25 2003-12-31 Brewer Science, Inc. Acides polyamiques/imides adaptes a la centrifugation, servant de liants polymeres a revelation en milieu humide pour revetements antireflets
WO2004053059A2 (fr) * 2002-12-06 2004-06-24 Pharmacia Corporation Polypeptide de « mitoneet » provenant de membranes mitochondriales, modulateurs correspondants, et leurs methodes d'utilisation
WO2005094783A2 (fr) 2004-03-28 2005-10-13 Novosom Ag Liposomes amphoteres a stabilite serique
US20050260135A1 (en) 2004-01-15 2005-11-24 Washington University High throughput pharmaceutical screening using drosophila
WO2006002991A1 (fr) 2004-07-07 2006-01-12 Gm Global Technology Operations, Inc. Ensemble essieu arriere de vehicule automobile comportant un essieu arriere a bras oscillant mixte et un faux-chassis supplementaire, essieu arriere a bras oscillant mixte et faux-chassis associe
WO2006048329A1 (fr) 2004-11-05 2006-05-11 Novosom Ag Ameliorations apportees a des compositions pharmaceutiques comprenant un oligonucleotide comme agent actif
WO2006053646A2 (fr) 2004-11-19 2006-05-26 Novosom Ag Ameliorations apportees ou se rapportant a des compositions pharmaceutiques destinees a une administration locale
WO2007008501A1 (fr) * 2005-07-06 2007-01-18 Merck & Co., Inc. Oxazolidinediones et thiazolidinediones antidiabétiques
WO2009038681A1 (fr) * 2007-09-14 2009-03-26 Metabolic Solutions Development Company Analogues de thiazolidinedione pour traiter l'hypertension
US20090220584A1 (en) 2005-12-01 2009-09-03 Neal Clifford Goodwin Amphoteric Liposome Formulation
WO2010105048A1 (fr) * 2009-03-12 2010-09-16 Metabolic Solutions Development Company Analogues de thiazolidinedione
US9610287B2 (en) 2011-06-20 2017-04-04 H. Lundbeck A/S Method of administration of 4-((1R,3S)-6-chloro-3-phenyl-indan-1-yl)-1,2,2-trimethyl-piperazine and the salts thereof in the treatment of schizophrenia

Patent Citations (83)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4534899A (en) 1981-07-20 1985-08-13 Lipid Specialties, Inc. Synthetic phospholipid compounds
US4426330A (en) 1981-07-20 1984-01-17 Lipid Specialties, Inc. Synthetic phospholipid compounds
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5506337A (en) 1985-03-15 1996-04-09 Antivirals Inc. Morpholino-subunit combinatorial library and method
US5139941A (en) 1985-10-31 1992-08-18 University Of Florida Research Foundation, Inc. AAV transduction vectors
US5010175A (en) 1988-05-02 1991-04-23 The Regents Of The University Of California General method for producing and selecting peptides with specific properties
US5354844A (en) 1989-03-16 1994-10-11 Boehringer Ingelheim International Gmbh Protein-polycation conjugates
US5108921A (en) 1989-04-03 1992-04-28 Purdue Research Foundation Method for enhanced transmembrane transport of exogenous molecules
US5416016A (en) 1989-04-03 1995-05-16 Purdue Research Foundation Method for enhancing transmembrane transport of exogenous molecules
US5227170A (en) 1989-06-22 1993-07-13 Vestar, Inc. Encapsulation process
US5527528A (en) 1989-10-20 1996-06-18 Sequus Pharmaceuticals, Inc. Solid-tumor treatment method
