JP2008509153A - Combination therapy using transferrin fusion protein containing GLP-1 - Google Patents

Abstract

The present invention provides a combination therapy comprising a transferrin fusion protein and a DPP-IV inhibitor and / or a neutral endopeptidase (NEP) inhibitor. Transferrin fusion proteins include therapeutic polypeptides or peptides useful in the treatment of diseases such as diabetes.
[Selection figure] None

Description

Related applications

  [0001] This application claims the benefit of US Provisional Application No. 60 / 598,031, filed Aug. 3, 2004, which is incorporated herein by reference in its entirety.

  [0002] This application is a continuation-in-part of US patent application Ser. No. 10 / 378,094 filed Mar. 4, 2003, PCT / US03 / 26818 filed Aug. 28, 2003. All of which are hereby incorporated by reference in their entirety.

Field of Invention

  [0003] The present invention relates to a transferrin fusion protein comprising an insulin secretagogue peptide with an extended therapeutic in vivo effective half-life. The present invention also relates to a combination therapy using a DPP-IV inhibitor and / or a neutral endopeptidase inhibitor (NEP) and an insulin secretagogue peptide.

Protease
[0004] Proteolytic enzymes play an important role in regulating physiological processes such as cell proliferation, differentiation and signal transduction processes by regulating protein turnover and processing. Proteolytic enzymes control the levels of important structural proteins, enzymes and regulatory proteins through proteolysis. Increased or decreased, uncontrolled proteolytic enzyme activity has been linked to various disease states, including inflammation, cancer, arteriosclerosis and degenerative disorders.

  [0005] The International Union of Biochemical Molecular Biology (IUBMB) recommended the use of the term "peptidase" for a subset of peptide bond hydrolases (subclass E.C3.4.). The widely used term protease is synonymous with peptidase. Peptidases comprise two groups of enzymes, endopeptidases and exopeptidases, which cleave peptide bonds at sites within the protein and sequentially remove amino acid sequences from either the N-terminus or C-terminus, respectively. . The term proteinase is synonymous with endopeptidase. Proteolytic enzymes are classified according to the mechanism of catalysis. Four mechanistic classes are recognized by IUBMB: serine protease, cysteine protease, aspartic protease and metalloprotease.

  [0006] Serine proteases are a large family of proteolytic enzymes that contain serine residues in the active catalytic site for protein cleavage. These are ubiquitous and have been found in viruses, bacteria and eukaryotes. Serine proteases have diverse substrate specificities and can be subdivided into subfamilies based on these specificities. Serine proteases have more than 20 subfamilies and are classified into 6 groups (SA, SB, SC, SE, SF and SG).

  [0007] Prolyl oligopeptidases are serine proteases classified into the SC group. This hydrolyzes the proline-containing peptide on the carboxyl side of the proline residue. Perhaps this is involved in the maturation and degradation of peptide hormones and neuropeptides (Wilk et al., 1983 Life Sci. 33, 2149-2157). Examples of prolyl oligopeptidases include dipeptidyl peptidase IV (DPP-IV), dipeptidyl peptidase II (DPP-II), fibroblast activation protein, and prolyl oligopeptidase. These enzymes show different specificities.

  [0008] Proline is present in many peptide hormones. This determines the structural properties of these peptides, such as their conformation and stability, and prevents degradation by non-specific proteases. There are several peptidases that attack proline binding. These peptidases are involved with low activity not only in the cleavage of X-Pro or Pro-X bonds, but also in the degradation of the corresponding alanyl bonds. Peptidases with remarkably specific effects on sequences containing proline are attractive targets for medicinal chemistry because some of them are implicated in the regulation of biological activity of natural peptide substrates. is there. For example, DPP-IV has been implicated in the treatment of diabetes through the regulation of glucagon-like peptide-1 (GLP-1) levels. DPP-IV activity is increased in various diseases such as rheumatoid arthritis, multiple sclerosis, Graves' disease and Hashimoto's thyroiditis, sarcoidosis, and cancer. DPP-IV activity is also increased in AIDS, Down's syndrome, anorexia / bulimia, pregnancy and hypogammaglobulinemia.

Dipeptidyl peptidase containing DPP-IV
[0009] Dipeptidyl aminopeptidase activity is peptidase activity that catalyzes the removal of dipeptides from the N-terminus of peptides, polypeptides and proteins. In general, dipeptidyl aminopeptidases can cleave dipeptide XY (where X and Y represent any amino acid residue) from an unsubstituted N-terminal amino group of a peptide, polypeptide, or protein. Examples of dipeptidyl peptidase (DPP) include dipeptidyl peptidase I (DPP-I), dipeptidyl peptidase II (DPP-II), dipeptidyl peptidase III (DPP-III) and dipeptidyl peptidase (DPP-IV). Can be mentioned.

  [0010] DPP-I, also known as cathepsin C, is a lysosomal cysteine protease that is expressed in most tissues. DPP-I has been implicated in the processing of granzyme, a neutral serine protease that is exclusively expressed in granules of activated cytotoxic lymphocytes. DPP-II is a serine protease present in lysosomes. Similar to DPP-IV, it cleaves peptide bonds containing proline. Indeed, DPP-II has a substrate specificity similar to DPP-IV, but is only active at acidic pH. Dipeptidyl peptidase III (DPP-III) is a metalloprotease.

  [0011] DPP-IV is a serine protease that contains the serine protease motif GWSYG and has a wide range of substrate specificities. This hydrolyzes the peptide in order from the amino terminus to liberate amino acids. However, hydrolysis is terminated when the amino acid residue before proline is reached. As a result, peptides with X-Pro-Y- (X and Y are any amino acids) bonds are cleaved to yield X-Pro and Y-. DPP-IV is also thought to cleave a dipeptide having an alanine in the second position from the end, but is less effective than a dipeptide having a proline (Yaron et al., 1993 Crit. Rev. Biochem. Mol. Biol. 28: 31-81). This enzyme is also thought to cleave other sequences, but is less efficient.

  [0012] DPP-IV has been shown to be very specific in releasing dipeptides from the N-terminus of bioactive peptides having a proline or alanine in the second position from the end of the N-terminal sequence of the peptide substrate. . A number of potential peptide substrates for DPP-IV have been identified. DPP-IV substrates include peptide hormones and chemokines. Examples of some peptide hormones are endomorphin-2, GLP-1, GLP-2, gastric inhibitory peptide (GIP), neuropeptide Y, growth hormone releasing hormone (GHRH) and substance P, and several Examples of chemokines are RANTES, GCP-2, SDF-1α, SDF-2β, MDC, MCP-1, MCP-2 and MCP-3. DPP-II has approximately the same substrate specificity as DPP-IV.

DPP-IV and diabetes
[0013] Insulin dependent diabetes (IDDM or type I diabetes) is currently being treated by administering insulin to patients. Non-insulin dependent diabetes mellitus (NIDDM or type II diabetes) is treated by diet, administration of sulfonylureas to promote insulin secretion, or with biguanides that increase glucose uptake. Resistant individuals may require insulin therapy. Standard therapy requires daily intravenous injection of insulin, which would treat acute symptoms, but long-term therapy results in vascular disease and nerve damage. Modern methods such as transplantation are expensive and require risky surgical intervention. Therefore, there is a need to develop alternatives for the treatment of diabetes that are extremely effective and low cost.

  [0014] In recent years, there has been increased interest in DPP-IV as a target for lowering blood glucose levels. Using an inhibitor to interfere with DPP-IV enzyme or DPP-IV-like enzyme activity in a subject's blood, endogenous or exogenously administered insulin secretion such as GIP, GLP-1, or analogs thereof The degradation of the facilitating peptide is reduced. GIP and GLP-1 are hormones that promote glucose-induced insulin secretion by the pancreas and are substrates for DPP-IV. Specifically, DPP-IV is unable to remove the amino-terminal His-Ala dipeptide of GLP-1 and induce glucose-dependent insulin secretion from islets GLP-1- (9-36) -In vivo inhibition of such DPP-IV or DPP-IV-like enzyme activity to produce amides will effectively suppress unwanted enzyme activity in the pathological state of mammalian organisms. .

  [0015] PCT / DE97 / 00820 discloses alanyl pyrrolidide and isoleucyl thiazolidide as inhibitors of DPP-IV or DPP-IV-like enzyme activity. DD296075 discloses pyrrolizide and isoleucyl thiazolidide hydrochloride. US Pat. No. 6,548,481 discloses dipeptide compounds formed from amino acids and thiazolidine or pyrrolidine groups and inhibitors similar to their salts. Although these are functional inhibitors of DPP-IV activity, the use of these inhibitors in certain patients or certain forms of disease can be problematic. This is because this enzyme is responsible for the activation or inactivation of a great variety of bioactive peptides, ie DPP-IV inhibitors lack specificity for the desired targets GIP and GLP-1. .

Protection of therapeutic peptides by modification
[0016] Another way to prevent therapeutic proteins and peptides such as GIP and GLP-1 from being cleaved by proteolytic enzymes is to modify those proteins and peptides themselves to prevent exposure to proteolytic enzymes It is to be. Protein modification has been shown to increase the stability, circulation time and biological activity of therapeutic polypeptides. Some general methods for modifying amino acids and peptides are described in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins-A Survey of Recipient Developments, edited by WeinD, MD. Inc. Publishing, New York (1983). Also, the review article by Francis (1992 Focus on Growth Factors 3; 4-10 (Mediscript, London)), incorporated herein by reference, describes protein modifications and fusion proteins.

  [0017] With the advance of recombinant DNA technology and automation technology, short, medium or long modified proteins can now be easily prepared in large quantities. Numerous small modified polypeptide hormones can be synthesized using automated peptide synthesizers, solid resin technology or recombinant technology. For example, modified substrates of dipeptidyl peptidases, such as DPP-IV substrates such as GLP-1, GIP, neuropeptide Y and bradykinin, can be made in large quantities using an automated peptide synthesizer.

Summary of the Invention

  [0018] The present invention provides a transferrin fusion protein comprising a therapeutic peptide or protein that is susceptible to protease cleavage. The invention also provides a transferrin fusion protein comprising a therapeutic peptide or protein that is sensitive, resistant, or partially resistant to protease cleavage. The protease may be DPP-IV or neutral endopeptidase (NEP). The present invention further provides a composition comprising a transferrin fusion protein and a second agent such as, but not limited to, an inhibitor of DPP-IV and / or NEP. Furthermore, these compositions can be pharmaceutical compositions used in the treatment of various diseases.

  [0019] The present invention provides a combination therapy comprising administering a transferrin fusion protein and at least one second agent in the treatment of various diseases. The transferrin fusion protein can be administered simultaneously with one or more second agents. Alternatively, the transferrin fusion protein is administered sequentially, either before or after administration of the second agent. Preferably, the second agent is an inhibitor of DPP-IV or NEP.

  [0020] The GLP-1 peptide portions of the present invention have been modified to include one or more mutations such that they are partially or fully resistant to protease cleavage, such as DPP-IV cleavage. obtain. The GLP-1 peptide may be GLP-1 (7-37) (SEQ ID NO: 32) or GLP-1 (7-36) (amino acids 1 to 30 of SEQ ID NO: 2). For example, these peptides can be modified by A8 to G and / or K34 to A mutations.

Detailed description

1. Overview
[0029] The present invention is based, in part, on the need to develop alternatives for the treatment of more effective and low cost diabetes. Insulin secretion-promoting peptides such as GLP-1 are promising therapeutic substances for treating type 2 non-insulin dependent diabetes and related metabolic disorders such as prediabetes, metabolic syndrome and obesity. Other useful insulin secretagogue peptides include exendin 3 and exendin 4. However, these insulin secretagogue peptides have a short plasma half-life in vivo, mainly due to rapid serum clearance and proteolysis. Much research has been done to inhibit DPP-IV, the enzyme responsible for degradation of GLP-1, or to modify GLP-1 in such a way that its degradation is slowed and at the same time still maintains biological activity. Was done. Despite these tremendous efforts, GLP-1 with long-term activity has not been produced. Therefore, GLP-1, Exendin 3, Exendin 4, and other insulin secretagogues to provide a longer duration of action in vivo while at the same time maintaining their low toxicity and therapeutic benefits The peptide needs to be modified.

2. Definition
[0030] As used herein, the term "derivative" refers to one of peptides by chemical means, with or without enzyme, for example, alkylation, acylation, ester formation, or amide formation. Or it means modification of a plurality of amino acid residues.

  [0031] As used herein, the term "derived from" means obtaining a molecule from a specified source, such as obtaining a molecule from a parent molecule.

  [0032] As used herein, the term "dipeptidyl aminopeptidase activity" means peptidase activity that cleaves a dipeptide from the N-terminus of a peptide, polypeptide or protein sequence. In general, dipeptidyl aminopeptidases are derived from an unsubstituted N-terminal amino group of a peptide, polypeptide or protein by dipeptide XY (where X or Y is Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, , His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val may represent any amino acid residue, but at least Ala, Arg, Asp and / or Gly) can be cut. Preferably Y is Pro or Ala. X and Y may all be different or the same. Examples of dipeptidyl aminopeptidases include, but are not limited to, DPP-I, DPP-II, DPP-III, and DPP-IV.

  [0033] As used herein, the terms "glucagon-like peptide-1 (GLP-1)" and "GLP-1 derivative" generally stimulate insulin secretion during hyperglycemia and regulate glucagon secretion. It refers to intestinal hormones that suppress, promote (pro) insulin biosynthesis, and slow gastric emptying and acid secretion. As disclosed in US Pat. No. 5,574,008, which is hereby incorporated by reference, some GLP-1 and GLP-1 derivatives promote glucose uptake by cells but do not stimulate insulin expression.

  [0034] As used herein, the term "insulin secretagogue peptide" means a peptide having insulin secretagogue activity. Insulin secretagogue peptides promote or cause insulin hormone synthesis or expression. Such peptides include precursors, analogs, fragments of peptides such as glucagon-like peptide 1, exendin 3 and exendin 4, and other peptides having insulin secretion promoting activity.

  [0035] As used herein, "pharmaceutically acceptable" is physiologically acceptable when administered to a human and is allergic or similar, such as stomach upset, dizziness, etc. By materials and compositions that do not normally cause adverse reactions. Generally, as used herein, the term “pharmaceutically acceptable” is approved by a federal or state government regulatory agency or used in animals, and more specifically in humans. Therefore, it is described in the United States Pharmacopeia or other generally recognized pharmacopoeias.

  [0036] As used herein, the term "pharmaceutical composition" means a composition comprising an agent, optionally with a pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable carriers and excipients are such that side effects from the drug compound are minimized and the ability of the compound is not counteracted or inhibited to the extent that treatment is ineffective. Selected.

  [0037] As used herein, a "physiologically effective amount" is an amount that is delivered to a subject to provide a desired symptomatic or therapeutic effect. This amount is unique for each drug and the final approved dosage level.

  [0038] As used herein, a "therapeutically effective amount" refers to a modified therapeutic polypeptide or peptide that is sufficient to effect treatment when administered to a subject in need. Means quantity. The amount of the modified therapeutic polypeptide or peptide corresponding to a “therapeutically effective amount” will vary depending on the therapeutic protein used, the severity of the condition or disease, and the age and weight of the subject being treated. A person of ordinary skill in the art can routinely determine by considering his / her own knowledge and the present disclosure.

  [0039] As used herein, "therapeutic protein" refers to a protein, polypeptide, antibody, peptide fragment or variant thereof having one or more therapeutic and / or biological activities. means. Therapeutic proteins included in the present invention include, but are not limited to, proteins, polypeptides, peptides, antibodies and biologics. The terms peptide, protein and polypeptide are used interchangeably herein. Further, the term “therapeutic protein” may mean an endogenous or naturally occurring correlator of the therapeutic protein. A polypeptide or peptide that exhibits “therapeutic activity” or a “therapeutically active” protein is one of the therapeutic proteins described herein or otherwise known in the art. It means a polypeptide, peptide or protein having one or more known biological and / or therapeutic activities associated with a therapeutic protein, such as species or species. By way of non-limiting example, a “therapeutic protein” is a protein, polypeptide or peptide useful for treating, preventing or ameliorating a disease, condition or disorder. Such diseases, conditions or disorders may be in humans or in non-human animals, for example veterinary applications.

  [0040] As used herein, the term "treatment" or "treating" means any administration of a compound of the invention and comprises the following steps: (1) suffering from the disease Preventing the occurrence of the disease in an animal that may be susceptible to the disease but has not yet experienced or presented with the disease state or symptom of the disease; (2) the disease state or symptom of the affected individual; Suppressing or preventing the disease in the experienced or presenting animal (ie, preventing further development of the condition and / or symptom); or (3) experiencing or presenting the condition or symptom of the affected individual. Ameliorating the disease in the living animal (ie, reversing the medical condition and / or overall symptoms).

  [0041] As used herein, the term "biological activity" means the ability to mediate biological function. “Biological activity” includes functional activity as well as structural activity.

  [0042] As used herein, the term "symptomatic" means the ability to alleviate or reduce the symptoms of a disease or disorder without affecting healing. For example, an agent that reduces pain without curing the condition or disease is a symptomatic substance.

  [0043] As used herein, the term "preventive" means having a protective effect, such as protecting against something, especially protecting against or preventing a disease or condition. .

  [0044] As used herein, "purified" protein or nucleic acid means a protein or nucleic acid that has been separated from cellular components. A “purified” protein or nucleic acid has been purified to a level of purity that does not occur in nature.

  [0045] As used herein, the term "substantially pure" protein or nucleic acid means a protein or nucleic acid preparation that lacks all other cellular components.

  [0046] As used herein, the term "therapeutic" means a curative, recovery-promoting or therapeutic effect. For example, a “therapeutic substance” or “therapeutic composition” has a curative effect.

3. Particular embodiments Dipeptidyl peptidase
[0047] A dipeptidyl peptidase is a hydrolase that removes a dipeptide from the unsubstituted N-terminal amino group of a peptide, polypeptide or protein. Examples of dipeptidyl peptidases include, but are not limited to, DPP-I, DPP-II, DPP-III, DPP-IV, attractin and fibroblast activation protein (FAP). New enzymes of this family or similar functions but different structures are emerging.

  [0048] Dipeptidyl peptidase I (DPP-I), also known as cathepsin C, is a lysosomal cysteine protease belonging to the papain family. DPP-I can sequentially remove dipeptides from the free amino terminus of various peptides and protein substrates and thus acts in the manner of an exopeptidase (specifically dipeptidyl peptidase). Cleavage is ineffective if the fragmented bond has a proline residue on either side, or if the N-terminal residue is lysine or arginine.

  [0049] DPP-II is a serine protease present in lysosomes and its function is unknown. Like DPP-IV, it mainly cleaves peptide bonds containing proline. In fact, DPP-II has a substrate specificity similar to DPP-IV, but is only active at acidic pH. Mammalian DPP-II and DPP-IV can be distinguished using the inhibitors puromycin and bacitracin. That is, puromycin is thought to inhibit only DPP-II, whereas bacitracin inhibits only DPP-IV (1988 J. Biol. Chem. 263, 6613-6618). Dipeptidyl peptidase III (DPP-III) is a metalloprotease. DPP-V releases N-terminal X-Ala, His-Ser and Ser-Tyr dipeptides.

  [0050] DPP-VII, also known as quiescent cell proline dipeptidase, is a proline-specific dipeptidase. DPP-VII and DPP-II were suggested to be the same protease (Araki et al., 2001 J. Biochem. Based on sequence comparison (78% match) between human DPP-VII and rat DPP-II. 129, 279-288).

  [0051] DPP-VIII is a human-derived post-proline dipeptidyl aminopeptidase homologous to DPP-IV and FAP (Abbott, CA, et al., 2000 European Journal of Biochemistry 267, 6140). Like DPP-IV, DPP-VIII is ubiquitous. The full length DPP-VIII cDNA encodes a 882 amino acid protein with about 27% identity and 51% similarity to DPP-IV and FAP, but with a transmembrane domain and N-linked or It does not encode O-linked glycosylation. The purified recombinant DPP-VIII hydrolyzed the DPP-IV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus, recombinant DPP-VIII shares postproline dipeptidyl aminopeptidase activity with DPP-IV and FAP. DPP-VIII enzyme activity had a neutral optimum pH, consistent with it being non-lysosomal. The similarities between DPP-VIII and DPP-IV in tissue expression patterns and substrates suggest a potential role for DPP-VIII in T cell activation and immune function, similar to DPP-IV.

