Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
  • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
  • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/521—Chemokines
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/605—Glucagons
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins

Definitions

• This invention relates to modified polypeptides that are resistant to dipeptidyl peptidase cleavage.
• this invention includes modified insulinotropic peptides that are protected from dipeptidyl peptidase IV.
• the methods of the invention include extending the effective therapeutic in vivo half life of the modified insulinotropic peptides.
• Proteolytic enzymes play an important role in regulating physiological processes such as cell proliferation, differentiation, and signaling processes by regulating protein turnover and processing.
• Proteolytic enzyme controls the levels of important structural proteins, enzymes, and regulatory proteins through proteolytic degradation. Uncontrolled proteolytic enzyme activity, either increased or decreased, has been implicated in a variety of disease conditions including inflammation, cancer, arteriosclerosis, and degenerative disorders.
• protease for the subset of peptide bond hydrolases (Subclass E.C 3.4.).
• protease is synonymous with peptidase.
• Peptidases comprise two groups of enzymes: the endopeptidases and the exopeptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively.
• proteinase is synonymous with endopeptidase.
• Proteolytic enzymes are classified according to their catalytic mechanisms. Four mechanistic classes have been recognized by the IUBMB: the serine proteases, cysteine proteases, aspartic proteases, and metalloproteases.
• Serine proteases are a large family of proteolytic enzymes containing a serine residue in the active catalytic site for protein cleavage. They are ubiquitous being found in viruses, bacteria, and eukaryotes. Serine proteases have a wide range of substrate specificities and can be subdivided into subfamilies on the basis of these specificities. There are over 20 subfamilies of serine proteases which are grouped into six clans (SA, SB, SC, SE, SF, and SG).
• Prolyl oligopeptidase is a serine protease grouped in the SC clan. It hydrolyzes proline containing peptides at the carboxyl side of proline residues. Presumably, it is involved in the maturation and degradation of peptide hormones and neuropeptides (Wilk et al. 1983 Life Sci. 33, 2149-2157).
• Examples of prolyl oligopeptidase include dipeptidyl peptidase IV (DPPIV), dipeptidyl peptidase II (DPPII), fibroblast activation protein, and prolyl oligopeptidase. These enzymes display distinct 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.
• Peptidases having highly specific actions on proline-containing sequences are attractive targets of medicinal chemistry because some of them have been linked to the modulation of the biological activity of natural peptide substrates. For example, DPPIV is linked to the treatment of diabetes through regulating the level of glucagon-like peptide-1 (GLP-1).
• GLP-1 glucagon-like peptide-1
• DPPIV activity is increased in various diseases such as rheumatoid arthritis, multiple sclerosis, Grave's disease, and Hashimoto's thyroiditis, sarcoidosis, and cancer. DPPIV activity is also increased in AIDS, Down's syndrome, anorexia/bulimia, pregnancy and hypogammaglobulinemia.
• Dipeptidyl aminopeptidase activity is peptidase activity which catalyzes the removal of dipeptides from the N-terminus of peptides, polypeptides, and proteins.
• a dipeptidyl aminopeptidase is capable of cleaving the dipeptide XY from the unsubstituted N-terminal amino group of a peptide, polypeptide or protein, wherein X and Y represent any amino acid residue.
• DPPs dipeptidyl peptidases
• DPPs dipeptidyl peptidases I
• DPPII dipeptidyl peptidase II
• DPPIII dipeptidyl peptidase III
• DPPIV dipeptidyl peptidase
• DPPI also known as cathepsin C
• cathepsin C is a lysosomal cysteine protease that is expressed in most tissues.
• DPPI has been implicated in the processing of granzymes, which are neutral serine proteases expressed exclusively in the granules of activated cytotoxic lymphocytes.
• DPPII is a serine protease found in lysosomes. Like DPPIV, it cleaves proline containing peptide bonds. In fact, DPPII has a similar substrate specificity to DPPIV but is only active at acidic pH.
• Dipeptidyl peptidase III (DPPIII) is a metalloprotease.
• DPPIV 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, the hydrolysis is terminated when an amino acid residue followed by proline is reached. As a result, a peptide having a bond of X-Pro- Y- (X and Y are optional amino acids) will be cleaved to yield X-Pro and Y-. DPPIV will also cleave dipeptides with alanine in the penultimate position, though less effectively than dipeptides with proline (Yaron et al, 1993 Crit. Rev. Biochem. Mol. Biol. 28:31-81). The enzyme will also cleave other sequences, but with still lower efficiency.
• DPPIV has been shown to be highly specific in 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 DPPIV have been identified.
• DPPIV substrates include peptide hormones and chemokines. Examples of some peptide hormones are endomo hin-2, GLP-
• GLP-2 gastric inhibitory peptide
• GHRH growth hormone releasing hormone
• substance P examples of some chemokines are RANTES, GCP-
• DPPII possesses almost identical substrate specificity to DPPIV.
• Insulin-dependent diabetes mellitus is currently treated through the administration of insulin to patients.
• Non-insulin-dependent diabetes mellitus is treated by diet, administration of sulphonylureas to stimulate insulin secretion or with biguanides to increase glucose uptake.
• Resistant individuals may need insulin therapy.
• Standard therapy requires daily intravenous injection of insulin which will treat the acute symptoms, but prolonged therapy results in vascular disease and nerve damage.
• Modern methods such as transplantation are expensive and require risky surgical intervention. Thus, there is a need to develop a highly effective, low cost alternative to the treatment of diabetes.
• DPPIV DPPIV-like enzyme activity
• endogenous or exogenously administered insulinotropic peptides such as, GIP, GLP-1 or analogs thereof.
• GIP and GLP-1 hormones that stimulate glucose-induced secretion of insulin by the pancreas, are substrates of DPPIV.
• DPPIV removes the amino-terminal His- Ala dipeptide of GLP-1 to generate GLP-1 -(9-36)-amide, which is unable to elicit glucose-dependent insulin secretion from the islets
• the inhibition of such DPPIV or DPPIV-like enzyme activity in vivo would effectively suppress undesired enzyme activity in pathological conditions in mammalian organisms.
• PCT/DE97/00820 discloses alanyl pyrrolidide and isoleucyl thiazolidide as inhibitors of DPPIV or DPPIV-like enzyme activity.
• DD 296075 discloses pyrrolidide and isoleucyl thiazolidide hydrochloride.
• U.S. Patent 6,548,481 discloses 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 DPPIV activities, the use of these inhibitors in certain patients or certain forms of the disease may be problematic since the enzyme is responsible for activation or inactivation of such a wide range of bioactive peptides, i.e. DPPIV inhibitors lack specificity for the desired targets GIP and GLP-1.
• An alternative way to prevent therapeutic proteins and peptides such as GIP or GLP-1 from being cleaved by proteolytic enzymes is to modify the proteins and peptides themselves to block their exposure to proteolytic enzymes. Protein modifications have been shown to increase therapeutic polypeptides' stability, circulation time, and biological activity. Some general methods of modifying amino acids and peptides are disclosed 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 is incorporated herein by reference. Also, the review article of Francis (1992 Focus on Growth Factors 3:4-10, (Mediscript, London)) describes protein modification and fusion proteins, which is incorporated herein by reference.
• modified polypeptides that are short, medium or long.
• a large number of modified small polypeptide hormones may be synthesized using automated peptide synthesizers, solid-state resin techniques, or recombinant techniques.
• modified substrates of dipeptidyl peptidase for example, the substrates of DPPIV such as GLP-1, GIP, neuropeptide Y, and bradykinin can be produced using an automated peptide synthesizer.
• the present invention provides modified therapeutic peptides and proteins that are resistant to dipeptidyl protease cleavage.
• the inventors discovered that modifying the amino terminus of dipeptidyl peptidase substrates by adding at least one additional N- terminal amino acid protects these peptide substrates from dipeptidyl peptidase activity.
• the inventors found that adding one or more amino acids to the amino terminus of the peptide substrates of DPPIV blocks DPPIV and DPPIV-like protease activity.
• Such modified substrates have enhanced biological stability in the blood of mammals and would be more effective as a therapeutic peptides or proteins.
• GLP-1 is a substrate of DPPIV activity. Modified GLP-1 peptide that are protected from DPPIV cleavage are more stable and more effective in lowering elevated blood glucose levels in mammals.
• the present invention provides modified therapeutic polypeptides and peptides that contain one to five additional amino acids at its N-terminus.
• the modified polypeptides and peptides are partially or substantially resistant to DPP cleavage.
• the modification reduces DPP cleavage activity by about 10%, about 30%, about 50%, about 70%, or about 90% as compared to the polypeptide prior to modification.
• the modified polypeptide or peptide has retained about 10%, about 30%>, about 50%, about 70%, and about 90%> of its activity and/or potency as compared to the polypeptide prior to modification.
• the present invention also provides modification of variants of therapeutic polypeptides or peptides that may have an increased or decreased functional activity as compared to their respective wild-type therapeutic polypeptide or peptide.
• the present invention provides fusion proteins comprising modified polypeptides or peptides linked to a second protein, for example transferrin or albumin, for increased stability.
• the present invention provides modified GLP-1 comprising one or more additional amino acids at its N-terminus.
• the present invention also provides fusion protein comprising modified GLP-1 resistant to DPP cleavage and transferrin.
• the GLP-1 peptide may be the wild-type peptide or a variant or analog thereof.
• the modified GLP-1 peptide may be fused to conjugated to a heterologous molecule such as a polyethylene glycol, a fatty acid, or fatty acid derivative.
• the present invention includes nucleic acids encoding the modified polypeptides or peptides.
• the invention provides vectors and host cells comprising the nucleic acids encoding the modified polypeptides or peptides.
• the present invention also disclose the use of the nucleic acid constructs for expression in vivo, for example in a mammal.
• the modified polypeptides and peptides of the present invention are useful for treating diseases. Specifically, the modified GLP-1 peptides are useful for treating diseases or conditions associated with abnormal blood glucose level .
• the modified GLP-1 peptides of the present invention are used to treat subjects with diabetes and obesity. The subjects may be mammals. The mammals may be humans.
• 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 restriction enzyme map of pREX0367.
• Figure 7 shows the restriction enzyme map of pREX0368.
• Figure 8 shows time course of incubation of GLP- 1 and H-GLP- 1 and DPP-IV.
• the graph shows the amount of active, full length peptide remaining, as measured by an ELISA specific for active GLP-1.
• Insulinotropic peptides such as GLP-1
• GLP-1 are promising therapeutic agents for the treatment of type 2 non-insulin-dependent diabetes mellitus as well as related metabolic disorders, such as obesity.
• Other useful insulinotropic peptides include exendin 3 and exendin 4.
• these insulinotropic peptides have short plasma half-lifes in vivo, mainly due to rapid serum clearance and proteolytic degradation. Extensive work has been done to inhibit DPPIV, the enzyme responsible for the degradation of GLP-1 or to modify GLP-1 in such a way that its degradation is slowed down while still maintaining biological activity.
• the present invention is based in part on the finding that modification of a dipeptidyl peptidase (DPP) substrate, by adding one or more amino acids at the N-terminus of the substrate renders the substrate resistant to DPP cleavage while maintaining biological activity. Specifically, the inventors discovered that adding one or more amino acids to GLP-1 substantially protects GLP-1 from DPPIV enzyme activity.
• the modified GLP-1 of the present invention may be useful in the treatment of diseases associated with abnormal level of blood glucose, such as diabetes.
• the present invention provides modification of the substrates of dipeptidyl peptidases including but not limited to DPPII, DPPIV, and prolyl oligopeptidase.