US5213804A (en) 1989-10-20 1993-05-25 Liposome Technology, Inc. Solid tumor treatment method and composition
US5013556A (en) 1989-10-20 1991-05-07 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5356633A (en) 1989-10-20 1994-10-18 Liposome Technology, Inc. Method of treatment of inflamed tissues
US5580575A (en) 1989-12-22 1996-12-03 Imarx Pharmaceutical Corp. Therapeutic drug delivery systems
US5469854A (en) 1989-12-22 1995-11-28 Imarx Pharmaceutical Corp. Methods of preparing gas-filled liposomes
US5459127A (en) 1990-04-19 1995-10-17 Vical, Inc. Cationic lipids for intracellular delivery of biologically active molecules
WO1991019735A1 (fr) 1990-06-14 1991-12-26 Bartlett Paul A Banques de peptides modifies resistant a la protease
WO1992000091A1 (fr) 1990-07-02 1992-01-09 Bioligand, Inc. Banque de bio-oligomeres aleatoires, son procede de synthese et son mode d'emploi
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5264221A (en) 1991-05-23 1993-11-23 Mitsubishi Kasei Corporation Drug-containing protein-bonded liposome
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5521291A (en) 1991-09-30 1996-05-28 Boehringer Ingelheim International, Gmbh Conjugates for introducing nucleic acid into higher eucaryotic cells
US5547932A (en) 1991-09-30 1996-08-20 Boehringer Ingelheim International Gmbh Composition for introducing nucleic acid complexes into higher eucaryotic cells
US5252479A (en) 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
WO1993020242A1 (fr) 1992-03-30 1993-10-14 The Scripps Research Institute Bibliotheques chimiques combinatoires codees
WO1993024510A1 (fr) 1992-05-25 1993-12-09 Centre National De La Recherche Scientifique (Cnrs) Composes biologiquement actifs de type phosphotriesters
WO1993024641A2 (fr) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Virus adeno-associe a sequences terminales inversees utilisees comme promoteur
US5288514A (en) 1992-09-14 1994-02-22 The Regents Of The University Of California Solid phase and combinatorial synthesis of benzodiazepine compounds on a solid support
US5583020A (en) 1992-11-24 1996-12-10 Ribozyme Pharmaceuticals, Inc. Permeability enhancers for negatively charged polynucleotides
WO1994013788A1 (fr) 1992-12-04 1994-06-23 University Of Pittsburgh Systeme porteur viral de recombinaison
US5556948A (en) 1993-01-22 1996-09-17 Mitsubishi Chemical Corporation Phospholipid derivatized with PEG bifunctional linker and liposome containing it
US5705188A (en) 1993-02-19 1998-01-06 Nippon Shinyaku Company, Ltd. Drug composition containing nucleic acid copolymer
US5395619A (en) 1993-03-03 1995-03-07 Liposome Technology, Inc. Lipid-polymer conjugates and liposomes
US5462854A (en) 1993-04-19 1995-10-31 Beckman Instruments, Inc. Inverse linkage oligonucleotides for chemical and enzymatic processes
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5770713A (en) 1993-05-12 1998-06-23 Centre National De La Recherche Scientifique Phosphorothioate triester oligonucleotides and method of preparation
WO1994026764A1 (fr) 1993-05-12 1994-11-24 Centre National De La Recherche Scientifique (Cnrs) Oligonucleotides phosphorothioates triesters et procede de preparation
