MX2007001424A - Combination therapy using transferrin fusion proteins comprising glp-1. - Google Patents

Abstract

The present invention provides combination therapy comprising transferrin fusion protein and DPP-IV inhibitors and/or neutral endopeptidase (NEP) inhibitors. The transferrin fusion protein comprises therapeutic polypeptides or peptides useful in the treatment of diseases such as diabetes.

Description

COMBINATION THERAPY USING TRANSFERRIN FUSION PROTEINS COMPRISING 1 TYPE GLUCAGON PEPTIDE FIELD OF THE INVENTION The present invention relates to transferrin fusion proteins comprising insulinotropic peptides with extended therapeutic effective in vivo half life. The present invention also relates to combination therapies using inhibitors of DPP-IV and / or neutral endopeptidase (NEP) inhibitors and insulinotropic peptides.

BACKGROUND OF THE INVENTION Proteases Proteolytic enzymes play an important role in the regulation of physiological processes such as cell proliferation, differentiation and signaling processes by regulating the development and processing of proteins. Proteolytic enzymes control the levels of important proteins, enzymes and structural regulatory proteins through proteolytic degradation. An uncontrolled proteolytic enzyme activity, either increased or reduced, has been implicated in a variety of disease conditions including inflammation, cancer, REF. : 179529 arteriosclerosis and degenerative disorders. The International Union of Biochemistry and Molecular Biology (IUBMB) has recommended the use of the term "peptidase" for the subset of peptide binding hydrolases (Subclass E.C. 3.4). The term widely used protease is synonymous with peptidase. The peptidases comprise two groups of enzymes: the endopeptidases and the exopeptidases, which cut peptide bonds at points within the protein and remove amino acids sequentially either of the N or C terminus respectively. The term "proteinase" is synonymous with endopeptidase. Proteolytic enzymes are classified according to their catalytic mechanisms. Four mechanical classes have been recognized by the IUBMB: serine proteases, cysteine proteases, aspartic proteases and metalloproteases. Serine proteases are a large family of proteolytic enzymes that contain a serine residue in the catalytic active site for protein cutting. They are ubiquitous because they are found in viruses, bacteria and eukaryotes. Serine proteases have a wide range of substrate specificities and can be subdivided into subfamilies based on these specificities. There are more than 20 subfamilies of serine proteases which are grouped into six clans (SA, SB, SC, SE, SF and SG). The prolyl oligopeptidase is a serine protease grouped in the SC clan. It hydrolyses proline-containing peptides on the carboxyl side of proline residues. It is presumably involved in the maturation and degradation of peptide and neuropeptide hormones (Wilk et al., 1983 Life Sci. 33, 2149-2157). Examples of prolyl oligopeptidase include dipeptidyl peptidase IV (DPP-IV), dipeptidyl peptidase II (DPP-II), fibroblast activation protein and prolyl oligopeptidase. These enzymes have different specificities. Proline is present in numerous peptide hormones. It determines certain structural properties of these peptides, such as conformation and stability of these peptides, preventing degradation by non-specific proteases. There are a number of peptidases that attack proline bonds. These peptidases are not only involved in the cleavage of X-Pro or Pro-X bonds, but also in the degradation of corresponding alanyl, with reduced activity. Peptidases that have highly specific actions in proline-containing sequences are attractive targets of medicinal chemistry since some of them have been linked to the modulation of the biological activity of natural peptide substrates. For example, DPP-IV is linked to the treatment of diabetes by regulating the level of glucagon-like peptide-1 (GLP-1). The activity of DPP-IV is increased in several diseases such as rheumatoid arthritis, multiple sclerosis, Grave's disease and Hashimoto's thyroiditis, sarcoidosis and cancer. The activity of DPP-IV is also increased in AIDS, Down syndrome, anorexia / bulimia, pregnancy and hypogammaglobulinemia.

Dipeptidyl Peptidases that Include DPP-IV The activity of dipeptidyl aminopeptidase is peptidase activity that catalyzes the removal of dipeptides from the N-terminus of peptides, polypeptides and proteins. Generally, a dipeptidyl aminopeptidase is capable of cleaving the XY dipeptide from the unsubstituted N-terminal amino group of a peptide, polypeptide or protein, wherein X and Y represent any amino acid residue. Examples of dipeptidyl peptidases (DPPs) include dipeptidyl peptidase I (DPP-I), dipeptidyl peptidase II (DPP-II), dipeptidyl peptidase III (DPP-III) and dipeptidyl peptidase IV (DPP-IV). 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 granzymes, which are neutral serine proteases expressed exclusively in activated cytotoxic lymphocyte granules. DPP-II is a serine protease found in lysosomes. Like DPP-IV, it cuts peptide bonds containing proline. In fact, DPP-II has a substrate specificity similar to DPP-IV but It is only active at acidic pH. Dipeptidyl peptidase III (DPP-III) is a metalloprotease. DPP-IV is a serine protease comprising the serine protease motif GWSYG and having broad substrate specificity. It hydrolyzes a peptide in sequence from the amino terminus to release an amino acid. However, hydrolysis is terminated when an amino acid residue followed by proline is reached. As a result, a peptide that has a X-Pro-Y- bond (X and Y are optional amino acids) will be cut to produce X-Pro and Y-DPP-IV will also cut dipeptides with alanine in the penultimate position, although less effectively than dipeptides with proline (Yaron et al., 1993 Crit. Rev. Biochem., Mol.

Biol. 28: 31-81). The enzyme will also cut other sequences, but with even lower efficiency. DPP-IV has been shown to be highly specific for releasing dipeptides from the N-terminal end of biologically active peptides with proline or alanine in the penultimate position of the N-terminal sequence of the peptide substrate. A large number of potential peptide substrates for DPP-IV have been identified. The DPP-IV substrates include peptide hormones and chemokines. Examples of some peptide hormones are endomorphine-II, GLP-1, GLP-2, gastric inhibitor peptide (GIP), neuropeptide Y, growth hormone-releasing hormone (GHRH) and substance P, and examples of some chemokines are RANTES, GCP-2, SDF-1, SDF-2β, MDC, MCP-1, MCP-2 and MCP-3. DPP-II has substrate specificity almost identical to that of DPP-IV.

DPP-IV and Diabetes Insulin-dependent diabetes mellitus (IDDM), or type I diabetes) is currently treated through the administration of insulin to patients. Non-insulin dependent diabetes mellitus (NIDDM), or type II diabetes) is treated with diet, administration of sulfonylureas to stimulate insulin secretion or with biguanides to increase glucose uptake. Resistant individuals may require insulin therapy. Standard therapy requires daily intravenous injection of insulin which will treat acute symptoms, but prolonged therapy results in vascular disease and nerve damage. Modern methods such as transplants are expensive and require a risky surgical intervention. Thus, there is a need to develop a low cost and highly effective alternative for the treatment of diabetes. In recent years, there has been a growing interest in DPP-IV as a goal to reduce blood glucose levels. The use of inhibitors to block DPP-IV enzyme or DPP-IV enzyme activity in the blood of subjects leads to reduced degradation of insulinotropic peptides endogenous or exogenously administered such as GIP, GLP-1 or analogs thereof. GIP and GLP-1, hormones that stimulate the glucose-induced secretion of insulin by the pancreas, are substrates of DPP-IV. Specifically, since DPP-IV removes the amino-terminal dipeptide of amino-terminal GLP-1 to generate GLP-1- (9-36) -amide, which is unable to develop glucose-dependent insulin secretion from the islets, the inhibition of this DPP-IV or DPP-IV type enzyme activity in vivo could effectively suppress undesired enzymatic activity under pathological conditions in mammalian organisms. PCT / DE97 / 00820 describes alanyl pyrrolidide and isoleucyl thiazolidide as inhibitors of DPP-IV or enzymatic activity type DPP-IV. DD 296075 describes pyrrolidide hydrochloride and isoleucyl thiazolidide. The patent of E.U.A. No. 6,548,481 describes inhibitors analogous to dipeptide compounds formed from an amino acid and a thiazolidine or pyrrolidine group, and salts thereof. Although these are functional inhibitors of DPP-IV activities, the use of these inhibitors in certain patients or certain forms of the disease can be problematic since the enzyme is responsible for the activation or inactivation of this broad range of bioactive peptides. say, DPP-IV inhibitors lack specificity for the desired GIP and GLP-1 targets.

Protection of Therapeutic Peptides by Modification An alternative way to prevent therapeutic proteins and peptides such as GIP or GLP-1 from being cut by proteolytic enzymes is to modify the proteins and peptides themselves to block their exposure to proteolytic enzymes. Modifications to proteins have been shown to increase the stability, circulation time and biological activity of the 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 Recent Developments (Weinstein, B., ed. Marcel Dekker, Inc., publ., New York 1983) which it is incorporated herein by way of reference. Also, Francis's review (1992 Focus on Growth Factors 3: 4-10, (Mediscript, London)) describes modification of proteins and fusion proteins, which is incorporated herein by reference. With the advancement of recombinant DNA technology and automated techniques, large quantities of modified polypeptides that are short, medium or long can now easily be prepared. A large number of modified small polypeptide hormones can be synthesized using automatic peptide synthesizers, solid state resin techniques or recombinant techniques. By For example, large quantities of modified dipeptidyl peptidase substrates, for example, DPP-IV substrates such as GLP-1, GIP, neuropeptide Y and bradykinin can be produced using an automatic peptide synthesizer.

Brief description of the invention The present invention provides transferrin fusion proteins comprising peptides or therapeutic proteins that are susceptible to protease cutting. The present invention also provides transferrin fusion proteins comprising peptides or therapeutic proteins that are sensitive, resistant or partially resistant to protease cleavage. The protease can be DPP-IV or neutral endopeptidase (NEP). Moreover, the present invention provides compositions comprising transferrin fusion protein and a second agent such as, but not limited to, DPP-IV and / or NEP inhibitors. In addition, the compositions may be pharmaceutical compositions used in the treatment of various diseases. The present invention provides combination therapies 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 concurrently with the one or more second agent. As an alternative, the protein of Transferrin fusion is administered sequentially, either before or after the administration of the second agent. Preferably, the second agent is an inhibitor of DPP-IV or NEP. The GLP-1 peptide portions of the present invention can be modified to contain one or more mutations such that they are partially or completely resistant to protease cleavage, such as cleaved by DPP-IV. The GLP-1 peptide can be GLP-I (7-37) (SEQ ID NO: 32) or GLP-1 (7-36) (amino acids 1-30 of SEQ ID NO: 2). For example, these peptides can be modified by mutating A8 to G and / or K34 to A.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the restriction enzyme map of pREX0094. Figure 2 shows the restriction enzyme map of plasmid pREX0198. Figure 3 shows the restriction enzyme map of pSAC35. Figure 4 shows the restriction enzyme map of plasmid pREX0240. Figure 5 shows the restriction enzyme map of pREX0052. Figure 6 shows the enzyme map of restriction of pREX0367. Figure 7 shows the restriction enzyme map of pREX0368. Figure 8 shows the incubation time course of GLP-1 and H-GLP-1 and DPP-IV. The graph shows the remaining amount of active peptide and full length, as measured by an ELISA specific for active GLP-1.

Detailed description of the invention 1. General description This invention is based, in part, on the need to develop a more effective and low-cost alternative for the treatment of diabetes. Insulinotropic peptides, such as GLP-1, are promising therapeutic agents for the treatment of non-insulin-dependent type II diabetes mellitus as well as related metabolic disorders, such as pre-diabetes, metabolic syndromes and obesity. Other useful insulinotropic peptides include exendin-3 and exendin-4. However, these insulinotropic peptides have short half-lives in vivo in plasma, mainly due to rapid elimination of serum and proteolytic degradation. Extensive work has been carried out to inhibit DPP-IV, the enzyme responsible for the degradation of GLP-1 or to modify GLP-1 in such a way that its degradation becomes slower while still maintaining the biological activity. Despite these extensive efforts, an active and long-lasting GLP-1 has not been produced. There is then a need to modify GLP-1, exendin 3, exendin 4 and other insulinotropic peptides to provide a longer in vivo duration of action, while retaining its low toxicity and therapeutic advantages. 2. Definitions As used herein, the term "derivative" refers to a modification of one or more amino acid residues of a peptide by chemical means, either with or without an enzyme, for example, by alkylation, acylation, formation of ester or amide formation. As used herein, the term "derivative of" refers to obtaining a molecule from a specified source such as obtaining a molecule of a progenitor molecule. As used herein, the term "dipeptidyl aminopeptidase activity" refers to a peptidase activity that cleaves N-terminal dipeptides from a sequence of peptides, polypeptides or proteins. Generally, dipeptidyl aminopeptidase is capable of cleaving the XY dipeptide from the unsubstituted N-terminal amino group of a peptide, polypeptide or protein, where X or Y can represent any amino acid residue selected from the group consisting of Ala, ARg, Asn, Asp, Cys, Gln, Glu, Gly, His, Lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val , but at least Ala, Arg, Asp and / or Gly. Preferably, Y is Pro or Ala. All of X and Y may be different or identical. Examples of dipeptidyl aminopeptidase include, but are not limited to DPP-I, DPP-II, DPP-III and DPP-IV. As used herein, the terms "Peptide 1 Type Glucagon (GLP-1)" and "GLP-1 derivatives" refer to intestinal hormones that generally stimulate insulin secretion during hyperglycemia, suppress glucagon secretion, stimulate insulin biosynthesis (pro) and slow down gastric emptying and acid secretion. Some GLP-ls and GLP-1 derivatives promote the uptake of glucose by cells but do not stimulate the expression of insulin as described in the patent of E.U.A. No. 5,574,008 which is incorporated herein by reference. As used herein, the term "insulinotropic peptides" refers to peptides with insulinotropic activity. Insulinotropic peptides stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin. These peptides include precursors, analogs, peptide fragments such as glucagon-like peptide 1, exendin 3 and exendin 4 and other peptides with insulinotropic activity. As used herein, "pharmaceutically acceptable" refers to materials and compositions that are physiologically tolerable and that typically do not produce an allergic reaction or similar unpleasant reaction, such as gastric discomfort, dizziness, and the like, when administered to a human. Typically, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the pharmacopoeia of E.U.A. or other pharmacopoeias generally recognized for use in animals, and more particularly in humans. As used herein, the term "pharmaceutical composition" refers to a composition comprising an agent together with a pharmaceutically acceptable carrier or diluent when required. The pharmaceutically acceptable carriers and additives are selected such that the side effects of the pharmaceutical compound are minimized and the yield of the compound is not canceled or inhibited to such an extent that the treatment is ineffective. As used herein, "physiologically effective amount" is that amount supplied to a subject to give the desired palliative or curative effect. This amount is specific for each drug and its level of final approved dose. As used herein, "therapeutically effective amount" refers to the amount of modified therapeutic polypeptide or peptide that, when administered to a subject that requires it, is sufficient to effect treatment. The amount of modified therapeutic polypeptide or peptide that constitutes a "therapeutically effective amount" will vary depending on the therapeutic protein used, the severity of the condition or disease and the age and body weight of the subject to be treated, but can be routinely determined by someone of Ordinary capacity in the technique referring to its own knowledge and its description. As used herein, "therapeutic protein" refers to proteins, polypeptides, antibodies, peptide fragments or variants thereof, which have one or more therapeutic and / or biological activities. The therapeutic proteins encompassed by the invention include but are not limited to proteins, polypeptides, peptides, antibodies and biologics. The terms peptides, proteins and polypeptide are used interchangeably herein. In addition, the term "therapeutic protein" may refer to the endogenous or naturally occurring correlate of a therapeutic protein. For a polypeptide or peptide that presents a "therapeutic activity" or a protein that is "therapeutically effective" is intended to mean a polypeptide, peptide or protein that possesses one or more known biological and / or therapeutic activities associated with a therapeutic protein such as one of the therapeutic proteins described herein or otherwise known in the art. As a non-limiting example, a "therapeutic protein" is a protein, polypeptide or peptide that is useful to treat, prevent or reduce a disease, condition or disorder. This disease, condition or disorder can be in humans or in a non-human animal, for example, veterinary use. As used herein, the term "treatment" or "treating" refers to any administration of a compound of the present invention and includes: 1) preventing the disease from occurring in an animal that may be predisposed to the disease but that still do not experience or present the pathology or symptomatology of the disease; 2) inhibit the disease in an animal that is experiencing or presenting the pathology or symptomatology of the disease (ie, stop further development of the pathology and / or symptomatology) or 3) decrease the disease in an animal that is experiencing or presenting the pathology or symptomatology of the disease (that is, reversing the pathology and / or symptomatology).

As used herein, the term "biological activity" refers to the ability to mediate a biological function. "Biological activity" includes functional activity as well as structural activity. As used herein, the term "palliative" refers to the ability to alleviate or reduce the symptoms of a disease or disorder without affecting a cure. For example, an agent that alleviates pain without curing the condition or disease is a palliative agent. As used herein, the term "prophylactic" refers to having a protective effect such as acting to defend against or prevent something, especially disease or condition. As used herein, "purified" nucleic acid or protein refers to a protein or nucleic acid that has been separated from a cellular component. The "purified" nucleic acids or proteins have been purified to a level of purity not found in nature. As used herein, the term "substantially pure" nucleic acid or protein refers to a preparation of proteins or nucleic acid that lacks all other cellular components. As used herein, the term "therapeutic" refers to having a curative effect, restorer or remedy. For example, a "therapeutic agent" or a "therapeutic composition" has a curative effect. 3. Specific Modalities Dipeptidyl Peptidases Dipeptidyl peptidases are hydrolases that remove dipeptides from the N-terminally unsubstituted amino group of a peptide, polypeptide or protein. Examples of dipeptidyl peptidases include but are not limited to DPP-1, DPP-II, DPP-III, DPP-IV, attractant and fibroblast activation protein (FAP). New enzymes of this family or with similar function but different structure are emerging. Dipeptidyl peptidase I (DPP-I), also known as cathepsin C, is a lysosomal cysteine protease that belongs to the papain family. DPP-IV is able to sequentially remove dipeptides from the free amino terminus of various peptide and protein substrates, then acting in the exopeptidase mode (specifically dipeptidyl peptidase). The cut is not effective if the fragmented bond has on either side a proline residue, or the N-terminal residue is lysine or arginine. DPP-II is a serine protease found in lysosomes with unknown function. Like DPP-IV, cuts predominantly proline-containing peptide bonds. In fact, DPP-II has a substrate specificity similar to DPP-IV but is only active at acidic pH. DPP-II and mammalian DPP-IV can be distinguished using the inhibitors puromycin and bacitracin; puromycin will inhibit DPP-II only while bacitracin inhibits DPP-IV alone (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. DPP-VII, also known as quiescent cell proline dipeptidase, is a proline-specific dipeptidase. It has been suggested that DPP-VII and DPP-II are identical proteases based on a sequence comparison of human DPP-VII and rat DPP-II (78% identity) (Araki et al., 2001 J. Biochem. , 279-288). DPP-VIII is a human post-proline dipeptidyl aminopeptidase which is homologous to DPP-IV and FAP (Abbott, C.A. et al., 2000 European Journal of Biochemistry 267, 6140). Similar to DPP-IV, DPP-VIII is ubiquitous. The full-length DPP-VIII cDNA codes for a protein of 882 amino acids that has approximately 27% identity and 51% similarity to DPP-IV and FAP, but no transmembrane domain and no N-linked or 0-linked glycosylation . Purified recombinant DPP-VIII hydrolyzed the DPP-IV substrates Ala-Pro, Arg-Pro and Gly-Pro. This Thus, recombinant DPP-VIII shares a postproline dipeptidyl aminopeptidase activity with DPP-IV and FAP. The activity of DPP-VIII enzyme had an optimum neutral pH consistent with that it is non-lysosomal. The similarities between DPP-VIII and DPP-IV in tissue expression pattern and substrates suggest a potential role for DPP-VIII in T cell activation and immune function similar to DPP-IV. Olsen C. et al. (2002 Gene 299, 185-93) report the identification and characterization of a new DPP-IV type molecule, called dipeptidyl peptidase DPP-IX type protein. Like DPP-IV, DPP-IX comprises the serine protease motif GWSYG (SEQ ID NO: 110). The presence of this motif and the conserved order and separation of the Ser, Asp and His residues that form the catalytic triad in DPP-IV, places DPP-IX in the family of DPP-IV genes. Attractiveness (DPPT-L) is a soluble glycoprotein of 175 kDa that is reported to hydrolyze Gly-Pro. The attractant contains a kelch repeat domain and does not share significant sequence homology with DPP-IV or any other peptidase. The fibroblast activation protein (FAP) is a cell surface bound protease of the prolyl oligopeptidase gene family expressed at sites of tissue remodeling. The prolyl endopeptidase (PEP), also called proline oligopeptidase (PO), was discovered for the first time by Walter et al. as an oxytocin degrading enzyme in the human uterus (Walter et al., Science 173, 827-829 (1971)). The enzyme cuts peptide bonds on the carboxy side of proline in peptides containing the sequence X-Pro-Y, where X is an N-terminal substituted peptide or amino acid and Y is a peptide, amino acid, amide or alcohol (Yoshimoto et al. ., J. Biol. Chem. 253, 3708-3716 (1979)). The enzyme has a high specificity for the trans conformation of the peptide bond on the imino side of proline (Lin &; Brandts, Biochemistry 22, 4480-4485 (1983)). The prolyl oligopeptidase hydrolyzes angiotensin I and angiotensin II which results in the release of angiotensin (1-7). Angiotensin (1-7) has vasodilatory activity and modulates the release of vasopressin, which is able to influence the memory process as demonstrated by injecting rats with specific PEP inhibitors. The injection reverses the amnesia induced by scopolamine. This experiment is not only an example that provides evidence for a possible physiological function of the enzyme, but has also led to the hypothesis that PEP inhibitors can influence the process of memory and against dementia (Yoshimoto et al., 1987 J. Pharmacobio-Dyn., 10, 730-735).