• the addition of one or more amino acids to the amino terminus of these substrates protects them from dipeptidyl peptidase cleavage.
• these modified substrates are more stable.
• 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, e.g., by alkylation, acylation, ester formation, or amide formation.
• the term "derived from” refers to obtaining a molecule from a specified source such as obtaining a molecule from a parent molecule.
• dipeptidyl aminopeptidase activity refers to a peptidase activity which cleaves dipeptides from the N-terminal end of a peptide, polypeptide, or protein sequence.
• the dipeptidyl aminopeptidase is capable of cleaving the dipeptide XY from the unsubstituted N-terminal amino group of a peptide, polypeptide, or protein, wherein X or Y may represent any amino acid residue selected from the group consisting of Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tip, Tyr, and Val, but at least Ala, Arg, Asp, and/or Gly. All of X and Y may be different or identical. Examples of dipeptidyl aminopeptidase include, but are not limited to DPPI, DPPII, DPPIII, and DPPIV.
• Glucagon-Like Peptide-1 GLP-1
• GLP-1 derivatives refer to intestinal hormones which generally simulate insulin secretion during hyperglycemia, suppresses glucagon secretion, stimulates (pro) insulin biosynthesis and decelerates gastric emptying and acid secretion.
• Some GLP-ls and GLP-1 derivatives promote glucose uptake by cells but do not simulate insulin expression as disclosed in U.S. Pat. No. 5,574,008 which is hereby incorporated by reference.
• insulinotropic peptides refers to peptides with insulinotropic activity. Insulinotropic peptides stimulate, or cause the stimulation of, the synthesis or expression of the hormone insulin. Such peptides include precursors, analogues, fragments of peptides such as Glucagon-like peptide, exendin 3 and exendin 4 and other peptides with insulinotropic activity.
• pharmaceutically acceptable refers to materials and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• composition refers to a composition comprising an agent together with a pharmaceutically acceptable carrier or diluent when needed.
• Pharmaceutically acceptable carriers and additives are chosen such that side effects from the pharmaceutical compound are minimized and the performance of the compound is not canceled or inhibited to such an extent that treatment is ineffective.
• physiologically effective amount is that amount delivered to a subject to give the desired palliative or curative effect. This amount is specific for each drug and its ultimate approved dosage level.
• therapeutically effective amount refers to that amount of modified therapeutic polypeptide or peptide which, when administered to a subject in need thereof, is sufficient to effect treatment.
• the amount of modified therapeutic polypeptide or peptide which 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 determined routinely by one of ordinary skill in the art having regard to his/her own knowledge and to this disclosure.
• therapeutic protein refers to proteins, polypeptides, antibodies, peptide fragments or variants thereof, having one or more therapeutic and/or biological activities.
• Therapeutic proteins encompassed by the invention include but are not limited to proteins, polypeptides, peptides, antibodies and biologies.
• the terms peptides, proteins, and polypeptides are used interchangeably herein.
• therapeutic protein may refer to the endogenous or naturally occurring correlate of a therapeutic protein.
• a polypeptide or peptide displaying a “therapeutic activity” or a protein that is “therapeutically active” is meant a polypeptide, peptide or protein that possesses one or more known biological and/or therapeutic activities associated with a therapeutic protein such as one or more of the therapeutic proteins described herein or otherwise known in the art.
• a "therapeutic protein” is a protein, polypeptide, or peptide that is useful to treat, prevent or ameliorate a disease, condition or disorder. Such a disease, condition or disorder may be in humans or in a non-human animal, e.g., veterinary use.
• treatment refers to any administration of a compound of the present invention and includes: (1) preventing the disease from occurring in an animal which may be predisposed to the disease but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology); or (3) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology).
• Dipeptidyl peptidases are hydrolases that remove dipeptides from the unsubstituted N-terminal amino group of a peptide, polypeptide, or protein.
• Examples of dipeptidyl peptidases include but are not limited to DPPI, DPPII, DPPIII, DPPIV, attractin, and fibroblast activation protein (FAP). New enzymes of this family or with similar function but different structure are emerging.
• Dipeptidyl peptidase I also known as cathepsin C, is a lysosomal cysteine protease belonging to the papain family.
• DPPI is capable of sequentially removing dipeptides from the free amino terminus of various peptide and protein substrates, thus acting in the exopeptidase (specifically dipeptidyl peptidase) mode. The cleavage is ineffective if the fragmented bond has on either side a proline residue, or the N-terminal residue is lysine or arginine.
• DPPII is a serine protease found in lysosomes with unknown function. Like DPPIV, it cleaves proline containing peptide bonds. In fact, DPPII has a similar substrate specificity as DPPIV but is only active at acidic pH. Mammalian DPPII and DPPIV can be distinguished using the inhibitors puromycin and bacitracin; puromycin will inhibit DPPII only while bacitracin inhibits DPPIV only (1988 J. Biol. Chem. 263, 6613-6618). Dipeptidyl peptidase III (DPPIII) is a metalloprotease. DPPV releases N-terminal X- Ala, His-Ser, and Ser-Tyr dipeptides.
• DPPIII Dipeptidyl peptidase III
• DPP- VII also known as quiescent cell proline dipeptidase, is a proline-specific dipeptidases. It has been suggested that DPPVII and DPPII are identical proteases based on a sequence comparison of human DPP- VII and rat DPP-II (78% identity) (Araki et al. 2001 J. Biochem. 129, 279-288).
• DPPVIII is a human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP (Abbott, CA. et al, 2000 European Journal of Biochemistry 267, 6140). Similar to DPPIV, DPPVIII is ubiquitous. The full-length DPPVIII cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Purified recombinant DPPVIII hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro.
• DPPVIII shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP.
• DPPVIII enzyme activity had a neutral pH optimum consistent with it being nonlysosomal.
• the similarities between DPPVIII and DPPIV in tissue expression pattern and substrates suggests a potential role for DPPVIII in T-cell activation and immune function similar to DPPIV.
• DPPIX dipeptidyl peptidase-like protein
• GWSYG serine protease motif GWSYG
• Attractin is a 175-kDa soluble glycoprotein reported to hydrolyze Gly- Pro. Attractin contains a kelch repeat domain and shares no significant sequence homology with DPPIV or any other peptidase.
• Fibroblast activation protein is a cell surface- bound protease of the prolyl oligopeptidase gene family expressed at sites of tissue remodelling.
• Prolyl endopeptidase also called proline oligopeptidase (PO) was first discovered by Walter and coworkers as an oxytocin-degrading enzyme in the human uterus (Walter et al, Science 173, 827-829 (1971)). The enzyme cleaves peptide bonds at the carboxy-side of proline in peptides containing the sequence X-Pro-Y, where X is a peptide or N-terminal substituted 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 at the imino-side of proline (Lin & Brandts, Biochemistry 22, 4480-4485 (1983)).
• Prolyl oligopeptidase hydrolyzes angiotensin I and angiotensin II which results in the relase of angiotensin (1-7).
• Angiotensin (1-7) has vasodilator activity and modulates the release of vasopressin, which is able to influence the process of memory as was shown by injecting rats with specific PEP-inhibitors. The injection reverses the scopolamine induced amnesia.
• DPPIV Dipeptidyl Peptidase
• DPPIV is a ubiquitously expressed molecule that has been implicated in the degradation of several peptides and hormones.
• Various types of DPPIV have been purified and the enzymological properties have been revealed.
• DPPIV has been isolated from rat liver (Hopsu-Havu V. K. et al, 1966 Histochem., 7:197-201), swine 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 K. M. et al 1981 Biochim. Biophys.
• DPPIV is identical to the T-cell surface antigen CD26 which is expressed by activated lymphocytes (T-, B-, and natural killer cells).
• CD26/DPPIV 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 on epithelial cells constitutively, but on T lymphocytes, it is expressed under tight cellular regulation, with expression upregulated upon cell activation.
• CD26/DPPIV has been shown to have dipeptidyl peptidase IV activity in its extracellular domain (Hegen et al, 1990 J.
• the circulating serum form has a molecular weight of 175 kDa, in contrast to the 105 kDa molecular weight of the membrane form, and it does not bind ADA- 1. Nevertheless, the circulating form expresses functional dipeptidylpeptidase IV activity and retains the ability to costimulate the T lymphocyte response to recall antigen.
• the proteolytic activity of DPPIV 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 a unique order which is different from the classical serine proteases such as chymotrypsin and subtilisin.
• Proline specific dipeptidyl peptidase activity alters the biological activity of a large number of bioactive proteins and polypeptides comprising, amongst others, GLP-1, the neurotransmitter substance P, human growth hormone-releasing factor, erythropoietin, interleukin 2 and many others.
• Potential DPPIV substrates are listed in Tables 1, 2 and 3.
• Modulation of these polypeptides to affect DPPIV cleavage may be useful in the treatment of clinical conditions including but not limited to diabetes, inflammation, vascular diseases, auto-immune disease, multiple sclerosis, joint diseases and diseases associated with benign and malign cell transformation.
• the present invention provides modified substrates of DPP comprising one or more additional amino acids at the N-terminus of the substrates to protect the substrates from DPP activity.
• the preferred substrates for modification according to the present invention are disclosed in Table 3.
• the substrates for modification comprise X-ProY, X-Ala-Y, X-Ser-Y, or X-Gly-Y at the amino terminus.
• the substrate for modification is GLP-1.
• modified polypeptides such as modified polypeptide substrates of DPP, comprising one or more additional amino acids at the N- terminus to protect the polypeptide substrates from DPP activity.
• the modified polypeptides have one additional amino acid at its N-terminus as compared to their wild-type polypeptides.
• the modified polyeptpides have five additional amino acids at its N-terminus.
• the modified polypeptides have between one and five additional amino acids at its N-terminus. Any one of the 20 amino acids may be added to the N-terminus of the polypeptide substrate or non-natural amino acids may be added.
• any pharmaceutical polypeptide having peptide bonds which would be subject to cleavage in the gastrointestinal tract or anywhere in vivo after administration would benefit from modification in accordance with the present invention because of the protection from DPP cleavage that is afforded by the present invention.
• the substrate's dipeptidyl peptidase sensitivity by at least about 30%, preferably at least about 50%, more preferably at least about 70%, still more preferably at least about 90%, and most preferably at least about 99% of the dipeptidyl peptidase sensitivity . It is also possible to completely remove the dipeptidyl aminopeptidase sensitivity using the methods of the present invention.
• the modified polypeptide or peptide substrates of the present invention are partially or substantially protected from DPP activity, the modified polypeptide substrates have retained at least about 30% , preferably at least about 50%, more preferably at least about 70%, and still more preferably at least about 90% , and most preferably at least about 99% of their functional activity and potency.
• the modified polypeptide or peptide substrates with lowered functional activity or potency will be useful.
• a modified polypeptide peptide substate with lowered functional activity or potency may be useful.
• the modified polypeptides or peptides have may increased potency as compared to the non-modified polypeptides or peptides.
• Modified polypeptide molecules of the invention are substantially protected from dipeptidyl peptidase cleavage as compared to an unmodified version of the same polypeptide. Qualification of this substantial protection may vary by the assay used to compare the modified versus unmodified polypeptide. In order to exhibit substantial protection, however, the modified polypeptide will exhibit a detectable level of resistance to dipeptidyl peptidase cleavage in the assay.
• assays include but are not limited to those disclosed 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).
• DPP stabilized polypeptide substrates of the present invention are also more stable in the presence of DPP in vivo than a non-stabilized polypeptide substrates.
• a DPP stabilized therapeutic polypeptide substrate generally has an increased activity half-life as compared to a non-stabilized peptide of identical sequence.