US5534259A (en) 1993-07-08 1996-07-09 Liposome Technology, Inc. Polymer compound and coated particle composition
US5543158A (en) 1993-07-23 1996-08-06 Massachusetts Institute Of Technology Biodegradable injectable nanoparticles
US5417978A (en) 1993-07-29 1995-05-23 Board Of Regents, The University Of Texas System Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5595756A (en) 1993-12-22 1997-01-21 Inex Pharmaceuticals Corporation Liposomal compositions for enhanced retention of bioactive agents
US5519134A (en) 1994-01-11 1996-05-21 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US5593853A (en) 1994-02-09 1997-01-14 Martek Corporation Generation and screening of synthetic drug libraries
US5539083A (en) 1994-02-23 1996-07-23 Isis Pharmaceuticals, Inc. Peptide nucleic acid combinatorial libraries and improved methods of synthesis
US5543152A (en) 1994-06-20 1996-08-06 Inex Pharmaceuticals Corporation Sphingosomes for enhanced drug delivery
US5525735A (en) 1994-06-22 1996-06-11 Affymax Technologies Nv Methods for synthesizing diverse collections of pyrrolidine compounds
US5549974A (en) 1994-06-23 1996-08-27 Affymax Technologies Nv Methods for the solid phase synthesis of thiazolidinones, metathiazanones, and derivatives thereof
US5591721A (en) 1994-10-25 1997-01-07 Hybridon, Inc. Method of down-regulating gene expression
US5512295A (en) 1994-11-10 1996-04-30 The Board Of Trustees Of The Leland Stanford Junior University Synthetic liposomes for enhanced uptake and delivery
US5569588A (en) 1995-08-09 1996-10-29 The Regents Of The University Of California Methods for drug screening
WO1997030731A2 (fr) 1996-02-21 1997-08-28 The Immune Response Corporation Procede de preparation de complexes porteurs de polynucleotides pour administration a des cellules
US6258792B1 (en) 1996-04-12 2001-07-10 University Of Pittsburgh Cationic cholesteryl derivatives containing cyclic polar groups
US5763239A (en) 1996-06-18 1998-06-09 Diversa Corporation Production and use of normalized DNA libraries
WO1998039352A1 (fr) 1997-03-07 1998-09-11 Takeshi Imanishi Nouveaux analogues de bicyclonucleoside et d'oligonucleotide
WO1999014226A2 (fr) 1997-09-12 1999-03-25 Exiqon A/S Analogues d'oligonucleotides
US6429200B1 (en) 1998-07-17 2002-08-06 Mirus Corporation Reverse micelles for delivery of nucleic acids
US6458382B1 (en) 1999-11-12 2002-10-01 Mirus Corporation Nucleic acid transfer complexes
US20020086356A1 (en) 2000-03-30 2002-07-04 Whitehead Institute For Biomedical Research RNA sequence-specific mediators of RNA interference
US20040120997A1 (en) 2001-02-21 2004-06-24 Steffan Panzner Amphoteric sterols and the use thereof
WO2002066012A2 (fr) 2001-02-21 2002-08-29 Novosom Ag Liposomes amphoteres et leur utilisation
WO2002066489A2 (fr) 2001-02-21 2002-08-29 Novosom Ag Sterols amphoteres et leur utilisation
US20030099697A1 (en) 2001-02-21 2003-05-29 Novosom Ag Amphoteric liposomes and their use
US20040131666A1 (en) 2001-02-21 2004-07-08 Steffan Panzner Sterol derivatives, liposomes comprising sterol derivatives and method for loading liposomes with active substances