  [0052] Olsen C.I. Et al. (2002 Gene 299, 185-93) report the identification and characterization of a novel DPP-IV-like molecule, termed dipeptidyl peptidase-like protein DPP-IX. Similar to DPP-IV, DPP-IX contains the serine protease motif GWSYG (SEQ ID NO: 110). Because of the presence of this motif and the conservation of the order and spacing of Ser, Asp, and His residues that form the catalytic triad in DPP-IV, DPP-IX is DPP-IV Placed in the gene family.

  [0053] Attractin (DPPT-L) is a 175-kDa soluble glycoprotein that has been reported to hydrolyze Gly-Pro. Attractin contains a Kerch repeat domain and does not share significant sequence homology with DPP-IV or any other peptidase. Fibroblast activating protein (FAP) is a cell surface-bound protease of the prolyl oligopeptidase gene family that is expressed at the site of tissue remodeling.

  [0054] Prolyl endopeptidase (PEP), also called proline oligopeptidase (PO), was first discovered by Walter and co-workers as an oxytocin-degrading enzyme in the human uterus (Walter et al., Science 173, 827-829). Page (1971)). This enzyme contains the sequence X-Pro-Y, where X is a peptide or an amino acid substituted at the N-terminus, and Y is a peptide, amino acid, amide or alcohol. The peptide bond is cleaved at the carboxy side (Yoshimoto et al., J. Biol. Chem. 253, 3708-3716 (1979)). This enzyme has a high specificity for the trans configuration of the peptide bond on the imino side of proline (Lin & Brandts, Biochemistry 22, 4480-4485 (1983)).

  [0055] Prolyl oligopeptidase hydrolyzes angiotensin I and angiotensin II resulting in the release of angiotensin (1-7). Angiotensin (1-7) has vasodilatory activity and modulates the release of vasopressin that can affect the memory process, as shown by injecting rats with specific PEP inhibitors. This injection reverses scopolamine-induced amnesia. This experiment is not only an example that provides evidence of the possible physiological function of this enzyme, but also led to the hypothesis that inhibitors of PEP can affect the memory process and prevent dementia ( Yoshimoto et al., 1987 J. Pharmacobio-Dyn. 10, 730-735).

Dipeptidyl peptidase (DPP-IV) and substrate
[0056] DPP-IV is a ubiquitously expressed molecule that has been implicated in the degradation of several peptides and hormones. Various types of DPP-IV have been purified and their enzymatic properties have been revealed. For example, DPP-IV can be used in rat liver (Hoppu-Havu V.K. et al., 1966 Histochem., 7: 197-201), porcine kidney (Barth A. et al., 1974 Biol. Med. Chem., 32 157-174), small intestine (Svensson B. 1978 Eur. J. Biochem., 90: 489-498), liver (Fukasawa KM et al. 1981 Biochim. Biophys. Acta, 657: 179-189). P.), Human submandibular gland (Oya H. et al., 1972 Biochim. Biophys. Acta, 258: 591-599), sheep kidney (Yoshimoto T. et al., 1977 Biochim. Biophys. Acta, 485: 391-401). Page; Yoshimo to T. et al., 1978 J. Biol. Chem., 253: 3708-3716), or microorganisms (Fukuzawa KM 1981 Biochem. Biophys., 210: 230-237; Yoshimoto T. 1982 J Biochem., 91: 1899-1906 (1982)).

  [0057] In the human immune system, DPP-IV is identical to the T cell surface antigen CD26 expressed by activated lymphocytes (T cells, B cells and natural killer cells). CD26 / DPP-IV is a type II membrane glycoprotein that has intrinsic dipeptidyl peptidase IV activity and the ability to bind to adenosine deaminase type I (ADA-1). It is constitutively expressed on epithelial cells, but is expressed under strict cell regulation on T lymphocytes and expression is upregulated when cells are activated. CA26 / DPP-IV has been shown to have dipeptidyl peptidase IV activity in its extracellular domain (Hegen et al., 1990 J. Immunol 144: 2908-2914; Ulmer et al., 1990 Scand. J Immunol.31: 429-435), and its costimulatory activity appears to depend to some extent on this enzyme activity (Tanaka et al., 1993 Proc. Natl. Acad. Sci. USA 90: 4586-4590). page). DPP-IV is involved in the regulation of chemokine function and may play an important role in HIV infection.

  [0058] US Pat. No. 6,265,551 discloses circulating soluble DPP-IV / CD26 isolated from human serum. Serotypes share enzyme and antigenic properties similar to ubiquitous membrane types. However, in some biochemical aspects, there are different differences. In particular, the circulating serotype has a molecular weight of 175 kDa and does not bind to ADA-1 in contrast to the membrane type having a molecular weight of 105 kDa. Nevertheless, the circulating form expresses functional dipeptidyl peptidase IV activity and retains the ability to costimulate a T lymphocyte response to a recall antigen.

  [0059] The proteolytic activity of DPP-IV is present in a stretch of about 200 amino acids located at the C-terminus of the protein. The catalytic residues (Ser-629, Asp-708, His-740) are arranged in a unique order different from classical serine proteases such as chymotrypsin and subtilisin. Dipeptidyl peptidase activity specific for proline includes numerous bioactive proteins and polypeptides, including, inter alia, GLP-1, neurotransmitter substance P, human growth hormone releasing factor, erythropoietin, interleukin 2 and many others. Modify the biological activity of Potential DPP-IV substrates are listed in Tables 1, 2, and 3. Modulating these polypeptides to affect DPP-IV cleavage includes, but is not limited to, diabetes, inflammation, vascular disease, autoimmune disease, multiple sclerosis, joint disease, and benign and malignant It can be useful in the treatment of clinical conditions, including diseases associated with cell transformation.

  [0060] The present invention may utilize a modified substrate of DPP that includes one or more additional amino acids at the N-terminus of the substrate to protect the substrate from DPP activity. Preferred substrates for modification according to the present invention are disclosed in Table 3.

[0061]

  [0062] Substrates for modification include X-ProY, X-Ala-Y, X-Ser-Y or X-Gly-Y at the amino terminus. Preferably, the modifying substrate is GLP-1.

Modified polypeptides protected from DPP activity
[0063] The present invention provides modified polypeptides, such as modified polypeptide substrates for DPP, that contain one or more additional amino acids at the N-terminus to protect the polypeptide substrate from DPP activity. In one embodiment, these modified polypeptides have one additional amino acid at the N-terminus compared to the wild-type polypeptide. In another embodiment, these modified polypeptides have 5 additional amino acids at the N-terminus. Alternatively, these modified polypeptides have 1 to 5 additional amino acids at the N-terminus. Any one of the 20 amino acids may be added to the N-terminus of the polypeptide substrate, or an unnatural amino acid may be added.

  [0064] Any pharmaceutical polypeptide having a peptide bond that will be cleaved in the circulation or anywhere in vivo after administration is not due to the protection from DPP cleavage provided by the present invention. It is anticipated that the invention will benefit from modifications.

  [0065] According to this aspect of the invention, at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 90%, most preferably at least at least dipeptidyl peptidase activity. About 99% can be lost. It is also possible to completely abolish dipeptidyl aminopeptidase activity using the method of the present invention.

  [0066] Similarly, the dipeptidyl peptidase sensitivity of the substrate is at least about 30%, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 90%, most preferably dipeptidyl peptidase sensitivity. Can be reduced by at least about 99%. It is also possible to completely abolish dipeptidyl aminopeptidase sensitivity using the method of the present invention.

  [0067] Although the modified polypeptides or peptide substrates of the invention are partially or substantially protected from DPP activity, the modified polypeptide substrates are at least about 10% of their functional activity and potency, Preferably at least about 30%, more preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 90%, and most preferably at least about 99%. In some cases, modified polypeptides or peptide substrates that have reduced functional activity or efficacy are useful. For example, when a modified polypeptide or peptide is fused to another polypeptide, such as transferrin, to form a fusion protein with improved serum stability and in vivo circulation half-life, a modification with reduced functional activity or efficacy Polypeptides or peptide substrates can be useful.

  [0068] In other cases, a modified polypeptide or peptide may have a higher potency than an unmodified polypeptide or peptide.

  [0069] Modified polypeptide molecules of the present invention are substantially protected from dipeptidyl peptidase cleavage compared to unmodified forms of the same polypeptide. This substantial protection capability may vary depending on the assay used to compare the modified polypeptide to the unmodified polypeptide. However, in order to present substantial protection, the modified polypeptide presents a detectable level of resistance to dipeptidyl peptidase cleavage in the assay. Such assays include, but are not limited to, Doyle et al. (2002 Endocrinology 142, 4462-4468), O'Harte et al. (1999 Diabetes 48, 758-765), and Siegel et al. (1999 Regulatory). Peptides 79, pages 93 to 102).

  [0070] Polypeptide substrates of the invention stabilized against DPP are also more stable than unstabilized polypeptide substrates in the presence of DPP in vivo. Therapeutic polypeptide substrates stabilized against DPP generally have a longer active half-life compared to unstabilized peptides of the same sequence. Peptidase stability can be measured by comparing the half-life of the unmodified polypeptide substrate in serum or blood to the half-life of the modified corresponding therapeutic peptide in serum or blood. The half-life can be determined by sampling serum or blood after administration of modified and unmodified peptides and measuring the activity of the peptide. In addition to measuring activity, the length of the polypeptide substrate can also be measured by HPLC or mass spectrometry.

  [0071] The present invention also includes amino and / or C-terminal amino acids that have an amino terminus modified in accordance with the present invention to protect against DPP cleavage and do not affect the functional activity or efficacy of the polypeptide. Also provided are modified polypeptides or peptides having the following changes: Although these modified polypeptides appear to have minor amino acid changes, usually conservative amino acid substitutions, non-conservative substitutions are also contemplated.

  [0072] Modified polypeptides or peptides of the invention may also have altered functional activity. For example, modified polypeptides or peptides with increased functional activity can be useful. Alternatively, modified polypeptides or peptides with reduced functional activity can also be used. Thus, a modified polypeptide or peptide of the invention also includes amino acid changes that affect functional activity or efficacy. For example, a GLP-1 analog with altered functional activity can be modified at its amino terminus to protect it from DPP cleavage.

  [0073] Examples of conservative amino acid substitutions include basic amino acids (such as arginine, lysine, histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids (such as glutamine and asparagine), hydrophobic amino acids (such as leucine, isoleucine, Valine, etc.), aromatic amino acids (phenylalanine, tryptophan, tyrosine, etc.), and small amino acids (glycine, alanine, serine, threonine, methionine, etc.) within the same group, such as substitutions.

  [0074] Non-conservative substitutions involve replacing one group of amino acids with another group of amino acids. For example, non-conservative substitutions may include replacing a hydrophobic amino acid with a polar amino acid. For an overview of nucleotide substitution, see, eg, Ford et al. (1991), Prot. Exp. Pur. 2: pp. 95-107.

  [0075] The present invention relates to naturally occurring mature forms of a polypeptide or peptide, allelic / sequence variants of a polypeptide, variants of a non-naturally occurring peptide derived recombinantly, and polypeptides or peptides Provided are variants of the amino acid sequence of modified polypeptides and peptides, such as orthologs and paralogs. Such variants can be readily generated using techniques known in the art in the fields of recombinant nucleic acid technology and protein biochemistry. Such variants can be easily identified / created using molecular techniques and sequence information. Furthermore, such variants can be readily distinguished from other peptides based on sequence and / or structural homology to the modified polypeptides or peptides of the invention.

  [0076] Preferably, the modified peptides of the present invention are GLP-1 and analogs thereof comprising one or more additional amino acids at the N-terminus.

  [0077] In some cases, a DPP, such as DPP-IV, may activate a peptide instead of inactivating the peptide by cleavage. In such cases, peptide modification should be able to substantially reduce, retard, or prevent peptide activation.

Nucleic acid encoding modified polypeptide
[0078] The present invention provides modified polypeptides and nucleic acid molecules encoding peptides that are partially or substantially protected from DPP cleavage and have functional activity and efficacy. In one embodiment, the nucleic acid molecules provided by the present invention encode modified polypeptides and peptides having at least one additional amino acid at its N-terminus compared to the wild-type unmodified polypeptide. In another embodiment, these nucleic acid molecules encode modified polypeptides and peptides having 5 additional amino acids at the N-terminus. Alternatively, these nucleic acid molecules encode modified polypeptides and peptides having 1 to 5 additional amino acids at the N-terminus. Preferably, the nucleic acid molecule encoding modified GLP-1 comprises a sequence encoding one or more additional amino acids at its N-terminus.

  [0079] The nucleic acid molecules of the present invention include both deoxyribonucleic acid (DNA), single-stranded and double-stranded deoxyribonucleic acids. However, they can also be ribonucleic acid (RNA), as well as RNA: DNA hybrid duplex molecules. Contemplated nucleic acid molecules also include genomic DNA, cDNA, mRNA, and antisense molecules. The nucleic acid molecules of the present invention also include natural or synthetic RNA, DNA or cDNA encoding a modified polypeptide, or a complementary strand thereof.

  [0080] To construct a modified polypeptide that is partially or substantially protected from DPP activity but has functional activity and / or efficacy as compared to a wild-type unmodified polypeptide. A nucleic acid encoding a wild-type unmodified polypeptide can be used as a starting point and modified to encode the desired modified polypeptide. Numerous methods are known for adding sequences or mutating nucleic acid sequences encoding certain polypeptides and confirming the function of the polypeptides encoded by these modified sequences.

  [0081] The present invention also includes amino and C-terminal amino acids that have an amino terminus modified to protect against DPP cleavage and do not substantially affect the functional activity or efficacy of the polypeptide. Also provided are nucleic acids encoding polypeptides and peptides having alterations. Although these modified polypeptides appear to have minor amino acid changes, usually conservative amino acid substitutions, non-conservative substitutions are also contemplated. Nucleotide substitution using techniques to achieve site-directed mutagenesis is well known in the art. Preferably, these nucleic acids encode GLP-1 analogs having one or more additional amino acids at the N-terminus.

  [0082] As is known in the art, "similarity" between two polynucleotides or polypeptides is the nucleotide or amino acid sequence of one polynucleotide or polypeptide and the conserved nucleotide or amino acid substitutions. The body is measured by comparison with the sequence of the second polynucleotide or polypeptide. Similarly, “identity”, which refers to the degree of sequence relatedness between two polypeptides or two polynucleotide sequences, is also known in the art and when matching two strands of these sequences. Measured by identity. Both identity and similarity can be easily calculated (Computational Molecular Biology, Edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Dm. Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, H. G. Ed., Humana Press, New Jersey, 1994; Heinj , G., Academic Press, 1987 years;.. And Sequence Analysis Primer, Gribskov, M and Devereux, J, eds., M Stockton Press, New York, 1991).

  [0083] Although there are several methods for measuring identity and similarity between the sequences of two polynucleotides or polypeptides, the terms "identity" and "similarity" Well known (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and DevreMux, 19 And Lipman, D., SIAM J. Applied Math., 48: 1073 (1988)). Commonly used methods for measuring identity or similarity between two sequences include, but are not limited to, Guide to Huge Computers, Martin J. et al. Bishop, Academic Press, San Diego, 1994, and Carillo, H .; And Lipman, D .; SIAM J .; Applied Math. 48: 1073 (1988).

  [0084] Preferred methods for measuring identity are designed to give the greatest match between the two sequences tested. Methods for measuring identity and similarity are organized in computer programs. Preferred computer program methods for measuring identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux et al., Nucleic Acids Research 12 (1): 387 (1984). ), BLASTP, BLASTN, FASTA (Atschul et al., J. Molec. Biol. 215; 403 (1990)). The degree of similarity or identity described above is measured as the degree of identity between two sequences, indicating the derivation of the first sequence from the second sequence. The degree of identity between two nucleic acid sequences can be determined by computer programs known in the art such as GAP (Needleman and Wunsch (1970) Journal of Molecular Biology 48: 443-453) provided in the GCG program package. Can be measured. To measure the degree of identity between two nucleic acid sequences for the present invention, GAP is used with the following settings: GAP generation penalty of 5.0 and GAP extension penalty of 0.3.

Codon optimization
[0085] The degeneracy of the genetic code allows for modification of the nucleotide sequence of the polypeptide that still produces a modified polypeptide comprising the same amino acid sequence as the polypeptide encoded by the first DNA sequence. A procedure known as “codon optimization” (described in US Pat. No. 5,547,871, which is incorporated herein by reference in its entirety) provides a means to design such altered DNA sequences. . Codon-optimized gene design includes frequency of codon usage in an organism, nearest neighbor frequency, RNA stability, possibility of secondary structure formation, synthetic pathway, and planned subsequent DNA manipulation of the gene Various factors should be taken into account. In particular, available methods can be used to alter the codon encoding a given fusion protein with the one that is most readily recognized by the yeast if a yeast expression system is used.

  [0086] The degeneracy of the genetic code allows the same amino acid sequence to be encoded and translated in many different ways. For example, leucine, serine, and arginine are each encoded by six different codons, and valine, proline, threonine, alanine, and glycine are each encoded by four different codons. However, the frequency of use of such synonymous codons varies from genome to genome between eukaryotes and prokaryotes. For example, the synonymous codon selection pattern between mammals is very similar, but yeast (S. cerevisiae), bacteria (such as E. coli), and insects (such as D. melanogaster) Evolutionary distant organisms, etc., clearly show different patterns of genomic codon usage (Grantham, R. et al., Nucl. Acids Res., 8, 49-62 (1980); Grantham, R. et al., Nucl. Acids Res., 9, 43-74 (1981); Maroyama, T. et al., Nucl.Acids Res., 151-197 (1986); Aota, S. et al., Nucl.Acids Res. 16, 315-402 (1988); Wa .. A, K other, Nucl. Acids Res, 19 Suppl, pp. 1981-1985 (1991); Kurland, C.G., FEBS Letters, pp 285,165～169 (1991)). These differences in codon selection patterns appear to contribute to the overall expression level of individual genes by regulating peptide elongation rates (Kurland, CG, FEBS Letters, 285, 165- 169 (1991); Pedersen, S., EMBO J., 3, 2895-2898 (1984); Sorensen, MA, J. Mol. Biol., 207, 365-377 (1989) Randall, LL et al., Eur. J. Biochem., 107, 375-379 (1980); Curran, J. F., and Yarus, M., J. Mol. Biol., 209, 65-77 (1989); Varenne, S. et al., J. Mol. Biol., 180, 549-5. 76 (1984), Varenne, S. et al., J. Mol. Biol., 180, 549-576 (1984); Garesl, JP, J. Theor. Biol., 43, 211-225. Ikeura, T., J. Mol. Biol., 146, 1-21 (1981); Ikemura, T., J. Mol. Biol., 151, 389-409 (1981) )).

  [0087] Preferred codon usage for synthetic genes is that of the exact (or as closely related as possible) genome of the cell / organism intended to be used for recombinant protein expression, particularly yeast species Should reflect the codon usage of the nuclear gene derived from. As mentioned above, in one preferred embodiment, the modified polypeptide is codon optimized before or after modification as described herein for yeast expression.

vector
[0088] An expression unit for use in the present invention generally comprises the following elements operatively linked in a 5 'to 3' orientation: a transcription promoter, a secretion signal sequence, a modified polypeptide. Contains the coding DNA sequence and transcription terminator. As noted above, any arrangement of modified polypeptides and peptides may be used in the vectors of the invention. The selection of the appropriate promoter, signal sequence, and terminator will be determined by the selected host cell and will be apparent to those skilled in the art and will be discussed more specifically below.

  [0089] Yeast vectors suitable for use in the present invention are described in US Pat. No. 6,291,212, YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76: 1035-1039, 1978). YEp13 (Broach et al., Gene 8; 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978), pPPC0005, pSeCHSA, pScNHSA, pC4, and derivatives thereof. . Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and Pichia vectors available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (YIps) and incorporate yeast selection markers HIS3, TRP1, LEU2, and URA3. Plasmid pRS413-416 is a yeast centromere plasmid (YCps).