Dipeptidyl Peptidase (DPP-IV) and Substrates DPP-IV is a ubiquitously expressed molecule that has been implicated in the degradation of several peptides and hormones. Several types of DPP-IV have been purified and the enzymatic properties have been revealed. For example, DPP-IV has been isolated from rat liver (Hopsu-Havu VK et al., 1966 Histochem., 7: 197-201), pig 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) , human submaxillary gland (Oya H., et al., 1972 Biochim Biophys. Acta, 258: 591-599), kidney of sheep (Yoshimoto T. et al., 1977 Biochim, Biophys. Acta, 485: 391-401 Yoshimoto T. et al., 1978 J. Biol. Chem., 253: 3708-3716) or microorganisms (Fukusawa KM 1981 Biochem. Biophys., 210: 230-237; Yoshimoto T. 1982 J. Biochem., 91: 1899-1906 (1982)). In the human immune system, DPP-IV is identical to the CD26 antigen on the surface of T cells that is expressed by activated lymphocytes (T cells, B cells and natural killers). CD26 / DPP-IV is a type II membrane glycoprotein with intrinsic dipeptidyl peptidase IV activity and the ability to bind adenosine deaminase type I (ADA-1). It is expressed in epithelial cells constitutively, but in T lymphocytes, it is expressed under tight regulation cellular, with regulated expression after cell activation. CD26 / 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 the costimulatory activity seems to partially depend on this enzymatic activity (Tanaka et al., 1993 Proc. Nati, Acad. Sci. USA 90: 4586-4590). DPP-IV is involved in the regulation of chemokine function and may play an important role in HIV infection. The patent of E.U.A. No. 6,265,551 describes a circulating and soluble form of DPP-IV / CD26 isolated from human serum. The serum form shares similar enzymatic and antigenic properties with the ubiquitous membrane form; however, in several biochemical aspects there are different differences. In particular, the circulating serum form has a molecular weight of 175 kDa, in contrast to the molecular weight of 105 kDa of the membrane form, and does not bind to ADA-1. However, the circulating form expresses functional dipeptidyl peptidase IV activity and retains the ability to co-stimulate the response of T lymphocytes to call antigens. The proteolytic activity of DPP-IV resides in a stretch of approximately 200 amino acids located at the C-terminal end of the protein. The catalytic residues (Ser-629, Asp-708, His-740) are arranged in an order unique that is different from classical serines proteases such as chymotrypsin and subtilisin. The proline-specific dipeptidyl peptidase activity alters the biological activity of a large number of bioactive proteins and polypeptide comprising, inter alia, GLP-1, the neurotransmitter substance P, human growth hormone releasing factor, erythropoietin, interleukin 2 and many others . Potential DPP-IV substrates are listed in Tables 1, 2 and 3. Modulation of these polypeptides to affect cleavage by DPP-IV could be useful in the treatment of clinical conditions including but not limited to diabetes, inflammation, diseases vascular diseases, autoimmune diseases, multiple sclerosis, diseases of the joints and diseases associated with the transformation of benign and malignant cells.

Table 1 Cytokines, neuro- and vasoactive human growth factors with a penultimate proline, which are putative substrates for DPP-IV Table 2 Human peptides and proteins with a penultimate alanine that are putative substrates for DPP-IV The present invention can use modified DPP substrates comprising one or more amino acids additional N-term substrates to protect substrates from DPP activity. The preferred substrates for the modification according to the present invention are described in Table 3 Table 3 Substrates for cutting with DPP-IV (CD26) The substrates for modification comprise X-ProY, X-Ala-Y, X-Ser-Y or X-Gly-Y at the amino terminus. Preferably, the substrate for the modification is GLP-1.

Modified Protected Polypeptides of Activity DPP The present invention provides modified polypeptides, such as modified DPP polypeptide substrates, comprising one or more additional amino acids at the N-terminus to protect substrates of DPP activity polypeptides. In one embodiment, the modified polypeptides have an additional amino acid at their N terminus in comparison to the wild type polypeptides. In another embodiment, the modified polypeptides have five Additional amino acids at their N terminus. Alternatively, the modified polypeptides have between one and five additional amino acids at their N terminus. Any of the 20 amino acids may be added to the N terminus of the polypeptide substrate or unnatural amino acids may be added. It is expected that any pharmaceutical polypeptide having peptide bonds that could be subjected to shear in the circulation or any in vivo after administration will benefit from the modification according to the present invention due to the protection of the DPP cut that is offered by the present invention. In accordance with this aspect of the invention, it is possible to remove at least about 30%, preferably at least about 50%, most preferably at least about 70%, even more preferably at least about 90% and more preferably about at least about 99% of the dipeptidyl peptidase activity. It is also possible to completely remove the dipeptidyl aminopeptidase activity using the methods of the present invention. Also, it is possible to reduce the sensitivity to dipeptidyl peptidase of the substrate by at least about 30%, preferably at least about 50%, most preferably at least about 70%, still more preferably at least about 90% and most preferably at least about 99% of the sensitivity to dipeptidyl peptidase. It is also possible to completely remove the sensitivity to dipeptidyl aminopeptidase using the methods of the present invention. Although the polypeptide or modified peptide substrates of the present invention are partially or substantially protected from DPP activity, the modified polypeptide substrates have retained at least about 10%, preferably at least about 30%, preferably at least about 50%, most preferably at least about 70% and more preferably at least about 90% and too preferably at least about 99% of its activity and functional potency. In some cases, substrates of modified polypeptides or peptides with reduced functional activity or potency will be useful. For example, when the polypeptide or modified peptide is fused to another polypeptide, such as transferrin, to form a fusion protein with increased serum stability and circulating half-life in vivo, a modified polypeptide or peptide substrate with functional activity or reduced potency. it could be useful. In other cases, the modified polypeptides or peptides may have increased potency compared to unmodified polypeptides or peptides.

The modified polypeptide molecules of the invention are substantially protected from cleavage by dipeptidyl peptidase as compared to an unmodified version of the same polypeptide. The rating of this substantial protection may vary by the assay used to compare the modified versus the unmodified polypeptide. To exhibit substantial protection, however, the modified polypeptide will exhibit a detectable level of cleavage resistance by dipeptidyl peptidase in the assay. These assays include but are not limited to those described in 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, 93-102). The polypeptide substrates stabilized by DPP of the present invention are also more stable in the presence of DPP in vivo than a non-stabilized polypeptide substrate. A therapeutic polypeptide substrate stabilized with DPP generally has an increased half-life of activity compared to an unstabilized peptide of identical sequence. Peptidase stability can be determined by comparing the half-life of the unmodified polypeptide substrate in serum or blood with the half-life of a counterpart therapeutic peptide modified in serum or blood. The half-life can be determined by sampling serum or blood after administration of the modified or unmodified peptides and determining the activity of the peptide. In addition to determining the activity, the length of the polypeptide substrates can also be measured by HPLC or mass spectrometry. The present invention also provides modified polypeptides or peptides having an altered amino terminus according to the invention, to protect against cleavage by DPP and having alterations of internal amino acids and / or in C terminus that do not affect the activity or functional potency of the polypeptide. These modified polypeptides would have minor amino acid changes that are normally conservative amino acid substitutions, although non-conservative substitutions are also contemplated. The modified polypeptides or peptides of the present invention may also have altered functional activity. For example, a modified polypeptide or peptide with increased functional activity may be useful. Alternatively, a modified polypeptide or peptide with reduced functional activity can be used. Thus, the modified polypeptides or peptides of the present invention also contain amino acid changes that do affect functional activity or potency. For example, GLP-1 analogs with altered functional activity can be modified at their amino terminus to protect against cleavage by DPP.

Examples of conservative amino acid substitutions are substitutions made within the same group as such within the group of 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), aromatic amino acids (such as phenylalanine, tryptophan, tyrosine) and small amino acids (such as glycine, alanine, serine, trionine, methionine). Non-conservative substitutions encompass substitutions with amino acids in one group for amino acids in another group. For example, a non-conservative substitution will include the substitution of a polar amino acid for a hydrophobic amino acid. For a general description of nucleotide substitution see for example, Ford et al. (1991), Prot. Exp. Pur. 2: 95-107. The present invention provides obvious variants of the amino acid sequence of the modified polypeptides or peptides, such as naturally occurring mature forms of polypeptides or variant allelic / sequence peptides of the polypeptides, recombinantly derived and non-naturally occurring variants of the peptides , and orthologs and paralogs of the polypeptides or peptides. These variants can be easily generated using techniques known in the art in the fields of recombinant nucleic acid technology and protein biochemistry.

These variants can be easily identified / made using molecular techniques and sequence information. In addition, these variants can be readily distinguished from other peptides based on sequence and / or structural homology to the modified polypeptides or peptides of the present invention. Preferably, the modified peptides of the present invention are GLP-1 and analogs thereof that comprise one or more additional amino acids at their N terminus. In some cases, DPP such as DPP-IV can activate a peptide instead of inactivating it through cutting.

In such cases, modification of the peptide could substantially reduce, delay or prevent the activation of the peptide.

Nucleic Acids Coding for Modified Polypeptides The present invention provides nucleic acid molecules that encode modified polypeptides or peptides that are partially or substantially protected from DPP cleavage and have activity and functional potency. In one embodiment, the nucleic acid molecules provided by the present invention encode polypeptides or peptides modified ones having at least one additional amino acid at their N-terminus compared to their unmodified wild-type polypeptide. In another modality, nucleic acid molecules encode modified polypeptides or peptides that have five additional amino acids at their N terminus. Alternatively, nucleic acid molecules encode modified polypeptides or peptides having between one and five additional amino acids at their N terminus. Preferably, the nucleic acid molecules encoding modified GLP-1 comprise a sequence encoding one or more additional amino acids at their N terminus. The nucleic acid molecules of the invention include deoxyribonucleic acids (DNAs), deoxyribonucleic acids both from a as double-stranded However, they can also be ribonucleic acids (RNAs), as well as double-stranded RNA: hybridized DNA molecules. Contemplated nucleic acid molecules also include genomic DNA, cDNA, mRNA and antisense molecules. The nucleic acid molecules of the present invention also include native or synthetic RNA, DNA or cDNA encoding a modified polypeptide, or the complementary strand thereof. To construct modified polypeptides that are partially or substantially protected from DPP activity but that have functional activity and / or potency compared to unmodified wild-type polypeptides, the acid The nucleic acid encoding the unmodified wild-type polypeptide can be used as a starting point and modified to encode the desired modified polypeptide. Numerous methods are known to add sequences or to mutate nucleic acid sequences encoding a polypeptide and to confirm the function of polypeptides modified by these modified sequences. The present invention also provides nucleic acids encoding polypeptides and peptides having a modified amino terminus for protection against cleavage by DPP and having alterations of internal amino acids and in the C terminus that do not substantially affect the activity or functional potency of the polypeptide. . These modified polypeptides may have minor amino acid changes that are usually conservative amino acid substitutions, although non-conservative substitutions are also contemplated. Nucleotide substitutions using techniques to achieve site-specific mutagenesis are well known in the art. Preferably, the nucleic acids encode GLP-1 analogs having one or more additional amino acids at their N terminus. As is known in the art the "similarity" between two polynucleotides or polypeptides is determined by comparing the nucleotide or amino acid sequence and the nucleotide or amino acid substitutes conserved from a polynucleotide or polypeptide with the sequence of a second polynucleotide or polypeptide. "Identity" is also known in the art which means the degree of sequence kinship between two polypeptide sequences or two polynucleotide sequences determined by the identity of the match between two strands of these sequences. Both identity and similarity can be easily calculated Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are a number of methods for measuring the identity and similarity between two polynucleotide or polypeptide sequences, the terms "identity" and "similarity" are well known to the skilled person (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48 : 1073 (1988) .The methods commonly used to determine identity or similarity between two sequences include, but are not limited to those described in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipman, D., SIAM J. Applied Math. 48: 1073 (1988). The preferred methods for determining identity are designed to give the greatest coincidence between the two sequences tested. Methods to determine identity and similarity are encoded in computer programs. The computer program methods that are preferred for determining the 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 referred to above is determined as the degree of identity between the two sequences indicating derivation of the first sequence from the second. The degree of identity between two nucleic acid sequences can be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Needleman and Wunsch (1970) Journal of Molecular Biology 48: 443-453). For purposes of determining the degree of identity between two nucleic acid sequences for the present invention, GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.

Codon Optimization The degeneracy of the genetic code allows for variations of the nucleotide sequence of polypeptides, still yielding a modified polypeptide comprising an amino acid sequence identical to that of the polypeptide encoded by a first DNA sequence. The method, known as "codon optimization" (described in U.S. Patent No. 5,547,871 which is incorporated herein by reference in its entirety) provides one with a means to design this altered DNA sequence. The design of genes optimized by codons must take into account a variety of factors, including the frequency of the use of codons in an organism, nearest adjacent frequencies, stability of the RNA, the potential for secondary structure formation, the synthesis route and the desired future DNA manipulations of that gene. In particular, the available methods can be used to alter the codons encoding a given fusion protein with those more readily recognized by yeast when using yeast expression systems. The degeneracy of the genetic code allows the same amino acid sequence to be encoded and translated into many different ways For example, leucine, serine and arginine are each encoded by six different codons, while valine, proline, trionine, alanine and glycine are each encoded by four different codons. However, the frequency of use of these synonymous codons varies from genome to genome between eukaryotes and prokaryotes. For example, patterns of choice of synonymous codons among mammals are very similar, while evolutionarily distinct organisms such as yeast (S. cerevisiae), bacteria (such as E. coli) and insects (such as D. nielanogaster) reveal a pattern clearly different from frequencies of use of genomic codons (Grantham, R., et al., Nucí Acids Res., 8, 49-62 (1980); Grantham, R., et al., Nucí. Acids Res., 9 , 43-74 (1981), Maroyama, T., et al., Nucí Acids Res., 14, 151-197 (1986); Aota, S., Et al., Nucí Acids Res., 16, 315 -402 (1988); Wada, K., et al., Nuci Acids Res., 19 Supp., 1981-1985 (1991); Kurland, CG, FEBS Letters, 285, 165-169 (1991)). These differences in codon selection patterns appear to contribute to the overall expression levels of individual genes by modulating the 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, JF, and Yarus, M., J. Mol.

Biol., 209, 65-77 (1989); Varenne, S., et al. , J. Mol, Biol., 180, 549-576 (1984), Varenne, S. , et al. , J. Mol, Biol., 180, 549-576 (1984); Garesl, J.-P., J. Theor. Biol., 43, 211-225 (1974); Ikemura, T., J. Mol. Biol., 146, 1-21 (1981); Ikemura, T., J. Mol. Biol., 151, 389-409 (1981)). The preferred codon usage frequencies for a synthetic gene should reflect the codon uses of nuclear genes derived from the exact (or most closely related) genome of the cell / organism to be used for expression of recombinant proteins, particularly that of the yeast species. As described above, in a preferred embodiment the modified polypeptide is optimized by codons, before or after modification as described herein for the expression of yeast.

Vectors Expression units for use in the present invention will generally comprise the following elements, operably linked in a 5 'to 3' orientation: a transcription promoter, a secretory signal sequence, a DNA sequence encoding a modified polypeptide and a transcription terminator. As indicated above, any arrangement of the polypeptide and modified peptide can be used in the vectors of the invention. The selection of suitable promoters, signal sequences and terminators will be determined by the selected host cell and will be apparent to one skilled in the art and described more specifically below. Yeast vectors suitable for use in the present invention are described in U.S. Pat. No. 6,291,212 and include YRp7 (Struhl et al., Proc. Nati, Acad. Sci. USA 76: 1035-1039, 1978), YEpl3 (Broach et al., Gene 8: 121-133, 1979), pJDB249 and pJDB219 (Beggs, Nature 275: 104-108, 1978), pPP00005, pSeCHSA, pScNHSA, pC4 and derivatives thereof. Useful yeast plasmid vectors also include pRS403-406, pRS413-416 and the Pichia vectors available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. The plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (YIps) and incorporate the selectable yeast markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-41.6 are Yeast Centromeres plasmids (YCps). These vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which there is a phenotypic assay to enable transformants to be selected. Selectable markers that are preferred are those that complement the auxotrophy of the host cell, provide resistance to antibiotics or make it possible for a cell to use specific carbon sources, and include LEU2 (Broach et al., cited above), URA3 (Botstein et al., Gene 8: 17, 1979), HIS3 (Struhl et al., cited above) or P0T1 (Kawasaki and Bell, EP 171, Í42). Other suitable selectable markers include the CAT gene, which confers resistance to chloramphenicol in yeast cells. Preferred promoters for use in yeast include promoters of yeast glycolytic genes (Hitzeman et al., J Biol. Chem. 225: 12073-12080, 1980; Alber and Kawasaki, J. Mol. Appl. Genet., 1: 419-434. , 1982; Kawasaki, U.S. Patent No. 4,599,311) or alcohol dehydrogenase genes (Young et al., In Genetic Engineering of Microorganisms for Chemicals, Hollaender et al., (Eds.), P 355, Plenum, NY, 1982 Ammerer, Meth. Enzymol. 101: 192-201, 1983). In this regard, the promoters that are particularly preferred are the TPI1 promoter (Kawasaki, U.S. Patent No. 4,599,311) and the ADH2-4C promoter (see U.S. Patent No. 6,291,212) (Russell et al., Nature 304: 652- 654, 1983). The expression units may also include a transcription terminator. A preferred transcription terminator is the TPI1 terminator (Alber and Kawasaki, cited above). Most preferably, the promoter is the PRB1 promoter described in EP 431880 and the terminator is the ADH1 terminator described in EP 60057, which is they are incorporated herein by reference in their entirety. In addition to yeast, the modified polypeptides and peptides of the present invention can be expressed in filamentous fungi, for example, species of the genus Aspergillus. Examples of useful promoters include those derived from glycolytic genes of Aspergillus nidulans, such as the ADH3 promoter (McKnight et al., EMBO 14: 2093-2099, 1985) and the tpiA promoter. An example of a suitable terminator is the ADH3 terminator (McKnight et al., Cited above). Expression units using these components can be cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus, for example. The mammalian expression vectors to be used in carrying out the present invention will include a promoter capable of directing the transcription of the modified polypeptides and peptides. Preferred promoters include viral promoters and cellular promoters. Preferred viral promoters include the major late promoter of adenovirus 2 (Kaufman and Sharp, Mol, Cell, Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subramani et al., Mol. Cell. Biol. : 854-864, 1981). Preferred cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al., Science 222: 809-814, 1983) and a promoter.