• Peptidase stability may be determined by comparing the half-life of the unmodified polypeptide substrate in serum or blood to the half-life of a modified counterpart therapeutic peptide in serum or blood.
• Half- life may be determined by sampling the serum or blood after administration of the modified and non-modified peptides and determining the activity of the peptide.
• the length of the polypeptide substrates may 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 DPP cleavage and having internal and/or C-terminus amino acid alterations that do not affect the functional activity or potency of the polypeptide.
• modified polypeptides would have minor amino acid changes that are usually conservative amino acid substitutions, although non- conservative substitutions are also contemplated.
• modified polypeptides or peptides of the present invention may also have altered functional activity.
• a modified polypeptide or peptide with increased functional activity may be useful.
• a modified polypeptide or peptide with decreased functional activity may be used.
• the modified polypeptides or peptides of the present invention also contain amino acid changes that do affect functional activity or potency.
• the analogs of GLP-1 with altered functional activity may be modified at its amino terminus to protect against DPP cleavage.
• conservative amino acid substitutions are substitutions made within the same group such as 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, threonine, methionine).
• 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
• small amino acids such as gly
• Non-conservative substitutions encompass substitutions of amino acids in one group by amino acids in another group.
• a non-conservative substitution would include the substitution of a polar amino acid for a hydrophobic amino acid.
• nucleotide substitution see e.g. 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 and peptides, such as naturally occurring mature forms of the polypeptides or peptides, allelic/sequence variants of the polypeptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the polypeptides or peptides.
• Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry.
• Such variants can readily be identified/made using molecular techniques and the sequence information. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the modified polypeptides or peptides of the present invention.
• the modified peptides of the present invention are GLP-1 and analogs thereof comprising one or more additional amino acids at their N-terminus.
• the DPP such as DPPIV may activate a peptide instead of inactivating it through cleavage.
• modification of the peptide could substantially reduce, delay, or prevent peptide activation.
• nucleic acid molecules encoding modified polypeptides and peptides that are partially or substantially protected from DPP cleavage and have functional activity and potency.
• nucleic acid molecules provided by the present invention encode modified polypeptides and peptides having at least one additional amino acid at its N-terminus as compared to their wild-type unmodified polypeptide.
• nucleic acid molecules encode modified polypeptides and peptides having five additional amino acids at their N-terminus.
• nucleic acid molecules encode modified polypeptides and peptides having between one and five additional amino acids at their N-terminus.
• the nucleic acid molecules encoding modified GLP-1 comprise sequence encoding one or more additional amino acids at its N-terminus.
• the nucleic acid molecules of the invention include deoxyribonucleic acids (DNAs), both single- and double-stranded deoxyribonucleic acids. However, they can also be ribonucleic acids (RNAs), as well as hybrid RNA:DNA double-stranded molecules. Contemplated nucleic acid molecules also include genomic DNA, cDNA, mRNA, and antisense molecules.
• the nucleic acids molecules of the present invention also include native or synthetic RNA, DNA, or cDNA that encode a modified polypeptide, or the complementary strand thereof.
• nucleic acid encoding the wild-type unmodified 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 that encode a polypeptide and to confirm the function of the polypeptides encoded by these modified sequences.
• the present invention also provides nucleic acids encoding polypeptides and peptides having a modified amino terminus for protection against DPP cleavage and having internal and C-terminus amino acid alterations that do not substantially affect the functional activity or potency of the polypeptide.
• modified polypeptides would have minor amino acid changes that are usually conservative amino acid substitutions, although non- conservative substitutions are also contemplated.
• Nucleotide substitutions using techniques for accomplishing site-specific mutagenesis are well-known in the art.
• the nucleic acids encode GLP-1 analogs having one or more additional amino acids at their N- terminus.
• similarity between two polynucleotides or polypeptides is determined by comparing the nucleotide or amino acid sequence and the conserved nucleotide or amino acid substitutes of one polynucleotide or polypeptide to the sequence of a second polynucleotide or polypeptide.
• identity also known in the art is “identity” which means the degree of sequence relatedness between two polypeptide or two polynucleotide sequences as determined by the identity of the match between two strings of such sequences. Both identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, A.
• identity and similarity are well known to skilled artisans (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). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to those disclosed 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).
• Preferred methods to determine identity are designed to give the largest match between the two sequences tested. Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, 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 a derivation of the first sequence from the second.
• the degree of identity between two nucleic acid sequences may 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).
• GAP is used with the following settings: GAP creation penalty of 5.0 and GAP extension penalty of 0.3.
• the degeneracy of the genetic code permits variations of the nucleotide sequence of polypeptides, while still producing a modified polypeptide comprising an identical amino acid sequence as the polypeptide encoded by a first DNA sequence.
• the procedure known as "codon optimization" (described in U.S. Patent 5,547,871 which is incorporated herein by reference in its entirety) provides one with a means of designing such an altered DNA sequence.
• the design of codon optimized genes should take into account a variety of factors, including the frequency of codon usage in an organism, nearest neighbor frequencies, RNA stability, the potential for secondary structure formation, the route of synthesis and the intended future DNA manipulations of that gene. In particular, available methods may be used to alter the codons encoding a given fusion protein with those most readily recognized by yeast when yeast expression systems are used.
• the degeneracy of the genetic code permits the same amino acid sequence to be encoded and translated in many different ways.
• leucine, serine and arginine are each encoded by six different codons
• valine, proline, threonine, alanine and glycine are each encoded by four different codons.
• the frequency of use of such synonymous codons varies from genome to genome among eukaryotes and prokaryotes.
• synonymous codon-choice patterns among mammals are very similar, while evolutionarily distant organisms such as yeast (S. cerevisiae), bacteria (such as E. coli) and insects (such as D.
• the preferred codon usage frequencies for a synthetic gene should reflect the codon usages of nuclear genes derived from the exact (or as closely related as possible) genome of the cell/organism that is intended to be used for recombinant protein expression, particularly that of yeast species.
• the modified polypeptide is codon optimized, before or after modification as herein described for yeast expression.
• Expression units for use in the present invention will generally comprise the following elements, operably linked in a 5' to 3' orientation: a transcriptional promoter, a secretory signal sequence, a DNA sequence encoding a modified polypeptide and a transcriptional terminator.
• a transcriptional promoter operably linked in a 5' to 3' orientation
• secretory signal sequence a DNA sequence encoding a modified polypeptide
• transcriptional terminator any arrangement of the modified polypeptide and peptide may be used in the vectors of the invention.
• suitable promoters, signal sequences and terminators will be determined by the selected host cell and will be evident to one skilled in the art and are discussed more specifically below.
• Suitable yeast vectors for use in the present invention are described in U.S. Patent 6,291,212 and include YR ⁇ 7 (Struhl et al, Proc. Natl. 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), pPPC0005, 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.
• Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, 7RPI, LEU2 and URA3.
• Plasmids pRS413-41.6 are Yeast Centromere plasmids (Ycps).
• Such vectors will generally include a selectable marker, which may be one of any number of genes that exhibit a dominant phenotype for which a phenotypic assay exists to enable transformants to be selected.
• selectable markers are those that complement host cell auxotrophy, provide antibiotic resistance or enable a cell to utilize specific carbon sources, and include LEU2 (Broach et al ibid.), URA3 (Botstein et al, Gene 8: 17, 1979), HIS3(Struhl et al, ibid.) or POT1 (Kawasaki and Bell, EP 171,142).
• Other suitable selectable markers include the CAT gene, which confers chloramphenicol resistance on yeast cells.
• promoters for use in yeast include promoters from 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. Pat. No. 4,599,311) or alcohol dehydrogenase genes (Young et al, in Genetic Engineering of Microorganisms for Chemicals, HoUaender et al, (eds.), p. 355, Plenum, N.Y., 1982; Ammerer, Meth. Enzymol. 101: 192-201, 1983).
• promoters are the TPI1 promoter (Kawasaki, U.S. Pat. No. 4,599,311) and the ADH2-4 C (see U.S. Patent 6,291,212) promoter (Russell et al, Nature 304: 652-654, 1983).
• the expression units may also include a transcriptional terminator.
• a preferred transcriptional terminator is the TPI1 terminator (Alber and Kawasaki, ibid.). More preferably, the promoter is the PRB1 promoter disclosed in EP 431880 and the terminator is the ADH1 terminator disclosed in EP 60057, which are herein incorporated by reference in their entirety.
• modified polypeptides and peptides of the present invention can be expressed in filamentous fungi, for example, species of the genus Aspergillus.
• useful promoters include those derived from Aspergillus nidulans glycolytic genes, such as the ADH3 promoter (McKnight et al, EMBO J. 4: 2093-2099, 1985) and the tpiA promoter.
• An example of a suitable terminator is the ADH3 terminator (McKnight et al, ibid.).
• the expression units utilizing such components may be cloned into vectors that are capable of insertion into the chromosomal DNA of Aspergillus, for example.
• Mammalian expression vectors for use 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 from adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2: 1304-13199, 1982) and the SV40 promoter (Subramani et al, Mol. Cell. Biol. 1: 854-864, 1981).
• Preferred cellular promoters include the mouse metallothionein-1 promoter (Palmiter et al, Science 222: 809-814, 1983) and a mouse VK (see U.S.
• Patent 6,291,212 promoter (Grant et al, Nuc. Acids Res. 15: 5496, 1987).
• a particularly preferred promoter is a mouse V H (see U.S. Patent 6,291,212) promoter.
• Such expression vectors may also contain a set of RNA splice sites located downstream from the promoter and upstream from the DNA sequence encoding the modified polypeptide or peptide. Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
• polyadenylation signal located downstream of the coding sequence of interest.
• Polyadenylation signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 E1B region and the human growth hormone gene terminator (DeNoto et al, Nuc. Acids Res. 9: 3719-3730, 1981).
• a particularly preferred polyadenylation signal is the V H (see U.S. Patent 6,291,212) gene terminator.
• the expression vectors may include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites.
• Preferred vectors may also include enliancer sequences, such as the SV40 enhancer and the mouse ⁇ (see U.S. Patent 6,291,212) enhancer (Gillies, Cell 33: 717-728, 1983).
• Expression vectors may also include sequences encoding the adenovirus VA RNAs.
• the expression vectors are also used for expressing fusion proteins comprising the modified polypeptide or peptide of the present invention fused to a second polypeptide or peptide, for example transferrin, to enhance the half-life of the modified polypeptide or peptide, as described below.
• the modified polypeptide or peptide may be fused to a tag and/or a cleavage site for expression and release of the modified polypeptide or peptide.
• Cloned DNA sequences comprising modified polypeptides and peptides of the invention may 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 may also be used.
• a selectable marker is generally introduced into the cells along with the gene or cDNA of interest.
• Preferred selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate.
• the selectable marker may be an amplifiable selectable marker.
• a preferred amphfiable selectable marker is the DHFR gene.
• a particularly preferred amplifiable marker is the DHFR r (see U.S. Patent 6,291,212) cDNA (Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80: 2495-2499, 1983).
• Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass.) and the choice of selectable markers is well within the level of ordinary skill in the art.
• the present invention also includes a cell, preferably a yeast cell transformed to express a modified polypeptides or peptides of the invention.
• the present invention also includes a culture of those cells, preferably a monoclonal (clonally homogeneous) culture, 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 away.
• Host cells for use in practicing the present invention include eukaryotic cells, and in some cases prokaryotic cells, capable of being transformed or transfected with exogenous DNA and grown in culture, such as cultured mammalian, insect, fungal, plant and bacterial cells.
• a vector comprising a nucleic acid sequence of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector. Integration is generally considered to be an advantage as the nucleic acid sequence is more likely to be stably maintained in the cell. Integration of the vector into the host chromosome may occur by homologous or non- homologous recombination.