[0090] Such vectors generally contain a selectable marker, which is one of a number of genes exhibiting a dominant phenotype that allows for the selection of transformants for which there is a phenotypic assay. Good. Preferred selectable markers are those that complement the auxotrophy of the host cell, confer antibiotic resistance, or allow the cell to utilize a specific carbon source, such as LEU2 (Broach et al., Ibid), URA3 ( Botstein et al., Gene 8; 17, 1979), HIS3 (Struhl et al., Ibid), or POT1 (Kawasaki and Bell, EP171142). Other suitable selectable markers include the CAT gene that confers chloramphenicol resistance to yeast cells. Preferred promoters for use in yeast include yeast glycolytic genes (Hitzeman et al., J Biol. Chem. 225: 12073-12080, 1980; Albert and Kawasaki, J. Mol. Appl. Genet. 1; 419 ˜434, 1982; Kawasaki, US Pat. No. 4,599,311) or alcohol dehydrogenase gene (Young et al., Genetic Engineering of Chemicals for Chemicals, Hollaender et al., Eds., Pp. 355, Plenum, N. 198; , Meth. Enzymol. 101: 192-201, 1983). In this regard, particularly preferred promoters are the TPI1 promoter (Kawasaki, US Pat. No. 4,599,311) and the ADH2-4 ^C (see US Pat. No. 6,291,212) promoter (Russell et al., Nature 304: 652-654, 1983). ). The expression unit may also include a transcription terminator. A preferred transcription terminator is the TPI1 terminator (Alber and Kawasaki, ibid.). More preferably, the promoter is the PRB1 promoter disclosed in EP431880 and the terminator is the ADH1 terminator disclosed in EP60057, which are hereby incorporated by reference in their entirety.

  [0091] In addition to yeast, the modified polypeptides and peptides of the present invention can also be expressed in filamentous fungi, for example, species of the genus Aspergillus. Examples of useful promoters include those derived from Aspergillus nidulans glycolytic genes such as the ADH3 promoter (McKnight et al., EMBO J. 4: 2093-2099, 1985) and the tpiA promoter. . An example of a suitable terminator is the ADH3 terminator (McKnight et al., Ibid). Expression units that utilize such components can be cloned into vectors that can be inserted into, for example, Aspergillus chromosomal DNA.

[0092] Mammalian expression vectors for use in practicing the present invention include modified polypeptides and promoters that can direct transcription of the peptides. Preferred promoters include viral promoters and cellular promoters. Preferred viral promoters include the major late promoter derived from adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. 1). : Pp. 854-864, 1981). Preferred cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., Science 222; 809-814, 1983) and the mouse Vκ (see US Pat. No. 6,291,212) promoter (Grant et al., Nuc. Acids Res. 15: 5496, 1987). A particularly preferred promoter is the mouse V _H (see US Pat. No. 6,291,212) promoter. Such expression vectors may also include a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the modified polypeptide or peptide. Preferred RNA splice sites can be obtained from adenovirus and / or immunoglobulin genes.

[0093] A polyadenylation signal located downstream of the coding sequence of interest is also included in the expression vector. Polyadenylation signals include early or late polyadenylation signals derived from SV40 (Kaufman and Sharp, ibid), adenovirus 5E1B region and human growth hormone gene terminator (DeNoto et al., Nuc. Acids Res. 9: 3719-3730. , 1981). A particularly preferred polyadenylation signal is the V _H (see US Pat. No. 6,291,212) gene terminator. The expression vector may comprise a non-coding viral leader sequence, such as the adenovirus 2 triple segment leader, located between the promoter and the RNA splice site. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse μ (see US Pat. No. 6,291,212) enhancer (Gillies, Cell 33: 717-728, 1983). The expression vector may also include sequences encoding adenovirus VA RNA.

  [0094] These expression vectors may also be modified according to the present invention fused to a second polypeptide or peptide, such as transferrin, to improve the half-life of the modified polypeptide or peptide, as described below. It is also used to express a polypeptide or a fusion protein comprising a peptide. Similarly, a modified polypeptide or peptide may be fused to a tag and / or cleavage site to express and release the modified polypeptide or peptide.

Transformation
[0095] Techniques for transforming fungi are well known in the literature, for example, Beggs (Id.), Hinnen et al. (Proc. Natl. Acad. Sci. USA 75: 1929-1933, 1978), Yelton et al., ( Proc. Natl. Acad. Sci. USA 81: 1740-1747 (1984), and Russell (Nature 301: 167-169, 1983). The host cell genotype generally comprises a genetic defect that is complemented by a selectable marker present on the expression vector. The selection of individual hosts and selectable markers is well within the level of ordinary skill in the art.

[0096] Cloned DNA sequences comprising the modified polypeptides and peptides of the invention can be obtained, for example, by transfection with calcium phosphate (Wigler et al., Cell 14: 725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7: 603). 1981; Graham and Van der Eb, Virology 52: 456, 1973). Other techniques for introducing cloned DNA sequences into mammalian cells such as electroporation (Neumann et al., EMBO J. 1: 841-845, 1982) or lipofection can also be used. In order to identify cells that have incorporated the cloned DNA, a selectable marker is generally introduced into those cells along with the gene or cDNA of interest. Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs such as neomycin, hygromycin and methotrexate. The selectable marker may be a selectable marker that can be amplified. A preferred amplifiable selectable marker is the DHFR gene. A particularly preferred amplifiable marker is DHFR ^r (see US Pat. No. 6,291,212) cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci, USA 80: 2495-2499, 1983). Selection markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.), And selection of selection markers is well within the level of one skilled in the art.

Host cell
[0097] The present invention also includes cells, preferably yeast cells, that have been transformed to express a modified polypeptide or peptide of the invention. In addition to the transformed host cells themselves, the present invention also provides a culture of those cells, preferably a monoclonal (clonally uniform) culture, or a culture derived from a monoclonal culture, in nutrient medium. Including goods. If the polypeptide is secreted, the medium contains the polypeptide with the cells or without the cells if they are removed by filtration or centrifugation.

  [0098] Host cells for use in practicing the present invention can be transformed or transfected with exogenous DNA, such as cultured mammalian cells, insect cells, fungal cells, plant cells, and bacterial cells, and cultured. Eukaryotic cells that can be grown in the state and in some cases prokaryotic cells. A vector containing a nucleic acid sequence of the invention is introduced into a host cell so that the vector is maintained as a chromosomal component or as an extrachromosomal vector that self-replicates. Integration is generally considered advantageous because the nucleic acid sequence tends to be stably maintained in the cell. Integration of the vector into the host chromosome can occur by homologous or non-homologous recombination.

  [0099] The choice of host cell will depend largely on the gene encoding the polypeptide and its source. The host cell may be a unicellular microorganism, such as a prokaryotic organism, or a non-unicellular microorganism, such as a eukaryotic organism. Either prokaryotic or eukaryotic organisms can be used. As prokaryotic host cells, commonly used cells such as Escherichia coli and Bacillus subtilis can be used.

  [00100] When prokaryotic cells are used as host cells, vectors that are replicable in the host cells may be used. A promoter, SD sequence (Shine-Dalgarno sequence), and initiation codon (eg, ATG) required to initiate protein synthesis are provided upstream of the gene of the present invention in the vector to control expression of that gene. An expression plasmid that facilitates may preferably be used. Examples of the vector include commonly used plasmids derived from E. coli such as pBR322, pBR325, pUC12, and pUC13. However, applicable vectors are not limited to these examples, and various known vectors can also be used. Examples of commercially available vectors that can be used in an expression system using E. coli include pGEX-4T (Amersham Pharmacia Biotech), pMAL-C2, pMAL-P2 (New England Biolabs), pET21 / lacq (Invitrogen), pBAD / His (Invitrogen) etc. are mentioned.

  [00101] Examples of eukaryotic host cells include yeast cells and the like. Examples of cranial cells that are preferably used include COS cells (monkey-derived cells) (1981 Cell, pages 23, 175), Chinese hamster ovary cells, and dihydrofolate reductase-deficient strains derived therefrom (1980). Proc. Natl. Acad. Sci., USA., Pages 77, 4216), and examples of yeast cells that are preferably used include Saccharomyces cerevisiae. However, the cells that can be used are not limited to these examples. Preferably, yeast cells are used to express the modified polypeptide or peptide.

  [00102] Fungal cells, including yeast species (eg, Saccharomyces species, Schizosaccharomyces species, Pichia species) may be used as host cells within the present invention. Examples of fungi, including yeast, that are expected to be useful in practicing the present invention as hosts for expressing the modified polypeptides or peptides of the present invention include Pichia (formerly Hansenula) ), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Toluropsis, Torulaspora, Fission yeast, Citeromyces (Citeromyces), Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium (Cep) Alosporium, Humicola, Mucor, Neurospora, Yarrowia, Yarrowia, Metschnikowia, Rhodosporidium, Rhodosporidium, Rhodospodium ), Endomycosis and the like. Examples of Saccharomyces spp. Cerevisiae, S. et al. Italicus, and S. Rouxii. Examples of Kluyveromyces spp. Fragilis, K.M. Lactis, and K. et al. Marxianus. Suitable Torlaspola spp. Delbrueckii. Examples of Pichia spp. Angusta (formerly H. polymorpha), P.A. Anomala (formerly H. anomala), and P. a. Pastoris.

  [00103] Host cells that are particularly useful for producing the modified polypeptides or peptides of the invention are methanol-utilizing Pichia pastoris (Steinlein et al. (1995) Protein Express. Purif. 6: 619-624. ). Pichia pastoris was developed to be an excellent host for producing heterologous proteins because its alcohol oxidase promoter was isolated and cloned, and its transformation was first reported in 1985. P. Pastoris can use methanol as a carbon source in the absence of glucose. P. In the Pastoris expression system, a methanol-inducible alcohol oxidase (AOX1) promoter that controls a gene encoding the expression of alcohol oxidase, which is an enzyme that catalyzes the first stage of methanol metabolism, can be used. This promoter has been characterized and a series of P. coli. It was incorporated into a Pastoris expression vector. P. Proteins produced in Pastoris are usually correctly folded and secreted into the medium, so that genetically engineered P. Pastoris fermentation is an excellent alternative to the E. coli expression system.

  [00104] A strain of the yeast Saccharomyces cerevisiae is another preferred host. In a preferred embodiment, yeast cells, or more specifically, Saccharomyces cerevisiae host cells that contain a genetic defect in a gene required for asparagine-linked glycosylation of glycoproteins are used. A number of available yeast strains have been modified to prevent or reduce glycosylation or excessive mannosylation, but S. coli having such deficiencies. S. cerevisiae host cells can be prepared using standard techniques of mutation and selection.

  [00105] In order to optimize the production of heterologous proteins, the host strain produces a reduction in proteolytic activity. It is also preferable to have a mutation such as a cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977). The gene encoding aspartyl protease yapsin 1 (YAP3) or gene encoding yapsin 2 (MKC7), or both have mutations (Copley et al. 1998 Biochem. J. 330, 1333-1340. As a result, it is particularly advantageous to use a host in which the proteolytic activity directed at basic residues is reduced or eliminated. Host strains containing mutations in other protease coding regions are particularly useful for making large quantities of the modified therapeutic polypeptides or peptides of the invention.

  [00106] Host cells containing the DNA constructs of the invention are grown in a suitable growth medium. As used herein, the term “appropriate growth medium” means a medium containing nutrients required for cell growth. Nutrients required for cell growth can include carbon sources, nitrogen sources, essential amino acids, vitamins, minerals, and growth factors. In growth media, cells containing the DNA construct are generally selected, for example, by selection with drugs, or lack of essential nutrients supplemented by a selectable marker on or with the DNA construct. For example, yeast cells are preferably grown in a known composition medium containing a non-amino acid nitrogen source, inorganic salts, vitamins, and essential amino acid supplements. The pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably pH 5.5-6.5. Methods for maintaining a stable pH preferably include buffering by addition of ammonia, ammonium hydroxide or sodium hydroxide and constant pH control. Preferred buffering agents include citric acid, phosphate, succinic acid and Bis-Tris (Sigma Chemical, St. Louis, MO). Yeast cells having a defect in a gene required for asparagine-linked glycosylation preferably grow in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol, which is added to the medium at a concentration of 0.1M to 1.5M, preferably 0.5M or 1.0M.

  [00107] Mammalian cultured cells generally grow in commercially available serum-containing or serum-free media. The selection of a suitable medium for the particular cell line used is within the level of ordinary skill in the art. Transfected mammalian cells are grown for a period of time, usually 1-2 days, to initiate expression of the DNA sequence of interest. Drug selection is then applied to select for the growth of cells that stably express the selectable marker. In the case of cells transfected with an amplifiable selectable marker, the drug concentration may be increased stepwise to select high copy number cloned sequences and thereby increase expression levels.

  [00108] Baculovirus / insect cell expression systems may also be used to make the modified therapeutic polypeptides or peptides of the invention. The “BacPAK ™” baculovirus expression system (BD Biosciences (Clontech)) expresses recombinant proteins at high levels in insect host cells by inserting the target gene into a transfer vector and linearizing it. The BacPAK6 DNA lacks an integral part of the baculovirus genome, and when this DNA recombines with the vector, its essential elements are repaired and the target gene is After recombination, several viral plaques are collected and purified to confirm the recombinant phenotype, then the newly isolated recombinant virus is amplified and infected with insect cell cultures Use to make desired tamper Can produce large quantities of soil.

Secretory signal sequence
[00109] The terms "secretory signal sequence" or "signal sequence" or "secretory leader sequence" are used interchangeably and include, for example, US Pat. No. 6,291,212, both of which are hereby incorporated by reference in their entirety. U.S. Pat. No. 5,547,871. The secretory signal sequence or signal sequence or secretory leader sequence encodes a secretory peptide. A secretory peptide is an amino acid sequence that serves to direct secretion of a mature polypeptide or protein from a cell. Secreted peptides are generally characterized by a core of hydrophobic amino acids and are usually (but not limited to) the amino terminus of newly synthesized proteins. Very often, secreted peptides are cleaved from the mature protein during secretion. The secretory peptide may include a processing site from which the signal peptide can be cleaved as the mature protein passes through the secretory pathway. The processing site may be encoded within the signal peptide or may be added to the signal peptide, for example by in vitro mutagenesis.

  [00110] Secretory peptides may be used to direct secretion of the modified polypeptides and peptides of the invention. One such secretory peptide that can be used in combination with other secretory peptides is the third domain of the yeast Barrier protein. Secretory signal sequences or signal sequences or secretory leader sequences are required for a complex series of post-translational processing steps that result in secretion of the protein. If the complete signal sequence is present, the expressed protein enters the lumen of the rough endoplasmic reticulum, then is transported through the Golgi apparatus to the secretory vesicle, and finally transported extracellularly. The In general, the signal sequence immediately follows the start codon and encodes the signal peptide at the amino terminus of the secreted protein. In most cases, the signal sequence is cleaved off by a specific protease called a signal peptidase. Preferred signal sequences improve the processing and export efficiency of recombinant protein expression using viral, mammalian, or yeast expression vectors. A preferred signal sequence is a mammalian or human transferrin signal sequence. In some cases, a natural substrate signal sequence may be used to express and secrete a modified polypeptide or peptide of the invention. In order to ensure efficient removal of the signal sequence, in some cases, a short propeptide sequence between the signal sequence and the mature protein, the C-terminal part of which contains a recognition site for a protease such as yeast kex2p protease. It may be preferable to include. Preferably, the propeptide sequence is about 2-12 amino acids in length, more preferably about 4-8 amino acids in length. Examples of such propeptides are Arg-Ser-Leu-Asp-Lys-Arg, Arg-Ser-Leu-Asp-Arg-Arg, Arg-Ser-Leu-Glu-Lys-Arg, and Arg-Ser- Leu-Glu-Arg-Arg (SEQ ID NO: 111-114, respectively).

Generation of modified polypeptide substrates protected from DPP cleavage
[00111] The modified polypeptides of the invention that are partially or substantially resistant to DPP activity may be produced by standard synthetic methods, recombinant DNA techniques, or any other method of preparing peptides and fusion proteins. May be prepared by

  [00112] Solid phase peptide synthesis methods are generally described in the following references: Merrifield, J. et al. Am. Chem. Soc. 888; 2149, 1963; Barany and Merrifield, the Peptides, E .; Gross and J.M. Meinenhofer, Academic Press, New York, 3: 285 (1980); B. H. Kent. Annu. Rev. Biochem. 57: 957 (1988). Solid phase peptide synthesis can produce peptides of the desired length and sequence through the stepwise addition of amino acids to the growing peptide chain that is covalently bound to solid resin particles. Automatic synthesis may be used in this method.

  [00113] As noted above, the modified polypeptides of the present invention can also be obtained by molecular biology techniques using nucleic acid sequences encoding those polypeptides. These sequences may be RNA or DNA and may be linked to regulatory sequences and / or inserted into a vector. The latter is then transfected into a host cell, such as a bacterium. Preparation of vectors and their production or expression in the host are performed by conventional molecular biology and genetic engineering techniques.

  [00114] Furthermore, the modified polypeptides of the invention can also be produced by recombinant techniques using DNA sequences that are readily synthesized in commercially available expression systems.

  [00115] The modified polypeptides of the present invention can be obtained by recombinant means comprising (a) culturing host cells under conditions that aid in polypeptide production; and (b) recovering the polypeptide. it can. These cells are cultured in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, shake flask culture, small scale or large scale fermentation (in laboratory or industrial fermenters) performed in a suitable medium and under conditions that allow the expression and / or isolation of the polypeptide. Cells can be cultured by continuous fermentation, batch fermentation, fed-batch fermentation, or solid phase fermentation). Culturing is carried out in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts using procedures known in the art (eg, references to bacteria and yeast; Bennett, JW and LaSure). , L. Edit, More Gene Manipulations in Fungi, Academic Press, California, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (eg, in the catalog of the American Type Culture Collection). If the modified polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the modified polypeptide is not secreted, it can be recovered from the cell lysate.

  [00116] By way of example, modified polypeptides or peptides of the present invention, including modified polypeptides or peptide fusion proteins, are disclosed in WO 0044772, which is incorporated herein by reference in its entirety. Can be made by fermentation methodologies.

  [00117] These modified polypeptides can be detected using methods known in the art specific for those polypeptides. These detection methods may include the use of specific antibodies that bind to specific receptors, the formation of enzyme products or loss of enzyme substrates, or the activity of specific receptors in cell-based assays. Can be detected. For example, enzymatic assays can be used to measure the activity of the modified polypeptide. The resulting modified polypeptide can be recovered by methods known in the art. For example, the modified polypeptide can be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or sedimentation.

  [00118] Polypeptides of the invention include, but are not limited to, chromatography (eg, ion exchange, affinity, hydrophobicity, chromatofocusing, and size exclusion), electrophoretic procedures (eg, preparative isoelectric focusing). Electrophoresis), differential solubility (eg, ammonium sulfate precipitation), SDS-PAGE or extraction can be purified by a variety of procedures (eg, Protein Purification, J.-C. Janson and Lars Ryden, VCH). Publishers, New York, 1989).

Fusion proteins and protein complexes
[00119] The present invention provides modified polypeptides or peptides attached to heterologous molecules by recombinant means or covalent bonds. The activity of a polypeptide or peptide modified by binding to a heterologous molecule, such as a plasma protein, is extended from days to weeks. In some cases, the administration of such a modified therapeutic polypeptide or peptide may be required only once during this period. Higher specificity can be achieved because the active compound is primarily bound to large molecules and is less likely to be taken up into cells and interfere with other physiological processes.

  [00120] In another embodiment, a modified polypeptide or peptide of the invention can be linked to a heterologous sequence by recombinant means to form a chimeric or fusion protein. Such chimeric or fusion proteins are partially or substantially protected from DPP cleavage and operably linked to a heterologous protein having an amino acid sequence that is not substantially homologous to the modified polypeptide or peptide. Includes modified polypeptides or peptides. “Operatively linked” indicates that the modified polypeptide or peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N-terminus or C-terminus of the modified polypeptide or peptide.