WO2003070220A1 (fr) 2002-02-19 2003-08-28 Novosom Ag Lipides cationiques sensibles au ph, liposomes et nanocapsules qui les contiennent
US20050164963A1 (en) 2002-02-19 2005-07-28 Frank Essler Components for producing amphoteric liposomes
WO2003070735A2 (fr) 2002-02-19 2003-08-28 Novosom Ag Elements permettant la preparation de liposomes amphoteres
WO2004000928A2 (fr) 2002-06-25 2003-12-31 Brewer Science, Inc. Acides polyamiques/imides adaptes a la centrifugation, servant de liants polymeres a revelation en milieu humide pour revetements antireflets
WO2004053059A2 (fr) * 2002-12-06 2004-06-24 Pharmacia Corporation Polypeptide de « mitoneet » provenant de membranes mitochondriales, modulateurs correspondants, et leurs methodes d'utilisation
US20050260135A1 (en) 2004-01-15 2005-11-24 Washington University High throughput pharmaceutical screening using drosophila
WO2005094783A2 (fr) 2004-03-28 2005-10-13 Novosom Ag Liposomes amphoteres a stabilite serique
WO2006002991A1 (fr) 2004-07-07 2006-01-12 Gm Global Technology Operations, Inc. Ensemble essieu arriere de vehicule automobile comportant un essieu arriere a bras oscillant mixte et un faux-chassis supplementaire, essieu arriere a bras oscillant mixte et faux-chassis associe
WO2006048329A1 (fr) 2004-11-05 2006-05-11 Novosom Ag Ameliorations apportees a des compositions pharmaceutiques comprenant un oligonucleotide comme agent actif
US20060216343A1 (en) 2004-11-05 2006-09-28 Steffen Panzner Pharmaceutical compositions comprising an oligonucleotide as an active agent
WO2006053646A2 (fr) 2004-11-19 2006-05-26 Novosom Ag Ameliorations apportees ou se rapportant a des compositions pharmaceutiques destinees a une administration locale
US20060159737A1 (en) 2004-11-19 2006-07-20 Steffen Panzner Pharmaceutical compositions for local administration
WO2007008501A1 (fr) * 2005-07-06 2007-01-18 Merck & Co., Inc. Oxazolidinediones et thiazolidinediones antidiabétiques
US20090220584A1 (en) 2005-12-01 2009-09-03 Neal Clifford Goodwin Amphoteric Liposome Formulation
WO2009038681A1 (fr) * 2007-09-14 2009-03-26 Metabolic Solutions Development Company Analogues de thiazolidinedione pour traiter l'hypertension
WO2010105048A1 (fr) * 2009-03-12 2010-09-16 Metabolic Solutions Development Company Analogues de thiazolidinedione
US9610287B2 (en) 2011-06-20 2017-04-04 H. Lundbeck A/S Method of administration of 4-((1R,3S)-6-chloro-3-phenyl-indan-1-yl)-1,2,2-trimethyl-piperazine and the salts thereof in the treatment of schizophrenia

Non-Patent Citations (122)

* Cited by examiner, † Cited by third party
Title
"Antisense Research and Applications", 1993, CRC PRESS, pages: 276 - 278
"Current Protocols in Molecular Biology", 2006, CURRENT PROTOCOLS
A. H. LIN ET AL., ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 41, no. 10, 1997, pages 2127 - 2131
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 10
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL ET AL., NUC. ACIDS RES., vol. 25, 1977, pages 3389 - 402
ANDERSON W F, NATURE, vol. 392, 1998, pages 25 - 30
ANDERSON; YOUNG, QUANTITATIVE FILTER HYBRIDIZATION, IN NUCLEIC ACID HYBRIDIZATION, 1985