• the choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
• the host cell may be a unicellular microorganism, e.g., a prokaryote, or a non-unicellular microorganism, e.g., a eukaryote. Either prokaryotes or eukaryotes can be used.
• prokaryotic host cells generally used cells such as Escherichia coli or Bacillus subtilis can be used.
• a vector replicable in the host cells may be used.
• An expression plasmid can be preferably used in which a promoter, an SD sequence (Shine-Dalgarno sequence), and an initiation codon (e.g. ATG) required for starting protein synthesis are provided in the vector upstream of the gene of the present invention to facilitate expression of the gene.
• Examples of the above vector include generally-used plasmids derived from E. coli such as pBR322, pBR325, pUC12, pUC13 and the like.
• applicable vectors are not limited to these examples and various known vectors can also be used. Examples of commercially available vectors usable in expression system using E.
• coli include pGEX-4T (Amersham Pharmacia Biotech), pMAL- C2, pMAl-P2 (New England Biolabs), ⁇ ET21/lacq (Invifrogen), pBAD/His (Invifrogen) and the like.
• Examples of eukaryotic host cells include yeast cells and the like.
• Examples of preferably used craniate cells include COS cell (cell from monkey) (1981 Cell, 23, 175), Chinese Hamster Ovary cells and the dihydrofolate reductase defective strain derived therefrom (1980 Proc. Natl. Acad. Sci., USA., 77, 4216) and the like, and examples of preferably used yeast cells include Saccharomyces cerevisiae or the like.
• a yeast cell is used to express the modified polypeptide or peptide.
• Fungal cells including species of yeast (e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp.) may be used as host cells within the present invention.
• yeasts e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp.
• yeasts contemplated to be useful in the practice, of the present invention as hosts for expressing the modified polypeptide or peptides of the inventions are Pichia (including species formerly classified as Hansenula), Saccharomyces, Kluyveromyces, Aspergillus, Candida, Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces, Pachysolen, Zygosaccharomyces, Debaromyces, Trichoderma, Cephalosporium, Humicola, Mucor, Neurospora, Yarrowia, Metschnikowia, Rhodo
• Saccharomyces spp. are S. cerevisiae, S. italicus and S. rouxii.
• Kluyveromyces spp. are K. fragilis, K. lactis andK. marxianus.
• a suitable Torulaspora species is T. delbrueckii.
• Examples of Pichia spp. are P. angusta (formerly H. polymorpha), P. anomala (formerly H. anomala) and P. pastoris.
• Particularly useful host cells to produce the modfied polypeptide or peptide of the invention are the methanoltrophic Pichia pastoris (Steinlein et al. (1995) Protein Express. Purif. 6:619-624). Pichia pastoris has been developed to be an outstanding host for the production of foreign proteins since its alcohol oxidase promoter was isolated and cloned; its transformation was first reported in 1985. P. pastoris can utilize methanol as a carbon source in the absence of glucose. The P.
• pastoris expression system can use the methanol- induced alcohol oxidase (AOX1) promoter, which controls the gene that codes for the expression of alcohol oxidase, the enzyme which catalyzes the first step in the metabolism of methanol.
• AOX1 methanol- induced alcohol oxidase
• This promoter has been characterized and incorporated into a series of P. pastoris expression vectors. Since the proteins produced in P. pastoris are typically folded correctly and secreted into the medium, the fermentation of genetically engineered P. pastoris provides an excellent alternative to E. coli expression systems.
• yeast Saccharomyces cerevisiae are another preferred host.
• a yeast cell or more specifically, a Saccharomyces cerevisiae host cell that contains a genetic deficiency in a gene required for asparagine-linked glycosylation of glycoproteins is used.
• S. cerevisiae host cells having such defects may be prepared using standard techniques of mutation and selection, although many available yeast strains have been modified to prevent or reduce glycosylation or hypermannosylation.
• the host strain carry a mutation, such as the S. cerevisiae pep4 mutation (Jones, Genetics 85: 23-33, 1977), which results in reduced proteolytic activity. It is particularly advantageous to use a host that carries a mutation in the gene encoding the aspartyl protease yapsin 1(YAP3) or the gene encoding yapsin 2(MKC7), or both (Copley et al. 1998 Biochem. J. 330, 1333-1340), such that the proteolytic activity directed to basic residues is reduced or eliminated. Host strains containing mutations in other protease encoding regions are particularly useful to produce large quantities of the modified therapeutic polypeptides or peptides of the invention.
• Host cells containing DNA constructs of the present invention are grown in an appropriate growth medium.
• appropriate growth medium means a medium containing nutrients required for the growth of cells.
• Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
• the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct.
• Yeast cells for example, are preferably grown in a chemically defined medium, comprising a non-amino acid nitrogen source, inorganic salts, vitamins and essential amino acid supplements.
• the pH of the medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 5.5 to 6.5.
• Methods for maintaining a stable pH include buffering and constant pH control, preferably tlirough the addition of ammonia, ammonium hydroxide or sodium hydroxide.
• Preferred buffering agents include citric acid, phosphate, succinic acid and Bis-Tris (Sigma Chemical Co., St. Louis, Mo.).
• Yeast cells having a defect in a gene required for asparagine-linked glycosylation are preferably grown in a medium containing an osmotic stabilizer.
• a preferred osmotic stabilizer is sorbitol supplemented into 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 grown in commercially available serum- containing or serum- free medium. Selection of a medium appropriate 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 sequence(s) of interest. Drug selection is then applied to select for growth of cells that are expressing the selectable marker in a stable fashion. For cells that have been transfected with an amplifiable selectable marker the drug concentration may be increased in a stepwise manner to select for increased copy number of the cloned sequences, thereby increasing expression levels.
• Baculovirus/insect cell expression systems may also be used to produce the modified therapeutic polypeptides or peptides of the invention.
• the BacPAKTM 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 the linearized BacPAK6 viral DNA.
• the BacPAK6 DNA is missing an essential portion of the baculovirus genome.
• the DNA recombines with the vector, the essential element is restored and the target gene is transferred to the baculovirus genome.
• a few viral plaques are picked and purified, and the recombinant phenotype is verified.
• the newly isolated recombinant virus can then be amplified and used to infect insect cell cultures to produce large amounts of the desired protein.
• secretory signal sequence or “signal sequence” or “secretion leader sequence” are used interchangeably and are described, for example in U.S. Pat. 6,291,212 and U.S. Pat 5,547,871, both of which are herein incorporated by reference in their entirety.
• Secretory signal sequences or signal sequences or secretion leader sequences encode 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 core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins.
• Secretory peptides may contain processing sites that allow cleavage of the signal peptide from the mature protein as it passes through the secretory pathway. Processing sites may be encoded within the signal peptide or may be added to the signal peptide by, for example, in vitro mutagenesis.
• Secretory peptides may be used to direct the secretion of modified polypeptides and peptides of the invention.
• One such secretory peptide that may be used in combination with other secretory peptides is the third domain of the yeast Barrier protein.
• Secretory signal sequences or signal sequences or secretion leader sequences are required for a complex series of post-translational processing steps which result in 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 is finally transported out of the cell.
• the signal sequence immediately follows the initiation codon and encodes a signal peptide at the amino-terminal end of the protein to be secreted. In most cases, the signal sequence is cleaved off by a specific protease, called a signal peptidase. Preferred signal sequences improve the processing and export efficiency of recombinant protein expression using viral, mammalian or yeast expression vectors. A preferred signal sequence is a mammalian or human transferrin signal sequence. In some cases, the native substrate signal sequence may be used to express and secrete modified polypeptide or peptides of the invention.
• the pro-peptide sequence is about 2-12 amino acids in length, more preferably about 4-8 amino acids in length.
• pro-peptides examples include 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: 111-114, respectively).
• modified polypeptides of this invention that are partially or substantially resistant to DPP activity, may be prepared by standard synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins.
• the modified polypeptide of the present invention may also be obtained using molecular biology techniques, employing nucleic acid sequences that encode those polypeptides. Those sequences may be RNA or DNA and may be associated with control sequences and/or inserted into vectors. The latter are then transfected into host cells, for example bacteria. The preparation of the vectors and their production or expression in a host is carried out by conventional molecular biology and genetic engineering techniques.
• 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 may be obtained by recombinant means comprising (a) cultivating a host cell under conditions conducive to production of the polypeptide; and (b) recovering the polypeptide.
• the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
• the cell may be cultivated by shake flask cultivation, small-scale or large- scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing 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 procedures known in the art (see, e.g., references for bacteria and yeast; Bennett, J. W. and LaSure, L., editors, More Gene Manipulations in Fungi, Academic Press, California, 1991).
• suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the modified polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the modified polypeptide is not secreted, it can be recovered from cell ly sates.
• modified polypeptides or peptides of the present invention including the modified polypeptide or peptide fusion protein may be made by the fermentation methodology disclosed in WO 0044772, which is herein incorporated by reference in its entirety.
• the modified polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate, binding to a specific receptor, or by detection of activation of a specific receptor in a cell- based assay. For example, an enzyme assay may be used to determine the activity of the modified polypeptide.
• the resulting modified polypeptide may be recovered by methods known in the art. For example, the modified polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
• polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing, differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).
• chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
• electrophoretic procedures e.g., preparative isoelectric focusing, differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. -C. Janson and Lars Ryden, editors, VCH
• the present invention provides modified polypeptides or peptides attached to a heterologous molecule via recombinant means or covalent attachment.
• the attachment to a heterologous molecule extends the activity of the modified polypeptides or peptides for days to weeks. In some instances, only one administration of such modified therapeutic polypeptide or peptide need be given during this period of time. Greater specificity can be achieved, since the active compound will be primarily bound to large molecules, where it is less likely to be taken up intracellularly to interfere with other physiological processes.
• the modified polypeptides or peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins via recombinant means.
• Such chimeric or fusion proteins comprise a modified polypeptide or peptide, partially or substantially protected from DPP cleavage, operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the modified polypeptide or peptide.
• "Operatively linked" indicates that the modified polypeptide or peptide and the heterologous protein are fused in-frame.
• the heterologous protein can be fused to the N-terminus or C-terminus of the modified polypeptide or peptide.
• the fusion protein does not affect the activity of the modified polypeptide of the invention per se.
• the fusion protein can include, but is not limited to, enzymatic fusion proteins, for example beta-galactosidase fusions, yeast two- hybrid GAL fusions, poly-His fusions, MYC-tagged, Hl-tagged and Ig fusions.
• enzymatic fusion proteins for example beta-galactosidase fusions, yeast two- hybrid GAL fusions, poly-His fusions, MYC-tagged, Hl-tagged and Ig fusions.
• Such fusion proteins, particularly poly-His fusions can facilitate the purification of recombinant modified polypeptide.
• the fusion protein comprises an amino acid sequence between the modified peptide of the invention and the other moiety, said amino acid sequence providing a recognition sequence that enables release of the modified peptide of the invention following chemical or enzymatic cleavage.
• expression and/or secretion of a protein can be increased by using a heterologous signal sequence.
• the modified polypeptide or peptide is fused to a molecule that will extend its serum stability or serum half-life, such as a plasma protein.
• the modified polypeptide or protein is fused to serum albumin, immunoglobulin, or a portion thereof such as the Fc domain.
• the modified polypeptide or peptide is fused to transferrin, lactotrasferrin, melanotransferrin, or hybrids thereof.
• Methods for making such fusion proteins are provided by U.S. Applications 10/231,494 and 10/378,094, and International Application PCT/US03/26818, which are herein incorporated by reference in their entirety.