  [00121] In one embodiment, the fusion protein does not affect the activity of the modified polypeptide of the invention itself. For example, fusion proteins include, but are not limited to, enzymatic fusion proteins such as β-galactosidase fusions, yeast two hybrid GAL fusions, poly-His fusions, MYC tagged fusions, HI tagged fusions. , And Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant modified polypeptides. In a further embodiment, the fusion protein comprises an amino acid sequence between the modified peptide of the present invention and other moieties, said amino acid sequence being recognized to allow release of the modified peptide of the present invention after chemical or enzymatic cleavage. Gives an array. In certain host cells (eg, mammalian host cells), heterologous signal sequences can be used to increase protein expression and / or secretion. In another embodiment, the modified polypeptide or peptide is fused to a molecule that would increase its serum stability or prolong serum half-life, such as plasma proteins. Preferably, the modified polypeptide or protein is fused to a portion thereof such as serum albumin, immunoglobulin or Fc domain. More preferably, the modified polypeptide or peptide is fused to transferrin, lactotransferrin, melanotransferrin or a hybrid thereof. Methods for making such fusion proteins are described in US patent application Ser. Nos. 10 / 231,494 and 10 / 378,094, which are incorporated herein by reference in their entirety, and international application PCT / US03 /. Provided by 26818.

  [00122] The transferrin bound to the modified polypeptide or peptide may be modified as discussed in these patent applications. Thereby, reduced glycosylation may be shown. The modified transferrin polypeptide can be selected from the group consisting of a single transferrin N domain, a single transferrin C domain, the N and C domains of transferrin, two transferrin N domains, and two transferrin C domains. .

  [00123] When the C domain of Tf is part of a fusion protein, two N-linked glycosylation sites, N413 and N611 (SEQ ID NO: 3 of PCT / US03 / 26818, which are incorporated herein by reference in their entirety) ) Can be mutated for expression in yeast systems to prevent glycosylation or excessive mannosylation and to increase the serum half-life of fusion and / or therapeutic proteins. (Asialo-Tf or optionally monoasialo-Tf or disialo-Tf is produced). In addition to the Tf amino acids corresponding to N413 and N611, there may be mutations in adjacent residues within the NXS / T glycosylation site to prevent or substantially reduce glycosylation. See U.S. Pat. No. 5,986,067 to Funk et al. It has also been reported that the N domain of Tf expressed in Pichia pastoris is O-linked glycosylated by a single hexose at S32 and can be mutated or modified to prevent such glycosylation.

  [00124] Thus, in one embodiment of the invention, transferrin fusion proteins comprise modified transferrin molecules that exhibit reduced glycosylation, including but not limited to Tf asialo, monosialo and disialo. . In another embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin variant mutated to prevent glycosylation. In another embodiment, the transferrin portion of the transferrin fusion protein comprises a recombinant transferrin variant that is fully glycosylated. In another embodiment, the transferrin portion of the transferrin fusion protein comprises at least one of Asn413 and Asn611 (SEQ ID NO: 3 of PCT / US03 / 26818, which is hereby incorporated by reference in its entirety) and is glycosylated. Includes recombinant human serum transferrin mutants mutated to prevent glycosylation that are mutated to non-occurring amino acids. In another embodiment, the transferrin portion of the transferrin fusion protein is a recombinant human serum transferrin variant mutated to prevent or substantially reduce glycosylation, wherein the transferrin portion is within the NXS / T glycosylation site. Including recombinant human serum transferrin variants that may have these mutations at adjacent residues. Furthermore, glycosylation may be reduced or prevented by mutating serine or threonine residues. Furthermore, it is known that glycosylation is inhibited by changing X to proline.

  [00125] Chimeric or fusion proteins can be made by standard recombinant DNA techniques. For example, DNA fragments encoding different protein sequences are ligated together in frame according to conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, an anchor primer can be used to generate a complementary overhang between two consecutive gene fragments, followed by PCR amplification of the gene fragment that is annealed and reamplified to produce a chimeric gene sequence. (See Ausubel et al., 1992 Current Protocols in Molecular Biology). In addition, many expression vectors are commercially available that pre-code a fusion moiety (eg, a GST protein). Nucleic acids encoding modified polypeptides or peptides can be cloned into such expression vectors such that the fusion moiety is linked in-frame to the modified polypeptide or peptide.

  [00126] In another embodiment, the modified therapeutic polypeptide or peptide is conjugated via a covalent bond to a heterologous molecule to increase its stability and protection from DPP activity.

[00127] By way of example, the modified polypeptide or peptide is via a covalent bond formed between the reactive group of the modified polypeptide or peptide and a blood component, with or without a linking group. Binds to blood components. The blood component may be fixed or mobile. Examples of stationary blood components are immobile blood components such as tissues, membrane receptors, stromal proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells, and their associated membranes and membrane receptors. The body, somatic cells, skeletal and smooth muscle cells, neuronal components, bone cells and osteoclasts, and all body tissues, particularly those related to the circulatory and lymphatic systems. An example of a mobile blood component is a blood component that is not fixed in position for any long period of time, usually no more than 5 minutes, more typically 1 minute. These blood components are not bound to the membrane, are present in the blood for an extended period of time, and are present at a minimum concentration of at least 0.1 μg / ml. The mobile blood components include serum albumin, transferrin, immunoglobulins such as IgM and IgG, alpha ₁ protease inhibitor, antithrombin III and alpha ₂ - antiplasmin, and the like. The half-life of mobile blood components is typically at least about 12 hours.

  [00128] Formation of a covalent bond between the blood component and the modified therapeutic polypeptide or peptide can occur in vivo or ex vivo. For ex vivo covalent bond formation, the modified polypeptide or peptide is added to a physiological saline solution containing blood, serum, or blood components such as human serum albumin or IgG, and the modified polypeptide or Allows covalent bond formation between peptides and blood components. Alternatively, the modified polypeptide peptide may be modified with maleimide or a similarly highly reactive chemical group and reacted with a blood component in a physiological saline solution. After the modified therapeutic polypeptide or peptide is reacted with a blood component to form a conjugate of the modified polypeptide or peptide, the conjugate may be administered to the patient. Alternatively, the modified therapeutic polypeptide or peptide may be administered directly to the patient such that a covalent bond is formed in vivo between the modified therapeutic polypeptide or peptide and the blood component. The same reaction may also be performed using a recombinant protein such as albumin.

  [00129] Various sites where chemically reactive groups of non-specific modified therapeutic polypeptides or peptides can react in vivo include cells, particularly red blood cells (erythrocytes) and platelets, and IgG And immunoglobulins including IgM, serum albumin, ferritin, steroid binding protein, transferrin, thyroxine binding protein, α-2-macroglobulin and the like.

  [00130] The modified polypeptide or peptide may contain, or may be chemically modified to contain, a reactive group that binds to a thiol. In one embodiment of the invention, the modified polypeptide or peptide may be conjugated to polyethylene glycol. Alternatively, the modified polypeptide or peptide may bind to a glycolipid modified with polyethylene glycol or a fatty acid modified with polyethylene glycol.

  [00131] In one aspect, the modified polypeptide or peptide may be conjugated to a fatty acid or fatty acid derivative to improve its stability. Examples of fatty acids include, but are not limited to, lauric acid, palmitic acid, oleic acid, and stearic acid. Examples of fatty acid derivatives include ethyl ester, propyl ester, cholesteryl ester, coenzyme A ester, nitrophenyl ester, naphthyl ester, monoglyceride, diglyceride, triglyceride, fatty alcohol, fatty alcohol acetate and the like.

  [00132] In another embodiment, the modified polypeptide or peptide may be reshaped into a drug affinity complex (DAC ™). A drug affinity complex has three parts: a drug component responsible for biological activity; a linkage that binds the drug component to a reactive chemical group; and a persistent binding of the construct to a specific target protein in the body. It is a reactive chemical group on the terminal side opposite to the connecting portion. For example, Kim et al. (2003, Diabetes 52 (3): 751) disclose a GLP-1-albumin drug affinity complex. Kim et al. Showed that albumin-bound DAC: GLP-1 bound to the GLP-1 receptor (GLP-1R) and activated cAMP formation in heterologous fibroblasts expressing that receptor. Is shown. These results suggest that albumin-bound DAC: GLP-1 mimics natural GLP-1. Kim et al. Provide a new approach to activate GLP-1R signaling for a longer time.

  [00133] The modified polypeptide or drug affinity complex of a peptide is designed to be administered by subcutaneous injection and then rapidly and selectively binds to albumin in vivo. The formed bioconjugate has the same therapeutic activity and similar efficacy as the endogenous polypeptide or peptide, but has a pharmacokinetic profile in animals that is closer to that of albumin.

Pharmaceutical composition
[00134] The present invention relates to pharmaceutical compositions comprising modified therapeutic polypeptides and peptides that are partially or substantially protected from DPP cleavage but substantially retain functional activity and efficacy. provide. Such a pharmaceutical composition can be administered orally, parenterally, such as intravascular (IV), intraarterial (IA), intramuscular (IM), subcutaneous (SC), intraperitoneal, transdermal, etc. . Where appropriate, it may be administered by infusion. In some cases, administration may be oral, nasal, rectal, transdermal, or aerosol, with the modified polypeptide being transferred to the vasculature. For example, when a modified polypeptide of the invention is fused or conjugated to a transferrin moiety, binding to a transferrin receptor as described in International Application PCT / US03 / 26778, which is incorporated herein by reference in its entirety. Via this, the modified polypeptide can be transported to the vasculature or across the blood brain barrier. Usually a single injection is used, but multiple injections may be used if desired. The modified therapeutic polypeptide or peptide may be administered by any convenient means such as a syringe, trocar, catheter or the like. The particular mode of administration will vary depending on the amount administered, such as single bolus administration or continuous administration. Preferably, it is administered intravascularly, where the site of introduction is not critical to the invention, but is preferably a site with fast blood flow, such as intravenous, peripheral or central veins. More preferably, these pharmaceutical compositions are administered subcutaneously. Other routes where administration is in conjunction with sustained release techniques or a protective matrix may also be used. The purpose is that the modified therapeutic peptide or polypeptide is effectively distributed, for example in the blood, so that it can react with blood or tissue components.

  [00135] In general, the present invention provides an effective amount of a modified therapeutic polypeptide or peptide of the present invention together with a pharmaceutically acceptable diluent, preservative, solubilizer, emulsifier, adjuvant and / or carrier. A pharmaceutical composition comprising. Such compositions include various buffer contents (eg Tris-HCl, acetate, phosphate), pH and ionic strength diluents; surfactants and solubilizers (eg polysorbate 80), antioxidants Additives such as agents (eg, ascorbic acid, sodium metabisulfite), preservatives (eg, Thimersol, benzyl alcohol), and bulking substances (eg, lactose, mannitol); polymer compounds such as polylactic acid, polyglycolic acid Incorporation of the material into a particulate preparation or liposome. Hyaluronic acid may also be used, which may have the effect of promoting prolonged duration in the blood circulation. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the proteins and derivatives of the invention. See, for example, Remington's Pharmaceutical Sciences, 18th Edition (1990, Mack Publishing Co., Easton, PA, 18042), pages 1435-1712, incorporated herein by reference.

  [00136] For example, the modified therapeutic polypeptide or peptide is, for example, deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, It can be administered in a physiologically acceptable medium such as mannitol, aqueous glucose solution, alcohol, vegetable oil. Other additives that may be included generally buffer the medium to a pH in the range of about 5-10 and generally have a concentration in the range of about 50-250 mM, typically in the range of about 5-500 mM. Salts, physiologically acceptable stabilizers and the like. Examples of physiological buffers, particularly for injection, include Hank's solution and Ringer's solution. The transdermal formulation may include penetrants such as bile salts and fusidate.

  [00137] The pharmaceutical composition is a tablet or dragee, sublingual tablet, sachet, packet, soft gelatin capsule, suppository, cream, ointment, skin gel, transdermal device, aerosol, drinkable Ampules and ampoules for injection may be prepared. These compositions may be prepared in a liquid form or may be a dried powder, such as a lyophilized form convenient for storage and transport. Implantable sustained release formulations are also contemplated.

Oral dosage form
[00138] In one embodiment, the present invention is generalized in Remington's Pharmaceutical Sciences (1990), 18th edition, (Mack Publishing Co., Easton, PA, 18042), incorporated herein by reference. A pharmaceutical composition comprising a modified therapeutic polypeptide or peptide in an oral solid dosage form as described in. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets, or pellets. Encapsulation with liposomes or proteinoids may also be used to prepare the compositions of the invention (eg, as proteinoid microspheres reported in US Pat. No. 4,925,673). Encapsulation with liposomes can also be used and these liposomes may be derivatized with various polymers (eg, US Pat. No. 5,013,556). Possible solid dosage forms for therapeutic substances are described in G.C., which is incorporated herein by reference. S. Banker and C.I. T.A. Marshall, K., edited by Rhodes. , Modern Pharmaceuticals (1979). In general, the formulation comprises a modified therapeutic polypeptide or peptide and inert ingredients that allow protection from the gastric environment and release of the bioactive material in the intestine.

  [00139] If necessary, the modified therapeutic polypeptide or peptide may be chemically modified so that oral delivery is efficacious. In general, a contemplated chemical modification is the addition of at least one moiety to the modified therapeutic polypeptide or peptide itself, which allows uptake from the stomach or intestine into the bloodstream . It is also desirable to increase the overall stability of the compound and increase circulation time in the body. A moiety useful as a covalently attached vehicle in the present invention may also be used for this purpose. Examples of such moieties include PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, and polyproline. See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme Products, Enzymes as Drugs (1981), Hocenberg and Roberts, Ed. Wiley-Interscience, New York, New York, pp. 367-83; Appl. Biochem. 4: 185-9. Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-thioxocane. As indicated above, preferred for pharmaceutical use is the PEG moiety.

  [00140] Similarly, a modified therapeutic polypeptide or peptide may be recombinantly fused to another polypeptide to increase its overall stability or improve oral delivery. For example, a modified therapeutic polypeptide or peptide may be fused to transferrin, melanotransferrin or lactoferrin. Methods for making such fusion proteins are described in US patent application Ser. No. 10 / 378,094, which is incorporated herein by reference in its entirety.

  [00141] In the case of oral delivery dosage forms, modified fats such as sodium N- (8- [2-hydroxybenzoyl] amino) caprylate (SNAC) as carriers to facilitate absorption of the therapeutic compounds of the invention It is also possible to use amino acid salts. The clinical efficacy of heparin formulations using SNAC was demonstrated in a phase 2 clinical trial conducted by Emisphere Technologies. See US Pat. No. 5,792,451 “Oral drug delivery composition and methods”, which is incorporated herein by reference in its entirety.

  [00142] The modified therapeutic polypeptides or peptides of the invention can be included in the formulation as fine multiparticulates in the form of granules or pellets having a particle size of about 1 mm. The formulation of the material for capsule administration may be a powder, a lightly compressed filling, or even a tablet. The therapeutic substance can be prepared by compression.

  [00143] Colorants and flavoring agents can all be included. For example, a modified therapeutic polypeptide or peptide may be formulated (such as by encapsulation with liposomes or microspheres) and then further included in an edible product such as a refrigerated beverage containing colorants and flavoring agents. Good.

  [00144] An inert material may be used to dilute or increase the volume of the pharmaceutical composition of the invention. These diluents can include carbohydrates, particularly mannitol, cc-lactose, anhydrous lactose, cellulose, sucrose, modified dextran and starch. Several inorganic salts may also be used as bulking agents, including tricalcium phosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx1500, Emcompress and Avicell.

  [00145] A disintegrant may be included when the therapeutic agent is incorporated into the solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including Explotab, a commercially available disintegrant based on starch. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acidic carboxymethylcellulose, natural sponges and bentonite may all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered rubbers may be used as disintegrants and binders, which can include powdered rubbers such as agar, karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

  [00146] Binders may be used to group modified therapeutic polypeptides or peptides together to form hard tablets, including materials derived from natural products such as gum arabic, tragacanth gum, starch and gelatin. It is done. Others include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (MC). Both polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can be used in alcoholic solutions to granulate the therapeutic substance.

  [00147] A lubricant may be included in the formulation of the pharmaceutical composition of the present invention to prevent sticking during the formulation process. Lubricants may be used as a layer between the modified therapeutic polypeptide or peptide and the mold wall, including, but not limited to, stearic acid, polytetra Mention may be made of fluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycols of various molecular weights, Carbowax 4000 and 6000 may also be used.

  [00148] Glidants may be added to improve the fluidity of the modified therapeutic polypeptide or peptide in the formulation and to aid rearrangement during compression. These glidants can include starch, talc, pyrogenic silica and hydrous silicoaluminate.

  [00149] A surfactant may be added as a wetting agent to facilitate dissolution of the modified therapeutic polypeptide or peptide of the invention in an aqueous environment. Examples of the surfactant include anionic surfactants such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic surfactants may be used and include benzalkonium chloride or benzethonium chloride. A list of promising nonionic surfactants that can be included in the formulation as surfactants is Lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, Polysorbates 40, 60, 65 and 80, fatty acid esters of sucrose, methylcellulose, and carboxymethylcellulose. These surfactants can be present in the protein or derivative formulation alone or as a mixture in different ratios.

  [00150] Modified therapeutic polypeptides and additives to improve uptake of peptides may also be included in the formulation. Additives potentially having this property are, for example, the fatty acids oleic acid, linoleic acid and linolenic acid.

  [00151] Controlled release formulations may also be desirable. The modified therapeutic polypeptide or peptide of the present invention can be incorporated into an inert matrix, such as rubber, that allows release by either diffusion or leaching mechanisms. Slowly degrading matrices such as alginate and polysaccharides may also be incorporated into the formulation. Another form of controlled release of the compounds of the present invention is the Oros treatment system (Alza Corp.), a semi-water that allows water to enter and push the drug out of a single small pore by osmotic action. By a method based on a system in which a drug is encapsulated in a permeable membrane. Some enteric coatings also have a delayed release effect.

  [00152] Other coating agents may be used for the formulation. These include various sugars that can be applied in the coating pan. Modified therapeutic polypeptides or peptides can also be given in film-coated tablets, in which case the materials used are divided into two groups. The first is a non-enteric material that includes methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxy-ethylcellulose, hydroxypropylcellulose, hydroxypropyl-methylcellulose, sodium carboxy-methylcellulose, providone, and polyethylene glycol. The second group consists of enteric materials that are generally esters of phthalic acid.

  [00153] Mixtures of materials may be used to provide optimal film coating. Film coating can be performed in a pan coating apparatus or fluidized bed, or by compression coating.

Pulmonary delivery form
[00154] In another embodiment, the invention also provides a pharmaceutical composition comprising a modified therapeutic polypeptide or peptide for pulmonary delivery. This pharmaceutical composition is delivered to the lungs of a mammal during inhalation and passes through the epithelial layer of the lungs into the bloodstream.

  [00155] The present invention is a variety designed to deliver therapeutic products to the lung, including but not limited to nebulizers, metered dose inhalers and powder inhalers, all well known to those skilled in the art. Provide the use of special machinery. Some specific examples of commercially available instruments suitable for practicing the present invention are described in Mallinckrodt, Inc. Ultravent nebulizer from St. Louis, MO; Acorn II nebulizer from Marquest Medical Products, Englewood, Colorado; Glaxo, Inc. Ventolin metered dose inhaler manufactured by Research Triangle Park, NC; and Fisons Corp. Spinbaler powder inhaler manufactured by the company (Bedford, Massachusetts).

  [00156] All such devices require the use of modified therapeutic polypeptides and formulations suitable for the administration of peptides. Typically, each formulation is specific to the type of device used and may involve the use of a suitable spray material in addition to diluents, adjuvants and / or carriers useful for therapy.

  [00157] The modified therapeutic polypeptide or peptide most advantageously has an average particle size of less than 10 μm, most preferably 0.5-5 μm, for the most effective delivery to the distal lung Should be prepared in particulate form.

  [00158] Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in the formulation can include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may also be used (further apart from its use in derivatizing proteins or analogs). Dextrans such as cyclodextran may also be used. Bile salts and other related accelerators may also be used. Cellulose and cellulose derivatives may also be used. Amino acids may also be used, such as use in buffer formulations.

  [00159] The use of liposomes, microcapsules or microspheres, inclusion complexes or other types of carriers is also contemplated.