ANDERSON; YOUNG: "Quantitative Filter Hybridization", NUCLEIC ACID HYBRIDIZATION, 1985
ATKINSON ET AL.: "OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH", pages: 35 - 81
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1987, WILEY INTERSCIENCE
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1995
AUSUBEL: "Current Protocols in Molecular Biology", 1987, WILEY INTERSCIENCE
BARANY, G.; MERRIFIELD, R. B.: "The Peptides", vol. 2, 1979, ACADEMIC PRESS, pages: 1 - 284
BERGE ET AL.: "Pharmaceutical Salts", J. OF PHARMA SCI., vol. 66, 1977, pages 1 - 19, XP002675560, DOI: doi:10.1002/jps.2600660104
BLOCK: "Pharmaceutical Dosage Forms", vol. 1, 1988, MARCEL DEKKER, INC., pages: 335
BLOCK: "Pharmaceutical Dosage Forms", vol. 2, 1988, MARCEL DEKKER, INC., pages: 335
BODANSZKY, M.; BODANSZKY, A.: "The Practice of Peptide Synthesis", 1984, SPRINGER-VERLAG
BOLTEN CHARLES W ET AL: "Insulin sensitizing pharmacology of thiazolidinediones correlates with mitochondrial gene expression rather than activation of PPAR gamma", GENE REGULATION AND SYSTEMS BIOLOGY, LIBERTAS ACADEMICA LTD, NZ, vol. 1, 1 January 2007 (2007-01-01), pages 73 - 82, XP002633659, ISSN: 1177-6250 *
BRAZMA; VILO, FEBS LETT., vol. 480, 2000, pages 17 - 24
BRUMMELKAMP T R ET AL., SCIENCE, vol. 296, 2002, pages 550 - 553
BRUNTON ET AL.: "Goodman & Gilman's The Pharmacological Basis of Therapeutics,9th Ed.", 1996, MCGRAW-HILL, pages: 934 - 935
BUDKER ET AL., NAT BIOTECHNOL., vol. 14, no. 6, 1996, pages 760 - 4
BUDKER ET AL., NAT. BIOTECHNOL., vol. 14, no. 6, 1996, pages 760 - 4
BUUR ET AL., J. CONTROL REL., vol. 14, 1990, pages 43 - 5 1
C. L. BASHFORD; D. A. HARRIS OXFORD: "Spectrophotometry And Spectrofluorometry", 1987, IRL PRESS
CARULLI ET AL., J. CELL BIOCHEM. SUPPL., vol. 31, 1998, pages 286 - 96
CELIS ET AL., FEBS LETT., vol. 480, 2000, pages 2 - 16
COLCA ET AL: "Insulin sensitizers may prevent metabolic inflammation", BIOCHEMICAL PHARMACOLOGY, PERGAMON, OXFORD, GB, vol. 72, no. 2, 14 July 2006 (2006-07-14), pages 125 - 131, XP027905479, ISSN: 0006-2952, [retrieved on 20060714] *
COLCA J R ET AL: "Identification of a novel mitochondrial protein (mitoNEET)", AMERICAN JOURNAL OF PHYSIOLOGY: ENDOCRINOLOGY AND METABOLISM, AMERICAN PHYSIOLOGICAL SOCIETY, BETHESDA, MD, US, vol. 286, 21 October 2003 (2003-10-21), pages 252 - 260, XP002997343, ISSN: 0193-1849, DOI: 10.1152/AJPENDO.00424.2003 *
CONSTANTINIDES ET AL., PHARMACEUTICAL RESEARCH, vol. 11, 1994, pages 1385
CONSTANTINIDES ET AL., PHARMACEUTICAL RESEARCH, vol. 11, 1994, pages 1385 - 1390
D. N. GLOVER,: "DNA Cloning: A Practical Approach", vol. I, II, IRL PRESS
DEVEREAUX ET AL., NUCL. ACIDS RES., vol. 12, 1984, pages 387 - 95
DIEFFENBACH AND DVEKSLER: "PCR Primer: A Laboratory Manual", 1995, COLD SPRING HARBOR LABORATORIES
DOMBURG R, GENE THERAP., vol. 2, 1995, pages 301 - 310
EDWARD B. ROCHE,: "Bioreversible Carriers in Drug Design", 1987, AMERICAN PHARMACEUTICAL ASSOCIATION AND PERGAMON PRESS
EGLITIS M A, BIOTECHNIQUES, vol. 6, 1988, pages 608 - 614
EL HARIRI ET AL., J. PHARM. PHANNACOL., vol. 44, 1992, pages 651 - 654