V? of mouse (see U.S. Patent No. 6,291,212) (Grant et al., Nuc.Aids Res. 15: 5496, 1987). A promoter that is particularly preferred is a mouse VH promoter (see U.S. Patent No. 6,291,212). These expression vectors may also contain a set of RNA splice site located towards the 3 'end from the promoter and towards the 5' end from the DNA sequence encoding the modified polypeptide or peptide. Preferred RNA splice sites can be obtained from adenovirus genes and / or immunoglobulin genes. A polyadenylation signal located towards the 3 'end of the coding sequence of interest is also contained in the expression vectors. Polyadenylation signals include the early or late polyadenylation signals of SV40 (Kaufman and Sharp, cited above), the polyadenylation signal of the E1B region of adenovirus 5 and the terminator of the human growth hormone gene (DeNoto et al., Nuc Acids Res. 9: 3719-3730, 1981). A polyadenylation signal that is particularly preferred is the VH gene terminator (see U.S. Patent No. 6,291,212). The expression vectors can include a non-coding viral leader sequence, such as the tripartite leader of adenovirus 2, located between the promoter and the RNA splice sites. Preferred vectors may also include enhancer sequences, such as the SV40 enhancer and the mouse μ enhancer (see U.S. Patent No. 6,291,212) (Gillies, Cell 33: 717-728, 1983). Expression vectors may also include sequences encoding VA adenovirus RNA molecules. Expression vectors are also used to express fusion proteins comprising the polypeptide or modified peptide of the present invention fused to a second polypeptide or peptide, eg, transferrin, to increase the half-life of the modified polypeptide or peptide, as described down. Also, the modified polypeptide or peptide can be fused to a marker and / or a cut-off site for the expression and release of the modified polypeptide or peptide.

Transformation Techniques for transforming fungi are well known in the literature, and have been described, for example, by Beggs (cited above), Hinnen et al. (Proc. Nati, Acad. Sci. E.U.A. 75: 1929-1933, 1978), Yelton et al. , (Proc. Nati, Acad. Sci. E.U.A. 81: 1740-1747, 1984) and Russell (Nature 301: 167-169, 1983). The genotype of the host cell will generally contain a genetic defect that is complemented by the selectable marker present in the expression vector. The choice of a guest and bookmark Particular selectable is within the level of ordinary skill in the art. The cloned DNA sequences comprising modified polypeptides and peptides of the invention can be introduced into cultured mammalian cells by, for example, calcium phosphate mediated transfection (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. To identify cells that have cloned DNA integrated, a selectable marker is generally introduced into the cells together with the gene or cDNA of interest. Selectable markers that are preferred to be used in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin and methotrexate. The selectable marker can be an amplifiable selectable marker. An amplifiable selectable marker that is preferred is the DHFR gene. An amplifiable marker that is particularly preferred is the cDNA of DHFRr (see U.S. Patent No. 6,291,212) (Simonsen and Levinson, Proc. Nati, Acad. Sci. USA 80: 2495-2499, 1983). Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stone ham, Mass.) And the choice of selectable markers is within the level of ordinary skill in the art.

Host cells The present invention also includes a cell, preferably a transformed yeast cell for expressing the modified polypeptides or peptides of the invention. In addition to the transformed host cells themselves, the present invention also includes a culture of those cells, preferably a monoclonal culture. (clonally homogeneous), or a culture derived from a monoclonal culture in a nutrient medium. If the polypeptide is secreted, the medium will contain the polypeptide, with the cells, or without the cells if they have been filtered or centrifuged. Host cells for use in carrying out the present invention include eukaryotic cells, and in some cases prokaryotic cells, capable of being transformed or transfected with exogenous DNA and cultured in culture, such as cultured cells of mammal, insect, fungal, plant and bacterial. A vector comprising a nucleic acid sequence of the present invention is introduced into a host cell such that the vector is maintained as a chromosomal integrant or as an extrachromosomal self-replicating vector. The integration it generally considers an advantage since the nucleic acid sequence is more likely to be stably maintained in the cell. The integration of the vector into the chromosome of the host can occur through homologous or non-homologous recombination. The choice of a host cell will largely depend on the gene encoding the polypeptide and its origin. The host cell may be a unicellular microorganism, for example, a prokaryote or a non-unicellular microorganism, for example, a eukaryote. They can be used either prokaryotes or eukaryotes. As prokaryotic host cells, the cells generally used can be used such as Escherichia coli or Bacillus subtilis. When prokaryotic cells are used as host cells, a replicable vector can be used in the host cells. An expression plasmid may preferably be used in which a promoter, an SD sequence (Shine-Dalgarno sequence) and a start codon (eg, ATG) required for the synthesis of starting protein are provided in the vector towards the 5 'end of the gene of the present invention to facilitate the expression of the gene. Examples of the above vector include plasmids used generally derived from E. coli such as pBR322, pBR325, pUC12, pUC13 and the like. However, the applicable vectors are not limited to these examples and are also they can use several known vectors. Examples of commercially available vectors that can be used in the expression system using E. coli include pGEX-4T (Amersham Pharmacia Biotech), pMALC2, pMAI-P2 (New England Biolabs), pET21 / lacq (Invitrogen), pBAD / His ( Invitrogen) and the like. Examples of eukaryotic host cells include yeast cells and the like. Examples of craniadas cells that are preferably used include COS cells (monkey cell) (1981 Cell, 23, 175), Chinese hamster ovary cells and the dihydrofolate reductase-defective strain derived therefrom (1980 Proc. Nati. Acad. Sci., USA, 77, 4216) and the like, and examples of yeast cells that are preferably used include Saccharomyces cerevisiae or the like. However, the cells that will be used are not limited to these examples. Preferably, a yeast cell is used to express the modified polypeptide or peptide. Fungal cells, including yeast species (eg, Saccharomyces spp., Schizosaccharomyces spp., Pi chia spp.) Can be used as host cells within the present invention. Examples of yeasts including fungi contemplated as useful in the practice of the present invention as hosts for expressing the modified polypeptide or peptide of the invention are Pichia (including species formerly classified as Hansenula), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Ci teromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschnikowia, Rhodosporidium, Leuco spo ridiuia, Botryoascus, Sporidiobolus, Endomycopsis, and the like). Examples of Sa ccharomyces spp. are S. cerevisiae, S. i tali cus and S. rouxii. Examples of Kluyveromyces spp. they are K fragilis, K. lactis and K marxianus. One suitable Torulaspora species is T. delckii. Examples of Pichia spp. they are P. angusta (formerly H. polyniorpha), anomalous P. (formerly anomalous H.) and P. pastoris. Particularly useful host cells for producing the polypeptide or modified peptide of the invention are Pichia pastoris methanoltrophic (Steinlein et al. (1995) Protein Express. Purif. 6: 619-624). Pichia pastoris has been developed to be a surprising host for the production of foreign proteins since its promoter of alcohol oxidase was isolated and cloned; its transformation was first reported in 1985. P. pastoris can use methanol as a carbon source in the absence of glucose. The expression system of P. pastoris can use the alcohol oxidase promoter induced by methanol (AOXl), which controls the gene that codes for the expression of alcohol oxidase, the enzyme that catalyses the first stage in the metabolism of methanol This promoter has been characterized and incorporated into a series of expression vectors of P. pastoris. Since the proteins produced in P. pastoris are typically correctly bent and secreted in the medium, the fermentation of P. pastoris genetically manipulated provides an excellent alternative to E. coli expression systems. Strains of the yeast Saccharomyces cerevisiae are another preferred host. In a preferred embodiment, a yeast cell, or more specifically, a Saccharomyces cerevisiae host cell that contains a genetic deficiency in a gene required for asparagine glycosylation of glycoproteins is used. S host cells. cerevisiae having these defects can be prepared using standard mutation and selection techniques, although many available yeast strains have been modified to prevent or reduce glycosylation or hypermanylation. To optimize the production of the heterologous proteins, it is also preferred that the host strain carry a mutation, such as the S mutation. cerevisiae pep4 (Jones, Genetics 85: 23-33, 1977), which results in reduced proteolytic activity. It is particularly appropriate to use a host carrying a mutation in the gene encoding aspartyl protease yapsin 1 (YAP3) or the gene that it encodes for yapsin 2 (MKC7), or both (Copley et al., 1998 Biochein, J. 330, 1333-1340), in such a way that the proteolytic activity directed to basic residues is reduced or eliminated. Host strains that contain mutations in other protease coding regions are particularly useful for producing large amounts of the modified therapeutic polypeptides or peptides of the invention. The host cells containing DNA constructs of the present invention are cultured in a suitable culture medium. As used herein, the term "suitable culture medium" means a medium that contains nutrients required for cell growth. The nutrients required for cell culture can include a carbon source, a source of nitrogen, essential amino acids, vitamins, minerals and culture factors. The growth medium will generally select cells containing the DNA construct for example by drug selection or deficiency in an essential nutrient that is complemented by the selectable marker in the DNA construct or co-transfected with the DNA construct. Yeast cells, for example, are preferably cultured in a chemically defined medium, comprising a nitrogen source that is not an amino acid, inorganic salts, vitamins and amino acid supplements. essentials The pH of the medium is preferably maintained at a pH of more than 2 and less than 8, preferably of pH 5.5 to 6.5. Methods for maintaining a stable pH include pH regulation and constant pH control, preferably through the addition of ammonia, ammonium hydroxide or sodium hydroxide. Preferred pH regulating agents include citric acid, phosphate, succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.). Yeast cells that have a defect in a gene required for glycosylation linked to asparagine are preferably cultured in a medium containing an osmotic stabilizer. A preferred osmotic stabilizer is sorbitol supplemented in the medium at a concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M. Cultured mammalian cells are generally cultured in medium containing serum or serum free medium commercially available 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 allowed to grow for a period of time, typically 1-2 days, to begin expressing the DNA sequences of interest. The drug selection is then applied to select the growth of cells that are expressing the selectable marker in a stable manner. For cells that have been transfected With an amplifiable selectable marker the drug concentration can be increased in a staggered manner to select the increased copy number of the cloned sequences, thereby increasing the expression levels. The insect cell / baculovirus expression systems can also be used to produce 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. The target gene is inserted into a transfer vector, which is cotransfected into insect host cells with linearized BacPAKd viral DNA. BacPAKd DNA lacks an essential portion of the baculovirus genome. When the DNA recombines with the vector, the essential element is re-established and the target gene is transferred to the baculovirus genome. After recombination, few viral plaques are harvested and purified, and the recombinant phenotype is verified. The freshly isolated recombinant virus can then be amplified and used to infect insect cell cultures and produce large quantities of the desired protein.

Secretory Signal Sequences The terms "secretory signal sequence" or "signal sequence" or "sequential secretory leader" are used interchangeably and are described, for example, in US Pat. No. 6,291,212 and patent of E.U.A. No. 5,547,871, both of which are hereby incorporated by reference in their entirety. Secretory signal sequences or signal sequences or leader secretion sequences code for secretory peptides. A secretory peptide is an amino acid sequence that acts to direct the secretion of a mature polypeptide or protein from a cell. Secretory peptides are generally characterized by a nucleus of hydrophobic amino acids and are typically (but not exclusively) found in the amino terminus of newly synthesized proteins. Most commonly the secretory peptide is cut off from the mature protein during secretion. The secretory peptides may contain processing sites that allow the cutting of the mature protein signal peptide while passing through the secretory pathway. The processing sites can be encoded within the signal peptide or can be added to the signal peptide by, for example, in vitro mutagenesis. The secretory peptides can be used to direct the secretion of modified polypeptides and peptides of the invention. One of these secretory peptides 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 leader secretion sequences are required for a complex series of post-translational processing steps that result in the secretion of a protein. If an intact signal sequence is present, the protein being expressed enters the lumen of the rough endoplasmic reticulum and is then transported through the Golgi apparatus to secretory vesicles and finally transported out of the cell. Generally, the signal sequence immediately follows the start codon and codes for a signal peptide at the amino terminus of the protein to be secreted. In most cases, the signal sequence is cut 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 signal sequence that is preferred is a mammalian or human transferrin signal sequence. In some cases, the native substrate signal sequence can be used to express and secrete modified polypeptides or peptides of the invention. To ensure efficient removal of the signal sequence, in some cases it may be preferred to include a short pro-peptide sequence between the signal sequence and the mature protein in which the C-terminal portion of the pro-peptide comprises a recognition site for a protease, such as the kex2p yeast protease. Preferably, the pro-peptide sequence is about 2-12 amino acids long, most preferably about 4-8 amino acids long. Examples of these pro-peptides 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 NOS: 111-11, respectively).