  [00160] Formulations suitable for use with jet or ultrasonic nebulizers typically dissolve in water at a concentration of about 0.1 to 25 mg of bioactive protein per mL of solution. Containing a compound of the present invention. The formulation may also include a buffer and a monosaccharide (eg, for protein stabilization and osmotic adjustment). The nebulizer formulation may also include a surfactant to reduce or prevent surface-induced protein aggregation caused by atomization of the solution in forming the aerosol.

  [00161] Formulations for use with metered dose inhalers generally comprise a finely divided powder comprising a compound of the invention suspended in a propellant with the aid of a surfactant. Propellants include chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or hydrocarbons, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, or combinations thereof Any conventional material used for this purpose may be used. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid can also be useful as a surfactant.

  [00162] A formulation for dispensing from a powder inhaler comprises a finely divided dry powder comprising a compound of the invention, and a bulking agent such as lactose, sorbitol, sucrose, mannitol, trehalose or xylitol from the device. An amount that promotes dispersion of the powder may be included, for example, in an amount of about 50-90% by weight of the formulation.

Nasal delivery form
[00163] Nasal delivery of a modified polypeptide or peptide pharmaceutical composition of the invention is also contemplated. Nasal delivery allows the protein to move directly into the bloodstream after the modified therapeutic polypeptide or peptide is administered to the nose, without the need to deposit the product in the lungs. Formulations for nasal delivery include those containing dextran or cyclodextran. Delivery via transport across other mucous membranes is also disclosed.

Dosage
[00164] The dosing regimen used in the method for treatment of the aforementioned conditions may include various factors that alter the action of the drug, such as the patient's age, condition, weight, sex and diet, and any infections. Determined by the attending physician, taking into account severity, time of administration, and other clinical factors. In general, the daily dosage should be in the range of 0.01 to 1000 micrograms of a compound of the invention per kilogram of body weight, preferably 0.1 to 150 micrograms per kilogram.

Treatment of diseases using modified therapeutic proteins
[00165] The present invention provides various transferrin fusion proteins that can be used in the treatment of various diseases. For example, a pharmaceutical composition comprising a fusion polypeptide or peptide of the present invention includes, but is not limited to, insulin resistance, hyperglycemia, hyperinsulinemia, or high blood levels of free fatty acids or glycerol, hyperlipidemia Disease, obesity, syndrome X, metabolic syndrome, inflammation, diabetic complications, glucose homeostasis, impaired glucose tolerance, type II diabetes, prediabetes, hypertriglyceridemia, atherosclerosis, nervous system disorder, congestion Can be used to treat diseases such as congenital heart failure, dyspepsia and irritable bowel syndrome. Modified polypeptides and peptides can also be used to activate the CNS or to induce an anxiolytic effect on the CNS for post-surgical treatment.

  [00166] The modified therapeutic polypeptides and peptides of the present invention are fused to transferrin or modified transferrin, or are partially or substantially protected from DPP activity, so that unmodified therapeutic polypeptides And more stable in vivo than peptides. Thus, smaller amounts of molecules may be administered for effective treatment. At low dosages, side effects can be reduced in some cases.

  [00167] In one embodiment, the modified therapeutic polypeptides and peptides of the present invention may be used as sedatives. Accordingly, the present invention provides a method of sedating a mammalian subject having an abnormality that results in increased central or peripheral nervous system activation using a modified polypeptide or peptide of the present invention. The method includes administering to the subject an amount of a modified therapeutic polypeptide or peptide sufficient to provide a sedative or anxiolytic effect to the subject. The modified therapeutic polypeptide or peptide may be administered intraventricularly, orally, subcutaneously, intramuscularly or intravenously. Such methods are useful for treating or ameliorating nervous system conditions such as anxiety, movement disorders, psychosis, aggression, seizures, panic attacks, hysteria and sleep disorders.

  [00168] Furthermore, the present invention also provides a method for increasing the activity of a mammalian subject comprising administering to the subject an amount of a modified therapeutic polypeptide or peptide sufficient to produce an activating effect on the subject. Include. The subject has a condition that results in reduced activation of the central or peripheral nervous system. Modified therapeutic polypeptides or peptides may include depression, schizophrenia, sleep apnea, poor concentration of attention deficit disorder to name a few situations where central nervous system arousal can be beneficial It is useful for treating or improving memory loss, amnesia and narcolepsy.

  [00169] Insulin resistance that occurs after certain types of surgery, palliative abdominal surgery, is most severe on the first day after surgery, lasts at least 5 days, and can take up to 3 weeks to normalize is there. Thus, post-operative patients may require administration of the modified insulin secretagogue peptide of the invention for a period of time following surgical trauma. Accordingly, the modified therapeutic polypeptides or peptides of the present invention can be utilized for post-surgical treatment. Patients need the modified insulin secretagogue peptides of the present invention for about 1 to 16 hours before surgery is performed on the patient, during the patient's surgery, and for a period of about 5 days or less after the patient's surgery.

  [00170] In addition, the modified therapeutic polypeptides and peptides of the present invention, such as insulin secretagogue peptides, may be utilized to treat insulin resistance separately from use in post-surgical treatment. Insulin resistance can be due to decreased binding of insulin to cell surface receptors or changes in intracellular metabolism. The first type, characterized as a decrease in insulin sensitivity, can usually be overcome by an increase in insulin concentration. The second type, characterized as reduced insulin responsiveness, cannot be overcome by large amounts of insulin. The insulin resistance that occurs following trauma can be overcome by doses of insulin proportional to the degree of insulin resistance and thus appears to be caused by a decrease in insulin sensitivity.

  [00171] Preferably, the present invention provides modified insulin secretagogue peptides for normalizing hyperglycemia through glucose-dependent, insulin-dependent, and insulin-independent mechanisms. Thus, the modified insulin secretagogue peptide is useful as a primary agent for the treatment of diabetes, particularly type II diabetes. The present invention relates to type I in that the action of the peptide is dependent on blood glucose concentration and thus the risk of side effects of hypoglycemia is greatly reduced than the risk of using current treatment methods. Particularly suitable for the treatment of patients with both type II and type II diabetes.

  [00172] The dose of a modified insulin secretagogue peptide effective to normalize a patient's blood glucose level may depend on several factors, including, but not limited to, the patient Gender, weight, and age, severity of blood glucose dysregulation, causes causing blood glucose dysregulation, whether glucose or another carbohydrate source is administered simultaneously, route of administration and biological use Performance, persistence in the body, formulation, and efficacy.

  [00173] Preferably, the modified therapeutic peptide of the present invention, such as an insulin secretagogue peptide, is impaired glucose tolerance, diabetes, hyperlipidemia, metabolic acidosis, diabetes, diabetic neuropathy and kidney Used for the treatment of disorders. More preferably, the modified peptide is modified GLP-1 and analogs thereof for the treatment of type II diabetes.

Monitoring the presence of modified therapeutic polypeptides and peptides
[00174] Modified therapeutic polypeptides and peptides can be monitored using assays to measure functional activity, HPLC-MS, or antibodies that target polypeptides or peptides. For example, the blood of a mammalian host can be monitored for the activity of the modified therapeutic polypeptide or peptide and / or the presence of the modified therapeutic polypeptide or peptide. Whether the modified therapeutic polypeptide or peptide has been bound to a long-lived blood component in an amount sufficient to have therapeutic activity by taking portions or samples of the host's blood at various time points, and The subsequent level of the modified therapeutic polypeptide or peptide in the blood can then be measured. If desired, it can also be determined to which blood component the modified therapeutic polypeptide or peptide, such as a modified insulin secretagogue peptide, is bound.

  [00175] As an example, assays for insulin secretagogue activity may be used to monitor the modified insulin secretagogue peptides of the invention. The modified insulin secretagogue peptide of the present invention has an insulin secretagogue activity that is at least equal to the insulin secretagogue activity of the unmodified insulin secretagogue peptide. The insulin secretion-promoting properties of the modified insulin secretagogue peptide are determined by providing the modified peptide to animal cells or injecting the peptide into the animal and monitoring the release of immunoreactive insulin into the culture medium or the animal's circulatory system, respectively. Can be measured. The presence of immunoreactive insulin is detected by use of a radioimmunoassay that can specifically detect insulin. Any radioimmunoassay capable of detecting the presence of IRI may be used, although Albano, J. et al., Which is incorporated herein by reference in its entirety. D. M.M. In addition, it is preferable to use a modified method of the assay method of (1972 Acta Endocrinol. 70: 487-509).

  [00176] The insulinotropic properties of a modified therapeutic polypeptide or peptide can also be measured by pancreatic infusion (Penhos, JC et al. 1969 Diabetes, incorporated herein by reference). 18: 733-738). The manner in which perfusion is performed, improved, and analyzed is described in Weir, G. et al., Incorporated herein by reference. C. Et al. (J. Clin. Investigat. 54: 1403-1412 (1974)).

  [00177] As is well known to those skilled in the art, HPLC coupled with mass spectrometry (MS) can also be used to analyze the presence of modified therapeutic polypeptides and peptides. Typically, two mobile phases are used, such as 0.1% TFA / water and 0.1% TFA / acetonitrile. The column temperature as well as the gradient conditions can also be changed.

  [00178] Another method for monitoring the presence of modified therapeutic polypeptides and peptides is to use antibodies specific for those modified therapeutic polypeptides and peptides. The use of monoclonal or polyclonal antibodies with specificity for a particular modified therapeutic polypeptide or peptide can help solve such problems. An antibody is made or derived from a host immunized with a specific modified therapeutic polypeptide or peptide, or with a synthetic immunogen corresponding to an immunogenic fragment of the agent or an antigenic determinant of the agent Can do. Preferred antibodies will have high specificity and affinity for the modified therapeutic polypeptide or peptide. Such antibodies can also be labeled with enzymes, fluorescent dyes, or radiolabels.

  [00179] These antibodies may be used to monitor the presence of modified therapeutic polypeptides and peptides in the bloodstream. Blood and / or serum samples may be analyzed by SDS-PAGE and Western blotting. Such techniques allow blood or serum analysis to measure the binding of the modified therapeutic polypeptide or peptide to blood components.

Glucagon-like peptide-1 (GLP-1)
[00180] Recombinant DNA technology was used to create new molecules with increased stability and biological activity. These molecules are a combination of biologically active proteins and peptides fused to stabilizing proteins with naturally long half-lives such as the Fc portion of immunoglobulins, albumin, and transferrin. These fusion molecules retain the biological activity of the active moiety with much longer pharmacokinetics than the unfused natural protein or peptide counterpart. Increased pharmacokinetics also improve biological activity, reduce unwanted side effects, and improve patient convenience. There are many examples of such fusion proteins such as interferon-albumin, interferon-Fc, BNP-albumin, GLP-1-albumin, GLP-1-transferrin.

  [00181] Although fusion proteins are stable and resistant to degradation, potential protease mechanisms that degrade the active moiety can result in slow inactivation of the molecule. Specifically, GLP-1, dynorphin (Berman YL, Juliano L, Devi LA J Biol Chem. Oct. 6, 1995; 270: 23845-50), enkephalin (Gu ZF, Menozzi D, Okamoto A, Maton PN, Bunnett NW Exp Physiol, January 1993; 78: 35-48), many peptides such as BNP, ANP, angiotensin, bradykinin, and PYY are proteases such as dipeptidyl peptidase IV and neutral endopeptidase. Very sensitive to These proteases, either individually or in combination, cause rapid inactivation of circulating peptides. The fusion of peptides to large proteins such as albumin, Fc, and transferrin provides significant resistance to proteases. However, the inactivation effect by protease cannot be completely excluded. Thus, the fusion protein and protease inhibitor combination may have a superior PK and PD over the fusion protein alone.

  [00182] The present invention provides a transferrin fusion protein comprising a therapeutic peptide that is resistant to proteases. Preferably, the modified therapeutic peptide of the present invention is a modified insulin secretagogue peptide that is partially or substantially protected from DPP activity. More preferably, the modified insulin secretagogue peptide is a modified GLP-1 peptide and analogs and fragments thereof. Modified GLP-1 peptides and analogs and fragments thereof are useful for treating diabetes, specifically type II diabetes. The N-terminal sequence of wild type GLP-1 is His-Ala-Glu. The modified GLP-1 polypeptides of the present invention are His-His-Ala-Glu (SEQ ID NO: 115), Gly-His-Ala-Glu (SEQ ID NO: 116), His-Gly-Glu, His-Ser-Glu, His. -Ala-Glu, His-Gly-Glu, His-Ser-Glu, His-His-Ala-Glu (SEQ ID NO: 82), His-His-Gly-Glu (SEQ ID NO: 83), His-His-Ser-Glu (SEQ ID NO: 84), Gly-His-Ala-Glu (SEQ ID NO: 85), Gly-His-Gly-Glu (SEQ ID NO: 86), Gly-His-Ser-Glu (SEQ ID NO: 87), His-X- Ala-Glu, His-X-Gly-Glu, and His-X-Ser-Glu (where X is any amino acid) It may include an N-terminal sequence selected from the group consisting of. As described below, other modifications may be made to reduce and prevent protease degradation, and these molecules may be used in the methods and compositions of the invention. Furthermore, any GLP-1 moiety or GLP-1 analog, derivative or mimetic (as a fusion protein) may be used in the methods and compositions of the invention.

  [00183] By adding one amino acid to the N-terminus of GLP-1, steric hindrance can contribute to prevent dipeptidyl peptidase from cleaving the second amino acid of GLP-1. . Therefore, GLP-1 is believed to remain functionally active. Any one of 20 amino acids or unnatural amino acids may be added to the N-terminus of GLP-1. Histidine is also a preferred amino acid. In some cases, uncharged or positively charged amino acids may be used, preferably with smaller amino acids such as glycine added. The modified GLP-1 with extra amino acids can then be fused to transferrin to make a fusion protein. In one embodiment, the GLP-1 peptide is modified to include at least one additional amino acid at its amino terminus. In another embodiment, the GLP-1 peptide is modified to include at least 5 additional amino acids at its amino terminus. Alternatively, the GLP-1 peptide is modified to contain 1 to 5 additional amino acids at its amino terminus.

  [00184] Glucagon-like peptide-1 (GLP-1) is a gastrointestinal hormone that regulates insulin secretion, belonging to the so-called enteropancreatic system. The intestinal pancreatic system refers to a group of hormones that are released from the gastrointestinal mucosa in response to the presence and absorption of nutrients in the intestine that promote early and enhanced release of insulin. The incretin effect, which is a stimulatory effect on insulin secretion, is probably essential for normal glucose tolerance. Since GLP-1 is responsible for the incretin effect, it is a physiologically important insulin secretion-promoting hormone.

  [00185] GLP-1 is the product of the proglucagon gene (Bell et al., Nature, 1983, 304; 368-371). It is synthesized in the intestinal endocrine cells as the two major molecular forms, GLP-1 (7-36) amide and GLP-1 (7-37). This peptide was first identified in the early 1980's following cloning of proglucagon cDNA and genes.

[00186] Early studies performed on the full-length peptide GLP-1 (1-37 and 1-36 ^amide ) concluded that the large GLP-1 molecule lacks biological activity. In 1987, three separate research groups demonstrated that removal of the first six amino acids resulted in GLP-1 molecules with improved biological activity.

[00187] The amino acid sequence of GLP-1 is disclosed by Schmidt et al. (1985 Diabetologia 28 704-707). Human GLP-1 is a 37 amino acid residue peptide derived from preproglucagon synthesized in L cells in the distal ileum, in the pancreas and in the brain. Processing of preproglucagon into GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2 occurs mainly in L cells. The amino acid sequence of GLP-1 (7-37) is SEQ ID NO: 32 (X = Gly), ie
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Lys-Gly-Arg-Gly.
In GLP-1 (7-36) amide, the terminal Gly is replaced by NH ₂ .

  [00188] GLP-1-like molecules have anti-diabetic activity in human subjects suffering from type II (non-insulin dependent diabetes mellitus (NIDDM)) and in some cases also type I diabetes. Treatment with GLP-1 only leads to activities such as increased insulin secretion and biosynthesis, reduced glucagon secretion, delayed gastric emptying, etc., only at high glucose levels, and therefore potentially far more than insulin or sulfonylureas. Provide safe therapy. The patient's postprandial glucose levels can be brought close to normal levels using appropriate GLP-1 therapy. There are also reports suggesting that GLP-1-like molecules have the ability to protect and even repair pancreatic β-cell function in type II patients.

  [00189] One or more at its amino terminus, including GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36) and GLP-1 (7-37) Any GLP-1 sequence can be modified by adding GLP-1 also has a strong action on the gastrointestinal tract. When infused in physiological amounts, GLP-1 potently inhibits pentagastrin-induced gastric acid secretion as well as diet-induced gastric acid secretion (Schjordager et al., Dig. Dis. Sci. 1989, 35 : 703-708; Wettergren et al., DigDis Sci 1993; 38: 665-673). It also suppresses gastric emptying rate and pancreatic enzyme secretion (Wettergren et al., Dig Dis Sci 1993; 38: 665-673). Perfusion of the ileum with solutions containing carbohydrates or lipids can induce similar inhibitory effects on gastric and pancreatic secretion and motility in humans (Layer et al., Dig Dis Sci 1995, 40: 1074-1082; Layer et al., Digestion 1993, 54: 385-38). Concomitantly, it was speculated that GLP-1 secretion was greatly stimulated and that GLP-1 may be involved, at least in part, in this so-called “ileal-brake” (Layer et al., Digestion 1993). Year 54: 385-38). Indeed, recent studies suggest that physiologically, the ileal braking effect of GLP-1 may be much more important than its effect on islets. Thus, in a dose-response study, GLP-1 affects gastric emptying rates at an infusion rate that is at least as slow as that required to affect islet secretion (Nuck et al., Gut 1995; 37 ( Addendum 2): A page 124).

[00190] GLP-1 appears to have an impact on food intake. Intracerebroventricular administration of GLP-1 significantly suppresses food intake in rats (Schick et al., Ditschuneit et al., Obesity in Europe, John Libey & Company Ltd, 1994; 363-367; Turton et al., Nature 1996, 379: 69-72). This effect appears to be very specific. Thus, NLP-extended GLP-1 (1-36 ^amide ) is inactive, and an appropriate dose of GLP-1 antagonist exendin 9-39 abolishes the effects of GLP-1 ( Tang-Christensen et al., Am. J. Physiol., 1996, 271 (4Pt2): R848-56). Acute peripheral administration of GLP-1 does not inhibit food intake in rats (Tang-Christensen et al., Am. J. Physiol., 1996, 271 (4Pt2): R848-56; Turton Et al., Nature 1996, 379: 69-72). However, there is still the possibility that GLP-1 secreted from intestinal L cells can also act as a satiety signal.

  [00191] In diabetic patients, the effect of GLP-1 on promoting insulin secretion and the effect of GLP-1 on the gastrointestinal tract has been maintained (Willms et al., Diabetelogia 1994; 37, Addendum 1: A118), While it can contribute to reducing the induced glucose level deviation, more importantly it can also affect food intake. Continuous intravenous administration of GLP-1 at 4 ng / kg / min for 1 week has been shown to dramatically improve glycemic control in NIDDM patients without significant side effects (Larsen et al., Diabetes 1996). Year; 45, Addendum 2: 233A).

  [00192] Modified GLP-1 and modified GLP-1 analogs that are partially or substantially protected from DPP activity are useful in the treatment of type 1 and type 2 diabetes and obesity.

  [00193] As used herein, the term "GLP-1 molecule" means GLP-1, a GLP-1 analog, or a GLP-1 derivative.

[00194] As used herein, the term "GLP-1 analog" is used as a molecule having one or more amino acid substitutions, deletions, inversions, or additions relative to GLP-1. Defined. Many GLP-1 analogs are known in the art, such as GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), Val ⁸ -GLP- ^{1 (7-37), Gln 9 -GLP1} (7-37), D-Gln 9 -GLP-1 (7-37), Thr 16 -Lys 18 -GLP-1 (7-37), and ^{Lys 18} - GLP-1 (7-37) (SEQ ID NO: 72) can be mentioned. US Pat. No. 5,118,666 discloses examples of GLP-1 analogs such as GLP-1 (7-34) and GLP-1 (7-35).

  [00195] The term "GLP-1 derivative" has the amino acid sequence of GLP-1 or a GLP-1 analog, but is one of its amino acid side chains, alpha-carbon atoms, terminal amino groups or terminal carboxylic acid groups. Alternatively, it is defined as a molecule further having a plurality of chemical modifications. Chemical modifications include, but are not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.