FEINSTEIN D L ET AL: "Receptor-independent actions of PPAR thiazolidinedione agonists: Is mitochondrial function the key?", BIOCHEMICAL PHARMACOLOGY, PERGAMON, OXFORD, GB, vol. 70, no. 2, 15 July 2005 (2005-07-15), pages 177 - 188, XP027715525, ISSN: 0006-2952, [retrieved on 20050715] *
FENG; DOOLITTLE, J. MOL. EVOL., vol. 35, 1987, pages 351 - 360
FIELDS ET AL., NATURE, vol. 340, 1989, pages 245 - 246
FIELDS ET AL., TRENDS IN GENETICS, vol. 10, 1994, pages 286 - 292
FISHER K J ET AL., J. VIROL., vol. 70, 1996, pages 520 - 532
FUCHS ET AL., ANAL. BIOCHEM., vol. 286, 2000, pages 91 - 98
GAIT: "Oligonucleotide Synthesis: A Practical Approach", 1984, IRL PRESS, pages: 1 - 22
GOING; GUSTERSON, EUR. J. CANCER, vol. 35, 1999, pages 1895 - 904
HELENE ET AL., NN. N.Y. ACAD. SCI., vol. 660, 1992, pages 27 - 36
HELENE, ANTICANCER DRUG DES., vol. 6, no. 6, 1991, pages 569 - 84
HENIKOFFAND HENIKOFF, PROC. NATL. ACAD. SCI. USA, vol. 89, 1989, pages 10915
HIGGINS; SHARP, CABIOS, vol. 5, 1989, pages 151 - 153
HIGUCHI ET AL.: "Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING CO., pages: 301
HIGUCHI: "V. Stella", vol. 14, A.C.S. SYMPOSIUM SERIES, article "Pro-drugs as Novel Delivery Systems"
HILL, CUR. OPINION DRUG DISC. DEV., vol. 1, 1998, pages 92 - 97
HO ET AL., J. PHARM. SCI., vol. 85, 1996, pages 138 - 143
HURST, H.C., BREAST CANCER RES, vol. 3, 2001, pages 395 - 398
IDSON: "Pharmaceutical Dosage Forms", vol. 1, 1988, MARCEL DEKKER, INC., pages: 199
IDSON: "Pharmaceutical Dosage Forms, Lieberman", vol. 1, 1988, MARCEL DEKKER, INC., pages: 199
J. F. RAMALHO ORTIGAO: "The Chemistry of Peptide Synthesis", KNOWLEDGE DATABASE OF ACCESS TO VIRTUAL LABORATORY WEBSITE
JARRETT, J., CHROMATOGR., vol. 618, 1993, pages 315 - 339
JILLIAN R. GUNTHER; YUHONG DU; ERIC RHODEN; IESTYN LEWIS; BRIAN REVENNAUGH; TERRY W. MOORE; SUNG HOON KIM; RAYMOND DINGLEDINE; HAI: "Energy Transfer Assays for the Discovery of Inhibitors of Estrogen Receptor-Coactivator Binding", JBIOMOL SCREEN, vol. 14, 2009, pages 181 - 193
JOHN R. W. MASTERS: "Animal Cell Culture: Practical Approach,Third Edition", 2000
JOSEPH R. LAKOWICZ: "Principles of Fluorescence Spectroscopy", 1983, PLENUM PRESS
JUNGBLUT ET AL., ELECTROPHORESIS, vol. 20, 1999, pages 2100 - 10
JURECIC; BELMONT, CURR. OPIN. MICROBIOL., vol. 3, 2000, pages 316 - 21
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, pages 5873 - 87
LARSON ET AL., CYTOOMETRY, vol. 41, 2000, pages 203 - 208
LARSSON ET AL., J. BIOTECHNOL., vol. 80, 2000, pages 143 - 57
LEE ET AL., CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, 1991, pages 92
LEE N S ET AL., NAT. BIOTECHNOL., vol. 20, 2002, pages 500 - 505
LEUNG; SHAH: "Controlled Release of Drugs: Polymers and Aggregate Systems", 1989, VCH PUBLISHERS, pages: 185 - 215
MADDEN ET AL., DRUG DISCOV. TODAY, vol. 5, 2000, pages 415 - 425
MAHER, BIOASSAYS, vol. 14, 1992, pages 807 - 15
MAORONG JIANG ET AL: "The molecular characterization of the brain protein 44-like (Brp44l) gene of Gekko japonicus and its expression changes in spinal cord after tail amputation", MOLECULAR BIOLOGY REPORTS ; AN INTERNATIONAL JOURNAL ON MOLECULAR AND CELLULAR BIOLOGY, KLUWER ACADEMIC PUBLISHERS, DO, vol. 36, no. 2, 18 November 2007 (2007-11-18), pages 215 - 220, XP019647202, ISSN: 1573-4978 *