Production of Modified Protected Polypeptide Substrates Protected from DPP The modified polypeptides of this invention that are partially or substantially resistant to DPP activity can be prepared by standard synthetic methods, recombinant DNA techniques or any other method for preparing peptides and fusion protein. . The solid phase peptide synthesis method is generally described in the following references: Merrifield, J. Am. Chem. Soc., 888: 2149, 1963; Barany and Merrifield, In the Peptides, E. Gross and J. Meinenhofer, Eds., Academic Press, New York, 3: 285 (1980); S. B. H. Kent. Annu. Rev. Biochem., 57: 957 (1988). By the peptide synthesis method in solid phase, a peptide of a The desired sequence length can be produced through the stepwise addition of amino acids to a growing peptide chain that is covalently bound to a solid resin particle. In this method, automatic synthesis can be used. As described above, the modified polypeptide of the present invention can also be obtained using molecular biology techniques, employing nucleic acid sequences that code for those polypeptides. The sequences can be RNA or DNA and can be associated with control sequences and / or inserted into vectors. The latter are then transferred to host cells, for example bacteria. The preparation of the vectors and their production or expression in a host is carried out by conventional techniques of molecular biology and genetic manipulation. Moreover, the modified polypeptides of the present invention can also be made by recombinant techniques using readily synthesized DNA sequences in commercially available expression systems. The modified polypeptides of the present invention can be obtained by recombinant means comprising (a) culturing a host cell under conditions that lead to polypeptide production and (b) recovering the polypeptide. The cells are cultured in a nutrient medium suitable for the production of the polypeptide using methods known in the art. For example, the cell can be cultivated by shake flask culture, small scale or large scale fermentation (including continuous, intermittent, intermittent or solid state fermentation) in laboratory or industrial fermentors carried out in a suitable medium and under conditions that allow the polypeptide to be expressed and / or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using methods known in the art (see, for example, references for bacteria and yeast; Bennett, JW and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, California, 1991). Suitable media are avble from commercial suppliers or can be prepared according to published compositions (for example, in catalogs of the American Collection of Crop Types). If the modified polypeptide is secreted in 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. As an example, the polypeptides or modified peptides of the present invention that include the Polypeptide or modified peptide fusion protein can be made by the fermentation methodology described in WO 0044772, which is incorporated herein by reference in its entirety. The modified polypeptides can be detected using methods known in the art that are specific for the polypeptides. These detection methods may include the use of specific antibodies, formation of an enzyme product or disappearance of an enzyme substrate, binding to a specific receptor, or detection of activation of a specific receptor in a cell-based assay. For example, an enzyme assay can be used to determine 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 methods including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation or precipitation. The polypeptides of the present invention can be purified by a variety of methods known in the art including, but not limited to, chromatography (e.g., ion exchange, hydrophobic affinity, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., isoelectric focusing) preparative, differential solubility (eg, precipitation with ammonium sulfate), SDS-PAGE or extraction (see, for example, Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Fusion Proteins and Protein Conjugates The present invention provides modified polypeptides or peptides linked to a heterologous molecule by recombinant means or covalent attachment. Fixation to a heterologous molecule, for example a plasma protein, extends the activity of the modified polypeptides or peptides for days to weeks. In some cases, only one administration of this modified therapeutic polypeptide or peptide has to be given during this time period. A greater specificity can be achieved, since the active compound will mainly bind to large molecules, where it is less likely to be picked up intracellularly to interfere with other physiological processes. In another embodiment, the modified polypeptides or peptides of the present invention can be linked to heterologous sequences to form chimeric or fusion proteins by recombinant means. These chimeric or fusion proteins comprise a polypeptide or modified peptide, partially or substantially protected from cutting with DPP, operably linked to a heterologous protein having an amino acid sequence that is not substantially homologous to that of the modified polypeptide or peptide. "Operably 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. In one embodiment, the fusion protein does not affect the activity of the modified polypeptide of the invention per se. For example, the fusion protein may include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast double-hybrid GAL fusions, poly-His fusions, MYC-labeled fusions, Hl-labeled fusions. and Ig fusions. These fusion proteins, particularly poly-His fusions, can facilitate the purification of the recombinant modified polypeptide. In a further example, the fusion protein comprises an amino acid sequence between the modified peptide of the invention and the other portion, the amino acid sequence provides a recognition sequence that makes possible the release of the modified peptide of the invention after the chemical cut or enzymatic. In certain host cells (e.g., mammalian host cells), the expression and / or secretion of a protein can be increased using a heterologous signal sequence. In another modality, the polypeptide or modified peptide is fused to a molecule that will extend its stability in serum or half-life in serum, such as a plasma protein. Preferably, the polypeptide or modified protein is fused to serum albumin, immunoglobulin or a portion thereof such as the Fc domain. Most preferably, the modified polypeptide or peptide is fused to transferrin, lactotrasferrin, melanotransferrin or hybrids thereof. Methods for making these fusion proteins are provided by the applications of E.U.A. 10 / 231,494 and 10 / 378,094 and international application PCT / US03 / 26818, which are hereby incorporated by reference in their entirety. As described in these applications, the transferrin to be bound to the modified polypeptide or peptide can be modified. It can exhibit reduced glycosylation. The modified transferrin polypeptide can be selected from the group consisting of a single N domain of transferrin, a single C domain of transferrin, a N and C domain of transferrin, two N domains of transferrin and two C domains of transferrin. When the Tf C domain is part of the fusion protein, the two N-linked glycosylation sites, amino acid residues corresponding to N413 and N611 (SEQ ID NO: 3 of PCT / US03 / 26818, which is incorporated in the present as a reference in its entirety) can be mutated for its expression in a yeast system to prevent glycosylation or hypermanosilation and extend the serum half-life of the fusion protein and / or the therapeutic protein (to produce asialo- or in some cases, monosialo-Tf or disialo-Tf) . In addition to the Tf amino acids corresponding to N413 and N611, mutations can be made to the adjacent residues within the glycosylation site N-X-S / T to prevent or substantially reduce glycosylation. See patent of E.U.A. No. 5,986,067 to Funk et al. It has also been reported that the N domain of Tf expressed in Pichia pastoris becomes glycosylated 0-linked with a single hexose in S32 which can also be mutated or modified to prevent this glycosylation. Accordingly, in one embodiment of the invention, the transferrin fusion protein includes a modified transferrin molecule in which transferrin exhibits reduced glycosylation, including but not limited to the asialo, monosialo and diasialo forms of Tf. In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant that is mutated to prevent glycosylation. In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant transferrin mutant that is fully glycosylated. In a further embodiment, the transferrin portion of the protein of Transferrin Fusion includes a recombinant human serum transferrin mutant that is mutated to prevent glycosylation, wherein at least one of Asn413 and Asndll (SEQ ID NO: 3 of PCT / US03 / 26818, which is incorporated herein by way of of reference in its entirety) is mutated to an amino acid that does not allow glycosylation. In another embodiment, the transferrin portion of the transferrin fusion protein includes a recombinant human serum transferrin mutant that is mutated to substantially prevent or reduce glycosylation, wherein the mutations can be in the adjacent residues within the glycosylation site NXS / T In addition, glycosylation can be reduced or prevented by mutating the serine or trionine residue. In addition, the change of X by proline is known to inhibit glycosylation. A chimeric or fusion protein can be produced by standard recombinant RNA techniques. For example, DNA fragments that code for 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 automatic DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary projections between two consecutive gene fragments which can be subsequently fixed and reamplified to generate a sequence of chimeric genes (see Ausubel et al., 1992 Current Protocols in Molecular Biology). Moreover, many expression vectors that already code for a fusion protein (eg, a GST protein) are commercially available. A modified polypeptide or peptide encoding nucleic acid can be cloned into this expression vector such that the fusion portion is bound in frame to the modified polypeptide or peptide. In another embodiment, the modified therapeutic polypeptide or peptide is conjugated by a covalent bond to a heterologous molecule via a covalent bond to increase its stability and protection from DPP activity. As an example, the polypeptide or modified peptide is conjugated to a blood component by means of a covalent bond formed between the reactive group of the modified peptide and a blood component, with or without a linking group. The blood components can be either fixed or mobile. Examples of fixed blood components are non-mobile blood components and include tissues, membrane receptors, interstitial proteins, fibrin protein, collagens, platelets, endothelial cells, epithelial cells and their membranes and membranous receptors. associates, cells of somatic bodies, smooth and skeletal muscle cells, neuronal components, osteocytes and osteoclasts and all body tissues especially those associated with the circulatory and lymphatic systems. Examples of mobile blood components are blood components that do not have a fixed site for any extended period of time, generally not exceeding 5, more usually 1 minute. These blood components are not associated with the membrane and are present in the blood for extended periods of time and are present in a minimum concentration of at least 0.1 μg / ml. The mobile blood components include serum albumin, transferrin, immunoglobulins such as IgM and IgG, inhibitor of OI protease, antithrombin III and oxy-antiplasmin. The average life of mobile blood components is typically at least about 12 hours. The formation of the covalent bond between the blood component and the modified therapeutic peptide or polypeptide can occur in vivo or ex vivo. For the formation of ex vivo covalent bonds, the modified polypeptide or peptide is added to blood, serum or saline containing the blood component, for example human serum albumin or IgG to allow the formation of covalent bonds between the polypeptide or modified peptide and the hematologic component. Likewise, the polypeptide or peptide modified can be modified with maleimide or a similarly reactive chemical group and reacted with a blood component in saline. Once the modified therapeutic polypeptide or peptide is reacted with the blood component to form a modified polypeptide or peptide conjugate, the conjugate can be administered to the patient. Alternatively, the modified therapeutic polypeptide or peptide can be administered to the patient directly such that the covalent bond is formed between the modified therapeutic polypeptide or peptide and the blood component in vivo. Likewise, the same reaction can be carried out with a recombinant protein, for example, albumin. The different sites with which the chemically reactive groups of the non-specific modified therapeutic peptide or polypeptide can react in vivo and include cells, particularly red blood cells (erythrocytes) and platelets, and proteins, such as immunoglobulins, including IgG and IgM, serum albumin , ferritin, steroid binding proteins, transferrin, thyroxine binding protein, -2-macroglobulin and the like. The polypeptide or modified peptide can contain or can be chemically modified to contain a reactive group to bind thiol. In one embodiment of the invention the modified polypeptide or peptide can be conjugated to polyethylene glycol. Alternatively, the modified polypeptide or peptide can be conjugated to a glycolipid modified with polyethylene glycol or fatty acid modified with polyethylene glycol. In one aspect, the modified polypeptide or peptide can 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, palmitic, oleic and stearic acids. Examples of fatty acid derivatives include ethyl esters, propyl esters, cholesteryl esters, coenzyme A esters, nitrophenol esters, naphthyl esters, monoglycerides, diglycerides and triglycerides, fatty alcohols, fatty alcohol acetates and the like. In another aspect, the modified polypeptide or peptide can be manipulated to create a drug affinity complex. (DAC ™). A drug affinity complex has three parts: a drug component that is responsible for the biological activity; a connector that links the drug component to the reactive chemistry group and a reactive chemistry group, at the opposite end of the connector, which is responsible for the permanent binding of the construct to certain target proteins in the body. For example, Kim et al. , (2003, Diabetes 52 (3): 751) describe a drug affinity complex GLP-1-albumin. Kim et al. , show that DAC: GLP-1 conjugated to albumin bound to the GLP-1 receptor (GLP-1R) and activated the formation of cAMP in heterologous fibroblasts that expressed the receptor. The results suggest that DAC: GLP-1 conjugated to albumin mimics native GLP-1. Kim et al. , provide a new approach for the prolonged activation of GLP-1R signaling. The drug affinity complex of the modified polypeptide or peptide is designed to be administered by subcutaneous injection and then rapidly and selectively binds in vivo to albumin. The bioconjugate formed has the same therapeutic activity and similar potency as the endogenous polypeptide or peptide but has a pharmacokinetic profile in animals that is closer to that of albumin.

Pharmaceutical Composition The present invention provides pharmaceutical compositions comprising therapeutic polypeptides or peptides modified partially or substantially protected from DPP cleavage, but which substantially retain their functional activity and potency. These pharmaceutical compositions can be administered orally, parenterally, such as intravascularly (IV), intraarterially (IA), intramuscularly (IM), subcutaneously (SC), intraperitoneally, transdermally or the like. The administration can in situations appropriate to be by transfusion. In some cases, the administration can be oral, nasal, rectal, transdermal or aerosolized, wherein the modified polypeptide allows transfer to the vascular system. For example, fusion or conjugation of a modified polypeptide of the invention to a portion of transferrin allows transport of the modified polypeptide to the vascular system or through the blood-brain barrier by binding to the transferrin receptor, as described in the PCT international application. / US03 / 26778, which is hereby incorporated by reference in its entirety. Normally, a single injection will be used although more than one injection can be used, if desired. The modified therapeutic polypeptides or peptides can be administered by any convenient means, including syringe, trocar, catheter or the like. The particular manner of administration will vary depending on the amount that will be administered, whether it is single bolus or continuous administration, or the like. Preferably, the administration will be intravascularly, where the introduction site is not critical for this invention, preferably at a site where there is rapid blood flow, for example, intravenously in peripheral or central vein. Most preferably, the pharmaceutical compositions will be administered subcutaneously. Other routes may find use when the administration is not coupled with slow release techniques or a protective matrix. The intent is that the modified therapeutic peptides or polypeptides are effectively distributed, for example, in the blood, so that they are thus able to react with the blood or tissue components. Generally, the invention encompasses pharmaceutical compositions comprising effective amounts of the modified therapeutic polypeptide or peptide of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or vehicles. These compositions may include diluents of various pH regulation contents (eg, tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol) and bulk substances (e.g., lactose, mannitol ); the incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or in liposomes. Hyaluronic acid can also be used, and this may have the effect of promoting prolonged duration in the circulation. These compositions can influence the physical state, stability, release rate in vivo and rate of in vivo elimination of the present proteins and derivatives. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, - Pa. 18042) pages 1435-1712 which are incorporated herein by reference. For example, the modified therapeutic polypeptides or peptides can be administered in a physiologically acceptable medium, for example, deionized water, pH regulated phosphate buffered saline solution (PBS), saline solution, aqueous ethanol or other alcohol, plasma, protein solutions, mannitol, aqueous glucose, alcohol, vegetable oil or the like. Other additives that can be included include pH regulators, wherein the media are generally pH regulated at a pH in the range of about 5 to 10, wherein the pH regulator will generally vary in concentration from about 50 to 250 mm, salt , wherein the salt concentration will generally vary from about 5 to 500 mm, physiologically acceptable stabilizers and the like. Examples of physiological pH regulators, especially for injection, include Hank's solution and Ringer's solution. The transdermal formulations may contain penetrants such as bile salts or fusidates. The pharmaceutical compositions can be prepared as tablets or lozenges, sublingual tablets, sachets, packages, soft gelatine capsules, suppositories, creams, ointments, dermal gels, transdermal devices, aerosols, drinkable and injectable ampoules. The compositions can also be prepared in liquid form, or they can be in dry powder form, such as convenient lyophilized form for storage and transport. Implantable prolonged-release formulations are also contemplated.

Oral dosage forms In one embodiment, the present invention provides pharmaceutical compositions comprising the modified therapeutic polypeptides or peptides in oral solid dosage forms, which are generally described in Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is incorporated herein by way of reference. Solid dosage forms include tablets, capsules, pills, troches or lozenges, sacs or granules. Likewise, liposomal or proteinoid encapsulation can also be used to formulate the present compositions (for example, as proteinoid microspheres reported in U.S. Patent No. 4, 925, 673). Liposomal encapsulation can be used and liposomes can be derived with various polymers (e.g., U.S. Patent No. 5,013,556). A description of possible solid dose forms for the therapeutic is given in Chapter 10 of Marshall, K., Modem Pharmaceutics (1979), edited by G.

S. Banker and C. T. Rhodes, incorporated herein by reference. In general, the formulation will include the modified therapeutic polypeptide or peptide, and inert ingredients that allow protection against the environment of the stomach, and the release of the biologically active material in the intestine. If necessary, the modified therapeutic polypeptide or peptide can be chemically modified such that oral delivery is effective. Generally, the contemplated chemical modification is the attachment of at least a portion to the therapeutic polypeptide or peptide itself, wherein the portion allows absorption into the bloodstream of the stomach or intestine. The increase in compound stability and an increase in circulation time in the body are also desired. Useful portions as vehicles covalently linked in this invention can also be used for this purpose. Examples of these portions 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 Adducts, Enzymes as Drugs (1981), Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4: 185-9. Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-thioxokane. For pharmaceutical use it is preferred, as indicated above, portions of PEG. Also, the modified therapeutic polypeptide or peptide can be recombinantly fused to another polypeptide to increase its total stability or improve oral delivery. For example, the modified therapeutic polypeptide or peptide can be fused to transferrin, melanotransferrin or lactoferrin. The methods for making these fusion proteins are described in the application of E.U.A. 10 / 378,094, which is hereby incorporated by reference in its entirety. For dosage forms of oral delivery, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N- (8- [2-hydroxybenzoyl] amino) carpilate (SNAC), as a vehicle for increasing the absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a phase II trial conducted by Emisphere Technologies. See patent of E.U.A. No. 5,792,451, "Composition and methods of oral delivery" which is hereby incorporated by reference in its entirety. The modified therapeutic polypeptides or peptides of this invention can be included in the formulation as fine microparticles in the form of granules or pellets of a particle size of about 1 mm.

The formulation of the material for capsule administration should also be as a powder, slightly compressed caps or even as tablets. The therapeutic can be prepared by compression. Dyes and flavoring agents can all be included. For example, the modified therapeutic peptide or polypeptide can be formulated (such as by encapsulation in liposomes and microspheres) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents. The volume of the pharmaceutical composition of the invention can be diluted or increased with an inert material. These diluents may include carbohydrates, especially mannitol, c-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts can also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell. Disintegrants may be included in the therapeutic formulation in a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial starch based disintegrant, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramilopectin, Sodium alginate, gelatin, orange peel, carboxymethylcellulose acid, natural sponge and bentonite can all be used. Another form of the disintegrants are insoluble cation exchange resins. Powdered gums can be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants. Binders can be used to keep the modified therapeutic peptide or polypeptide bound to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methylclulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethylcellulose (HPMC) can both be used in alcoholic solutions to granulate the therapeutic. Any anti-friction agent can be included in the formulation of the pharmaceutical composition of the invention to prevent adhesion during the formulation process. Lubricants with a layer between the modified therapeutic polypeptide or peptide and the die wall can be used, and these can include but are not limited to: stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, oils vegetables and waxes. Soluble lubricants such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000 can also be used. Slides can be added which can improve the flow properties of the modified therapeutic polypeptide or peptide during the formulation and to assist to redisposition during compression can. Glidants may include starch, talc, fumed silica and hydrated silicoaluminate. To aid in the dissolution of the modified therapeutic polypeptide or peptide of this invention in the aqueous environment, a surfactant may be added as a wetting agent. The surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents can be used and can include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that can be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, hydrogenated polyoxyethylene castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, Fatty acid ester of sucrose, methylcellulose and carboxymethylcellulose. These surfactants can be present in the formulation of the protein or derivative either alone or as a mixture in different ratios. Additives may also be included in the formulation to increase the absorption of the modified therapeutic polypeptide and peptide. The additives that potentially have this property are, for example, the fatty acids of oleic acid, linoleic acid and linolenic acid. A controlled release formulation may also be desirable. The modified therapeutic polypeptide or peptide of this invention can be incorporated into an inert matrix that allows its release either by diffusion or leaching mechanisms, for example gums. Matrices of slow degeneration can also be incorporated into the formulation, for example, alginates, polysaccharides. Another form of controlled release of the compounds of this invention is by a method based on the Oros (Alza Corp.) therapeutic system, ie, the drug is enclosed in a semipermeable membrane that allows water to enter and push the drug out. through a single small opening due to osmotic defects. Some enteric coatings also have a prolonged release effect. Other coatings can be used for the formulation. These include a variety of sugars that can be applied in a coating vat. He The modified therapeutic peptide or polypeptide can also be given in a film-coated tablet and the materials used in this case are divided into two groups. The first are monoenther materials and include methylcellulose, ethylcellulose, hydroxyethylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, povidone and the polyethylene glycols. The second group consists of the enteric materials that are commonly phthalic acid esters. A mixture of materials can be used to provide the optimum film coating. The film coating can be carried out in a coating tank or in a fluidized bed or by compression coating.

Forms of pulmonary delivery In another embodiment, the present invention also provides pharmaceutical compositions comprising the modified therapeutic polypeptides or peptides for pulmonary delivery. The pharmaceutical composition is delivered to the lungs of a mammal while inhaling and through pulmonary epithelial lining into the bloodstream. The present invention provides the use of a wide range of mechanical devices designed for the pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices that are suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Coló .; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C. and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass. All of these devices require the use of formulations suitable for the delivery of the modified therapeutic polypeptide and peptide. Typically, each formulation is specific to the type of device employed and may include the use of a suitable propellant material, in addition to diluents, adjuvants and / or vehicles useful in therapy. The modified therapeutic polypeptide or peptide must be very adequately prepared in the form of particles with an average particle size of less than 10 μm, most preferably 0.5 to 5 μm, for a very effective delivery to the distal lung.

Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants can be used. PEG can be used (even apart from its use to derive the protein or analogue). Dextrans such as cyclodextran can be used. Bile salts and other related enhancers can be used. Cellulose and cellulose derivatives can be used. Amino acids such as use in a pH regulating formulation can be used. Likewise, the use of liposomes, microcapsules or microspheres, inclusion complexes or other types of vehicles is contemplated. Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the compound of the invention dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation may also include a pH regulator and a simple sugar (eg, for protein stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface-induced aggregation of the protein caused by atomization of the solution when forming the aerosol . Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material used for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1, -tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid surfactant may also be useful as an oleic acid surfactant. Formulations for delivery from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulk agent, such as lactose, sorbitol, sucrose, mannitol, trehalose or xylitol in amounts that facilitate powder dispersion of the device, for example, about 50 to 90% by weight of the formulation.

Nasal delivery forms Nasal delivery of the pharmaceutical composition of the modified polypeptide or peptide of the present invention is also contemplated. The nasal delivery allows the passage of the protein into the bloodstream directly after the modified therapeutic polypeptide or peptide is administered to the nose, without the need for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. Delivery is also described by means of transport through other mucous membranes.

Dosage The dose regime involved in a method to treat the conditions described above will be determined by the physician attending, considering several factors that modify the action of the drug, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the compound of the invention per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.