  [00196] As used herein, the term "compound related to GLP-1" means any compound that falls within the definition of GLP-1, GLP-1 analogs or GLP-1 derivatives. To do.

  [00197] WO 91/11457 discloses analogs of active GLP-1 peptides 7-34, 7-35, 7-36 and 7-37 that may also be useful as GLP-1 moieties. ing.

[00198] In EP 0708179-A2 (Eli Lilly & Co.), a GLP-1 analog comprising an N-terminal imidazole group and optionally an unbranched C ₆ -C ₁₀ acyl group attached to a lysine residue at position 34 Bodies and derivatives are disclosed.

  [00199] EP 0699686-A2 (Eli Lilly & Co.) discloses several N-terminal truncated fragments of GLP-1 that have been reported to have biological activity.

  [00200] In US Pat. No. 5,545,618, essentially GLP-1 (7-34), GLP-1 (7-35), GLP-1 () having at least one modification selected from the group consisting of: 7-36), or a GLP-1 molecule consisting of GLP-1 (7-37), or an amide form thereof, and a pharmaceutically acceptable salt thereof: (a) positions 26 and / or 34 Glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine; or arginine at position 36 is glycine, serine, cysteine, Threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, Substitution with methionine, phenylalanine, lysine or D-arginine (SEQ ID NO: 73); (b) substitution of tryptophan at position 31 with an oxidation resistant amino acid (SEQ ID NO: 74); (c) substitution of valine at position 16 with tyrosine Replacing serine at position 18 with lysine; replacing glutamic acid at position 21 with aspartic acid; replacing glycine at position 22 with serine; replacing glutamine at position 23 with arginine; replacing alanine at position 24 with arginine; and position 26 And (d) alanine at position 8 is replaced with glycine, serine, or cysteine; glutamic acid at position 9 is aspartic acid, glycine, serine, Cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, a Replacing leucine, leucine, methionine, or phenylalanine; replacing glycine at position 10 with serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine; and aspartic acid at position 15 Substitution with glutamic acid (SEQ ID NO: 76); and (e) histidine at position 7 is glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, Or substituted with phenylalanine, or a D- or N-acylated or alkylated form of histidine (SEQ ID NO: 77); wherein (a), (b), (d), and (e) Well D type sometimes amino acids conversion is and the amino acid to be substituted in position 7 may be a N-acylated-type or N-alkylated form in some cases.

  [00201] US Pat. No. 5,118,666 discloses GLP-1 molecules having insulin secretagogue activity. Such a molecule has the amino acid sequence His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu- Phe-Ile-Ala-Trp-Leu-Val-Lys (GLP-1, 7-34, see SEQ ID NO: 32) or His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val -Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly (GLP-1, 7-35, SEQ ID NO: 32 Selected from the group consisting of peptides having derivatives thereof, and derivatives of said peptides, wherein The peptide comprises a pharmaceutically acceptable acid addition salt of the peptide; a pharmaceutically acceptable carboxylate of the peptide; a pharmaceutically acceptable lower alkyl ester of the peptide; and an amide, a lower alkyl amide, and a lower dialkyl amide. Selected from the group consisting of pharmaceutically acceptable amides of the peptide.

[00202] US Pat. No. 6,277,819 teaches a method of reducing mortality and morbidity after myocardial infarction comprising administering to a patient GLP-1, a GLP-1 analog, and a GLP-1 derivative. ing. Following structural formula (SEQ ID _{**): R 1 -X 1 -Glu} -Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-X 2 -Gly-Gln-A1a- _{A1a-Lys-X 3 -Phe-} Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R 2 ( SEQ ID NO: 78) wherein, _{R 1} is L- histidine, D- histidine, desamino - histidine , 2-amino-histidine, β-hydroxy-histidine, homohistidine, α-fluoromethyl-histidine and α-methyl-histidine; X ₁ is Ala, Gly, Val, Thr, Ile, and α - it is selected from the group consisting of methyl -Ala; _{X 2} is Glu, Gln, Ala, Thr, from the group consisting of Ser and Gly Is-option; _{X 3} is Glu, Gln, Ala, Thr, selected from the group consisting of Ser and Gly; _{R 2} is selected from the group consisting of NH ₂ and Gly-OH. However, the GLP-1 analog has an isoelectric point in the range of about 6.0 to about 9.0, R ₁ is His, X ₁ is Ala, and X ₂ is Glu. , X ₃ is Glu, R ₂ must be NH ₂ . And a pharmaceutically acceptable salt thereof.

[00203] Ritzel et al. (Journal of Endocrinology, 1998, 159: 93-102), [Ser ⁸ ] GLP-1 is a GLP-1 analog in which the second alanine from the N-terminus is replaced by serine. Disclosure. This modification did not reduce the insulin secretagogue of the peptide, but produced an analog with higher plasma stability compared to GLP-1.

  [00204] In US Pat. No. 6,429,197, GLP-1 treatment after acute stroke or bleeding, preferably intravenous administration, optimizes insulin secretion, increases brain anabolism, and suppresses glucagon Teaching that it can be an ideal treatment to increase the sexiness and realize the means to maintain normoglycemia or mild hypoglycemia without the risk of severe hypoglycemia and other harmful side effects Yes. The present invention optimizes insulin secretion and suppresses glucagon antagonism after acute stroke or bleeding to increase insulin efficacy and maintain normoglycemia or mild hypoglycemia without the risk of severe hypoglycemia To do so, a method for treating ischemic or reperfused brain with GLP-1 or an analog thereof having biological activity is provided.

  [00205] In US Pat. No. 6,277,819, a compound selected from the group consisting of GLP-1, GLP-1 analogs, GLP-1 derivatives and pharmaceutically acceptable salts thereof is used to normalize blood glucose. A method is provided for reducing mortality and morbidity after myocardial infarction comprising administering to a patient in need thereof at a dose that is effective.

  [00206] In US Pat. No. 6,191,102, a period of at least 4 weeks effective to effect weight loss, glucagon-like peptide (GLP-1), glucagon-like peptide analog (GLP-1 analog), glucagon Administering a composition comprising a peptide-like peptide derivative (GLP-1 derivative) or a pharmaceutically acceptable salt thereof to a subject in need of weight loss at a dose sufficient to cause weight loss. A method of reducing weight is disclosed.

  [00207] GLP-1 is sufficiently active after subcutaneous administration (Ritzel et al., Diabetologia 1995; 38: 720-725), but is rapidly degraded primarily due to degradation by dipeptidyl peptidase IV-like enzymes (Deacon et al., J Clin Endocrinol Metab 1995, 80; 952-957; Deacon et al., 1995, Diabetes 44: 1126-1131). Thus, unfortunately, the plasma half-life in many humans of GLP-1 and its analogs is short (Orskov et al., Diabetes 1993; 42; 658-661). Accordingly, it is an object of the present invention to provide a modified GLP-1 or analog thereof having a longer action profile than GLP-1 (7-37). It is a further object of the present invention to provide derivatives of GLP-1 and its analogs that have a lower clearance than GLP-1 (7-37). Furthermore, it is also an object of the present invention to provide a pharmaceutical composition comprising a modified GLP-1 or GLP-1 analog with improved stability. Furthermore, the present invention includes the use of modified GLP-1 or GLP-1 analogs to treat diseases associated with GLP-1, such as, but not limited to, those described above.

  [00208] In one aspect of the invention, pharmaceutical compositions comprising modified GLP-1 and GLP-1 analogs are prepared, for example, as described in Remington's Pharmaceutical Sciences, 1985. Can be prepared by any of the established methods. The composition may be in a form suitable for systemic injection or infusion and may therefore be prepared with a suitable liquid vehicle such as sterile water, isotonic saline or glucose solution. These compositions may be sterilized by conventional sterilization techniques well known in the art. The resulting aqueous solution may be placed in a container for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being mixed with a sterile aqueous solution prior to administration. The The composition may optionally contain pharmaceutically acceptable auxiliary substances such as buffers, isotonic agents, eg sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc., in order to approximate physiological conditions. May be included.

  [00209] The modified GLP-1 and GLP-1 analogs of the present invention may be adapted for nasal, transdermal, pulmonary or rectal administration. The pharmaceutically acceptable carrier or diluent used in the composition may be any conventional solid carrier. Examples of solid carriers are lactose, clay, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

  [00210] It may be particularly advantageous to provide the compositions of the invention in the form of sustained release formulations. Thus, modified GLP-1 or GLP-1 encapsulated by or dispersed in a suitable pharmaceutically acceptable biodegradable polymer such as polylactic acid, polyglycolic acid, or lactic acid / glycolic acid copolymer The composition may be prepared as microcapsules or microparticles containing the analog.

  [00211] For nasal administration, the preparation may comprise a modified GLP-1 or GLP-1 analog dissolved or suspended in a liquid carrier, particularly an aqueous carrier, for aerosol application. The carrier may contain additives such as solubilizers such as propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) and cyclodextrins or preservatives such as parabens.

  [00212] Generally, a modified polypeptide or peptide of the invention is dosed in unit dosage form with a pharmaceutically acceptable carrier per unit dosage.

  [00213] Furthermore, the present invention provides a modified GLP- for the manufacture of a pharmaceutical product that can be used in the treatment of diseases with high glucose levels (metabolic diseases) such as, but not limited to, those described above. The use of 1 and GLP-1 analogs is also contemplated. Specifically, the present invention relates to modified GLP-1 and GLP-1 analogs for the treatment of diabetes, including type II diabetes, obesity, severe burns, and heart failure, including congestive heart failure, and acute coronary syndromes. Contemplate use of the body.

  [00214] The present invention also provides modified exendin-3 and exendin-4 peptides that are partially and substantially protected from DPP activity. Exendin-3 and exendin-4 are insulin secretion-promoting peptides containing 39 amino acids (the second and third residues are different) and have about 53% homology to GLP-1. . The exendin-3 sequence is HSDGFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 79) and the exendin-4 sequence is HGEGFTSDLSKSKMEEEAVRLFIEWLKNGGPSSGAPPPS (SEQ ID NO: 80). The invention also encompasses a modified exendin-4 fragment comprising an amino acid sequence such as exendin-4 (1-31) HGEGFTSDDLSKQMEEAVRLFIEWLKNGGPY (SEQ ID NO: 81). Furthermore, the present invention includes modified analogs of exendin-3 and exendin-4 peptides.

Modified GLP-1 fusion protein or conjugate for treating diabetes, prediabetes, or obesity
[00215] The modified GLP-1 may be fused to a heterologous molecule to increase the overall stability in vivo. The modified GLP-1 can be fused to the heterologous molecule by recombinant means or covalently linked to the heterologous molecule by methods well known in the art. The modified GLP-1 can be fused or covalently bound to, for example, a plasma protein such as serum albumin or transferrin, a portion thereof such as an immunoglobulin or Fc domain. More preferably, the modified polypeptide or peptide is fused to transferrin, lactotransferrin or melanotransferrin. A method for making such fusion proteins is provided by US patent application Ser. No. 10 / 378,094, which is incorporated herein by reference in its entirety.

[00216] Provides greater physical distance and gives greater spatial mobility between their fusion proteins, thereby maximizing the accessibility of therapeutic proteins, for example to bind to their cognate receptors To do so, the GLP-1 molecule may be attached to the heterologous protein via a linker of variable length. The linker peptide can consist of flexible or harder amino acids. For example, a linker such as polyglycine stretch may be used. The linker may be less than about 50, 40, 30, 20, 10, 10 or 5 amino acid residues. The linker can be covalently linked to and between the heterologous protein and GLP-1. Preferably, the linker is combined with 1 Ser residue, 2 Ser residues, peptide Ser-Ser-Gly, peptide PEAPTD, peptide (PEAPTD) ₂ , peptide PEAPTT in combination with IgG hinge linker, and IgG hinge linker Peptide (PEAPTD) ₂ may be sufficient. These linkers may be used to link GLP-1 to transferrin.

  [00217] The transferrin bound to the modified polypeptide or peptide may be modified. This may indicate a reduction in glycosylation. The modified transferrin polypeptide can be selected from the group consisting of a single transferrin N domain, a single transferrin C domain, the N and C domains of transferrin, two transferrin N domains, and two transferrin C domains. .

  [00218] As noted above, GLP-1 activates and regulates important endocrine hormone systems in the body and plays a vital administrative role in glucose metabolism. Unlike all other diabetes treatments on the market, GLP-1 has the potential to drive recovery by acting as a beta cell growth factor and thereby improving the ability of the pancreas to secrete insulin, and It also has the potential to make existing insulin levels work more efficiently by improving sensitivity and making glucose levels more stable. This alleviates the burden of daily monitoring of glucose levels and potentially delays serious long-term side effects caused by blood glucose fluctuations due to diabetes. Furthermore, GLP-1 can reduce appetite and reduce weight. Obesity is a unique outcome of inadequate control of glucose metabolism, which only serves to exacerbate the pathology of diabetes.

  [00219] Since natural GLP-1 is rapidly degraded in the blood circulation (half-life is a few minutes), its clinical application is limited. Maintaining therapeutic levels in the blood circulation requires constant administration of high doses using pumps or application devices, which increases treatment costs. This is particularly inconvenient for long-term continuous use in combination with all other medications to treat diabetes and monitor glucose levels. The modified GLP-1 fusion protein retains GLP-1 activity, but has the long half-life (14-17 days), solubility and biodistribution properties of transferrin. Due to these characteristics, low cost, small volume, monthly s. c. (Subcutaneous) injection can be realized and this type of product is definitely needed for long-term continuous use.

  [00220] The modified GLP-1 may be covalently bound to a blood component to increase its stability. For example, the modified GLP-1 can be covalently linked to serum albumin, transferrin, immunoglobulin or the Fc portion of an immunoglobulin. In one embodiment, the modified GLP-1 can be conjugated to a fatty acid or fatty acid derivative. In another embodiment, the modified GLP-1 can be reshaped into a drug affinity complex (DAC). As discussed above, Kim et al. (2003, Diabetes 52 (3): 751) disclose a GLP-1-albumin drug affinity complex. Kim et al. Show that albumin-bound DAC: GLP-1 mimics native GLP-1. Kim et al. Provide a new approach to activate GLP-1R signaling for a longer time.

  [00221] Upon subcutaneous administration, DAC: modified GLP-1 binds rapidly and selectively to albumin in vivo. The formed bioconjugate has the same therapeutic activity and similar potency as endogenous GLP-1, but has a pharmacokinetic profile that is closer to albumin.

Modified GLP-1 and fusion proteins thereof in combination with other therapeutic substances
[00222] In one aspect of the present invention, the modified GLP-1 peptide of the present invention and fusion proteins thereof, such as GLP-1-Tf fusion protein, may be type II diabetes, obesity and other diseases or conditions with abnormal glucose levels. Used in combination with at least one second therapeutic molecule such as “Glucophage®” (metformin hydrochloride tablets) and “Glucophage® XR” (metformin hydrochloride sustained release tablets). The

  [00223] "Glucophage®" and "Glucophage® XR" are oral antihyperglycemic agents for the treatment of type II diabetes. “Glucophage® XR” is a sustained release formulation of Glucophage. Therefore, “Glucophage® XR” may be taken once a day because the drug is slowly released from the dosage form. “Glucophage®” contributes to reducing glucose production from the liver by the body. Thus, “Glucophage®” is effective in controlling a patient's blood glucose level. “Glucophage®” rarely causes low blood glucose (hypoglycemia) because it does not cause more insulin production by the body.

  [00224] “Glucophage®” also contributes to lowering triglycerides and cholesterol, fatty blood components that are often high in people with type II diabetes. Metformin has been shown to contribute to weight loss of a few pounds if it reduces appetite and people begin to take the drug.

  [00225] Metformin has been approved for combination therapy with sulfonylurea, or combination therapy with insulin, or as a monotherapy (by itself). Metformin is used to dissolve blood clots (in combination with t-PA-derivatives) (International Publication No. 9108763, International Publication No. 9108766), hypertension in patients with insulin resistance (International Publication No. 9112003-Upjohn). Brochure, WO 9108767, and WO 9108765-Boehringer Mannheim), ischemia and tissue anoxia (EP283369-Lipha), atherosclerosis (DE 1936274-Brunnenglaber & Co., DE 2357875-Hurka). And U.S. Pat. No. 4,050,087-ICI) for use in treating various cardiovascular diseases. In addition, it has also been proposed to use metformin as a coronary vasodilator and in combination with cyclopentane derivatives similar to prostaglandins to reduce blood pressure (US Pat. No. 4,182,772—Hoechst). Metformin is also substituted with a 2-hydroxy-3,3,3-trifluoropropionic acid derivative (US Pat. No. 4,107,329-ICI), a 1,2-diarylethylene derivative (US Pat. No. 4,067,172-Hoechst), Aryloxy-3,3,3-trifluoro-2-propionic acid, esters and salts (US Pat. No. 4,055,595-ICI), substituted hydroxyphenyl-piperidones (US Pat. No. 4,024,267—Hoechst), It has also been proposed to be used in combination with partially hydrogenated 1H-indeno- [1,2B] -pyridine derivatives (US Pat. No. 3,980,656—Hoechst) to lower cholesterol.

  [00226] Montanari et al. (Pharmacological Research, Vol. 25, No. 1, 1992) used metformin in an amount of 500 mg twice a day (bid) and metformin 850 mg three times a day ( the increase in post-ischemic blood flow in a manner similar to ti.d.). Sirtori et al. (J. Cardiovas. Pharm., 6: 914-923, 1984) used metformin in an amount of 850 mg three times a day (tid) for patients with peripheral vascular disease. Disclosed increased arterial flow.

  [00227] The present invention provides for the treatment of various diseases comprising the modified GLP-1 of the present invention or a fusion protein thereof in combination with one or more therapeutic substances such as metformin. In one embodiment, the modified GLP-1 or fusion protein thereof in combination with metformin is used to treat diseases and conditions with abnormal blood glucose levels such as diabetes. Preferably, the GLP-1 / mTf fusion protein in combination with metformin is used to treat type II diabetes or obesity.

  [00228] Other therapeutic substances that can be used in combination with the modified GLP-1 and fusion proteins thereof of the present invention include, but are not limited to, sulfonylureas and sulfonylurea-like substances, thiazolidinediones, peroxisome proliferation Factor activated receptor (PPAR) gamma modulator, PPARα modulator, protein tyrosine phosphatase-1B inhibitor, insulin receptor tyrosine kinase activator, 11β-hydroxysteroid dehydrogenase inhibitor, glycogen phosphorylase inhibitor, glucokinase activation Factors, beta-3 adrenergic agonists, and glucagon receptor agonists.

DPP-IV inhibitor
[00229] Inhibitors of DPP-IV have been shown to be promising in treating various conditions mediated by DPP-IV. For example, inhibitors of DPP-IV are a very promising approach in the treatment of impaired glucose tolerance and in diseases associated with hyperglycemia such as type II diabetes and obesity. Furthermore, DPP-IV has been shown to play a role in immune responses such as transplant rejection (Transplantation 1997, 63 (10): 1495-1500). Thus, DPP-IV can also be useful in preventing transplant rejection. In addition, inhibitors of DPP-IV are useful in the treatment of cancer and the prevention of cancer metastasis because pulmonary endothelial DPP-IV binds to fibronectin in cancerous cells to promote metastasis of those cells. (J. Biol. Chem. 1998, 273 (37): 24207-24215). DPP-IV also plays an important role in the pathogenesis of periodontitis (Infect.Immun.2000, 68 (2), 716-724), and GLP-2, an intestine after extensive resection It is thought to govern the inactivation of factors that promote restoration. Therefore, DPP-IV inhibitors may be useful in intestinal restoration.