• the transferrin to be attached to the modified polypeptide or peptide may be modified. It may exhibit reduced glycosylation.
• the modified transferrin polypeptide may be selected from the group consisting of a single transferrin N domain, a single transferrin C domain, a transferrin N and C doman, two transferrin N domains, and two transferrin C domains.
• a chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques.
• the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see Ausubel et al. 1992 Current Protocols in Molecular Biology).
• many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein).
• a modified polypeptide or peptide encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the modified polypeptide or peptide.
• the modified therapeutic polypeptide or peptide is conjugated via a covalent bond to a heterologous molecule via a covalent bond to increase its stability and protection from DPP activity.
• the modified polypeptide or peptide is conjugated to a blood component via a covalent bond formed between the reactive group of the modified peptide and a blood component, with or without a linking group.
• Blood components may be either fixed or mobile.
• fixed blood components are non-mobile blood components and include tissues, membrane receptors, interstitial proteins, fibrin proteins, collagens, platelets, endothelial cells, epithelial cells and their associated membrane and membraneous receptors, somatic body cells, skeletal and smooth muscle cells, neuronal components, osteocytes and osteoclasts and all body tissues especially those associated with the circulatory and lymphatic systems.
• Example of mobile blood components are blood components that do not have a fixed situs for any extended period of time, generally not exceeding 5, more usually one minute. These blood components are not membrane- associated and are present in the blood for extended periods of time and are present in a minimum concentration of at least 0.1 ⁇ g/ml.
• Mobile blood components include serum albumin, transferrin, immunoglobulins such as IgM and IgG, protease inhibitor, antithrombin III and ⁇ 2 -antiplasmin.
• the half-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 polypeptide or peptide may occur in vivo or ex vivo.
• the modified polypeptide or peptide is added to blood, serum or saline solution containing the blood component, e.g. human serum albumin or IgG to permit covalent bond formation between the modified polypeptide or peptide and the blood component.
• the modified polypeptide peptide may be modified with maleimide or a similarly reactive chemical group and reacted with a blood component in saline solution.
• the conjugate may be administered to the patient.
• the modified therapeutic polypeptide or peptide may be administered to the patient directly so that the covalent bond forms between the modified therapeutic polypeptide or peptide and the blood component in vivo.
• the same reaction may be carried out with a recombinant protein, for example, albumin.
• the various sites with which the chemically reactive groups of the non-specific modified therapeutic polypeptide or peptide may react in vivo 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, thyroxin binding protein, ⁇ -2-macroglobulin, and the like.
• cells particularly red blood cells (erythrocytes) and platelets
• proteins such as immunoglobulins, including IgG and IgM, serum albumin, ferritin, steroid binding proteins, transferrin, thyroxin binding protein, ⁇ -2-macroglobulin, and the like.
• the modified polypeptide or peptide may contain or may be chemically modified to contain a reactive group for binding to thiol.
• the modified polypeptide or peptide may be conjugated to polyethylene glycol.
• the modified polypeptide or peptide may be conjugated to a polyethylene glycol modified glycolipid or polyehtylene glycol modified fatty acid.
• the modified polypeptide or peptide may be conjugated to a fatty acid or fattv acid derivative to improve its stabilitv.
• fattv acids include, but are not limited to, lauric, palmitic, oleic, and stearic acids.
• fatty acid derivatives include ethyl esters, propyl esters, cholesteryl esters, coenzyme A esters, nifrophenyl esters, naphthyl esters, monoglycerides, diglycerides, and friglycerides, fatty alcohols, fatty alcohol acetates, and the like.
• the modified polypeptide or peptide may be engineered into into a drug affinity complex (DACTM).
• a drug affinity complex has three parts: a drug component which is responsible for biological activity; a connector attaching the drug component to the reactive chemistry group; and a reactive chemistry group, at the the oppposite end of the connector, which is responsible for the permanent bonding of the construct to certain target proteins in the body.
• Kim et al disclose a GLP-1-albumin drug affinity complex.
• Kim et al. show that the albumin-conjugated DAC: GLP-1 bound to the GLP-1 receptor (GLP-1 R) and activated cAMP formation in heterologous fibroblasts expressing the receptor.
• the results suggest that the albumin-conjugated DAC: GLP-1 mimics the native GLP-1.
• Kim et al. provide a new approach for prolonged activation of GLP-1 R signaling.
• the modified polypeptide or peptide drug affinity complex is designed to be administered by subcutaneous injection and then rapidly and selectively bonds in vivo to albumin.
• the bioconjugate formed has the same therapeutic activity and similar potency as endogenous polypeptide or peptide but has a pharmacokinetic profile in animals that is closer to that of albumin.
• compositions comprising modified therapeutic polypeptides and peptides partially or substantially protected from DPP cleavage, but substantially retaining their functional activity and potency.
• Such pharmaceutical compositions may be be administered orally, parenterally, such as intravascularly (IV), intraarterially (IA), intramuscularly (IM), subcutaneously (SC), intraperitoneally, transdermally, or the like. Administration may in appropriate situations be by transfusion. In some instances, administration may be oral, nasal, rectal, transdermal or aerosol, where the modified polypeptide allows for transfer to the vascular system.
• fusion or conjugation of a modified polypeptide of the invention to a transferrin moiety allows for transport of the modified polypeptide to the vascular system or across the blood-brain barrier via binding to the transferrin receptor, as described in International Application PCT/US03/26778, which is herein incorporated by reference in its entirety.
• a single injection will be employed although more than one injection may be used, if desired.
• the modified therapeutic polypeptides or peptides may be administered by any convenient means, including syringe, trocar, catheter, or the like. The particular manner of administration will vary depending upon the amount to be administered, whether a single bolus or continuous administration, or the like.
• the administration will be infravascularly, where the site of introduction is not critical to this invention, preferably at a site where there is rapid blood flow, e.g., intravenously, peripheral or central vein. More preferably, the pharmaceutical compositions will be administered subcutaneously. Other routes may find use where the administration is coupled with slow release techniques or a protective matrix. The intent is that the modified therapeutic peptides or polypeptides be effectively distributed, for example, in the blood, so as to be able to react with the blood or tissue components.
• the invention encompasses pharmaceutical compositions comprising effective amounts of modified therapeutic polypeptide or peptide of the invention together with pharmaceutically acceptable diluents, preservatives, solubihzers, emulsifiers, adjuvants and/or carriers.
• compositions may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
• Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
• compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference.
• the modified therapeutic polypeptides or peptides may be administered in a physiologically acceptable medium, e.g., deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetable oil, or the like.
• a physiologically acceptable medium e.g., deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol or other alcohol, plasma, proteinaceous solutions, mannitol, aqueous glucose, alcohol, vegetable oil, or the like.
• Other additives which may be included include buffers, where the media are generally buffered at a pH in the range of about 5 to 10, where the buffer will generally range in concentration from about 50 to 250 mM, salt, where the concentration of salt will generally range from about 5 to 500 mM, physiologically acceptable stabilizers, and the like.
• physiological buffers
• compositions may be prepared as tablets or dragees, sublingual tablets, sachets, paquets, soft gelatin capsules, suppositories, creams, ointments, dermal gels, transdermal devices, aerosols, drinkable and injectable ampoules.
• the compositions may also be prepared in liquid form, or may be in dried powder, such as lyophilized form convenient for storage and transport. Implantable sustained release formulations are also contemplated.
• the present invention provides pharmaceutical compositions comprising the modified therapeutic polypeptides or peptides in oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa. 18042, which is herein incorporated by reference.
• Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets.
• liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673).
• Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat.
• the formulation will include the modified therapeutic polypeptide or peptide, and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
• the modified therapeutic polypeptide or peptide may be chemically modified so that oral delivery is efficacious.
• the chemical modification contemplated is the attachment of at least one moiety to the modified therapeutic polypeptide or peptide itself, where said moiety permits uptake into the blood stream from the stomach or intestine.
• the increase in overall stability of the compound and increase in circulation time in the body are also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
• the modified therapeutic polypeptide or peptide may be recombinantly fused to another polypeptide to increase its overall stability or improve oral delivery.
• the modified therapeutic polypeptide or peptide may be fused to transferrin, melanotransferrin, or lactoferrin. Methods for making such fusion proteins are described in U.S. Application 10/378,094, which is herein incorporated by reference in its entirety.
• a salt of a modified aliphatic amino acid such as sodium N-(8-[2-hydroxybenzoyl] amino) caprylate (SNAC)
• SNAC sodium N-(8-[2-hydroxybenzoyl] amino) caprylate
• the modified therapeutic polypeptides or peptides of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm.
• the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
• the therapeutic could be prepared by compression.
• the modified therapeutic polypeptide or peptide may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
• an edible product such as a refrigerated beverage containing colorants and flavoring agents.
• These diluents could include carbohydrates, especially mannitol, cc-lactose, anhydrous lactose, cellulose, sucrose, modified dexfrans and starch.
• Certain inorganic salts may 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 formulation of the therapeutic into a solid dosage form.
• Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
• Another form of the disintegrants are the insoluble cationic exchange resins.
• Powdered gums may 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 may be used to hold the modified therapeutic polypeptide or peptide together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.
• MC methyl cellulose
• EC ethyl cellulose
• CMC carboxymethyl cellulose
• PVP polyvinyl pyrrolidone
• HPMC hydroxypropylmethyl cellulose
• An antifrictional agent may be included in the formulation of the pharmaceutical composition of the invention to prevent sticking during the formulation process.
• Lubricants may be used as a layer between the modified therapeutic polypeptide or peptide and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
• Glidants that might improve the / flow properties of the modified therapeutic polypeptide or peptide during formulation and to aid rearrangement during compression might be added.
• the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
• surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
• Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride.
• the list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could 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 enhance uptake of the modified therapeutic polypeptide and peptide. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
• Controlled release formulation also may be desirable.
• the modified therapeutic polypeptide or peptide of this invention could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms e.g., gums.
• Slowly degenerating matrices may also be incorporated into the formulation, e.g., alginates, polysaccharides.
• Another form of a controlled release of the compounds of this invention is by a method based on the Oros therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.
• Other coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan.
• the modified therapeutic polypeptide or peptide could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups.
• the first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
• the second group consists of the enteric materials that are commonly esters of phthalic acid.
• a mix of materials might be used to provide the optimum film coating.
• Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.
• 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 traverses across the lung epithelial lining to the blood stream.
• the present invention provides the use of a wide range of mechanical devices designed for 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 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, Colo.; 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.
• each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.
• the modified therapeutic polypeptide or peptide should most advantageously be prepared in particulate form with an average particle size of less than 10 ⁇ m, most preferably 0.5 to 5 ⁇ m, for most 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 may be used.
• PEG may be used (even apart from its use in derivatizing the protein or analog).
• Dexfrans such as cyclodexfran, may be used.
• Bile salts and other related enhancers may be used.
• Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation.
• Formulations suitable for use with a nebulizer will typically comprise the inventive compound 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 buffer and a simple sugar (e.g., 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 in 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 employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tefrafluoroethane, or combinations thereof.
• Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.
• Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., about 50 to 90% by weight of the formulation.
• a bulking agent such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., about 50 to 90% by weight of the formulation.
• Nasal delivery of the pharmaceutical composition of the modified polypeptide or peptide of the present invention is also contemplated.
• Nasal delivery allows the passage of the protein to the blood stream directly after administering the modified therapeutic polypeptide or peptide to the nose, without the necessity for deposition of the product in the lung.
• Formulations for nasal delivery include those with dexfran or cyclodexfran. Delivery via transport across other mucous membranes is also disclosed.