Treatment of Diseases with Modified Therapeutic Proteins The present invention provides various transferrin fusion proteins that can be used in the treatment of a variety of diseases. For example, the pharmaceutical compositions comprising the fusion polypeptides or peptides of the present invention can be used to treat diseases such as, but not limited to, insulin resistance, hyperglycemia, hyperinsulinemia or elevated blood levels of free fatty acids or glycerol, Hyperlipidemia, obesity, Syndrome X, dysmetabolic syndrome, inflammation, diabetic complications, impaired glucose homeostasis, impaired glucose tolerance, type II diabetes, prediabetes, hypertriglyceridemia, atherosclerosis, nervous system disorders, congestive heart failure, dyspepsia and irritable bowel syndrome . The polypeptides and modified peptides can also be used to induce an anxiolytic effect in the CNS, to activate the CNS or for post-surgical treatment. The modified therapeutic polypeptides and peptides of the present invention are more stable in vivo than unmodified therapeutic polypeptides and peptides since they are fused to transferrin or modified transferrin or are partially or substantially protected from DPP activity. Consequently, smaller amounts of the molecule can be administered for an effective treatment. A lower dose amount may in some cases alleviate side effects. In one embodiment, the modified therapeutic polypeptides and peptides of the present invention can be used as a sedative. Accordingly, the present invention provides a method for sedating a mammalian subject with an abnormality that results in increased activation of the central or peripheral nervous system using the modified polypeptides or peptides of the invention. The method comprises administering a therapeutic modified polypeptide or peptide to the subject in an amount sufficient to produce a sedative or anxiolytic effect in the subject. Modified therapeutic polypeptides or peptides can be administered intracerebroventricularlyorally, subcutaneously, intramuscularly or intravenously. These methods are useful for treating or decreasing nervous system conditions such as anxiety, movement disorder, aggression, psychosis, attacks, panic attacks, hysteria and sleep disorders. Furthermore, the present invention encompasses a method for increasing the activity of a mammalian subject, which comprises administering a modified therapeutic polypeptide or peptide to the subject in an amount sufficient to produce an activating effect on the subject. The subject has a condition that results in reduced activation of the central or peripheral nervous system. The modified therapeutic polypeptides or peptides are useful in the treatment or reduction of depression, schizoaffective disorders, sleep apnea, attention deficit syndromes with poor concentration, memory loss, forgetfulness and narcolepsy, to name but a few conditions in which the activation of the central nervous system could be adequate. Also, insulin resistance after a particular type of surgery, elective abdominal surgery, is deeper on the first postoperative day, lasts at least five days, and can take up to three weeks to normalize. Thus, the postoperative patient may be in need of administration of the modified insulinotropic peptides of the present invention for a period of time following the trauma of surgery. Accordingly, the modified therapeutic polypeptides or peptides of the invention can be used for postsurgical treatments. A patient is in need of the modified insulinotropic peptides of the present invention for about 1-16 hours before the surgery is performed on the patient, during the patient's surgery and after the patient's surgery for no more than a period of approximately 5 days. In addition, the therapeutic polypeptides and peptides modified, such as the insulinotropic peptides of the invention can be used to treat insulin resistance independently of its use in postoperative treatment. Insulin resistance may be due to a reduction in the binding of insulin to cell surface receptors, or alterations in intracellular metabolism. The first type, characterized as a reduction in insulin sensitivity, can typically be overcome by an increased insulin concentration. The second type, characterized with a reduction in the insulin response, can not be overcome by large amounts of insulin. Insulin resistance after trauma can be overcome by insulin doses that are proportional to the degree of insulin resistance, and in this way is apparently caused by a reduction in insulin sensitivity. Preferably, the present invention provides modified insulinotropic peptides to normalize hyperglycemia through insulin-dependent, insulin-dependent, glucose-dependent mechanisms. In this way, the modified insulinotropic peptides are useful as primary agents for the treatment of diabetes, especially type II diabetes mellitus. The present invention is especially suitable for the treatment of patients with diabetes, both type I and type II, since the action of the peptide depends on the concentration of glucose in the blood, and in this way the risk of hypoglycemic side effects is greatly reduced on the risks in the use of current methods of treatment. The dose of modified insulinotropic peptides effective to normalize a patient's blood glucose level will depend on a number of factors, including, without limitation, the patient's sex, weight and age, the severity of inability to regulate the blood glucose, the underlying causes of inability to regulate blood glucose, either glucose, or another source of carbohydrates, if administered simultaneously, the route of administration and bioavailability, persistence in the body, formulation and potency. Preferably, the modified therapeutic peptides such as the insulinotropic peptides of the present invention are used for the treatment of impaired glucose tolerance, glycosuria, hyperlipidemia, metabolic acidosis, diabetes mellitus, diabetic neuropathy and nephropathy. More preferably, the modified peptides are modified GLP-1 and analogs thereof for the treatment of type II diabetes.

Monitoring the Presence of Modified Therapeutic Polypeptides and Peptides Modified therapeutic polypeptides and peptides can be monitored using assays to determine functional activity, HPLC-MS or antibodies directed against the polypeptide or peptide. For example, the blood of the 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. By taking a portion or sample of host blood at different times, it can be determined whether the modified therapeutic polypeptide or peptide has been bound to the long-lived blood components in sufficient quantity to be therapeutically active and, subsequently, the level of polypeptide or modified therapeutic peptide in the blood. If desired, it can also be determined to which of the components of the blood the modified therapeutic polypeptide or peptide, such as a modified insulinotropic peptide, binds. As an example, assays for insulinotropic activity can be used to monitor the modified insulinotropic peptides of the present invention. The modified insulinotropic peptides of the present invention have an insulinotropic activity that at least equals the insulinotropic activity of the peptides unmodified insulinotropics. The insulinotropic property of a modified insulinotropic peptide can be determined by providing that modified peptide to animal cells, or by injecting that peptide into animals and monitoring the release of immunoreactive insulin in the animal's circulatory system or medium, respectively. The presence of immunoreactive insulin is detected through the use of a radioimmunoassay that can specifically detect insulin. Although any radioimmunoassay capable of detecting the presence of IRI can be employed, it is preferable to use a modification of the assay method of Albano, J. D. M., et al. (1972 Act Endorcinol 70: 487-509), which is incorporated herein by reference in its entirety. The insulinotropic property of a modified therapeutic polypeptide or peptide can also be determined by pancreatic infusion (Penhos, J. C, et al., 1969 Diabetes 18: 733-738, which is incorporated herein by reference). The manner in which perfusion is performed, modified and analyzed follows preferably the methods of Weir, G.C., et al. (J. Clin Investigat., 54: 1403-1412 (1974)), which is incorporated herein by reference. HPLC coupled with mass spectrometry (MS) can be used to test the presence of polypeptides and modified therapeutic peptides as is well known to the skilled person. Typically, two mobile phases are used, such as 0.1% TFA / water and 0.1% TFA / acetonitrile. The temperatures of the column can be varied as well as the conditions of gradients. Another method for monitoring the presence of modified therapeutic peptides and polypeptides is to use antibodies specific for the modified therapeutic polypeptides and peptides. The use of antibodies, either monoclonal or polyclonal, which have specificity for particular modified therapeutic peptides or polypeptides, can help mediate any of these problems. The antibody can be generated or derived from a host immunized with the particular modified therapeutic peptide or polypeptide, or with an immunogenic fragment of the agent, or a synthesized immunogen corresponding to an antigenic determinant of the agent. The antibodies that are preferred will have high specificity and affinity for the modified therapeutic polypeptide or peptide. These antibodies can also be labeled with enzymes, fluorochromes, or radiolabels. The antibodies can be used to monitor the presence of modified therapeutic polypeptides and peptides in the bloodstream. Blood and / or serum samples can be analyzed by SDS-PAGE and western blotting.

These techniques allow the analysis of the blood or serum to determine the binding of the modified therapeutic polypeptides or peptides to blood components.

Peptide 1 Type Glucagon (GLP-1) Recombinant DNA technology has been used to create new molecules with increased stability and biological activity. These molecules are combinations of biologically active proteins and peptides fused to a stabilizing protein with naturally long half-life such as Fc portion of immunoglobin, albumin and transferrin. These fusion molecules retain the biological activity of the active portion with much greater pharmacokinetics than their natural unfused protein or peptide counterparts. The increase in pharmacokinetics also improves biological activity, reduces unwanted side effects and improves convenience for patients. There are many examples of these fusion proteins such as interferon-albumin, interferon-Fc, BNP-albumin, GLP-1-albumin, GLP-1-Transferrin. Although the fusion proteins are stable and resistant to degradation, the underlying protease mechanism that degrades the active portion may result in a slow inactivation of the molecule. Specifically, many peptides such as GLP-1, dynorphin (Berman YL, Juliano L, Devi LA J Biol. Chem. 1995 Oct 6; 270: 23845-50), enkephalin (Gu ZF, Menozzi D, Okamoto A, Bully PN, Bunnett NW Exp Physiol., 1993 Jan; 78:35 -48), BNP, ANP, angiotensin, bradykinin and PYY are very susceptible to proteases such as dipeptidyl-peptidase IV, neutral endopeptidase. These proteases individually or in combination cause a rapid inactivation of the peptides in the circulation. The fusion of peptides to large proteins such as albumin, Fc and transferrin confers a significant resistance to protease. However, it may not completely eliminate the effect of protease inactivation. Therefore, the combination of the fusion proteins and protease inhibitors may have better PK and PD than the fusion protein alone. The present invention provides transferrin fusion proteins comprising therapeutic peptides that are resistant to protease. Preferably, the modified therapeutic peptides of the present invention are modified insulinotropic peptides partially or substantially protected from DPP activity. Most preferably, the modified insulinotropic peptides are modified GLP-1 peptides and analogs and fragments thereof. Modified GLP-1 peptides and analogues and fragments thereof are useful for treating diabetes, specifically type II diabetes. The N-terminal sequence of GLP-1 type wild is His-Ala-Glu; the modified GLP-1 polypeptides of the invention may comprise an N-terminal sequence selected from the group consisting of: His-His-Ala-Glu (SEQ ID NO: 115), Gy-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 IDNO: 84), Gly-His-Ala-Glu (SEQ IDNO: 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 As described below, other modifications can be made to reduce and prevent the degradation of proteases and these molecules can be used in the methods and compositions of the invention. In addition, any portion of GLP-1 or analogs, derivatives or mimetics of GLP-1 can be used (as a fusion protein) in the methods and compositions of the invention. The addition of an amino acid to the N-terminus of GLP-1 can prevent the dipeptidyl peptidase from cleaving into the second amino acid of GLP-1 due to steric hindrance. Therefore, GLP-1 will remain functionally active. Any of the 20 amino acids or a non-natural amino acid can be added to the N-terminus of GLP-1. Histidine is also an amino acid that is preferred. In some cases, an uncharged or positively charged amino acid can be used and preferably, a smaller amino acid such as glycine. The GLP-1 modified with the additional amino acid can then be fused to transferrin to make a fusion protein. In one embodiment, the GLP-1 peptide is modified to contain at least one additional amino acid at its amino terminus. In another embodiment, the GLP-1 peptide is modified to contain at least five additional amino acids at its amino terminus. Alternatively, the GLP-1 peptide is modified to contain between one and five additional amino acids at its amino terminus. Glucagon-like peptide 1 (GLP-1) is a gastrointestinal hormone that regulates the secretion of insulin that belongs to the so-called enteroinsular axis. The enteroinsular axis designates a little hormones, released from the gastrointestinal mucosa in response to the presence and absorption of nutrients in the intestine, which promote an early and enhanced release of insulin. The effect of incretin, which is the increasing effect on insulin secretion, is probably essential for a normal glucose tolerance. GLP-1 is a physiologically important insulinotropic hormone since it is responsible for the incretin effect. GLP-1 is a product of the proglucagon gene (Bell, et al., Nature, 1983, 304: 368-371). It is synthesized in intestinal endocrine cells in two major major molecular forms, such as GLP-1 (7-36) amide and GLP-1 (7-37). The peptide It was first identified after the cloning of cDNA molecules and genes for proglucagon in the early 80's. Initial studies carried out on the full-length peptide GLP-I (l-37 and l-36amide) concluded that the larger GLP-1 molecules are free of biological activity. In 1987, three independent research groups demonstrated that the removal of the first six amino acids resulted in a GLP-1 molecule with increased biological activity. The amino acid sequence of GLP-1 is described by Schmidt et al (1985 Diabetologia 28 704-707). Human GLP-1 is a peptide of 37 amino acid residues that originate from preproglucagon that is synthesized in the L cells in the distal ileum, in the pancreas and in the brain. The processing of preproglucagon to GLP-1 (7-36) amide, GLP-1 (7-37) and GLP-2 occurs mainly in the L cells. The amino acid sequence of GLP-I (7-37) is SEQ ID NO: 32 (X = Gly): 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 NH2. GLP-1 type molecules possess antidiabetic activity in human subjects suffering from type diabetes II (non-insulin dependent diabetes mellitus (NIDDM)) and, in some cases, even type I diabetes. Treatment with GLP-1 develops activity, such as increased insulin secretion and biosynthesis, reduced glucagon secretion, delayed gastric emptying, only a high glucose levels and thus provides a potentially much safer therapy than insulin or sulfonylureas. Post-prandial and glucose levels in patients can move to normal levels with adequate GLP-1 therapy. There are also reports suggesting that GLP-1 type molecules possess the ability to conserve and even restore the function of pancreatic beta cells in type II patients. Any GLP-1 sequence can be modified by adding one or more amino acids at its amino terminus, including GLP-K7-34), GLP-K7-35), GLP-1 (7-36) and GLP-1 (7-37 ). GLP-1 also has powerful actions in the gastrointestinal tract. Infused in physiological quantities, GLP-1 potentially inhibits the gastric acid secretion induced by pentagastrin as well as that induced by food (Schjoldager et al., Dig. Dis. Sci. 1989, 35: 703-708; Wettergren et al., Dig. Dis Sci 1993; 38: 665-673). It also inhibits the rate of gastric emptying and the secretion of pancreatic enzymes (Wettergren et al., Dig Dis Sci 1993; 38: 665-673). Similar inhibitory effects on secretion Gastric and pancreatic changes and motility can develop in humans after ileal perfusion with solutions containing carbohydrates or lipids (Layer et al., Dig Dis Sci 1995, 40: 1074-1082; Layer et al. , Digestion 1993, 54: 385-38). Concomitantly, the secretion of GLP-1 is widely stimulated, and it has been speculated that GLP-1 may be at least partially responsible for this so-called "ileal brake" effect (Layer et al., Digestion 1993; 54: 385- 38). In fact, recent studies suggest that, physiologically, the effects of ileal brake of GLP-1 may be more important than its effects on pancreatic islets. Thus, in GLP-1 dose response studies, it influences gastric emptying velocity at infusion rates at least as low as those required to influence islet secretion (Nauck et al., Gut 1995; 37 (suppl.2): A124). GLP-1 seems to have an effect on the consumption of food. Intraventricular administration of GLP-1 profoundly inhibits feed intake in rats (Schick et al., In Ditschuneit et al. (Eds.), Obesity in Europe, John Libbey &Company ltd., 1994, pp. 363-367; Turton et al., Nature 1996, 379: 69-72). This effect seems to be highly specific. Thus, N-terminally extended GLP-1 (1-36am: Lda) is inactive and adequate doses of the GLP-1 antagonist, exendin 9-39, arrest the effects of GLP-1 (Tang-Christensen et al., Am. J. Physiol., 1996, 271 (4 Pt 2): R848-56). The Peripheral and acute administration of GLP-1 does not inhibit food intake acutely in rats (Tang-Christensen et al., Am. J. Physiol, 1996, 271 (4 Pt 2): R848-56; Turton et al. , Nature 1996, 379: 69-72). However, it remains a possibility that GLP-1 secreted from intestinal L cells could also act as a signal of satiety. In diabetic patients, the insulinotropic effects of GLP-1 and the effects of GLP-1 in the gastrointestinal tract are preserved (Will s et al., Diabetologia 1994; 37, suppl.1: A118), which can help reduce excursions of glucose induced by food, but, more importantly, can also influence the consumption of food. Administered intravenously, continuously for one week, GLP-1 at 4 ng / kg / min has been shown to dramatically improve glycemic control in NIDDM patients without significant side effects (Larsen et al., Diabetes 1996; 45, suppl.2: 233A. ). Modified GLP-1 partially or substantially protected from DPP activity and modified GLP-1 analogs are useful in the treatment of Type 1 and Type 2 diabetes and obesity. As used herein, the term "GLP-1 molecule" means GLP-1, a GLP-1 analog or GLP-1 derivative.

As used herein, the term "GLP-1 analog" is defined as a molecule having one or more amino acid substitutions, deletions, inversions or additions as compared to GLP-1. Many GLP-1 analogs are known in the art and include, for example, GLP-1 (7-34), GLP-1 / 7-35), GLP-I (7-36), Val8-GLP-1 ( 7-37), Gln9-GLPl (7-37), D-Gln9-GP-l (7-37), Thr16-Lys18-GLP-1 (7-37) and Lys18-GLP-K7-37) (SEQ ID NO: 72), the US patent 5,118,666 describes examples and analogs of GLP-1 such as GLP-1 (7-34) and GLP-1 (7-35). The term "GLP-1 derivative" is defined as a molecule having the amino acid sequence of GLP-1 or a GLP-1 analog, but which additionally has chemical modification of one or more of its side groups of amino acids, atoms of carbon a, terminal amino group or terminal carboxylic acid group. A chemical modification includes, but is not limited to, adding chemical portions, creating new bonds, and removing chemical portions. As used herein, the term "GLP-1-related compound" refers to any compound that falls within the definition of GP-1, GLP-1 analog or GLP-1 derivative. WO 91/11457 discloses analogues of the active GLP-1 peptides 7-34, 7-35, 7-36 and 7-37 which may also be useful as GLP-1 portions.

EP 0708179-A2 (Eli Lilly &Co.) discloses GLP-1 analogs and derivatives that include an N-terminal imidazole group and optionally an unbranched C6-C? 0 acyl group attached to the lysine residue at position 34 EP 0699686-A2 (Eli Lilly &Co.) discloses certain N-terminal truncated fragments of GLP-1 that are reported to be biologically active. The patent of E.U.A. 5,545,618 discloses GLP-1 molecules consisting essentially of GLP-I (7-34), GLP1 (7-35), GLP-K7-36) or GLP-I (7-37), or the amide forms of the same, and pharmaceutically acceptable salts thereof, having at least one modification selected from the group consisting of: (a) substitution with glycine, serine, cysteine, threonine, asparagine, glutanin, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine or D-lysine by lysine at position 26 and / or position 34; or substitution with glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine or a D-arginine by arginine at position 36 (SEQ ID NO: 73); (b) substitution with an amino acid resistant to oxidation by tryptophan at position 31 (SEQ ID NO: 74); (c) substitution with at least one of: tyrosine by valine at position 16; lysine by serine in position 18; aspartic acid by glutamic acid in position 21; serine by glycine in position 22; arginine by glutamine at position 23; arginine for alanine in position 24 and glutamine for lysine in position 26 (SEQ ID NO: 75) and (d) substitution with at least one of: glycine, serine or cysteine for alanine in position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine by glutamic acid in position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine by glycine in position 10 and glutamic acid by aspartic acid in position 15 (SEQ ID NO: 76) and (e) substitution with glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine or phenylalanine, or the D- or N-acylated or alkylated form of histidine by histidine at position 7 (SEQ ID NO: 77); wherein, in substitutions (a), (b), (d) and (e), the substituted amino acids may optionally be in the D form and the amino acids substituted in the 7-position may optionally be in the N-acylated form or N-alkylated. The patent of E.U.A. No. 5,118,666 discloses a GLP-1 molecule having insulinotropic activity. This molecule is selected from the group consisting of a peptide having 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-Le-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, see SEQ ID NO: 32); and a peptide derivative and wherein the peptide is selected from the group consisting of: a pharmaceutically acceptable acid addition salt of the peptide; a pharmaceutically acceptable carboxylate salt of the peptide; a pharmaceutically acceptable lower alkyl ester of the peptide and a pharmaceutically acceptable amide of the peptide selected from the group consisting of amide, lower alkylamide and lower dialkylamide. The patent of E.U.A. 6,277,819 teaches a method for reducing mortality and morbidity after myocardial infarction, which comprises administering to the patient GLP-1, GLP-1 analogs and GLP-1 derivatives. The GLP-1 analog that is being represented by the following structural formula (SEQ ID NO: **); R? -X? -Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-X2-Gly-Gln-Ala-Ala-Lys-X3-Phe-Ile-Ala- Trp-Leu-Val-Lys-Gly-Arg-R2 (SEQ ID NO: 78) and pharmaceutically acceptable salts thereof, wherein: Ri is selected from the group consisting of L-histidine, D-histidine, deamino-histidine, 2-amino-histidine, beta-hydroxy-histidine, homohistidine, alpha-fluoromethyl-histidine and alpha-methyl-histidine; Xi is selected from the group that consists of Ala, Gly, Val, Thr, lie and alphamethyl-Ala; X2 is selected from the group consisting of Glu, Gln, Ala, Thr, Ser and Gly; X3 is selected from the group consisting of Glu, Gln, Ala, Thr, Ser and Gly; R2 is selected from the group consisting of NH2 and Gly-OH; as long as the GLP-l analog has an isoelectric point on the scale of about 6.0 to about 9.0 and also provided that when Ri is His, Xi is Ala, X2 is Glu and X3 is Glu, R2 must be NH2 . Ritzel et al. (Journal of Endocrinology, 1998, 159: 93-102) describe an analog of GLP-1, [Ser8] GLP-1, in which the second N-terminal alanine is replaced with serine. The modification did not impair the insulinotropic action of the peptide but produced an analog with increased plasma stability compared to GLP-1. The patent of E.U.A. 6,429,197 teaches that treatment with GLP-I after an acute embolism or hemorrhage, preferably intravenous administration, can be an ideal treatment since it provides a means to optimize insulin secretion, increase cerebral anabolism, increase the effectiveness of insulin by suppressing glucagon and maintaining euglycemia or mild hypoglycemia without risk of severe hypoglycaemia or other adverse side effects. The present invention provides a method for treating ischemic or reperfused brain with GLP-1 or its biologically active analogues after acute embolism or hemorrhage to optimize insulin suppression, to increase the effectiveness of insulin by suppressing glucagon antagonism, and to maintain mild euglycemia or hypoglycemia without risk of severe hypoglycaemia. The patent of E.U.A. 6,277,819 provides a method for reducing mortality and morbidity after myocardial infarction, which comprises administering to a patient in need thereof, a compound selected from the group consisting of GLP-1, GLP-1 analogs, GLP-1 derivatives and salts pharmaceutically acceptable thereof, at an effective dose to normalize blood glucose. The patent of E.U.A. 6,191,102 discloses a method for reducing body weight in a subject that requires reduction in body weight by administering to the subject a composition comprising a glucagon-like peptide 1 (GLP-1), a glucagon-like peptide analogue (GLP-1 analogue). ), a glucagon-like peptide derivative (GLP-1 derivative) or a pharmaceutically acceptable salt thereof in a dose sufficient to cause reduction in body weight over an effective period of time to produce weight loss, this time being at least four weeks. GLP-1 is fully active after subcutaneous administration (Ritzel et al., Diabetologia 1995; 38: 720-725), but rapidly degrades mainly due to degradation by dipeptidyl peptidase type IV enzymes (Deacon et al., J Clin Endocrinol Metab 1995, 80: 952-927; Deacon et al., 1995, Diabetes 44: 1126-1131).