  [00230] WO 95/15309 discloses several peptide derivatives that are inhibitors of DPP-IV and are therefore useful for treating several DPP-IV mediated processes. ing. WO 95/13069 discloses several cyclic amine compounds that are useful for stimulating the release of natural or endogenous growth hormone. EP 555824 discloses several benzimidazolyl compounds that prolong thrombin time and inhibit thrombin and serine related proteases. Archives of Biochemistry and Biophysics, 323, 1, 148-154 (1995) discloses several aminoacylpyrrolidine-2-nitriles that are useful as DPP-IV inhibitors. Journal of Neurochemistry, 66, 2105-2112 (1996) discloses several Fmoc-aminoacylpyrrolidine-2-nitriles that are useful for inhibiting prolyl oligopeptidases. Bulletin of the Chemical Society of Japan, Vol. 50, No. 7, pp. 1827-1830 (1977), aminohexapeptide, Z-Val-Val-lmPro-Gly-Phe-Phe-OMe, and related The synthesis of aminopeptides is disclosed. In addition, the antibacterial properties of the compounds were examined. WO 90/12005 discloses several amino acid compounds that inhibit prolyl endopeptidase activity and are therefore useful for treating dementia or amnesia. In Derwent Abstract 95: 302548, several N- (aryl (alkyl) carbonyl) substituted, high cholinesterase activators with high peripheral selectivity useful for treating conditions resulting from reduced cholinesterase activity. Heterocyclic compounds are disclosed. Chemical Abstracts 84: 177689 discloses some 1-acyl-pyrrolidine-2-carbonitrile compounds that are useful as intermediates for proline compounds exhibiting angiotensin converting enzyme (ACE) inhibitory activity. Chemical Abstracts 96: 116353 discloses several 3-amino-2 mercapto-propyl-proline compounds that are Ras farnesyl-transferase inhibitors useful for treating various carcinomas or myeloid leukemias. WO 95/34538 describes a number of pyrrolizides, phosphonates, azetidines, peptides and azaprolines that inhibit DPP-IV and are therefore useful in treating conditions ameliorated by DPP-IV inhibition. Disclosure. In WO 95/29190 several compounds characterized by a multiple KPR-type repeating pattern supported by a peptide matrix that allows multiple presentations to the enzyme DPP-IV and has affinity for it And these compounds show the ability to inhibit the entry of HIV into cells. WO 91/16339 discloses several tetrapeptide boronic acids that are DPP-IV inhibitors useful for treating autoimmune diseases and conditions mediated by IL-2 suppression. Yes. WO 93/08259 discloses several polypeptide boronic acids that are DPP-IV inhibitors useful for treating autoimmune diseases and conditions mediated by IL-2 suppression. Yes. WO 95/11689 discloses several tetrapeptide boronic acids that are DPP-IV inhibitors useful for blocking HIV cell entry. East German Patent No. 158109 discloses several N-protected peptidyl-hydroxamic acids and nitrobenzoyloxamides that are particularly useful as DPP-IV inhibitors. WO 95/29691 specifically discloses some dipeptide proline phosphonates that are DPP-IV inhibitors useful in the treatment of immune system disorders. German patent DD296075 discloses several amino acid amides that inhibit DPP-IV. In Biochimica et Biophysica Acta, 1293, pp. 147-153, several di- and tripeptide p-nitroanilides are studied to study the effect of side chain modification on DPP-IV and PEP catalyzed hydrolysis. The preparation of is disclosed. Bioorganic and Medicinal Chemistry Letters, Vol. 10, No. 10, 1163-1166 (1996) discloses several 2-cyanopyrrolidines that are DPP-IV inhibitors. J. et al. Med. Chem. 39, 2087-2094 (1996) disclose several proline boronic acid-containing dipeptides that are inhibitors of DPP-IV. Diabetes 44, 1126-1131 (September 1996) demonstrates that GLP-I amide is rapidly degraded when administered by subcutaneous or intravenous routes to diabetic and non-diabetic subjects. It is intended for research.

  [00231] In US Pat. No. 6,727,261, novel DPP-IV inhibition useful for the treatment and / or prevention of diseases associated with DPP-IV, such as diabetes, especially non-insulin dependent diabetes, and impaired glucose tolerance Pyrido [2,1-a] isoquinoline derivatives are provided as agents. These compounds are also useful for the treatment and / or prevention of bowel disease, ulcerative colitis, Crohn's disease (Morbus Crohn), obesity and / or metabolic syndrome.

  [00232] US Pat. No. 6,716,843 provides α-amino acid sulfonyl compounds useful as inhibitors of DPP-IV.

  [00233] US Pat. No. 6,645,995 discloses 2-substituted unsaturated heterocyclic compounds in which the nitrogen atom in the heterocycle is attached to an amino acid or amino acid derivative via an amide bond or a peptide bond. . These compounds are potent and selective inhibitors of DPP-IV and are effective in treating conditions that can be modulated or normalized by inhibition of DPP-IV.

  [00234] US Pat. No. 6,617,340 discloses the use of N- (substituted glycyl) pyrrolidine, and said compounds in the inhibition of dipeptidyl peptidase-IV. US Pat. No. 6,124,305 discloses N- (substituted glycyl) -2-cyanopyrrolidines that inhibit DPP-IV. These compounds are effective in treating conditions mediated by DPP-IV.

[00235] Compound 1-[[[[2-[(5-Cyanopyridin-2-yl) amino] ethyl] amino] acetyl] -2-cyano- (S) -pyrrolidine (NVP DPP728) from Novartis Was administered to 93 type 2 diabetic patients (HbA _1c mean 7.4%) over 4 weeks, and plasma glucose, insulin and HbA _1c levels decreased over the 4-week study period (Diabetes Care 2002) Year 25 (5): 869-875).

Combination therapy using inhibitors of DPP-IV
[00236] In one aspect, the invention involves the use of a transferrin fusion protein comprising a therapeutic protein, polypeptide or peptide in combination with one or more DPP-IV inhibitors for the treatment of various conditions. provide. The present invention provides a pharmaceutical composition comprising a transferrin fusion protein and one or more DPP-IV inhibitors. Transferrin conjugated to a therapeutic protein, polypeptide or peptide may be modified as disclosed in US patent application Ser. No. 10 / 378,094, which is incorporated herein by reference in its entirety. This may indicate a reduction in glycosylation. The modified transferrin polypeptide can be selected from the group consisting of a single transferrin N domain, a single transferrin C domain, the N and C domains of transferrin, two transferrin N domains, and two transferrin C domains. . The therapeutic protein or peptide bound to transferrin may be natural or modified. Preferably, the transferrin fusion protein comprises GLP-1 as a therapeutic peptide linked to a modified transferrin molecule as described in US patent application Ser. No. 10 / 378,094. In addition, the combination therapy of the present invention comprises a GLP-1 / mTf fusion protein, one or more DPP-IV inhibitors, and another therapeutic molecule. Such molecules are “Glucophage®” or “Glucophage® XR”.

  [00237] In another aspect, the invention provides the use of a modified protein or peptide or fusion protein thereof that is resistant to dipeptidyl protease cleavage in combination with one or more DPP-IV inhibitors. To do. The present invention discloses a pharmaceutical composition comprising a modified protein or peptide or fusion protein thereof in combination with one or more DPP-IV inhibitors. Preferably, the modified peptide is modified GLP-1 and the fusion protein is a modified GLP-1 / mTf protein. Further, the combination therapy of the present invention comprises a modified GLP-1 or modified GLP-1 / mTf fusion protein, one or more DPP-IV inhibitors, and “Glucophage®” or “Glucophage®”. Also includes other therapeutic molecules such as "XR".

  [00238] DPP-IV inhibitors can be used in the methods of the invention for treating any related disease. For example, the GLP-1-transferrin fusion proteins described herein are 1-[[[2-[(5-cyanopyridin-2-yl) for treating pre-diabetes, diabetes, obesity or diabetic conditions. It can be used in combination with a DPP-IV inhibitor such as amino] ethyl] amino] acetyl] -2-cyano- (S) -pyrrolidine (NVP DPP728). These therapeutic substances may be administered sequentially or simultaneously.

[00239] The transferrin fusion protein may comprise a GLP-1 (7-37) peptide (SEQ ID NO: 32) or a GLP-1 (7-36) peptide (amino acids 1-30 of SEQ ID NO: 32). More preferably, the GLP-1 (7-37) peptide or GLP-1 (7-36) peptide comprises an A8 to G and K34 to A mutation. The transferrin protein may also include a linker between the GLP-1 (7-37) peptide or GLP-1 (7-36) peptide and the transferrin molecule. Preferably, the linker is (PEAPTD) ₂ peptide.

Improvement of pharmacokinetics and pharmacodynamics of fusion proteins by combination therapy with endopeptidase inhibitors
[00240] The invention also provides a combination therapy using a neutral endopeptidase (NBP) inhibitor and a transferrin fusion protein. Furthermore, the present invention also includes combination therapy using NBP inhibitors and DPP-IV inhibitors and transferrin fusion proteins. The NEP inhibitor and DPP-IV inhibitor may be administered simultaneously or sequentially. In addition, these inhibitors and transferrin fusion proteins may also be administered simultaneously or sequentially. These inhibitors may be administered before or after the transferrin fusion protein is administered.

[00241] The transferrin fusion protein comprises a GLP-1 (7-37) peptide (SEQ ID NO: 32) or a GLP-1 (7-36) peptide (amino acids 1-30 of SEQ ID NO: 32). More preferably, the GLP-1 (7-37) peptide or GLP-1 (7-36) peptide comprises an A8 to G and K34 to A mutation. The transferrin protein may also include a linker between the GLP-1 (7-37) peptide or GLP-1 (7-36) peptide and the transferrin molecule. Preferably, the linker is (PEAPTD) ₂ peptide.

  [00242] Neutral endopeptidase (NEP), also known as enkephalinase, neprilysin and atriopeptidase, is a membrane-bound membrane present in many tissues including the brain, kidney, lung, gastrointestinal tract, heart and peripheral vasculature Type zinc metalloendopeptidase. NEP plays an important role in natriuretic peptide clearance by degrading circulating natriuretic peptides, thereby preventing their effects on vasodilation, blood pressure and blood volume. NEP is associated with hypertension, heart failure and renal failure by degrading and inactivating the natriuretic peptide.

  [00243] In addition to degrading the circulating natriuretic peptide, NEP is a circulating bradykinin; adrenomedullin, renal vasodilatory and natriuretic peptide; and / or urodilatin, ANP. It also degrades other vasodilatory substances, including the kidney type.

  [00244] NEP is also involved in the degradation of the vasoconstrictor endothelin isoform ET1, and may also be involved in the formation of ET-1 (Brunner-La Rocca et al., Cardiovascular Research 51 (2001) 510-520). NEP is also a potent vasoconstrictor, angiotensin II, endomorphin, and bradykinin, GLP-1 (Hupe-Sodmann, K., McGregor, GP, Bridenbaugh, R et al. Regulatory Peptides 1995; 58: 149- 56, Hupe-Sodmann, K, Goeke, R., Goeke, B et al. Peptides 1997; 18: 625-32), PYY (Mediiros MD, Turner AJ., Endocrinology. May 1994); : 2088-94), and glucagon (Trebien R et al. Am J Physiol Endocrinol Metab. September 2004; 287 (3): E431-8) Some peptides involved in the degradation are also degraded.

  [00245] Some NEP inhibitors, such as phosphoramidon and NEP / ACE inhibitors (omapatrilat disclosed in US Pat. No. 5,508,272, gempatrilato disclosed in US Pat. No. 5,552,297, US Pat. No. 5,430,145 Have been reported in the literature as being useful for monotherapeutic treatment of hypertension and heart failure, for example. Natsuiwan et al., “A Review of Vasepeptidase Inhibitors: A New Modality in the Treatment of Hypertension and Chronic Failure,” page 42, Pharm. Candoxatril and ecadotril are two highly specific NEP inhibitors currently in clinical trials as future drugs for heart failure. Both compounds are prodrugs that are metabolized in the body and have the same type of activity. Candoxatril is activated in the liver to become candoxatrilat, whereas ecadotolyl is converted to S-thiorphan with its similar activity.

  [00246] Some examples of ACE / NEP inhibitors are described in U.S. Patent Nos. 5,508,272, 5,362,727, 5,366,973, 5,225,401, 4,722,810, 5,223,516, 5,552,597, 4,749,688, Nos. 5,504,080, 5,612,359, and 5,525,723, and European Patent Applications Nos. 0481,522, 0534363 A2, 534,396, and 534,492.

  [00247] The present invention provides combination therapies comprising transferrin fusion proteins and DPP-IV inhibitors and / or ACE / NEP inhibitors for the treatment of various diseases or conditions. Such diseases or conditions include, but are not limited to, diabetes, preferably type II diabetes, congestive heart failure, obesity, hypertension and irritable bowel syndrome.

Transgenic animals
[00248] The generation of transgenic non-human animals that express a modified polypeptide or peptide that is protected from DPP activity is contemplated in one embodiment of the invention. In some embodiments, transgenic non-human animals that express a fusion protein comprising a modified polypeptide or peptide with high stability are contemplated.

  [00249] For example, US Pat. No. 6,291,740 (issued September 18, 2001); US Pat. No. 6,281,408 (issued August 28, 2001), which is incorporated herein by reference in its entirety; and US In several patents and publications such as patent 6271436 (issued August 7, 2001), successful transgenic non-human animal production has been described.

  [00250] The ability to modify the genetic properties of animals such as domesticated mammals, including dairy cows, pigs, goats, horses, cows and sheep, allows for several commercial applications. For these applications, production of animals that express large amounts of exogenous protein in an easily recovered form (ie, expression in milk or blood), weight gain, feed efficiency, carcass composition, milk production or yield, Includes the production of animals with increased disease resistance and resistance to infection by certain microorganisms, as well as the production of animals with improved growth rate or fertility. Animals that contain exogenous DNA sequences in their genome are called transgenic animals.

  [00251] The most widely used method for producing transgenic animals is microinjection of DNA into the pronucleus of fertilized embryos (Wall et al., J. Cell. Biochem. 49: 113 [1992]. ]). Other methods for producing transgenic animals include infection of embryos with retroviruses or retroviral vectors. Infection of both pre- and post-implantation mouse embryos with wild-type or recombinant retroviruses has been reported (Janenich, Proc. Natl. Acad. Sci. USA 73: 1260 [1976]; Janenich; Cell 24: 519 [1981]; Stuhlmann et al., Proc. Natl. Acad. Sci. USA 81: 7151 [1984]; Jahner et al., Proc. Natl. Acad. Sci. USA 82: 6927 [ 1985]; Van der Putten et al., Proc. Natl. Acad. Sci. USA 82: 6148-6152 [1985]; Stewart et al., EMBO J. 6: 383-388 [1987]).

  [00252] An alternative means of infecting embryos with retroviruses is the injection of virus or virus-producing cells into the cleavage space of mouse embryos (Jahner, D. et al., Nature 298: 623 [1982]). ). The introduction of transgenes into the germline of mice using intrauterine retroviral infection of mid-gestation mouse embryos has been reported (Jahner et al., Supra [1982]). Infection of bovine and sheep embryos with retroviruses or retroviral vectors to produce transgenic animals has been reported. These protocols involve microinjection of retroviral particles, or cells that have been proliferated (ie, treated with mitomycin C) that drop the retroviral particles into the follicular cavity of a fertilized or early embryo (PCT International Application) WO 90/08832 [1990]; and Haskell and Bowen, Mol. Reprod. Dev., 40: 386 [1995]). PCT International Application No. WO 90/08832 describes the injection of wild-type feline leukemia virus B into the follicular cavity of 2-8 cell stage sheep embryos. Fetuses derived from injected embryos have been shown to contain multiple integration parts.

  [00253] In US Pat. No. 6,291,740 (issued September 18, 2001), premature maturation using retroviral vectors (eg, vectors derived from murine leukemia virus [MLV]) that transduce dividing cells. The production of transgenic animals is described by introducing exogenous DNA into oocytes and mature unfertilized oocytes (ie, pre-fertilized oocytes). This patent also describes methods and compositions for cytomegalovirus promoter driven expression of various recombinant proteins, as well as expression by mouse breast cancer LTR.

  [00254] US Pat. No. 6,281,408 (issued Aug. 28, 2001) describes a method for producing transgenic animals using embryonic stem cells. Briefly, embryonic stem cells are used in cell co-cultures mixed with morulae to produce transgenic animals. Exogenous genetic material is introduced into embryonic stem cells prior to co-culture, for example, by electroporation, microinjection or retroviral delivery. ES cells transfected in this manner are selected for gene integration by a selectable marker such as neomycin.

  [00255] US Pat. No. 6,271,436 (issued August 7, 2001), isolating primordial germ cells, culturing these cells to produce a cell line derived from primordial germ cells, Describes the production of transgenic animals using a method comprising transforming both cells and cultured cell lines, and generating transgenic animals using these transformed cells and cell lines. Yes. The efficiency with which transgenic animals occur is greatly increased, which allows the use of homologous recombination in creating transgenic non-rodent species.

Gene therapy
[00256] It is contemplated in one embodiment of the invention to use a modified polypeptide or peptide construct of the invention for gene therapy. The polypeptide or peptide was modified to be protected from DPP activity by adding one or more additional amino acids at the N-terminus. For example, a nucleic acid construct encoding GLP-1 containing an additional His residue at the N-terminus is provided for gene therapy. Nucleic acid constructs encoding modified GLP-1 / transferrin fusion proteins are also provided for gene therapy. The modified GLP-1 constructs of the present invention are protected from DPP activity, are more stable and are therefore ideally suited for gene therapy treatments.

  [00257] Briefly, gene therapy by injection of an adenoviral vector comprising a gene encoding a soluble fusion protein consisting of the cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglobulin G1 is disclosed in Ijima (June 10, 2001) Human Gene Therapy (USA) 12/9: 1063-77 recently. In this application of gene therapy, a mouse model of type II collagen-induced arthritis has been successfully treated by intra-articular injection of the vector.

  [00258] Gene therapy is described in US Pat. No. 6,225,290 (issued May 1, 2001); US Pat. No. 6,187,305 (issued February 13, 2001); and US Pat. No. 6,140,111 (Oct. 31, 2000). And is also described in several US patents.

  [00259] US Pat. No. 6,225,290 provides methods and constructs for genetically modifying intestinal epithelial cells of a mammalian subject to operably incorporate a gene that expresses a protein having a desired therapeutic effect. Intestinal cell transformation is accomplished by administration of a preparation composed primarily of naked DNA, which can be administered orally. Oral or other routes of administration within the gastrointestinal tract provide a simple method of administration, while simultaneously using naked nucleic acids avoids complications associated with the use of viral vectors to perform gene therapy. The expressed protein is secreted directly into the gastrointestinal tract and / or blood stream, resulting in a therapeutic blood level of the protein, thereby treating patients in need of the protein. Transformed intestinal epithelial cells provide short-term or long-term therapeutic recovery for diseases associated with a deficiency of a specific protein or to which treatment by overexpression of the protein is applicable.

  [00260] US Pat. No. 6,187,305 provides a method for gene or DNA targeting in cells of vertebrate, particularly mammalian origin. Briefly, DNA is transferred into primary or secondary cells of vertebrate origin by homologous recombination or by targeting DNA introduced into preselected sites in the genomic DNA of primary or secondary cells. be introduced.

  [00261] US Pat. No. 6,140,111 (issued Oct. 31, 2000) describes a retroviral gene therapy vector. The disclosed retroviral vectors contain an insertion site for the gene of interest and are capable of expressing high levels of proteins derived from the gene of interest in a wide variety of transfected cell types. Also disclosed are retroviral vectors suitable for human gene therapy in the treatment of various disease states that lack a selectable marker and thus do not involve co-expression of marker products such as antibiotics. These retroviral vectors are particularly suitable for use in certain packaging cell lines. Retroviral vectors are particularly promising candidates for use in gene therapy of human and animal genetic diseases because they can be inserted into the genome of mammalian cells. Gene therapy typically involves (1) adding new genetic material to patient cells in vivo, or (2) removing patient cells from the body, adding new genetic material to those cells, and the body Including reintroducing them, ie in vitro gene therapy. A discussion of how to perform gene therapy in a variety of cells using retroviral vectors can be found, for example, in US Pat. No. 4,868,116 issued September 19, 1989 and No. 25,1998 issued December 25, 1990. No. 4980286 (epithelial cells), WO 89/07136 published on August 10, 1989 (hepatocytes), EP 378576 (fibroblasts) published on July 25, 1990, and 1989. It can be confirmed in International Publication No. 89/05345 pamphlet published on June 15 and International Publication No. 90/0697 pamphlet (endothelial cells) published on June 28, 1990. Are incorporated herein by reference.