• the dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors.
• the daily regimen should be in the range of 0.01-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.
• the present invention provides various modified therapeutic polypeptides or peptides that could be used in the treatment of a variety of diseases.
• the pharmaceutical compositions comprising the modified therapeutic polypeptides or peptides of the present invention could 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, hypertriglyceridemia atherosclerosis, nervous system disorders.
• the modified polypeptides and peptides could also be used to induce an anxiolytic effect on the CNS, to activate the CNS or for post surgery treatment.
• modified therapeutic polypeptides and peptides of the present invention are more stable in vivo than the nonmodif ⁇ ed therapeutic polypeptides and peptides because they are partially or substantially protected from DPP activity. Accordingly, smaller amounts of the molecule may be administered for effective treatment. A lower dosage amount may in some instances alleviate side effects.
• the modified therapeutic polypeptides and peptides of the present invention may be used as a sedative. Accordingly, the present invention provides a method of sedating a mammalian subject with an abnormality resulting in increased activation of the central or peripheral nervous system using the modified polypeptides or peptides of the invention. The method comprises administering a modified therapeutic polypeptides or peptides to the subject in an amount sufficient to produce a sedative or anxiolytic effect on the subject.
• the modified therapeutic polypeptides or peptides may be administered intracerebroventriculary, orally, subcutaneously, intramuscularly, or intravenously. Such methods are useful to treat or ameliorate nervous system conditions such as anxiety, movement disorder, aggression, psychosis, seizures, panic attacks, hysteria and sleep disorders.
• the present invention encompasses a method of increasing the activity of a mammalian subject, comprising administering a modified therapeutic polypeptides or peptides to the subject in an amount sufficient to produce an activating effect on the subject.
• the subject has a condition resulting in decreased activation of the central or peripheral nervous system.
• the modified therapeutic polypeptides or peptides are useful in the treatment or amelioration of depression, schizoaffective disorders, sleep apnea, attention deficit syndromes with poor concentration, memory loss, forgetfulness, and narcolepsy, to name just a few conditions in which arousal of the central nervous system may be advantageous.
• the post-operative 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.
• the modified therapeutic polypeptides or peptides of the invention may be utilized for post surgery treatments.
• a patient is in need of the modified insulinotropic peptides of the present invention for about 1-16 hours before surgery is performed on the patient, during surgery on the patient, and after the patient's surgery for a period of not more than about 5 days.
• the modified therapeutic polypeptides and peptides, such as the insulinotropic peptides, of the invention may be utilized to treat insulin resistance independently from their use in post surgery treatment.
• Insulin resistance may be due to a decrease in binding of insulin to cell-surface receptors, or to alterations in intracellular metabolism.
• the first type characterized as a decrease in insulin sensitivity
• the second type characterized as a decrease in insulin responsiveness, cannot be overcome by large quantities of insulin. Insulin resistance following trauma can be overcome by doses of insulin that are proportional to the degree of insulin resistance, and thus is apparently caused by a decrease in insulin sensitivity.
• the present invention provides modified insulinotropic peptides to normalize hyperglycemia through glucose-dependent, insulin-dependent and insulin- independent mechanisms.
• the modified insulinofropic peptides are useful as primary agents for the treatment of diabetes, especially type II diabetes mellitus.
• the present invention is especially suited for the treatment of patients with diabetes, both type I and type II, in that the action of the peptide is dependent on the glucose concentration of the blood, and thus the risk of hypoglycemic side effects are greatly reduced over the risks in using current methods of treatment
• modified insulinotropic peptides effective to normalize a patient's blood glucose level will depend on a number of factors, among which are included, without limitation, the patient's sex, weight and age, the severity of inability to regulate blood glucose, the underlying causes of inability to regulate blood glucose, whether glucose, or another carbohydrate source, is simultaneously administered, the route of administration and bioavailability, the persistence in the body, the formulation, and the potency.
• the modified therapeutic peptides such as the insulinotropic peptides, of the present invention are used for the treatment of impaired glucose tolerance, glycosuria, hyperlipidaemia, metabolic acidoses, 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.
• the modified therapeutic polypeptides and peptides may be monitored using assays for determining functional activity, HPLC-MS, or antibodies directed against the polypeptide or peptide.
• the blood of the mammalian host may be monitored for the activity of the modified therapeutic polypeptide or peptide and/or presence of the modified therapeutic polypeptide or peptide.
• the modified therapeutic polypeptide or peptide has become bound to the long-lived blood components in sufficient amount to be therapeutically active and, thereafter, the level of modified therapeutic polypeptide or peptide in the blood. If desired, one may also determine to which of the blood components the modified therapeutic polypeptide or peptide, such as a modified insulinotropic peptide, is bound.
• assays for insulinotropic activity may be used to monitor the modified insulinotropic peptides of the present invention.
• the modified insulinofropic peptides of the present invention have an insulinotropic activity that at least equals the insulinotropic activity of the non-modified insulinotropic peptides.
• the insulinotropic property of a modified insulinotropic peptide may be determined by providing that modified peptide to animal cells, or injecting that peptide into animals and monitoring the release of immunoreactive insulin into the media or circulatory system of the animal, respectively. The presence of immunoreactive insulin is detected through the use of a radioimmunoassay which can specifically detect insulin.
• radioimmunoassay capable of detecting the presence of IRI
• the insulinotropic property of a modified therapeutic polypeptide or peptide may also be determined by pancreatic infusion (Penhos, J. C, et al. 1969 Diabetes 18:733-738, which is hereby incorporated by reference).
• pancreatic infusion Penhos, J. C, et al. 1969 Diabetes 18:733-738, which is hereby incorporated by reference.
• the manner in which perfusion is performed, modified, and analyzed preferably follows the methods of Weir, G. C, et al, (J. Clin. Investigat. 54:1403-1412 (1974)), which is hereby incorporated by reference.
• HPLC coupled with mass spectrometry can be utilized to assay for the presence of modified therapeutic polypeptide and peptides as is well known to the skilled artisan.
• MS mass spectrometry
• two mobile phases such as 0.1% TF A/water and 0.1% TFA/acetonitrile.
• Column temperatures can be varied as well as gradient conditions.
• Another method to monitor the presence of modified therapeutic polypeptides and peptides is to use antibodies specific to the modified therapeutic polypeptides and peptides.
• the use of antibodies, either monoclonal or polyclonal, having specificity for particular modified therapeutic polypeptides or peptides, can assist in mediating any such problem.
• the antibody may be generated or derived from a host immunized with the particular modified therapeutic polypeptide or peptide, or with an immunogenic fragment of the agent, or a synthesized immunogen corresponding to an antigenic determinant of the agent.
• Preferred antibodies will have high specificity and affinity for the modified therapeutic polypeptide or peptide.
• Such antibodies can also be labeled with enzymes, fluorochromes, or radiolabels.
• the antibodies may be used to monitor the presence of modified therapeutic polypeptides and peptides in the blood stream.
• Blood and/or serum samples may be analyzed by SDS-PAGE and western blotting. Such techniques permit the analysis of the blood or serum to determine the bonding of the modified therapeutic polypeptides or peptides to blood components.
• the modified therapeutic peptides of the present invention are modified insulinotropic peptides partially or substantially protected from DPP activity. More preferably, the modified insulinotropic peptides are modified GLP-1 peptides and analogs and fragments thereof.
• the modified GLP-1 peptides and analogs and fragments thereof are useful for treating diabetes, specifically type II diabetes.
• the N-terminal sequence of wild- type GLP-1 is His-Ala-Glu; preferred modified GLP-1 polypeptides of the invention comprise an N-terminal sequence selected from the group consisting of: His-His-Ala-Glu (SEQ ID NO: 115), Gly-His-Ala-Glu (SEQ ID NO: 116), His-Gly-Glu, His-Ser-Glu, His- Ala-Glu, His-Gly-Glu, His-Ser-Glu, His-His-Ala-Glu (SEQ ID NO: 82), His-His-Gly-Glu (SEQ ID NO: 83), His-His-Ser-Glu (SEQ ID NO: 84), Gly-His-Ala-Glu (SEQ ID NO: 85), Gly-His-Gly-Glu (SEQ ID NO: 86), Gly-His-Ser-Glu (SEQ ID NO: 87), His-X-Ala-Glu, His-X-Gly-Glu, and His-X-S
• the C-terminus of GLP-1 is normally amidated. In yeast, amidation does not occur.
• an extra amino acid is added on the N-terminus of GLP-1.
• the addition of an amino acid to the N-terminus of GLP-1 may prevent dipeptidyl peptidase from cleaving at the second amino acid of GLP-1 due to steric hindrance. Therefore, GLP-1 will remain functionally active. Any one of the 20 amino acids or a non- natural amino acid may be added to the N-terminus of GLP-1. Histidine is also a preferred amino acid.
• an uncharged or positively charged amino acid may be used and preferably, a smaller amino acid such as Glycine is added.
• the modified GLP-1 with the extra amino acid can then be fused to transferrin to make a fusion protein.
• the GLP-1 peptide is modified to contain at least one additional amino acid at its amino terminus.
• the GLP-1 peptide is modified to contain at least five additional amino acids at its amino terminus.
• the GLP-1 peptide is modified to contain between one and five additional amino acids at its amino terminus.
• Glucagon-Like Peptide-1 is a gastrointestinal hormone that regulates insulin secretion belonging to the so-called enteroinsular axis.
• the enteroinsular axis designates a group of hormones, released from the gastrointestinal mucosa in response to the presence and absorption of nutrients in the gut, which promote an early and potentiated release of insulin.
• the incretin effect which is the enhancing effect on insulin secretion is probably essential for a normal glucose tolerance.
• GLP-1 is a physiologically important insulinotropic hormone because it is responsible for the incretin effect.
• GLP-1 is a product of proglucagon (Bell, et al, Nature, 1983, 304: 368-371). It is synthesized in intestinal endocrine cells in two principal major molecular forms, as GLP- l(7-36)amide and GLP-1 (7-37). The peptide was first identified following the cloning of cDNAs and genes for proglucagon in the early 1980s.
• GLP-1 The amino acid sequence of GLP-1 is disclosed by Schmidt et al. (1985 Diabetologia 28 704-707).
• Human GLP-1 is a 37 amino acid residue peptide originating from preproglucagon which is synthesized in the L-cells in the distal ileum, in the pancreas, and in the brain. Processing of preproglucagon to GLP-1 (7-36)amide, GLP-l(7-37) and GLP-2 occurs mainly in the L-cells.
• GLP-1 like molecules possesses anti-diabetic activity in human subjects suffering from Type II (non-insulin-dependent diabetes mellitus (NIDDM)) and, in some cases, even Type I diabetes.
• Treatment with GLP-1 elicits activity, such as increased insulin secretion and biosynthesis, reduced glucagon secretion, delayed gastric emptying, only at elevated glucose levels, and thus provides a potentially much safer therapy than insulin or sulfonylureas.
• Post-prandial and glucose levels in patients can be moved toward normal levels with proper GLP-1 therapy.
• GLP-1 -like molecules possess the ability to preserve and even restore pancreatic beta cell function in Type-II patients.
• GLP-1 sequence may be modified by adding one or more amino acids at its amino terminus, including GLP-l(7-34), GLP-l(7-35), GLP-l(7-36), and GLP-l(7-37).
• GLP-1 also has powerful actions on the gastrointestinal tract. Infused in physiological amounts, GLP-1 potently inhibits pentagastrin-induced as well as meal-induced gastric acid secretion (Schjoldager et al, Dig. Dis. Sci. 1989, 35:703-708; Wettergren et al, Dig Dis Sci 1993; 38:665-673). It also inhibits gastric emptying rate and pancreatic enzyme secretion (Wettergren et al, Dig Dis Sci 1993; 38:665-673).