Unfortunately, then, GLP1 and many of its analogs have a short half-life in human plasma (Orskov et al., Diabetes 1993; 42: 658-661). Accordingly, an object of the present invention is to provide modified GLP-I or analogs thereof having a prolonged profile of action in relation to GLP-I (7-37). A further object of the invention is to provide GLP-1 derivatives and analogs thereof which have a lower elimination than GLP-1 (7-37). Moreover, an object of the invention is to provide pharmaceutical compositions comprising modified GLP-1 or GLP-1 analogs with improved stability. In addition, the present invention includes the use of modified GLP-1 or GLP analogs to treat diseases associated with GLP-1 such as but not limited to those described above. In one aspect of the present invention, pharmaceutical compositions comprising modified GLP-1 and GLP-1 analogs can be formulated by any of the established methods to formulate pharmaceutical compositions, for example as described in Remington's Pharmaceutical Sciences, 1985. The composition may be in a form suitable for injection or systemic infusion and it can, as such, be formulated with a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution. The compositions can be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting agents, tonicity adjusting agents and the like, for example sodium acetate, sodium lactate, sodium chloride, potassium chloride. , calcium chloride, etc. The modified GLP-1 and GLP-1 analogs of the present invention can also be adapted for nasal, transdermal, pulmonary or rectal administration. The pharmaceutically acceptable carrier or diluent employed in the composition can be any conventional solid carrier. Examples of solid carriers are lactose, white earth, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Similarly, the carrier or diluent may include any prolonged release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. It may be of particular advantage to provide the composition of the invention in the form of a sustained release formulation. In this manner, the composition can be formulated as microcapsules or microparticles containing the modified GLP-1 or GLP-1 analogs encapsulated by or dispersed in a suitable pharmaceutically acceptable biodegradable polymer such as polylactic acid, polyglycolic acid or a lactic acid copolymer. Glycolic Acid. For nasal administration, the preparation can contain modified GLP-1 or GLP-1 analogues dissolved or suspended in a liquid vehicle, in particular an aqueous vehicle, for aerosol application. The vehicle may contain additives such as solubilizing agents, for example propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens. Generally, the modified polypeptides or peptides of the present invention are delivered in unit dosage form together with a pharmaceutically acceptable carrier per unit dose. Moreover, the present invention contemplates the use of modified GLP-1 and GLP-1 analogs in the manufacture of a medicinal product that can be used in the treatment of diseases associated with elevated glucose level (metabolic disease), such as but not limited to those described above. Specifically, the present invention contemplates the use of 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 syndrome. The present invention also provides peptides Exendin-3 and Exendin-4 modified partially and substantially protected from DPP activity. Exendin-3 and Exendin-4 are insulinotropic peptides comprising 39 amino acids (differing in residues 2 and 3) which are approximately 53% homologous to GLP-1. The sequence of exendin-3 is HSDGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS (SEQ ID NO: 79), and the sequence of Exendin-4 is HGEGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS (SEQ ID NO: 80). The invention also encompasses the modified Exendin-4 fragments comprising the amino acid sequences such as Exendin-4 (1-31) HGEGTFTSDLSKQMEEAVRLFIEWLKNGGPY (SEQ ID NO: 81). In addition, the present invention includes modified analogues of peptides Exendin-3 and Exendin-4.

Protein or GLP-I Fusion Conjugate Modified to Treat Diabetes, Prediabetes or Obesity Modified GLP-l can be fused to a heterologous molecule for general stability in vivo. The modified GLP-1 can be fused to a heterologous molecule by recombinant means or covalently linked to a heterologous molecule by methods well known in the art. Modified GLP-1 can be fused or covalently linked, for example to a plasma protein such as serum albumin or transferrin, an immunoglobulin or a portion thereof such as the Fc domain. Most preferably, the modified polypeptide or peptide is fused to transferrin, lactotransferrin or melanotransferrin. Methods for making these fusion proteins are provided by the application of E.U.A. 10 / 378,094, which is hereby incorporated by reference in its entirety. The GLP-1 molecule can be linked to the heterologous protein by means of a variable length linker to provide greater physical separation and allow more spatial mobility between the fused proteins and thereby maximize the accessibility of the therapeutic protein, for example , to join your cognate receiver. The linker peptide may consist of amino acids that are flexible or more rigid. For example, a linker such as a stretch of Polyglycine can be used. The linker may have less than about 50, 40, 30, 20, 10 or 5 amino acid residues. The linker can be covalently linked to and between the heterologous protein and GLP-1. Preferably, the linker can be a Ser residue, two Ser residues, the Ser-Ser-Gly peptide, the PEAPTD peptide, the peptide (PEAPTD) 2, the PEAPTD peptide in combination with pivot linker or hinge of IgG and the peptide (PEAPTD) 2 in combination with pivot linker or hinge of IgG. These linkers can be used to link GLP-1 to transferrin. The transferrin to be bound to the modified polypeptide or peptide can be modified. It can exhibit reduced glycosylation. The modified transferrin polypeptide can be selected from the group consisting of a single N domain of transferrin, a single C domain of transferrin, a N and C domain of transferrin, two N domains of transferrin and two C domains of transferrin. As described above, GLP-1 activates and regulates important endocrine hormone systems in the body and plays a critical administration role in glucose metabolism. Unlike all other diabetic treatments in the market, GLP-l has the potential to be a restorer by acting as a growth factor for β-cells, thereby improving the ability of the pancreas to secrete insulin and also to make the levels of existing insulin act more efficiently by improving sensitivity and better stabilize glucose levels. This reduces the burden on daily monitoring of glucose levels and potentially offers a delay in the serious long-term side effects caused by fluctuations in blood glucose due to diabetes. Furthermore, GLP-l can reduce appetite and reduce weight. Obesity is an inherent consequence of a poor control of glucose metabolism and this only serves to aggravate the diabetic condition. The clinical application of natural GLP-l is limited since it rapidly degrades in the circulation (the half-life is several minutes). To maintain therapeutic levels in the circulation requires the constant administration of high doses using pumps or patch devices that help and which is added to the cost of treatment. This is inconvenient for chronic long-term use especially in conjunction with all other medications to treat diabetes and glucose level monitoring. The modified GLP-1 fusion proteins retain the capacity of GLP-1 but have a long half-life (14-17 days), solubility and properties of transferrin biodistribution. These properties could provide a subcutaneous (subcutaneous) injection of low cost, small volume and monthly and this type of product is required Absolutely for chronic long-term use. Modified GLP-1 can also be covalently linked to a blood component to increase its stability. For example, the modified GLP-1 can be covalently linked to serum albumin, transferrin, immunoglobulin or to the Fc portion of the immunoglobulin. In one embodiment, the modified GLP-1 can be linked to a fatty acid or a fatty acid derivative. In another embodiment, the modified GLP-1 can be manipulated to create a drug affinity complex (DAC). As described above, Kim et al. (2003, Diabetes 52 (3): 751) describes a drug affinity complex of GLP-1 albumin. Kim et al. show that the DAC: GLP-l conjugated albumin mimics the native GLP-l. Kim et al. provide a new approach for the prolonged activation of GLP-1R signaling. After subcutaneous administration, the DAC: modified GLP-1 binds rapidly and selectively in vivo to albumin. The bioconjugate formed has the same therapeutic activity and similar potency as endogenous GLP-1 but has a pharmacokinetic profile that is closer to that of albumin.

Modified GLP-1 and its Fusion Protein in Combination with Other Therapeutic Agents In one aspect of the invention, the GLP-1 peptide modified and its fusion protein, eg, GLP-1-Tf fusion protein, of the present invention are used in combination with at least one second therapeutic molecule such as Glucophage® (metformin hydrochloride tablets) or Glucophage® XR ( prolonged-release metformin hydrochloride tablets) to treat type II diabetes, obesity, and other diseases or conditions associated with abnormal glucose levels. Glucophage® and Glucophage® XR are oral antihyperglycaemic drugs for the management of type II diabetes. Glucophage® XR is an extended-release formulation of Glucophage®. Consequently, Glucophage® XR can be taken once a day whenever the drug is slowly released from the dosage form. Glucophage® helps the body produce less glucose from the liver. Consequently, Glucophage® is effective in controlling blood sugar levels in a patient. Glucophage® rarely causes low blood glucose (hypoglycemia) since it does not cause the body to make more insulin. Glucophage® also helps reduce the blood components of fat, triglycerides and cholesterol, which are commonly high in people with type II diabetes. Metformin has been shown to reduce appetite and help people lose a few pounds when they start taking the medicine. Metformin has been approved for treatment with sulfonylureas, or with insulin, or as monotherapy (by itself). Metformin has been suggested for use in the treatment of various cardiovascular diseases such as hypertension in insulin resistant patients (WO 9112003-Upjohn), to dissolve blood clots (in combination with a t-PA derivative) (WO 9108763, WO 9108766 , WO 9108767 and WO 9108765-Boehringer Mannheim), ischemia and tissue anoxia (EP 283369-Lipha), atherosclerosis (DE 1936274-Brunnengraber &Co., DE 2357875-Hurka and US Patent No. 4, 205, 087-ICI ). Furthermore, it has been suggested to use metformin in combination with prostaglandin-like cyclopentane derivatives such as coronary dilators and for blood pressure reduction (U.S. Patent No. 4,182,772-Hoechst). Metformin has also been suggested for use in cholesterol reduction when used in combination with 2-hydroxy-3, 3, 3-trifluoropropionic acid derivatives (US Patent No. 4, 107, 329-ICI), 1,2-diarylethylene derivatives (U.S. Patent No. 4, 061, 772-Hoechst), acids, esters and aryloxy-3, 3, 3- salts substituted trifluoro-2-propionics, (U.S. Patent No. 4, 055, 595-ICI), substituted hydroxyphenylpiperidones (U.S. Patent No. 4, 024, 267-Hoechst) and derivatives of lH-indeno- [1, 2B] partially hydrogenated pyridine (US Patent No. 3,980,656-Hoechst). Montanari et al. (Pharmacological Research, vol. 25, No. 1, 1992) describe that the use of metformin in amounts of 500 mg twice a day (b.i.d.) increased post-ischemia blood flow in a manner similar to 850 mg metformin three times a day (t.i.d.). Sirtori et al. (J. Cardiovas, Pharm., 6: 914-923, 1984), describes that metformin in 850 mg three times a day (t.i.d.) increased arterial flow in patients with peripheral vascular disease. The present invention provides for the treatment of various diseases comprising modified GLP-1 of the present invention or its fusion protein in combination with one or more therapeutic agents such as metformin. In one embodiment, the modified GLP-I or its fusion protein in combination with metformin is used to treat diseases and conditions associated 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. Other therapeutic agents can be used in combination with the modified GLP-1 of the present invention and their fusion proteins include but are not limited to sulfonylurea and sulfonylurea-like agents, thiazolidinediones, range modulators of Peroxisome Proliferator Activated Receptor (PPAR), modulators. of PPAR alpha, tyrosine phosphatase-lB protein inhibitors, tyrosine activators insulin receptor kinase, llbeta-hydroxysteroid dehydrogenase inhibitors, glycogen phosphorylase inhibitors, glucokinase activators, beta-3-adrenergic agonists and glucagon receptor agonists.

DPP-IV Inhibitors DPP-IV inhibitors have shown promise for treating various conditions mediated by DPP-IV. For example, DPP-IV inhibitors are an extremely promising approach in the treatment of glucose intolerance and in disorders associated with hyperglycemia, such as type II diabetes or obesity. Furthermore, DPP-IV has been shown to play a part in the immune response, such as rejection of transplants (Transplantation 1997, 63 (10): 1495-1500). Consequently, DPP-IV can be useful in the prevention of transplant rejection. Likewise, DPP-IV inhibitors can be useful in the treatment of cancer and in the prevention of cancer metastasis, since the binding of endothelial DPP-IV of the lung to fibronectin of cancer cells promotes the metastasis of those cells (J. Biol .. Chem. 1998, 273 (37: 24207-24215) Also, it is believed that DPP-IV plays an important part in the pathogenesis of periodontitis (Infect. Immun., 2000, 68 (2), 716-724) and which is responsible for the inactivation of GLP-2, a factor that facilitates the recovery of the intestine after a major resection (J. Surg. Res. 1999, 87 (1), 130-133). Consequently, DPP-IV inhibitors are also potentially useful in bowel recovery. WO 95/15309 discloses certain peptide derivatives that are inhibitors of DPP-IV and, therefore, are useful for treating a number of processes mediated by DPP-IV. WO 95/13069 discloses certain cyclic amine compounds which are useful for stimulating the release of natural or endogenous growth hormone. European Patent 555,824 describes certain benzimidazolyl compounds which prolong the thrombin time and inhibit thrombin and serine related proteases. Archives of Biochemistry and Biophysics, vol. 323, No. 1, p. 148-154 (1995) describes certain aminoacylpyrrolidin-2-nitriles which are useful as inhibitors of DPP-IV. Journal of Neurochemistry, vol. 66, pgs. 2105-2112 (1996) discloses certain Fmoc-aminoacylpyrrolidin-2-nitrols which are useful for inhibiting prolyl oligopeptidase. Bulletin of the Chemical Society of Japan, vol. 50, No. 7, pgs. 1827-1830 (1977) describes the synthesis of an aminohexapeptide, in particular Z-Val-Val-ImPro-Gly-Phe-Phe-OMe, and its related aminopeptides. In addition, the antimicrobial properties of the compounds were examined. WO 90/12005 discloses certain amino acid compounds which inhibit prolylendopeptidase activity and, therefore, are useful for treating dementia or amnesia. WO 90/12005 discloses certain heterocyclic N- (aryl (alkyl) carbonyl) substituted compounds which are cholinesterase activators with increased peripheral selectivity useful for treating conditions caused by a reduction in cholinesterase activity. Chemical Abstracts 84: 177689 discloses certain 1-acyl-pyrrolidine-2-carbonitrile compounds that are useful as intermediates for proline compounds that exhibit angiotensin-converting enzyme (ACE) inhibitory activity. Chemical Abstracts 96: 116353 discloses certain 3-amino-2-mercapto-propyl-proline compounds which are Ras farnesyl transferase inhibitors useful for treating various carcinomas or myeloid leukemias. WO 95/34538 describes certain pyrrolidides, phosphonates, azetidines, peptides and azaprolines which inhibit DPP-IV and, therefore, are useful for treating reduced conditions by the inhibition of DPP-IV. WO 95/29190 discloses certain compounds characterized by a plurality of KPR-type repeat patterns carried by a peptide matrix that makes possible their multiple presentation to, and having an affinity for, the DPP-IV enzyme, compounds that exhibit the ability to inhibit the entry of HIV into cells. WO 91/16339 discloses certain tetrapeptide boronic acids which are DPP-IV inhibitors useful for treating autoimmune diseases and conditions mediated by suppression of IL-2. WO 93/08259 describes certain boronic acids polypeptides which are DPP-IV inhibitors useful for treating autoimmune diseases and conditions mediated by suppression of IL-2. WO 95/11689 discloses certain tetrapeptide boronic acids which are DPP-IV inhibitors useful for blocking the entry of HIV into cells. The East German Patent 158109 describes certain N-protected peptidyl-hydroxamic acids and nitrobenzoyloxamides which are useful as, inter alia, DPP-IV inhibitors. WO 95/29691 discloses, among others, certain proline dipeptide phosphonates which are DPP-IV inhibitors useful in the treatment of disorders of the immune system. German patent DD 296075 discloses certain amino acid amides that inhibit DPP-IV. Biochimica et Biophysica Acta, vol. 1293, p. 147-153 describes the preparation of certain p-nitroanilides di- and tri-peptides to study the influence of side chain modifications on their hydrolysis catalyzed by DPP-IV and PEP. Bioorganic and Medicinal Chemistry Letters, vol. 6, No. 10, p. 1163-1166 (1996) describes certain 2-cyanopyrrolidines which are DPP-IV inhibitors. J. Med. Chem., Vol. 39, pgs. 2087-2094 (1996) describes certain dipeptides containing prolinboronic acid which are inhibitors of DPP-IV. Diabetes, vol. 44, pgs. 1126-1131 (September 1996) is directed to a study that shows that GLP-1 amide degrades rapidly when administered by routes subcutaneous or intravenous to diabetic and non-diabetic subjects. The patent of E.U.A. 6,727,261 provides pyrido [2, 1-a] isoquinoline derivatives as novel DPP-IV inhibitors useful for the treatment and / or prophylaxis of diseases that are associated with DPP-IV, such as diabetes, particularly non-insulin-dependent diabetes mellitus, and tolerance to impaired glucose.

These compounds are also useful in the treatment and / or prophylaxis of intestinal diseases, ulcerative colitis, Morbid Crohn, obesity and / or metabolic syndrome. The patent of E.U.A. 6,716,843 provides alpha-amino acid sulfonyl compounds useful as inhibitors for DPP-IV. The patent of E.U.A. No. 6,645,995 describes 2-substituted unsaturated heterocyclic compounds in which a nitrogen atom in the heterocyclic ring is linked via an amide bond or a peptide bond to an amino acid or an amino acid derivative. These compounds are potent and selective inhibitors of DPP-IV, and are effective to treat conditions that can be regulated or normalized by the inhibition of DPP-IV. The patent of E.U.A. 6,617,340 describes N- (substituted glycyls) -pyrrolidines, and the use of these compounds to inhibit dipeptidyl peptidase-IV. The patent of E.U.A. 6124.305 describes N- (glycyl substituted) -2-cyanopyrrolidines which inhibit DPP-IV. These compounds are effective to treat conditions mediated by DPP-IV. Administration of the Novartis compound l - [[[2- [(5-cyanopyridin-2-yl) amino] ethyl] amino] acetyl] -2-cyano- (S) -pyrrolidine (NVP DPP728) over a period of 4 hours Weeks to 93 patients with type 2 diabetes (mean HbA? c of 7.4%) reduced plasma levels of glucose, insulin and HbAic during the study period of 4 weeks (see Diabetes Care 2002, 25 (5): 869-875) .