MARTIN ET AL., HELV. CHIM. ACTA, vol. 78, 1995, pages 486 - 504
MERRIFIELD, R. B., J. AM. CHEM. SOC., vol. 85, 1963, pages 2149 - 2154
MILLER A D, HUM GENE THERAP., vol. 1, 1990, pages 5 - 14
MIYAGISHI M ET AL., NAT. BIOTECHNOL., vol. 20, 2002, pages 497 - 500
MIYAO ET AL., ANTISENSE RES. DEV., vol. 5, 1995, pages 115 - 121
MURANISHI, CRITICAL REVIEWS IN THERAPEUTIC DRUG CARRIER SYSTEMS, vol. 7, 1990, pages 1 - 33
MUSSELMANN, L.P. ET AL.: "A high-sugar diet produces obesity and insulin resistance in wild-type Drosophila", DISEASE MODELS AND MECHANISMS, vol. 4, 2011, pages 842 - 849
NEEDLEMAN; WUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1497 - 1500
PADDISON P J ET AL., GENES DEV., vol. 16, 2002, pages 948 - 958
PAUL C P ET AL., NAT. BIOTECHNOL., vol. 20, 2002, pages 505 - 508
PEARSON; LIPMAN, PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444
PRASHAR; WEISSMAN, METHODS ENZYMOL., vol. 303, 1999, pages 258 - 72
RIEGER: "Pharmaceutical Dosage Forms", 1988, MARCEL DEKKER, INC., pages: 285
RIEGER: "Pharmaceutical Dosage Forms", vol. 1, 1988, MARCEL DEKKER, INC., pages: 285
RIEGER: "Pharmaceutical Dosage Forms, Lieberman", vol. 1, 1988, MARCEL DEKKER, INC., pages: 285
RITSCHEL, METH. FIND. EXP. CLIN. PHARMACOL., vol. 13, 1993, pages 205
ROGERS, DRUG DISCOVERY TODAY, vol. 2, 1997, pages 156 - 160
ROSOFF: "Pharmaceutical Dosage Forms", vol. 1, 1988, MARCEL DEKKER, INC., pages: 245
SAMBROOK ET AL.: "Molecular cloning, A laboratory manual", 1989, COLD SPRING HARBOR LABORATORY
SAMBROOK ET AL.: "Molecular Cloning: Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORIES
SAMBROOK ET AL.: "Molecular Cloning-A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY
SAMBROOK: "Molecular Cloning: Molecular Cloning: A Laboratory Manual, Third Edition", 2001, COLD SPRING HARBOR LABORATORIES
SAMBROOK; FRITSCH; MANIATIS: "Molecular Cloning: A Laboratory Manual,Second Edition", vol. I, II, I, 1989, COLD SPRING HARBOR LABORATORIES
SAMULSKI R ET AL., J. VIROL., vol. 61, 1987, pages 3096 - 3101
SAMULSKI R ET AL., J. VIROL., vol. 63, 1989, pages 3822 - 3826
SCHOTT: "Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING CO., pages: 271
SCOPES: "Protein purification: principles and practice, Third Edition", 1994, SPRINGER VERLAG
SMITH; WATERMAN, ADV. APPL. MATH., vol. 2, 1970, pages 482
SPROAT ET AL.: "OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH", pages: 83 - 115
SUTCLIFFE ET AL., PROC. NATL. ACAD. SCI. U. S. A., vol. 97, 2000, pages 1976 - 81
SWINYARD: "Remington's Pharmaceutical Sciences, 18th Ed.,", 1990, MACK PUBLISHING CO., pages: 782 - 783
TAKAKURA ET AL., ANTISENSE & NUCL. ACID DRUG DEV., vol. 6, 1996, pages 177 - 183
TIJSSEN: "Techniques in Biochemistry and Molecular Biology-Hybridisation with Nucleic Probes", 1993, article "Overview of principles of hybridization and the strategy of nucleic acid assays"
TO, COMB. CHEM. HIGH THROUGHPUT SCREEN, vol. 3, 2000, pages 235 - 41