• GLP-1 seems to have an effect on food intake. Intraventricular administration of GLP-1 profoundly inhibits food 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-l(l-36 a ⁇ de ) is inactive and appropriate doses of the GLP-1 antagonist, exendin 9-39, abolish the effects of GLP-1 (Tang-Christensen et al, Am. J.
• GLP-1 secreted from the intestinal L-cells may also act as a satiety signal.
• GLP-1 In diabetic patients, GLP-1 's insulinotropic effects and the effects of GLP-1 on the gastrointestinal tract are preserved (Willms et al, Diabetologia 1994; 37, suppl.1: Al 18), which may help curtail meal-induced glucose excursions, but, more importantly, may also influence food intake. Administered intravenously, continuously for one week, GLP-1 at 4 ng/kg/min has been demonstrated to dramatically improve glycaemic control in NIDDM patients without significant side effects (Larsen et al, Diabetes 1996; 45, suppl. 2: 233A.). [00186] 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.
• GLP-1 molecule means GLP-1, a GLP-1 analog, or GLP-1 derivative.
• GLP-1 analog is defined as a molecule having one or more amino acid substitutions, deletions, inversions, or additions compared with GLP-1.
• Many GLP-1 analogs are known in the art and include, for example, GLP-1 (7-34), GLP- 1(7-35), GLP-l(7-36), Val 8 -GLP-l(7-37), Gln 9 -GLPl(7-37), D-Gln 9 -GLP-l(7-37), Thr 16 - Lys 18 -GLP- 1(7-37), and Lys 18 -GLP-l(7-37) (SEQ ID NO: 72).
• U.S. Patent 5,118,666 discloses examples of GLP-1 analogs such as GLP-l(7-34) and GLP-l(7-35).
• GLP-1 derivative is defined as a molecule having the amino acid sequence of GLP-1 or a GLP-1 analog, but additionally having chemical modification of one or more of its amino acid side groups, ⁇ -carbon atoms, terminal amino group, or terminal carboxylic acid group.
• a chemical modification includes, but is not limited to, adding chemical moieties, creating new bonds, and removing chemical moieties.
• GLP-1 related compound refers to any compound falling within the GLP-1, GLP-1 analog, or GLP-1 derivative definition.
• WO 91/11457 discloses analogs of the active GLP-1 peptides 7-34, 7-35, 7-36, and 7-37 which can also be useful as GLP-1 moieties.
• EP 0708179- A2 (Eli Lilly & Co.) discloses GLP-1 analogs and derivatives that include an N-terminal imidazole group and optionally an unbranched C 6 -C 10 acyl group in attached to the lysine residue in position 34.
• EP 0699686-A2 (Eli Lilly & Co.) discloses certain N-terminal truncated fragments of GLP-1 that are reported to be biologically active.
• U.S. Patent 5,545,618 discloses GLP-1 molecules consisting essentially of GLP- 1(7-34), GLPl(7-35), GLP-l(7-36), or GLP-1 (7-37), or the amide forms thereof, and pharmaceutically-acceptable salts thereof, having at least one modification selected from the group consisting of: (a) substitution of glycine,serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, arginine, or D-lysine for lysine at position 26 and/or position 34; or substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, lysine, or a
• U.S. Pat. No. 5,118,666 discloses a GLP-1 molecule having insulinotropic activity.
• Such 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-Leu-Val-Lys (GLP-1, 7-34, see SEQ ID NO: 32) or His-Ala- Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe- Ile-Ala-Trp-Leu-Val-Lys-Gly (GLP-1, 7-35, see SEQ ID NO: 32); and
• U.S. Patent 6,277,819 teaches a method of reducing mortality and morbidity after myocardial infarction comprising administering GLP-1, GLP-1 analogs, and GLP-1 derivatives to the patient.
• the GLP-1 analog being represented by the following structural formula (SEQ ID NO: **): R X Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Xz- Gly-Gln-Ala-Ala-Lys- X 3 -Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R 2 (SEQ ID NO: 78) and pharmaceutically-acceptable salts thereof, wherein: R t is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, .beta.-hydroxy-histidine, homohistidine, al
• U.S. Patent 6,429,197 teaches that GLP-1 treatment after acute stroke or hemorrhage, preferably intravenous administration, can be an ideal treatment because it provides a means for optimizing insulin secretion, increasing brain anabolism, enhancing insulin effectiveness by suppressing glucagon, and maintaining euglycemia or mild hypoglycemia with no risk of severe hypoglycemia or other adverse side effects.
• the present invention provides a method of treating the ischemic or reperfused brain with GLP- 1 or its biologically active analogues after acute stroke or hemorrhage to optimize insulin secretion, to enhance insulin effectiveness by suppressing glucagon antagonism, and to maintain euglycemia or mild hypoglycemia with no risk of severe hypoglycemia.
• U.S. Patent 6,277,819 provides a method of reducing mortality and morbidity after myocardial infarction, comprising administering to a patient in need thereof, a compound selected from the group consisting of GLP-1, GLP-1 analogs, GLP-1 derivatives and pharmaceutically-acceptable salts thereof, at a dose effective to normalize blood glucose.
• U.S. Patent 6,191,102 discloses a method of reducing body weight in a subject in need of body weight reduction by administering to the subject a composition comprising a glucagon-like peptide-1 (GLP-1), a glucagon-like peptide analog (GLP-1 analog), a glucagon-like peptide derivative (GLP-1 derivative) or a pharmaceutically acceptable salt thereof in a dose sufficient to cause reduction in body weight for a period of time effective to produce weight loss, said time being at least 4 weeks.
• GLP-1 glucagon-like peptide-1
• GLP-1 analog glucagon-like peptide analog
• GLP-1 derivative a glucagon-like peptide derivative
• GLP-1 is fully active after subcutaneous adminisfration (Ritzel et al, Diabetologia 1995; 38: 720-725), but is rapidly degraded mainly due to degradation by dipeptidyl peptidase IV-like enzymes (Deacon et al, J Clin Endocrinol Metab 1995, 80: 952-957; Deacon et al, ⁇ 995, Diabetes 44: 1126-1131).
• GLP-1 and many of its analogues have a short plasma half-life in humans (Orskov et al, Diabetes 1993; 42:658- 661).
• modified GLP-1 or analogues thereof which have a protracted profile of action relative to GLP-1 (7-37). It is a further object of the invention to provide derivatives of GLP-1 and analogues thereof which have a lower clearance than GLP-l(7-37). Moreover, it is an object of the invention to provide pharmaceutical compositions comprising modified GLP-1 or GLP-1 analogs with improved stability. Additionally, the present invention includes the use of modified GLP-1 or GLP-1 analogs to treat diseases associated with GLP-1 such as but not limited to those described above.
• the pharmaceutical compositions comprising modified GLP-1 and GLP-1 analogs may be formulated by any of the established methods of formulating pharmaceutical compositions, e.g. as described in Remington's Pharmaceutical Sciences, 1985.
• the composition may be in a form suited for systemic injection or infusion and may, as such, be formulated with a suitable liquid vehicle such as sterile water or an isotonic saline or glucose solution.
• the compositions may be sterilized by conventional sterilization techniques which are well known in the art.
• the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration.
• the composition may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
• the modified GLP-1 and GLP-1 analogs of the present invention may also be adapted for nasal, transdermal, pulmonal or rectal administration.
• the pharmaceutically acceptable carrier or diluent employed in the composition may be any conventional solid carrier. Examples of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
• the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
• composition of the invention in the form of a sustained release formulation.
• the composition may 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/glycolic acid copolymer.
• the preparation may contain modified GLP-1 or GLP-1 analogs dissolved or suspended in a liquid carrier, in particular an aqueous carrier, for aerosol application.
• a liquid carrier in particular an aqueous carrier
• the carrier may contain additives such as solubilizing agents, e.g. propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabenes.
• modified polypeptides or peptides of the present invention are dispensed in unit dosage form together with a pharmaceutically acceptable carrier per unit dosage.
• the present invention contemplates the use of the modified GLP-1 and GLP-1 analogs for the manufacture of a medicinal product which can be used in the treatment of diseases associated with elevated glucose level (metabolic disease), such as but not to limited to those described above.
• 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 modified Exendin-3 and Exendin-4 peptides partially and substantially protected from DPP activity.
• Exendin-3 and Exendin-4 are insulinotropic peptides comprising 39 amino acids (differing at residues 2 and 3) which are approximately 53% homologous to GLP-1.
• the Exendin-3 sequence is HSDGTFTSDLSKQMEEEAVRLFIEWLKNGG PSSGAPPPS (SEQ ID NO: 79), and the Exendin-4 sequence 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). Additionally, the present invention includes modified analogs of Exendin-3 and Exendin-4 peptides.
• the modified GLP-1 may be fused to a heterologous molecule for increased overall stability in vivo.
• the modified GLP-1 may be fused to a heterologous molecule by recombinant means or covalently attached to a heterologous molecule by methods well known in the art.
• Modified GLP-1 may be fused or covalently attached, for example to a plasma protein such as serum albumin or fransferrin, an immunoglobulin, or a portion thereof such as the Fc domain. More preferably, the modified polypeptide or peptide is fused to transferrin, lactofransferrin, or melanotransferrin. Methods for making such fusion proteins are provided by U.S. Application 10/378,094, which is herein incorporated by reference in its entirety.
• the GLP-1 molecule may be attached to the heterologous protein via a linker of variable length to provide greater physical separation and allow more spatial mobility between the fused proteins and thus maximize the accessibility of the therapeutic protein, for instance, for binding to its cognate receptor.
• the linker peptide may consist of amino acids that are flexible or more rigid.
• a linker such as a poly-glycine stretch may be used.
• the linker can be 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.
• the linker may be one Ser residue, two Ser residues, or the peptide Ser-Ser-Gly. These linkers may be used to link GLP-1 to transferrin.
• the transferrin to be attached to the modified polypeptide or peptide may be modified. It may exhibit reduced glycosylation.
• the modified transferrin polypeptide may be selected from the group consisting of a single fransferrin N domain, a single transferrin C domain, a transferrin N and C doman, two fransferrin N domains, and two transferrin C domains.
• GLP-1 activates and regulates important endocrine hormone systems in the body and plays a critical management role in the metabolism of glucose. Unlike all other diabetic treatments on the market GLP-1 has the potential to be restorative by acting as a growth factor for ⁇ -cells thus improving the ability of the pancreas to secrete insulin and also, to make the existing insulin levels act more efficiently by improving sensitivity and better stabilizing 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-1 can reduce appetite and reduce weight. Obesity is an inherent consequence of poor control of glucose metabolism and this only serves to aggravate the diabetic condition.
• GLP-1 Clinical application of natural GLP-1 is limited because it is rapidly degraded in the circulation (half-life is several minutes). To maintain therapeutic levels in the circulation requires constant adminisfration of high doses using pumps or patch devices which adds to the cost of treatment. This is inconvenient for long term chronic use especially in conjunction with all the other medications for treating diabetes and monitoring of glucose levels.
• the modified GLP-1 fusion proteins retain the activity of GLP-1 but have the long half-life (14-17 days), solubility, and biodistribution properties of transferrin. These properties could provide for a low cost, small volume, monthly s.c. (subcutaneous) injection and this type of product is absolutely needed for long term chronic use.
• the modified GLP-1 also may be covalently attached to a blood component to increase its stability.
• the modified GLP-1 may be covalently attached to serum albumin, transferrin, immunoglobulin, or the Fc portion of the immunoglobulin.