Combination Therapy Using DPP-IV Inhibitors In one aspect, the present invention provides 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. The present invention also provides pharmaceutical compositions comprising the transferrin fusion protein and one or more DPP-IV inhibitors. As described in the application of E.U.A. No. 10 / 378,094, which is incorporated herein by reference in its entirety, the transferrin to be bound to the protein, polypeptide or therapeutic peptide can be modified. It may exhibit reduced glycosylation. The transferrin polypeptide The modified one may be selected from the group consisting of a single N domain of transferrin, a single C domain of transferrin, a N and C domain of transferrin, two N domains of transferrin and two C domains of transferrin. The therapeutic protein or peptide that will be bound to transferrin can be in its native or modified form. Preferably, the transferrin fusion protein comprises GLP-1 as the therapeutic peptide, linked to a modified transferrin molecule, as described in the application of E.U.A. No. 10 / 378,094. Furthermore, the combination therapy of the present invention comprises GLP-1 / mTf fusion protein, one or more DPP-IV inhibitors, and another therapeutic molecule. This molecule can be Glucophage® or Glucophage® XR. In another aspect, the present invention provides the use of a modified protein or peptide that is resistant to cleavage by dipeptidyl protease or its fusion protein in combination with one or more DPP-IV inhibitors. The present invention describes pharmaceutical compositions comprising the modified protein or peptide or its fusion protein in combination with one or more DPP-IV inhibitors. Preferably, the modified peptide is modified GLP-1 and the fusion protein is modified GLP-1 / mTf protein. In addition, the combination therapy of the present invention comprises modified GLP-1 or modified GLP-1 / mTf protein, one or more DPP-IV inhibitors and another therapeutic molecule such as Glucophage or Glucophage® XR. DPP-IV inhibitors can be used in methods of the invention to treat any relevant disease. For example, a GLP-1-transferrin fusion protein as described herein can be combined with a DPP-IV inhibitor, such as [[[2- [(5-cyanopyridin-2-yl) amino] ethyl] ] amino] acetyl] -2-cyano- (S) -pyrrolidine (NVP DPP728) to treat prediabetes, diabetes, obesity or a diabetic symptom. The therapeutic agents may be administered sequentially or concurrently. The transferrin fusion protein may comprise the peptide GLP-I (7-37) (SEQ ID NO: 32) or the peptide GLP-I (7-36) (amino acids 1-30 of SEQ ID NO: 32). More preferably, the peptide GLP-1 / 7-37) or GLP-I peptide (7-36) comprises an A8 to G mutation and K34 to A. The transferrin protein may also comprise a linker between the GLP-1 peptide (7). -37) or GLP-1 peptide (7-36) and the transferrin molecule. Preferably, the linker is the peptide (PEAPTD) 2.

Increasing the Pharmacokinetics and Pharmacodynamics of Fusion Proteins with Combination Therapy using Endopeptidase Inhibitors The present invention also provides combination therapy using neutral endopeptidase inhibitors (NEP) and transferrin fusion proteins. In addition, the present invention includes a combination therapy using NEP inhibitors and DPPIV and transferrin fusion proteins. The NEP and DPPIV inhibitors can be administered concurrently or sequentially. Moreover, inhibitors and transferrin fusion proteins can be administered concurrently or sequentially. Inhibitors can be administered before or after administration of the transferrin fusion proteins. The transferrin fusion protein comprises the peptide GLP-I (7-37) (SEQ ID NO: 32) or the peptide GLP-I (7-36) (1-30 amino acids of SEQ ID NO: 32). Most preferably, the peptide GLP-I (7-37) or peptide GLP-I (7-36) comprises a mutation of A8 to G and K34 to A. The transferrin protein can also comprise a linker between the GLP-1 peptide ( 7-37) or GLP-I peptide (7-36) and the transferrin molecule. Preferably, the linker is the peptide (PEAPTD) 2. Neutral endopeptidase (NEP), which is also known as enkephalinase, neprilysin and atriopeptidase, is a membrane-bound zinc metalloendopeptidase found in many tissues including the brain, kidney, lungs, gastrointestinal tract, heart and peripheral vasculature. NEP plays a large role in the elimination of natriuretic peptides by degrading circulating natriuretic peptides, thus preventing their effects on vasodilation, blood pressure and volume. NEP, by degrading and inactivating the natriuretic peptides, is associated with hypertension, heart failure and renal failure. In addition to degrading circulating natriuretic peptides, NEP also degrades other vasodilator substances including circulating bradykinins; adrenomedullin, diuretic-natriuretic peptide and renal vasodilator; and / or urodilatin, a renal form of ANP. NEP is also involved in the degradation of the endothelin isoform ET-1, a vasoconstrictor, and may be involved in the formation of ET-1 (Brunner-La Rocca et al., Cardiovascular Research 51 (2001) 510-520). NEP also degrades angiotensin II, a potent vasoconstrictor, endomorphins and a number of peptides involved in metabolism such as bradykinin, GLP-1 (Hupe-Sodmann, K., McGregor, G P., Bridenbaugh, R et al Regulatory Peptides 1995; : 149-56, Hupe-Sodmann, K, Goeke, R., Goeke, B et al Peptides 1997; 18: 625-32), PYY (Medeiros MD, Turner AJ., Endocrinology, 1994 May; 134 (5): 2088-94) and glucagon (Trebbien R et al Am J Physiol Endocrinol Metab. 2004 Sep; 287 (3): E431-8). A number of NEP inhibitors such as phosphoramidon or NEP / ACE inhibitors (including omapatrilat described in U.S. Patent No. 5,508,272, gempatrilat described in the patent of E.U.A. No. 5,552,397, sampatrilat and MDL100240 described in the U.S. patent. No. 5,430,145) have been reported in the literature as useful for the immunotherapeutic treatment of, for example, hypertension and heart failure. Nathisuwan et al., "A Review of Vasopeptidase Inhibitors: A New Modality in the Treatment of Hypertension and Chronic Heart Failure", Pharmacotherapy, vol. 22 (1), pgs. 27-42 (2002). Candoxatril and ecadotril are the two highly specific inhibitors of NEP that are currently being tested as future drugs for heart failure. Both compounds are prodrugs that are metabolized in the body to active congeners. Candoxatril is activated in the liver by candoxatrilat, while ecadotril is converted into its active congener, S-thiorfan. Some examples of ACE / NEP inhibitors described in the patents of E.U.A. Nos. 5,508,272, 5,362,727, 5,366,973, 5,225,401, 4,722,810, 5,223,516, 5,552,397, 4,749,688, 5,504,080, 5,612,359 and 5,525,723, and European patent applications 0481,522, 0534363A2, 534,396 and 534,492. The present invention provides a combination therapy comprising transferrin fusion proteins and DPP-IV inhibitors and / or ACE / NEP inhibitors for the treatment of various diseases or conditions. These 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 The production of transgenic non-human animals expressing modified polypeptide or peptide that is protected from DPP activity is contemplated in an embodiment of the present invention. In some embodiments, transgenic non-human animals expressing fusion proteins comprising a modified polypeptide or peptide and having increased stability are contemplated. Successful production of non-human transgenic animals has been described in a number of patents and publications, such as, for example, U.S. Pat. 6,291,740 (published September 18, 2001); patent of E.U.A. 6,281,408 (published August 28, 2001) and patent of E.U.A. 6,271,436 (published on August 7, 2001) the contents of which are incorporated herein by reference in their totals. The ability to alter the genetic makeup of animals, such as domesticated mammals including cows, pigs, goats, horses, cattle and sheep, allows a number of commercial applications. These applications they include the production of animals that express large amounts of exogenous proteins in an easily harvested manner (for example, expression in milk or blood), the production of animals with increased weight gain, feeding efficiency, carcass composition, production or milk content, resistance to diseases and resistance to infections by specific microorganisms and the production of animals that have increased growth rates or reproductive performance. Animals that contain exogenous DNA sequences in their genome are referred to as transgenic animals. The most widely used method for the production of transgenic animals is the microinjection of DNA in the pronuclei of fertilized embryos (Wall et al., J. Cell, Biochem 49: 113

[1992]). Other methods for the production of transgenic animals include infection of embryos with retroviruses or with retroviral vectors. Detection of mouse embryos both pre- and post-implantation with either wild-type or recombinant retroviruses has been reported (Janenich, Proc. Nati. Acad. Sci. USA 73: 1260

[1976]; Janenich et al. , Cell 24: 519

[1981]; Stuhlmann et al. , Proc. Nati Acad. Sci. USA 81: 7151

[1984]; Jahner et al. , Proc. Nati Acad Sci. USA 82: 6927

[1985]; Van der Putten et al. , Proc. Nati Acad Sci. USA 82: 6148-6152

[1985]; Stewart et al. , EMBO J. 6: 383-388

[1987]).

An alternative means for infecting embryos with retroviruses is the injection of virus or virus-producing cells into the blastocoel of mouse embryos (Jahner, D. et al., Nature 298: 623

[1982]). The introduction of transgenes into the germline of mice has been reported using intrauterine retroviral infection of the mid-gestation mouse embryo (Jahner et al., Cited above

[1982]). It has been reported the infection of bovine and ovine embryos with retroviruses or retroviral vectors to create transgenic animals. These protocols include the microinjection of retroviral particles or arrested growth cells (ie, treated with mitomycin C) which shed retroviral particles in the perivitelline space of fertilized eggs or early embryos (PCT International Application WO 90/08832

[1990] and Haskell and Bowen, Mol. Reprod Dev., 40: 386

[1995] The international application of PCT WO 90/08832 describes the injection of wild type feline leukemia virus B into the perivitelline space of sheep embryos in the stage of 2 to 8 cells Fetuses derived from injected embryos were found to contain several integration sites US patent 6,291,740 (published September 18, 2001) describes the production of transgenic animals by introducing exogenous DNA into pre-mature oocytes and oocytes mature and not fertilized (ie, pre-fertilization oocytes) using retroviral vectors which transduce dividing cells (eg, vectors derived from murine leukemia virus [MLV]). This patent also describes methods and compositions for the LTR expression of mammary tumors driven by promoters as well as mouse of several recombinant proteins. The patent of E.U.A. 6,281,408 (published August 28, 2001) describes methods for producing transgenic animals using embryonic stem cells. Briefly, embryonic stem cells are used in a mixed cell co-culture with a morula to generate transgenic animals. Foreign genetic material is introduced into the embryonic stem cells before co-culture by, for example, electroporation, delivery by electroporation, microinjection or retroviral. ES cells transfected in this manner are selected for gene integrations by a selection marker such as neomycin. The patent of E.U.A. 6,271,436 (published August 7, 2001) describes the production of transgenic animals using methods that include the isolation of primordial germ cells, the cultivation of these cells to produce cell lines derived from primordial germ cells, the transformation of primordial germ cells and the lines of cultured cells and the use of these cells and lines of transformed cells to generate transgenic animals. The efficiency at which the transgenic animals are generated is greatly increased, thus allowing the use of homologous recombination to produce transgenic non-rodent animal species.

Gene Therapy The use of polypeptide or modified peptide constructs of the present invention for gene therapy is contemplated in one embodiment of this invention. The polypeptide or peptide has been modified to protect it from DPP activity by the addition of one or more additional amino acids at its N terminus. For example, the nucleic acid construct encoding GLP-1 comprises an additional His residue at its N terminus. provided for gene therapy. Also, the nucleic acid construct encoding the modified GLP-I fusion protein / transferrin is provided for gene therapy. The modified GLP-1 constructions of the present invention are protected from DPP activity and are more stable; in this way, they are ideally suited for gene therapy treatment. Briefly, gene therapy by injection of an adenovirus vector containing a gene encoding a soluble fusion protein consisting of the antigen 4 of cytotoxic lymphocyte (CTLA4) and the Fc portion of human immunoglobulin Gl was recently shown in Ijima et al. (June 10, 2001) Human Gene Therapy (United States) 12/9: 1063-77. In this gene therapy application, a murine model of arthritis induced by type II collagen was successfully treated by intraarticular injection of the vector. Gene therapy is also described in a number of US patents. including the patent of E.U.A. 6,225,290 (published May 1, 2001); patent of E.U.A. 6,187,305 (published February 13, 2001); and patent of E.U.A. 6,140,111 (published on October 31, 2000). The patent of E.U.A. 6,225,290 provides methods and constructions with which intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate a gene that expresses a protein having a desired therapeutic effect. The transformation of intestinal cells is achieved by the administration of a formulation composed mainly of naked DNA, and the DNA can be administered orally. Oral administration routes or other routes of intragastrointestinal administration provide a simple method of administration, while the use of naked nucleic acid avoids the complications associated with the use of viral vectors to achieve gene therapy. The Expressed protein is secreted directly into the gastrointestinal tract and / or bloodstream to obtain therapeutic blood levels of the protein thereby treating the patient requiring the protein. Transformed intestinal epithelial cells provide short and long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are prone to treatment by overexpression of a protein. The patent of E.U.A. 6,187,305 provides methods of gene or DNA targeting in cells of vertebrate origin, particularly mammalian. Briefly, DNA is introduced into primary or secondary cells of vertebrate origin through homologous recombination and DNA direction, which is introduced into genomic DNA of the primary or secondary cells at a preselected site. The patent of E.U.A. 6,140,111 (published October 31, 2000) describes vectors for retroviral gene therapy. The retroviral vectors described include an insertion site for genes of interest and are capable of expressing high levels of the protein derived from the genes of interest in a wide variety of transfected cell types. Retroviral vectors lacking a selectable marker are also described, thus making them suitable for human gene therapy in the treatment of a variety of disease states without the co-expression of a marker product, such as an antibiotic. These retroviral vectors are especially suitable for use in certain lines of packaging cells. The ability of retroviral vectors to insert them into the genome of mammalian cells has made them particularly promising candidates for use in the genetic therapy of genetic diseases in humans and animals. Genetic therapy typically includes (1) adding new genetic material to patient cells in vivo, or (2) removing patient cells from the body, adding new genetic material to the cells and reintroducing them into the body, ie, gene therapy in vi tro. Discussions about how to carry out gene therapy in a variety of cells using retroviral vectors can be found, for example, in the patents of E.U.A. Nos. 4,868,116, published September 19, 1989 and 4,980,286, published December 25, 1990 (epithelial cells), WO 89/07136 published August 10, 1989 (hepatocyte cells), EP 378,576 published July 25. of 1990 (fibroblast cells) and WO 89/05345 published June 15, 1989 and WO / 90/06997, published June 28, 1990 (endothelial cells), the disclosures of which are hereby incorporated by reference. Without additional description, it is believed that someone from Ordinary skill in the art can, using the above description and the following illustrative examples, make and use the claimed invention. The following working examples therefore specifically indicate preferred embodiments of the present invention, and should not be considered as limiting in any way the remainder of the description. All articles, publications, patents and documents mentioned throughout this application are hereby incorporated by reference in their entirety.

Examples Example 1 Modified GLP-1 Having Protection Against Dipeptidyl-Peptidase IV This example describes modified GLP-1 peptides protected from DPP-IV activity. The following peptides were synthesized using standard solid phase Fmoc chemistry and purified by reverse phase HPLC using a C18 column and quantified by absorbance at 220 mm. The purified peptides were analyzed by mass spectrometry (MALDI-TOF): GLP-l NH2-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-COOH (1-30 amino acids of SEQ ID NO: 32) GLP-l (A8G) NH2-His-Gly-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-COOH (SEQ ID NO: 90) H-GLP-1 NH2-His-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-COOH (SEQ ID NO: 91) H-GLP-1 (A8G) NH2-His-His-Gly-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-COOH (SEQ ID NO: 92) HH-GLP-1 NH2-His-His-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-COOH (SEQ ID NO: 93) G-GLP-1 NH2-Gly-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-Gy-Arg-COOH (SEQ ID NO: 94) H-Exendin-4 NH2-His-His-Gly-Glu-gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gin-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu -Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-COOH (SEQ ID NO: 95) Treatment with Dipeptidylpeptidase-IV Equimolar concentrations of each peptide (6 μM) were treated with 2 μg of recombinant human DPP-IV (1 μg / μL, R &D Systems, Minneapolis, MN) in 25 mM Tris-Cl (pH 8.0 ). Control reactions that excluded DPP-IV were installed in parallel for each peptide. The digestions were incubated at room temperature for two hours, after which the reactions were diluted 10 times in the Krebs-Ringer pH regulator (Biosource International, Camarillo, CA) supplemented with lmM of 3-isobutyl-1-methylxanthine (IBMX, Calbiochem, San Diego, CA). The peptides were then analyzed to determine the activating activity of the residual GLP-l receptor, as described below.

Cyclic AMP Stimulation Assay Four 96-well tissue culture plates were seeded with CH0-GLP1R cells (Montrose-Rafizadeh, et al., 1997 J. Biol. Chem. 272, 21201-21206) at a density of 2 x 104 cells / well in RPMI medium / 10% FBS one day before treatment. The next day the cells appeared uniformly distributed with an approximate confluence of 60-80 percent. One day after seeding the culture plates the cells were washed twice with Krebs-Ringer pH regulator (KRB) followed by incubation in KRB for one hour at 37 ° C to reduce the intracellular levels of cAMP. This was followed by incubation for 10 minutes in KRB / IBMX to inhibit intracellular enzymes that degrade cAMP. Dilutions of each test compound were prepared in KRB / IBMX and triplicate wells of CH0-GLP1R cells were treated with 50 μL of test compound per well exactly for 20 minutes at 37 ° C. The treatment was stopped when washing the cultures twice are saline pH regulated with ice-cold phosphate. The lysates were prepared by adding 0.1 ml of pH lysis buffer IB (Amersham Biosciences cAMP Biotrak EIA) for 10 minutes at room temperature. The complete volume of each cell extract was then tested for cAMP concentration using the Biotrak Enzyme Immunoassay System cAMP (Amersham Biosciences Corporation, Piscataway, NJ, product code RPN225) according to the equipment instructions. It was found that the peptides of the invention were more resistant to DPP-IV than the non-modified forms.