  [00262] Without further explanation, one of ordinary skill in the art will be able to implement and utilize the claimed invention using the foregoing description and the following illustrative examples. Accordingly, the following examples illustrate preferred embodiments of the present invention, but should not be construed as limiting the remainder of the disclosure in any way. All articles, publications, patents, and documents mentioned throughout this application are hereby incorporated by reference in their entirety.

Example 1: Modified GLP-1 with dipeptidyl-peptidase IV protection
[00263] This example illustrates a modified GLP-1 peptide protected from DPP-IV activity. The following peptides were synthesized using standard solid phase Fmoc chemistry, purified by reverse phase HPLC using a C18 column and quantified by absorbance at 220 nm. The purified peptide was analyzed by mass spectrometry (MALDI-TOF):

Dipeptidyl peptidase IV treatment
[00264] Equimolar concentrations of each peptide (6 μM) were treated with 2 μg of recombinant human DPP-IV (1 μg / μL, R & D Systems, Minneapolis, Minn.) In 25 mM Tris-Cl (pH 8.0). A control reaction with the exception of DPP-IV was set up for each peptide simultaneously. These digests were incubated at room temperature for 2 hours and at 2 hours the reactions were added to Krebs Ringer buffer (Biosource) supplemented with 1 mM 3-isobutyl-1-methylxanthine (IBMX, Calbiochem, San Diego, Calif.). International Inc., Camarillo, Calif.). These peptides were then analyzed to measure the remaining GLP-1 receptor activation activity, as described below.

Cyclic AMP stimulation assay
[00265] One day prior to treatment, CHO-GLP1R cells (Montrose-Rafizadeh et al., 1997 J. Biol. Chem. 272, 21201-21206) were placed in RPMI / 10% FBS medium at 2 × 10 ⁴ cells / Four 96-well tissue culture plates were seeded at a well density. The next day, the cells appeared to be uniformly distributed with an estimated confluence of 60-80%. One day after seeding on the culture plate, the cells were washed twice with Krebs Ringer buffer (KRB) and subsequently incubated in KRB at 37 ° C. for 1 hour to reduce the intracellular level of cAMP. This was followed by incubation in KRB / IBMX for 10 minutes to inhibit intracellular enzymes that disrupt cAMP. Dilutions of each test compound were prepared in KRB / IBMX and three wells of CHO-GLP1R cells were treated with 50 μl of test compound per well at 37 ° C. for exactly 20 minutes. The treatment was stopped by washing these cultures twice with ice-cold phosphate buffered saline. Lysates were prepared by adding 0.1 ml of lysis buffer 1B (Amersham Biosciences cAMP Biotrak EIA kit) for 10 minutes at room temperature. The total volume of each cell extract was then analyzed to determine the cAMP concentration using cAMP Biotrak Enzyme Immunoassay System (Amersham Biosciences Corporation, Piscataway, NJ, product code RPN225) according to the kit instructions. It was found that the peptides of the present invention are more resistant to DPP-IV than the unmodified form.

ELISA specific for active GLP-1
[00266] Alternatively, GLP-1, which is specific for fully active GLP-1 and has the two N-terminal amino acids removed due to the action of DPP-IV, ie GLP-1 (9-36 Or GLP-1 and GLP-1 of the present invention using an ELISA system (glucagon-like peptide-1 [activity] ELISA kit [Linco Research, Inc., St. Charles, MO]) that does not recognize 9-37) DPP-IV degradation of the derivatives was analyzed. Equimolar concentrations of GLP-1 and H-GLP-1 (1200 pM) were used with recombinant human DPP-IV (200 ng / μL, R & D Systems, Minneapolis, MN) in 25 mM Tris-Cl (pH 8.0). The reaction was stopped by treatment and dilution in assay buffer supplied with the kit containing protease inhibitors.

  [00267] The kit includes a 96 well microtiter plate coated with anti-GLP-1 monoclonal antibody. The plates were washed (4 times with plate washer, ThermoLabsystems Ultrawash Plus, 25 mM borate buffered saline) and then incubated with peptide samples (300 pM and 10-fold serial dilutions down the plate) for 3 hours at room temperature. After washing as described above, the plates were incubated for 2 hours at room temperature with anti-GLP antibody conjugated with alkaline phosphatase (provided as a ready-to-use component of the kit). After washing, 4-methylumbelliferyl phosphate (MUP) substrate (1: 200 dilution in 50 mM Borate pH 9.5) was added to all wells and incubated at room temperature in the dark for 30 minutes. . The plate was read on a SpectraMax Gemini EM fluorescent plate reader with an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Since H-GLP-1 was less likely to bind to the monoclonal antibody than GLP-1 itself, the concentration of active H-GLP-1 remaining after DPP-IV treatment was determined using the H-GLP-1 standard curve. Decided. FIG. 8 shows that H-GLP-1 is substantially more resistant than GLP-1 to the action of DPP-IV.

Example 2: Modified GLP-1 fusion protein
[00268] This example describes a fusion protein comprising a modified GLP-1 protected from DPP-VI activity fused to a modified transferrin molecule.

これらのプライマーの位置を以下に示す。

プライマー（８μＬ、濃度２０ｐｍｏｌ）を混合し、５分間６５℃まで加熱し、次いでアニーリング反応を起こさせ、室温までゆっくり放冷させた。 [00269] The following overlapping primers were designed to construct sequences encoding the N-terminal portion of GLP-1 and transferrin following the transferrin secretion leader.

The positions of these primers are shown below.

Primers (8 μL, concentration 20 pmol) were mixed, heated to 65 ° C. for 5 minutes, then allowed to undergo an annealing reaction and allowed to cool slowly to room temperature.

[00270] After adding T4 DNA ligase to the annealing reaction and incubating for an additional 2 hours at room temperature, 1 μL of the reaction was removed and used in a PCR reaction to complete the insert using outer primers P0236 and P0251. Amplified. PCR conditions were as follows.
94 ° C for 5 minutes 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 1 minute 25 cycles 72 ° C for 7 minutes maintained at 4 ° C

  [00271] The resulting PCR product was digested with AflII and KpnI and ligated into pREX0094 (FIG. 1) previously digested with AflII and KpnI. Ligation was used to transform E. coli. The resulting DNA from the clone was sequenced and a clone with the correct length of the AflII / KpnI insert was selected and named pREX0198 (FIG. 2). Next, pREX0198 was digested with NotI and PvuI and inserted into pSAC35 (FIG. 3) to create pREX0240 (FIG. 4).

[00272] To generate a plasmid encoding H-GLP-1 (7-36) fused to a natural transferrin secretion leader followed by a modified transferrin (mTf), an extra N-terminus to the sequence encoded by pREX0198 Overlapping primers P0424 and P0425 were designed to add histidine.

  [00273] pREX0198 was used as a template for the first PCR reaction using these two mutagenic overlapping primers and two outer primers, P0424 and P0012 and P0425 and P0025, in separate reactions. The products of these reactions were then used as templates in the second round of PCR using only the outer primers, P0012 and P0025, to bind them. Both rounds of PCR reaction conditions consisted of 94 ° C for 1 minute once, 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 1 minute 20 times, and 72 ° C for 7 minutes to complete. 1 time.

  [00274] The PCR product obtained from the final reaction was digested with AflII and KpnI and ligated into pREX0052 (FIG. 5) digested with AflII / KpnI to create pREX0367 (FIG. 6). To confirm the insertion of the codon for extra histidine, the DNA sequence of the construct was determined.

  [00275] pREX0367 was then digested with NotI and PvuI (the latter to destroy the ampicillin resistance gene) and ligated into pSAC35 previously digested with NotI to create pREX0368 (FIG. 7).

  [00276] pREX0368 was transformed into the host Saccharomyces cerevisiae strain by electroporation, and transformed colonies were selected based on leucine prototrophy on buffered minimal media plates. After selecting a single colony, the yeast transformant was placed in 40% trehalose and stored at -70 ° C. Expression was measured by growth in liquid minimal medium buffered to pH 6.5 and analysis of the supernatant by SDS-PAGE, Western blot and ELISA.

[00277] GLP-1 / mTf as described in US patent application Ser. No. 10 / 378,094 filed Mar. 4, 2003, which is incorporated herein by reference in its entirety. An encoding plasmid (pREX0100) and a plasmid encoding H-GLP-1 / mTf were constructed. The amino acid sequences of GLP-1 (7-36) and GLP-1 (7-37) can be used to make GLP-1 / mTf fusion proteins.

[00278] For example, the peptide sequence of GLP-1 (7-36) can be back-translated into DNA and codons optimized for yeast.

[00279] These primers form 5'XbaI and 3'KpnI cohesive ends after annealing and into pREX0052 cleaved with XbaI / KpnI, just 5 'to the end of the leader sequence and mTf. It was specifically designed to allow direct ligation at the N-terminus. Alternatively, other sticky ends may be artificially created for ligation into other vectors.

  [00280] After annealing and ligation, clones were sequenced to confirm correct insertion. This vector was called pREX0094. The cassette was excised from pREX0094 using NotI and subcloned into the yeast vector pSAC35 cut with NotI to produce pREX0010.

  [00281] This plasmid was then electroporated into the host Saccharomyces yeast strain, and transformants were selected based on leucine prototrophy on minimal media plates. Expression was measured by growth in liquid minimal medium and analysis of the supernatant by SDS-PAGE, Western blot, and ELISA.

  [00282] GLP-1 / mTf and H-GLP-1 / mTf were expressed and purified from fermentation cultures grown under standard conditions by cation exchange and anion exchange chromatography.

Dipeptidyl peptidase IV treatment
[00283] Equimolar concentrations of GLP-1 / mTf and H-GLP-1 / mTF (2 μM) were added to recombinant human DPP-IV (1 μg / μL, R & D Systems) in 25 mM Tris-Cl solution (pH 8.0). Company). A control reaction with the exception of DPP-IV was set up simultaneously for each fusion protein. These digests were incubated at room temperature for 2 hours, and the reaction was diluted 20-fold in Krebs Ringer buffer (Biosource International) supplemented with 1 mM IBMX (Calbiochem) at 2 hours.

Cyclic AMP stimulation assay
[00284] One day before treatment, CHO-GLP1R cells were seeded in tissue culture plates (24 wells) at a density of 1 x 10 ⁵ cells / well in RPMI / 10% FBS medium. The next day, the cells appeared to be uniformly distributed with an estimated confluence of 60-80%. One day after seeding on the culture plate, the cells were washed twice with Krebs Ringer buffer (KRB) and subsequently incubated in KRB at 37 ° C. for 1 hour to reduce the intracellular level of cAMP. This was followed by incubation in KRB / IBMX for 10 minutes to inhibit intracellular enzymes that disrupt cAMP. Dilutions of each test compound were prepared in KRB / IBMX and three wells of CHO-GLP1R cells were treated with 0.15 ml of test compound per well at 37 ° C. for exactly 50 minutes. The treatment was stopped by washing these cultures twice with ice-cold phosphate buffered saline. Lysates were prepared by adding 0.2 ml of lysis buffer 1B (Amersham Biosciences cAMP Biotrak EIA kit) for 10 minutes at room temperature, then cAMP Biotrak Enzyme Immunoassay System (Amersham Biosciences Bios Handbook). Were used to analyze 100 μl of each cell extract to determine cAMP concentration.

  [00285] H-GLP-1 / mTf was found to be more resistant to DPP-IV than GLP-1 / mTf.

Example 3: Modified GLP-1 / mTf for the treatment of diabetes
[00286] In this example, the modified GLP-1 / mTf of the present invention is used as a therapeutic substance to treat diabetes. Modified GLP-1 / mTf is administered to Zucker rats, a standard animal model of type II diabetes. Zucker rats have abnormally high blood glucose levels. Treatment of these animals with GLP-1 has been shown to induce insulin secretion and lower blood glucose.

  [00287] Zucker rats are fasted overnight and then treated with H-GLP-1 or H-GLP-1 fused to transferrin (H-GLP-1 / mTf). Thirty minutes after subcutaneous injection of H-GLP-1 or H-GLP-1 / mTf, these animals are subjected to a glucose tolerance test (GTT). For this test, fasted animals are given a glucose solution (1.5 mg / g body weight) and blood glucose is measured at appropriate time intervals. Immediately after glucose administration, blood glucose levels in untreated animals rose and slowly declined towards baseline, whereas H-GLP-1 or H-GLP-1 / mTf was injected. Animals show faster normalization of blood glucose levels due to the contribution of GLP-1 to the insulinotropic effect.

  [00288] In another experiment, modified H-GLP-1 or H-GLP-1 / mTf is used to normalize high fasting blood glucose in Zucker rats without glucose administration. Blood glucose levels remain high in untreated animals, whereas a significant reduction is observed in animals treated with H-GLP-1 or modified H-GLP-1 / mTf.

Example 4: Modified glucagon with protection from dipeptidyl-peptidase IV
[00289] This example describes a modified glucagon molecule protected from DPP-IV activity.

[00290] The following peptides were synthesized using standard solid phase Fmoc chemistry, purified by reverse phase HPLC using a C18 column and quantified by absorbance at 220 nm. The purified peptide was analyzed by mass spectrometry (MALDI-TOF):

  [00291] These peptides were pretreated with DPP-IV as described above and then analyzed for the ability to activate the glucagon receptor using a recombinant cell line expressing the cloned glucagon receptor. .

Example 5: Modified GIP with protection from dipeptidyl-peptidase IV
[00292] This example provides a modified GIP molecule protected from DPP-IV activity.

[00293] The following peptides were synthesized using standard solid phase Fmoc chemistry, purified by reverse phase HPLC using a C18 column and quantified by absorbance at 220 nm. The purified peptide was analyzed by mass spectrometry (MALDI-TOF):

  [00294] These peptides were pretreated with DPP-IV as described above and then analyzed for the ability to activate the GIP receptor using recombinant cell lines expressing the cloned GIP receptor. .

  [00295] It is to be understood that the foregoing discussion and examples only present a detailed description of certain preferred embodiments. Accordingly, it should be apparent to those skilled in the art that various modifications can be made and equivalents can be made without departing from the spirit and scope of the invention. All journal articles, other references, patents, and patent applications identified in this patent application are incorporated by reference in their entirety.

It is a figure which shows the restriction enzyme map of pREX0094. It is a figure which shows the restriction enzyme map of plasmid pREX0198. It is a figure which shows the restriction enzyme map of pSAC35. It is a figure which shows the restriction enzyme map of plasmid pREX0240. It is a figure which shows the restriction enzyme map of pREX0052. It is a figure which shows the restriction enzyme map of pREX0367. It is a figure which shows the restriction enzyme map of pREX0368. It is a graph which shows a time-dependent change of incubation with GLP-1 and H-GLP-1 and DPP-IV. The graph shows the amount of remaining active full-length peptide as determined by ELISA specific for active GLP-1.

Claims (52)

  A method of treating a disease or condition in a patient comprising administering an effective amount of a transferrin (Tf) fusion protein and at least one second agent.   The method of claim 1, wherein the second agent is selected from the group consisting of a DPP-IV inhibitor and a neutral endopeptidase (NEP) inhibitor.   The method of claim 1, wherein the Tf fusion protein is susceptible to DPP-IV clearance.   2. The method of claim 1, wherein the Tf fusion protein is sensitive, resistant or partially resistant to DPP-IV clearance.   2. The method of claim 1, wherein the Tf fusion protein comprises one or more GLP-1 peptides fused to a Tf molecule.   6. The method of claim 5, wherein the Tf fusion protein further comprises a linker. The method of claim 6, wherein the linker is PEAPTD or (PEAPTD) ₂ .   6. The method of claim 5, wherein the GLP-1 peptide is at the N-terminus of the fusion protein.   6. The method of claim 5, wherein the GLP-1 peptide is GLP-1 (7-37) or GLP-1 (7-36) having SEQ ID NO: 32 (amino acids 1-30 of SEQ ID NO: 32).   10. The method of claim 9, wherein the GLP-1 peptide has been modified by an A8 to G mutation.   10. The method of claim 9, wherein the GLP-1 peptide has been modified by a K34 to A mutation.   10. The method of claim 9, wherein the GLP-1 peptide has been modified by A8 to G and K34 to A mutations. 13. The method of claim 12, wherein the GLP-1 peptide is linked to the Tf molecule via a linker (PEAPTD) ₂ .   6. The method of claim 5, wherein the Tf molecule has been modified to exhibit less glycosylation as compared to a fully glycosylated Tf molecule.   6. The method of claim 5, wherein the Tf molecule has been modified so as not to exhibit glycosylation.   6. The method of claim 5, wherein the Tf molecule has been modified to include at least one mutation that prevents glycosylation.   6. The method of claim 5, wherein the Tf molecule has been modified to exhibit a lower affinity for the transferrin receptor (TfR) compared to a wild type Tf molecule.   6. The method of claim 5, wherein the Tf molecule has been modified so that it does not bind to TfR.   6. The method of claim 5, wherein the Tf molecule has been modified to exhibit a lower affinity for iron compared to a wild type Tf molecule.   6. The method of claim 5, wherein the Tf molecule has been modified so that it does not bind to iron.   6. The method of claim 5, wherein the Tf molecule is lactoferrin or melanotransferrin.   The method of claim 1, wherein the disease is a metabolic disease.   23. The method of claim 22, wherein the metabolic disease is prediabetes, diabetes or obesity.   24. The method of claim 23, wherein the diabetes is type II diabetes.   2. The method of claim 1, wherein the disease is congestive heart failure.   The method of claim 1, wherein the disease is irritable bowel syndrome or dyspepsia.   The method of claim 1, wherein there are two second agents.   28. The method of claim 27, wherein the two second agents are a DPP-IV inhibitor and a NEP inhibitor.   30. The method of claim 29, wherein the two second agents are administered simultaneously.   30. The method of claim 1 or 29, wherein the transferrin fusion protein and the one or more second agents are administered sequentially.   30. The method of claim 1 or 29, wherein the transferrin fusion protein and the one or more second agents are administered simultaneously.   A composition comprising a transferrin (Tf) fusion protein and at least one second agent.   35. The composition of claim 32, wherein the second agent is selected from the group consisting of a DPP-IV inhibitor and a neutral endopeptidase (NEP) inhibitor.   33. The composition of claim 32, wherein the Tf fusion protein is susceptible to DPP-IV clearance.   35. The composition of claim 32, wherein the Tf fusion protein is sensitive, resistant or partially resistant to DPP-IV clearance.   33. The composition of claim 32, wherein the Tf fusion protein comprises one or more GLP-1 peptides fused to a Tf molecule.   40. The composition of claim 36, wherein the Tf fusion protein further comprises a linker. 38. The composition of claim 37, wherein the linker is PEAPTD or (PEAPTD) ₂ .   37. The composition of claim 36, wherein the GLP-1 peptide is at the N-terminus of the fusion protein.   37. The composition of claim 36, wherein the GLP-1 peptide is GLP-1 (7-37) or GLP-1 (7-36) having SEQ ID NO: 32 (amino acids 1-30 of SEQ ID NO: 32). .   41. The composition of claim 40, wherein the GLP-1 peptide has been modified by an A8 to G mutation.   41. The composition of claim 40, wherein the GLP-1 peptide is modified by a K34 to A mutation.   41. The composition of claim 40, wherein the GLP-1 peptide is modified by A8 to G and K34 to A mutations. 44. The composition of claim 43, wherein the GLP-1 peptide is linked to the Tf molecule via a linker (PEAPTD) ₂ .   35. The composition of claim 32, wherein the Tf molecule has been modified to exhibit less glycosylation as compared to a fully glycosylated Tf molecule.   35. The composition of claim 32, wherein the Tf molecule has been modified so as not to exhibit glycosylation.   33. The composition of claim 32, wherein the Tf molecule has been modified to include at least one mutation that prevents glycosylation.   33. The composition of claim 32, wherein the Tf molecule has been modified to exhibit a lower affinity for the transferrin receptor (TfR) compared to a wild type Tf molecule.   33. The composition of claim 32, wherein the Tf molecule has been modified so that it does not bind to TfR.   33. The composition of claim 32, wherein the Tf molecule has been modified to exhibit a lower affinity for iron compared to a wild type Tf molecule.   33. The composition of claim 32, wherein the Tf molecule has been modified so that it does not bind to iron.   33. The composition of claim 32, wherein the Tf molecule is lactoferrin or melanotransferrin.

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