TOTOWA N.J.: "Protocols for Oligonucleotide Conjugates", 1994, HUMANA PRESS
TOTOWA, N.J.: "Protocols for Oligonucleotide Conjugates", 1994, HUMANA PRESS
TUSCHL, T. ET AL.: "The siRNA User Guide", 11 October 2002
TUSCHL, T., NAT. BIOTECHNOL, vol. 20, 2002, pages 446 - 448
WACHECK ET AL.: "Chemosensitisation ofmalignant melanoma by BCL2 antisense therapy", LANCET, vol. 356, 2000, pages 1728 - 1733
WEBB ET AL.: "BCL-2 antisense therapy in patients with non-Hodgkin lymphoma", LANCET, vol. 349, 1997, pages 1137 - 1141, XP004267082, DOI: doi:10.1016/S0140-6736(96)11103-X
WIEBOLDT ET AL., ANAL. CHEM., vol. 69, 1997, pages 1683 - 1691
WU ET AL.: "OLIGONUCLEOTIDE SYNTHESIS: A PRACTICAL APPROACH", pages: 135 - 151
XIA H ET AL., NAT. BIOTECH., vol. 20, 2002, pages 1006 - 1010
YAMAMOTO ET AL., J. PHARM. EXP. THER., vol. 263, 1992, pages 25
YAMASHITA ET AL., J. PHARM. PHARMACOL., vol. 39, 1987, pages 621 - 626
YAMASHITA ET AL., J. PHARM. SCI., vol. 79, 1990, pages 579 - 583
ZHANG; MADDEN, GENOME RES., vol. 7, 1997, pages 649 - 656

Similar Documents

Publication Publication Date Title
JP7030685B2 (ja) Ｄｃｒ３又はｄｃｒ３ネットワーク遺伝子に遺伝子変異を有する患者における自己免疫状態を治療する方法
Hudson et al. Identification, classification, and expression of RAGE gene splice variants
KR101835889B1 (ko) 지질 수송 및 대사 유전자에 대한 천연 안티센스 전사체의 억제에 의해 지질 수송 및 대사 유전자 관련된 질환의 치료
US20160282354A1 (en) Compositions and methods for selecting a treatment for b-cell neoplasias
WO2006108581A2 (fr) Genes marqueurs humains et agents associes pour le diagnostic, le traitement et la prophylaxie de troubles cardio-vasculaires et d'atherosclerose
JP7636338B2 (ja) コルチコステロイドを含む、ｔｔｒ遺伝子編集およびａｔｔｒアミロイドーシスを治療するための組成物および方法、またはその使用
Fellows et al. AUF1/hnRNP D represses expression of VEGF in macrophages
KR20240033237A (ko) 류신이 풍부한 반복 키나아제 2(LRRK2) iRNA 제제 조성물 및 이의 사용 방법
KR102584661B1 (ko) 염증 촉진 인자 발현 억제제, 그 유효 성분의 스크리닝 방법, 그 방법에 유용한 발현 카세트, 진단약 및 진단 방법
US20120135879A1 (en) Method and Composition for Cancer Diagnosis and Treatment
WO2013063412A2 (fr) Méthodes de diagnostic et de traitement d'états associés au stress du réticulum endoplasmique (re)
US20140248628A1 (en) Isoforms of the human sst5 receptor originated by alternative splicing and oligonucleotide pairs to detect them by pcr
US20040253728A1 (en) New method
WO2012109495A1 (fr) Cibles cellulaires de thiazolidinediones
WO2003055507A1 (fr) Medicaments contre l'anorexie ou des affections liees au mode de vie et procede de recherche systematique de ces medicaments
CN102816225B (zh) 一种转移肿瘤缺失蛋白抑制剂多肽
US7884084B2 (en) Oligonucleotides which inhibit expression of the OB-RGRP protein
Miller Identification of potent and selective inhibitors of the epithelial sodium channel d
KR101099650B1 (ko) 인테그린알파５를 이용한 항암제 또는 ｓｉｐ１／ｚｅｂ２ 저해제의 스크리닝 방법
CA2906695A1 (fr) Variant de tcf7l2 et ses procedes d'utilisation dans des essais de diagnostic et de criblage de medicament
Contarelli The biological role of the IGF-1 pool in breast cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12709415

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12709415

Country of ref document: EP

Kind code of ref document: A1