Specific GLP-1 ELISA As an alternative, the degradation by DPP-IV of GLP-1 and GLP-1 derivatives of the invention was tested using an ELISA system (Glucagon-Like Peptide-1 [Active] ELISA [Lineo Research , Inc., St. Charles, MO]) which is specific for intact and active GLP-1 and does not recognize GLP-1 in which the two N-terminal amino acids have been removed due to the action of DPP-IV, ie , GLP-1 (9-36 or 9-37). Equimolar concentrations of GLP-1 and H-GLP-1 (1200 pM) were treated with recombinant human DPP-IV (200 ng / μL, R &D Systems, Minneapolis, MN) in 25 mM Tris-Cl (pH 8.0) and the reaction was stopped by dilution in the assay pH regulator supplemented with the kit, which contains protease inhibitors. The equipment comprises a microtiter plate of 96 wells coated with an anti-GLP-1 monoclonal antibody. The plate was washed (25 mM saline pH regulated with borate x4 in a plate washer, ThermoLabsystems Ultrawash Plus), then incubated with peptide samples (300 pM and 10-fold serial dilutions). below the plate) for three hours at room temperature. After washing as described above, the plate was incubated with anti-GLP antibody conjugated to alkaline phosphatase (supplied as a ready-to-use component of the kit) for two hours at room temperature. After washing, substrate of 4-methylumbelliferyl phosphate (MUP) (1: 200 dilution in 50 mM borate, pH 9.5) was applied to all wells, and incubated in the dark at room temperature for 30 minutes. The plate was read at an excitation of 355 and emission wavelengths of 460 nm in a SpectraMax Gemini EM fluorescence plate reader. Since H-GLP-1 bound less easily to the monoclonal antibody than GLP-1 itself, the concentration of active H-GLP-1 that remained after treatment with DPP-IV was determined using a standard curve for H-GLP. -1. Figure 8 shows that H-GLP-1 is substantially more resistant to the action of DPP-IV than GLP-1.

Example 2 Modified GLP-I Fusion Protein This example describes a fusion protein comprising a modified GLP-1 protected from DPP-IV activity fused to a modified transferrin molecule. To construct a sequence that codes for the transferrin secretion leader followed by GLP-1 and the N-terminal part of transferrin, the following superposition primers were designed: P0236-TTCCCATACAAACTTAAGAGTCCAATTAGCTTCATCGCCA (SEQ ID NO: 96) P0237- GGTTTAGCTTGTTTTTTTATTGGCGATGAAGCTAATTGGACTCTTAAGTTTGTATGGGAA (SEQ ID NO: 97) P0244-ATAAAAAAACAAGCTAAACCTAATTCTAACAAGCAAAGATGAGGCTCGCCGTGGGAGCCC (EQ ID NO: 98) P0245- CAGGACGGCGCAGACCAGCAGGGCTCCCACGGCGAGCCTCATCTTTGCTTGTTAGAATTA (SEQ ID NO: 99) P0248-TGCTGGTCTGCGCCGTCCTGGGGCTGTGTCTGGCGCATGCTGAAGGTACTTTTTACTACTGA TGTTTCTTC (SEQ ID NO: 100) P0249- AATTCTTTAGCAGCTTGACCTTCCAAATAAGAAGAAACATCAGAAGTAAAAGTACCTTCAG CATGCGCCAGACACAGCCC (SEQ ID NO: 101) P0250- TTATTTGGAAGGTCAAGCTGCTAAAGAATTTATTGCTTGGTTGGTTAAAGGTAGGGTACCT GATAAAACT (SEQ ID NO: 102) P0251- AGTTTTATCAGGTACCCTACCTTTAACCAACCAAGCAATA (SEQ ID NO: 103) The positions of these primers are shown below. AflII - + v g a l l v a v í g l c l l a »... GLP-l > h a ß g t > > P0248 »» P0250 > > «P0249« < < .P0251 < < > GLP-l > f t s d v B ß y l e g q a a e f i a Kpnl + 961 gtgagatggt gtgcagtgtc ggagcatgag cactctacca cacgtcacag cctcgtactc > .... GLP-l .... > > 1 v k g r > > mTf > v p k t v r c a v s e h e (SEQ ID NO: 104 is the coding thread; SEQ ID NO: 105 is the encoded protein). The primers (8 μL of 20 picomolar) were combined and heated at 65 ° C for 5 minutes and then the binding reaction was allowed to cool slowly to room temperature. After adding T4 DNA ligase to the fixation reaction and incubation for two more hours at room temperature, 1 μL of the reaction was removed and used in a PCR reaction to amplify the insert completed with the outer primers P0236 and P0251. The PCR conditions were the following: 5 min. at 94 ° C 25 cycles of: 30 sec. at 94 ° C 30 sec. at 50 ° C 1 min. at 72 ° C 7 min. at 72 ° C maintenance at 4 ° C The resulting PCR product was digested AflII and f? pril and ligated into pREX0094 (Figure 1) which had previously been digested with Afl11 and .Kpnl. The ligation was used to transform E. coli. The DNA from the resulting clones was sequenced and a correct clone the length of the AflI I / Kpnl insert was selected and designed pREX0198 (figure 2). Next, pREX0198 was digested with Notl and Pvul and inserted into pSAC35 (figure 3) to create pREX0240 (figure 4). To create a plasmid encoding the natural transferrin secretion leader followed by H-GLP-1 (7-36) fused to a modified transferrin (mTf), overlay primers P0424 and P0425 were designed to add the N-terminal histidine additional to the sequence encoded by pREX0198. P0424 5 'to 3' CTGTGTCTGGCGCATCATGCTGAAG (SEQ ID NO: 106) P0425 5 'to 3' CTTCAGCATGATGCGCCAGACACAG (SEQ ID NO: 107) pREX0198 was used as the template for the initial PCR reactions using the two mutagenic primers in superposition and two primers external and separate reactions, that is, P0424 plus P0012 and P0425 more P0025 The products of these reactions were then used as templates in a second round of PCR just with the outer primers, ie P0012 plus P0025, to join them in this way together. The reaction conditions for rounds of PCR rounds were 1 x 94 ° C for 1 minute, 20 x 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 1 minute and 1 x 72 ° C for 7 minutes to end. The PCR product of the final reaction was digested with Afill and Kpnl and ligated into pREX0052 digested with AflI I / Kpnl (Figure 5) to create pREX0367 (Figure 6). The Construction was sequenced in its DNA to confirm the insertion of the codon for the additional histidine. pREX0367 was then digested with Notl and Pvul (the last to destroy the ampicillin resistance gene) and ligated into pSAC35 previously digested with Notl to create pREX0368 (Figure 7). ? REX0368 was transformed into the Saccharomyces cerevisiae host strain by electroporation and transformed colonies selected based on the prototrophy of leucine in plates of minimum medium regulated in pH. After selection of individual colonies, yeast transformants were stored in 40% trehalose and stored at -70 ° C. The expression was determined by growth in liquid minimum medium regulated at pH 6.5 and analysis of supernatant by SDS-PAGE, western blot and ELISA. Plasmids encoding GLP-1 / mTf (pREXOlOO) and H-GLP-1 / mTf were constructed as described in the application of E.U.A. 10 / 378,094, filed on March 4, 2003, which is hereby incorporated by reference in its entirety. To produce the GLP-1 / mTf fusion protein, the amino acid sequence of GLP-1 (7-36) and GLP-1 (7-37) can be used. haegtftsdvssylegqaakefiawlvkgr (1-30 amino acids of SEQ ID? O: 32) haegtftsdvssylegqaakefiawlvkgrg (SEQ ID NO: 32) For example, the peptide sequence of GLP-I (7-36) can be retro-translated into DNA and the codon optimized for yeast: catgctgaaggtacttttacttctgatgtttcttcttatttggaaggtcaagctgctaaagaa h a e g t f t s d v s ß and l e g q a a k e tttattgcttggttggttaaaggtaga (SEQ ID N0: 117) f i a w l v k g r (1-30 amino acids of SEQ ID NO: 32) The primers were specifically designed to form sticky ends 5 'Xbal and 3' .Kpnl after fixation and to make direct ligation possible in pREX0052 cut with Xbal / Kpnl, just 5 'from the end of the leader sequence and at the N terminus. of mTf. Alternatively, other sticky ends can be manipulated for ligations in other vectors. Xbal - + __ __ __ 1 aggtct ^ ag "agaaaaggca *" gcígaaggt ao tttact o gatgtttc Vtcttatttaj tccagagatc ^ tcttttccg aagácttcca ífaaaatcfaa gac kcaaag aagaataa ^ »PL '. »R s 1 e k r» QLP-1 > h a e g t f t s d v ß s and l Kpnl + v p d SEQ ID NOs: 118 and 119 After the fixation and ligation, the clones were sequenced to confirm the correct insertion.

This vector was designated pREX0094. The cassette was cut out of pREX0094 with Notl and sub-cloned in Notl-cut yeast vector, pSAC35, to make pREXOlOO. The plasmid was then electroporated into the Saccharomyces yeast host strains and transformants were selected for leucine prototrophy in plates of minimum medium. Expression was determined by growth in minimal liquid medium and analysis of the supernatant by SDS-PAGE, western blot and ELISA. GLP-1 / mTf and H-GLP-1 / mTf were expressed and purified from fermentation cultures, allowed to grow under standard conditions by exchange and by cation exchange chromatography and anion exchange.

Treatment with Dipeptidylpeptidase-IV Equimolar concentrations of GLP-1 / mTf and H-GLP / 1-mTF (2 μM) were treated with recombinant human DPP-IV (lμg / μL, R &L) Systems) in a 25 mM solution of Tris-Cl (pH 8.0). Control reactions that excluded DPP-IV were installed in parallel for each fusion protein. The digestions were incubated at room temperature for two hours, time after which the reactions were diluted 20 times in Krebs-Ringer pH regulator (Biosource International) supplemented with 1 mM of IBMX (Calbiochem).

Cyclic AMP Stimulation Assay Tissue culture plates (24 wells) were seeded with CHO-GLP1R cells at a density of 1 x 10 5 cells per well in RPMI / 10% FBS medium one day before treatment. The next day the cells appeared uniformly distributed with an approximate confluence of 60-80 percent. One day after seeding the culture dishes the cells were washed twice with Krebs-Ringer's pH regulator (KRB) followed by incubation in KRB for one hour at 37 ° C to reduce the intracellular levels of cAMP. This was followed by incubation for 10 minutes in KRB / IBMX to inhibit intracellular enzymes that degrade cAMP. Dilutions of each test compound were prepared in KRB / IBMX and triplicate wells of CHO-GLP1R cells were treated with 0.15 ml of test compound per well exactly for 50 minutes at 37 ° C. The treatment was stopped by washing the cultures twice with saline and pH regulated with ice-cold phosphate. The lysates were prepared by the addition of 0.2 ml of pH lysis buffer IB (Amersham Biosciences cAMP Biotrak EIA) for 10 minutes at At room temperature, then 100 μl of each cell extract was assayed to determine the concentration of cAMP using the Biotrak cAMP Enzyme Immunoassay System (Amersham Biosciences) according to the instructions of the kit. It was found that H-GLP-1 / mTf was more resistant to DPP-IV than GLP-1 / mTf.

Example 3 GLP-1 / mTf Modified for the Treatment of Diabetes In this example, modified GLP-1 / mTf of the present invention is used as a therapeutic agent to treat diabetes. Modified GLP-1 / mTf is administered to Zucker rats, a standard animal model for type II diabetes. Zucker rats have abnormally high blood glucose levels. It has been shown that the treatment of these animals with GLP-1 induces insulin secretion and reduces blood glucose. 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 the subcutaneous injection of H-GLP-1 or H-GLP-1 / mTf, the animals are subjected to Glucose Tolerance Test (GTT). For this test, fasted animals are fed glucose solution (1.5 mg / g body weight), and glucose is measured in blood at appropriate time intervals. Right after the administration of glucose, the blood glucose level of the untreated animals rises and falls slowly towards the baseline while the animals that are injected with H-GLP-1 or H-GLP-1 / mTf show a faster normalization of the blood glucose level due to the insulinotropic effect of GLP-1. In a further experiment, H-GLP-1 or H-GLP-1 / mTf is used to normalize the high fasting glucose of the Zucker rats without glucose administration. While blood glucose levels remain high in untreated animals, a significant drop is observed in animals treated with H-GLP-1 or modified H-GLP-1 / mTf.

Example 4 Modified Glucagon Having Dipeptidyl-Peptidase IV Protection This example describes modified glucagon molecules protected from DPP-IV activity. The following peptides are synthesized using standard solid phase Fmoc chemistry and purified by reverse phase HPLC using a C18 column quantified by absorbance at 220 nm. The purified peptides are analyzed by mass spectrometry (MALDI-TOF): Glucagon NH2-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln -Trp-Leu-Met-Asn-Thr-COOH (SEQ ID NO: 35) H-Glucagon NH2-His-His-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Val-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe -Val-Gln-Trp-Leu-Met-Asn-Thr-COOH (SEQ ID NO: 108) The peptides are pre-treated with DPP-IV as described above and then assayed to verify the ability to activate the glucagon receptor using a recombinant cell line that expresses a cloned glucagon receptor.

Example 5 Modified GIP having Protection against Dipeptidyl-Peptidase IV This example provides modified GIP molecules protected from DPP-IV activity. The following peptides are synthesized using standard solid phase Fmoc chemistry and purified by reverse phase HPLC using a C18 column and quantified by absorbance at 220 nm. The peptides purified are analyzed by mass spectrometry (MALDI-TOF): GIP NH2-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe-Val-Asn -Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln-COOH (SEQ ID NO: 31) Y-GIP NH2-Tyr-Tyr-Ala-Glu-Gly-Thr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-His-Gln-Gln-Asp-Phe -Val-Asn-Trp-Leu-Leu-Ala-Gln-Lys-Gly-Lys-Lys-Asn-Asp-Trp-Lys-His-Asn-Ile-Thr-Gln-COOH (SEQ ID NO: 109) The peptides are pre-treated with DPP-IV as described above and then assayed to verify the ability to activate the GIP receptor using a recombinant cell line that expresses a cloned GIP receptor. It should be understood that the above description and examples simply present a detailed description of certain preferred embodiments. It should therefore be apparent to those of ordinary skill in the art that various equivalent modifications can be made without departing from the spirit and scope of the invention. All the bulletin articles, other references, patents and patent applications that are identified in this patent application are incorporated by reference in their entirety. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (52)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. A method for treating a disease or condition in a patient, characterized in that it comprises administering an effective amount of a fusion protein to transferrin (Tf) and at least a second agent. 2. The method according to claim 1, characterized in that the second agent is selected from the group consisting of a DPP-IV inhibitor and a neutral endopeptidase inhibitor (NEP). 3. The method of compliance with the claim 1, characterized in that the fusion protein Tf is susceptible to elimination by DPP-IV. 4. The method according to claim 1, characterized in that the fusion protein Tf is sensitive, resistant or partially resistant to elimination by DPP-IV. The method according to claim 1, characterized in that the Tf fusion protein comprises one or more GLP-1 peptides fused to a Tf molecule. 6. The method of compliance with the claim 5, characterized in that the fusion protein Tf also comprises a linker. 7. The method of compliance with the claim 6, characterized in that the linker is PEAPTD or (PEAPTD) 2. 8. The method according to the claim 5, characterized in that the GLP-1 peptide is at the N-terminus of the fusion protein. 9. The method according to claim 5, characterized in that the peptide is GLP-I (7-37) having SEQ ID? O: 32 O GLP-I (7-36) (amino acids 1-30 of SEQ ID? O: 32). The method according to claim 9, characterized in that the GLP-1 peptide has been modified by mutating A8 to G. 11. The method according to claim 9, characterized in that the GLP-1 peptide has been modified by mutating K34 to A. 12. The method according to claim 9, characterized in that the GLP-I peptide has been modified by mutating A8 to G and K34 to A. 13. The method according to claim 12, characterized in that the GLP-l peptide is linked to the Tf molecule by means of the linker (PEAPTD) 2. The method according to claim 5, characterized in that the Tf molecule has been modified to exhibit reduced glycosylation in comparison with a fully glycosylated Tf molecule. 15. The method according to claim 5, characterized in that the Tf molecule has been modified so as not to exhibit glycosylation. 16. The method of compliance with the claim 5, characterized in that the Tf molecule has been modified to comprise a mutation that prevents glycosylation. 17. The method according to claim 5, characterized in that the Tf molecule has been modified to have reduced affinity for a transferrin receptor (TfR) compared to a wild type Tf molecule. 18. The method according to claim 5, characterized in that the Tf molecule has been modified to have no binding to a TfR. 19. The method according to the claim 5, characterized in that the Tf molecule has been modified to have reduced affinity for iron as compared to a wild type Tf molecule. 20. The method according to claim 5, characterized in that the Tf molecule has been modified so as not to have iron binding. 21. The method according to claim 5, characterized in that the Tf molecule is lactoferrin or melanotransferrin. 22. The method of compliance with the claim 1, characterized in that the disease is a metabolic disease. 23. The method according to the claim 22, characterized in that the metabolic disease is prediabetes, diabetes or obesity. 24. The method of compliance with the claim 23, characterized in that diabetes is type II diabetes. 25. The method according to claim 1, characterized in that the disease is congestive heart failure. 26. The method according to claim 1, characterized in that the disease is irritable bowel syndrome or dyspepsia. 27. The method according to claim 1, characterized in that there are two second agents. 28. The method according to claim 27, characterized in that the two second agents are a DPP-IV inhibitor and a NEP inhibitor. 29. The method according to claim 28, characterized in that the two second agents are administered concurrently. 30. The method according to claim 1 or 29, characterized in that the transferrin fusion protein and the one or more second agents are administered sequentially. 31. The method according to claim 1 or 29, characterized in that the transferrin fusion protein and the one or more second agents are administered concurrently. 32. A composition characterized in that it comprises a transferrin-fusion protein (Tf) and at least one second agent. The composition according to claim 32, characterized in that the second agent is selected from the group consisting of a DPP-IV inhibitor and a neutral endopeptidase inhibitor (NEP). 34. The composition according to claim 32, characterized in that the Tf fusion protein is susceptible to elimination by DPP-IV. 35. The composition according to claim 32, characterized in that the Tf fusion protein is sensitive, resistant or partially resistant to elimination by DPP-IV. 36. The composition according to claim 32, characterized in that the fusion protein Tf comprises one or more GLP-1 peptides fused to a Tf molecule. 37. The composition according to claim 36, characterized in that the Tf fusion protein further comprises a linker. 38. The composition according to claim 37, characterized in that the linker is PEAPTD or (PEAPTD) 2. 39. The composition according to claim 36, characterized in that the GLP-1 peptide is at the N-terminus of the fusion protein. 40. The composition according to claim 36, characterized in that the peptide is GLP-1 (7-37) having SEQ ID NO: 32 or GLP-1 (7-36) (amino acids 1-30 of SEQ ID NO: 32). 41. The composition according to claim 40, characterized in that the GLP-1 peptide has been modified by mutating A8 to G. 42. The composition according to claim 40, characterized in that the GLP-1 peptide has been modified by mutating K34 to A. 43. The composition according to claim 40, characterized in that the GLP-1 peptide has been modified by mutating A8 to G and K34 to A. 44. The composition according to claim 43, characterized in that the peptide is GLP-l is linked to the Tf molecule by means of the linker (PEAPTD) 2. 45. The composition according to claim 32, characterized in that the molecule of Tf has been modified to exhibit reduced glycosylation compared to a fully glycosylated Tf molecule. 46. The composition according to claim 32, characterized in that the Tf molecule has been modified so as not to exhibit glycosylation. 47. The composition according to claim 32, characterized in that the Tf molecule has been modified to comprise a mutation that prevents glycosylation. 48. The composition according to claim 32, characterized in that the Tf molecule has been modified to have reduced affinity for a transferrin receptor (TfR) compared to a wild type Tf molecule. 49. The composition according to claim 32, characterized in that the Tf molecule has been modified to have no binding to a TfR. 50. The composition according to claim 32, characterized in that the Tf molecule has been modified to have reduced affinity for iron compared to a wild type Tf molecule. 51. The composition according to claim 32, characterized in that the Tf molecule has been modified to have no binding to iron. 52. The composition according to claim 32, characterized in that the Tf molecule is lactoferrin or melanotransf errin.