• the modified GLP-1 may be attached to a fatty acid or a fatty acid derivative.
• the modified GLP-1 may be engineered into a drug affinity complex (DAC).
• DAC drug affinity complex
• the DAC:modified GLP-1 Upon subcutaneous administration, the DAC:modified GLP-1 rapidly and selectively bonds 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 albumin.
• the modified GLP-1 peptide and its fusion protein for example, GLP/mTf 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 (metformin hydrochloride extended-release tablets) to treat type II diabetes, obesity, and other diseases or conditions associated with abnormal glucose levels.
• at least one second therapeutic molecule such as Glucophage® (metformin hydrochloride tablets) or Glucophage® XR (metformin hydrochloride extended-release tablets) to treat type II diabetes, obesity, and other diseases or conditions associated with abnormal glucose levels.
• Glucophage® and Glucophage® XR are oral antihyperglycemic drugs for the mangagement of type II diabetes.
• Glucophage® XR is an extended release formulation of Glucophage. Accordingly, Glucophage® XR may be taken once daily because the drug is released slowly from the dosage form.
• Glucophage® helps the body produce less glucose from the liver. Accordingly, Glucophage® is effective in controlling blood sugar level in a patient. Glucophage® rarely causes low blood glucose (hypoglycemia) because it does not cause the body to make more insulin.
• Glucophage® also helps lower the fatty blood components, friglycerides and cholesterol, that are often high in people with Type II diabetes. Metformin has been shown to decrease the appetite and help people lose a few pounds when they starting 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 treating various cardiovascular diseases such as hypertension in insulin resistant patients (WO 9112003- Upjohn), for dissolving 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 U.S. Pat. No. 4,205,087-ICI).
• metformin in combination with prostaglandin-analogous cyclopentane derivatives as coronary dilators and for blood pressure lowering
• Metformin has also been suggested for use in cholesterol lowering when used in combination with 2-hydroxy-3,3,3-trifluoropropionic acid derivatives (U.S. Pat. No. 4,107,329-ICI), 1,2-diarylethylene derivatives (U.S. Pat. No. 4,061, 772-Hoechst), substituted aryloxy-3,3,3-trifluoro-2-propionic acids, esters and salts (U.S. Pat. No.
• the present invention provides 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.
• the modified GLP-1 or its fusion protein in combination with metformin is used to treat diseases and conditions associated with abnormal blood glucose level, such as diabetes.
• the GLP-1/mTf fusion protein in combination with metformin is used to treat type II diabetes or obesity.
• modified GLP-1 of the present invention and its fusion proteins include but are not limited to sulfonylurea and sulfonylurea-like agents, thiazolidinediones, Peroxisome Proliferator- Activated Receptor (PPAR) gamma modulators, PPAR alpha modulators, Protein Tyrosine Phosphatase-IB inhibitors, Insulin Receptor Tyrosine Kinase activators, 1 lbeta-hydroxysteroid dehydrogenase inhibitors, glycogen phosphorylase inhibitors, glucokinase activators, beta-3 adrenergic agonists, and glucagon receptor agonists.
• PPAR Peroxisome Proliferator- Activated Receptor
• transgenic non-human animals that express a modified polypeptide or peptide that is protected from DPP activity is contemplated in one embodiment of the present invention.
• transgenic non-human animals expressing fusion proteins comprising a modified polypeptide or peptide and having increased stability is contemplated.
• transgenic animals The most widely used method for the production of transgenic animals is the microinjection of DNA into the pronuclei of fertilized embryos (Wall et al, J. Cell. Biochem. 49: 113 [1992]).
• Other methods for the production of transgenic animals include the infection of embryos with refroviruses or with retroviral vectors. Infection of both pre- and post-implantation mouse embryos with either wild-type or recombinant retroviruses has been reported (Janenich, Proc. Natl. Acad. Sci. USA 73:1260 [1976]; Janenich et al, Cell 24:519 [1981]; Sruhlmann et al, Proc. Natl. Acad. Sci.
• An alternative means for infecting embryos with retroviruses is the injection of virus or virus-producing cells into the blastocoele of mouse embryos (Jahner, D. et al, Nature 298:623 [1982]).
• the introduction of transgenes into the germline of mice has been reported using infrauterine retroviral infection of the midgestation mouse embryo (Jahner et al, supra [1982]).
• Infection of bovine and ovine embryos with retroviruses or retroviral vectors to create transgenic animals has been reported.
• PCT International Application WO 90/08832 [1990]; and Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995].
• PCT International Application WO 90/08832 describes the injection of wild-type feline leukemia virus B into the perivitelline space of sheep embryos at the 2 to 8 cell stage. Fetuses derived from injected embryos were shown to contain multiple sites of integration.
• U.S. Patent 6,291,740 (issued September 18, 2001) describes the production of transgenic animals by the introduction of exogenous DNA into pre-maturation oocytes and mature, unfertilized oocytes (i.e., pre-fertilization oocytes) using retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]).
• retroviral vectors which transduce dividing cells (e.g., vectors derived from murine leukemia virus [MLV]).
• MMV murine leukemia virus
• U.S. Patent 6,281,408 (issued August 28, 2001) describes methods for producing transgenic animals using embryonic stem cells. Briefly, the 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 prior to co-culturing by, for example, electroporation, microinjection or retroviral delivery. ES cells transfected in this manner are selected for integrations of the gene via a selection marker such as neomycin.
• a selection marker such as neomycin.
• U.S. Patent 6,271,436 (issued August 7, 2001) describes the production of transgenic animals using methods including isolation of primordial germ cells, culturing these cells to produce primordial germ cell-derived cell lines, transforming both the primordial germ cells and the cultured cell lines, and using these transformed cells and cell lines to generate transgenic animals.
• the efficiency at which transgenic animals are generated is greatly increased, thereby allowing the use of homologous recombination in producing transgenic non-rodent animal species.
• modified polypeptide or 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.
• the nucleic acid construct encoding GLP-1 comprising an additional His residue at its N-terminus is provided for gene therapy.
• the nucleic acid construct encoding modified GLP-1 /transferrin fusion protein is provided for gene therapy.
• the modified GLP-1 constructs of the present invention are protected from DPP activity and are more stable; thus, they are ideally suited to gene therapy treatments.
• adenovirus vector containing a gene encoding a soluble fusion protein consisting of cytotoxic lymphocyte antigen 4 (CTLA4) and the Fc portion of human immunoglubulin GI was recently shown in Ijima et al. (June 10, 2001) Human Gene Therapy (United States) 12/9:1063-77.
• CTL4 cytotoxic lymphocyte antigen 4
• a murine model of type II collagen-induced arthritis was successfully treated via intraarticular injection of the vector.
• U.S. Patent 6,225,290 provides methods and constructs whereby intestinal epithelial cells of a mammalian subject are genetically altered to operatively incorporate a gene which expresses a protein which has a desired therapeutic effect.
• Intestinal cell transformation is accomplished by administration of a formulation composed primarily of naked DNA, and the DNA may be administered orally.
• Oral or other intragastrointestinal routes of administration provide a simple method of adminisfration, while the use of naked nucleic acid avoids the complications associated with use of viral vectors to accomplish gene therapy.
• the expressed protein is secreted directly into the gastrointestinal tract and/or blood stream to obtain therapeutic blood levels of the protein thereby treating the patient in need of the protein.
• the transformed intestinal epithelial cells provide short or long term therapeutic cures for diseases associated with a deficiency in a particular protein or which are amenable to treatment by overexpression of a protein.
• U.S. Pat. 6,187,305 provides methods of gene or DNA targeting in cells of vertebrate, particularly mammalian, origin. Briefly, DNA is introduced into primary or secondary cells of vertebrate origin through homologous recombination or targeting of the DNA, which is introduced into genomic DNA of the primary or secondary cells at a preselected site.
• U.S. Pat. 6,140,111 (issued October 31, 2000) describes retroviral gene therapy vectors.
• the disclosed retroviral vectors 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, thus rendering 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 suited for use in certain packaging cell lines.
• the ability of retroviral vectors to insert 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 involves (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, i.e., in vitro gene therapy.
• Discussions of how to perform gene therapy in a variety of cells using retroviral vectors can be found, for example, in U.S. Pat. Nos. 4,868,116, issued Sep. 19, 1989, and 4,980,286, issued Dec. 25, 1990 (epithelial cells), WO 89/07136 published Aug. 10, 1989 (hepatocyte cells) , EP 37 f 8,576 published Jul. 25, 1990 (fibroblast cells), and WO 89/05345 published Jun. 15, 1989 and WO/90/06997, published Jun. 28, 1990 (endothelial cells), the disclosures of which are incorporated herein by reference.
• This Example describes modified GLP-1 peptides protected from DPPIV activity.
• the following peptides were synthesized using standard solid phase Fmoc chemistry and purified by reverse phase HPLC using a C18 column and quantitated by absorbance at 220nm.
• the purified peptides were analyzed by mass spectrometry (MALDI-TOF):
• GLP-1 NH 2 -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 (amino acids 1-30 of SEQ ID NO: 32)
• Dilutions of each test compound were prepared in KRB/IBMX and triplicate wells of CHO-GLP1R cells were treated with 50 ⁇ l of test compound per well for exactly 20 minutes at 37°C. The treatment was halted by washing the cultures twice with ice-cold phosphate-buffered saline. Lysates were prepared by the addition of 0.1ml lysis buffer IB (Amersham Biosciences cAMP Biotrak EIA kit) for 10 minutes at room temperature. The entire volume of each cell extract was then assayed to determine the cAMP concentration using the cAMP Biotrak Enzyme Immunoassay System (Amersham Biosciences Corporation, Piscataway, NJ, product code RPN225) according to kit instructions. Peptides of the invention were found to be more resistant to DPP-IV than the unmodified forms.
• DPP-IV degradation of GLP-1 and GLP-1 derivatives of the invention was assayed using an ELISA system (Glucagon-Like Peptide-1 [Active] ELISA kit [Linco Research, Inc., St. Charles, MO]) that is specific for intact, active GLP-1 and does not recognize GLP-1 in which the N-terminal two amino acids have been removed due to the action of DPP-IV, i.e. GLP-1 (9-36 or 9-37).
• ELISA system Glucagon-Like Peptide-1 [Active] ELISA kit [Linco Research, Inc., St. Charles, MO]
• the kit comprises a 96-well microtitre plate coated with anti-GLP-1 monoclonal antibody.
• the plate was washed (25mM Borate-buffered Saline x4 in a plate washer, ThermoLabsystems Ultrawash Plus), then incubated with peptide samples (300pM and 10- fold serial dilutions down the plate) for 3 hours at room temperature. After washing as described above, the plate was incubated with Alkaline-Phosphatase-conjugated anti-GLP antibody (supplied as a ready-to-use component of the kit) for 2 hours at room temperature.
• Example 2 Modified GLP-1 Fusion Protein
• This Example describes a fusion protein comprising a modified GLP-1 protected from DPPIV activity fused to a modified fransferrin molecule.
• CTTCTGATGTTTCTTC SEQ ID NO: 100
• SEQ ID NO: 104 is the coding strand; SEQ ID NO: 105 is the encoded protein.
• the primers (8 ⁇ L of 20pmol cone.) were combined and heated to 65°C for 5 min. and then the annealing reaction was allowed to cool slowly to room temperature.
• CTGTGTCTGGCGCATCATGCTGAAG SEQ ID NO: 1036
• pREX0198 was used as the template for the initial PCR reactions using the two overlapping mutagenic primers and two outer primers in separate reactions, i.e. P0424 plus P0012 and P0425 plus P0025. The products of these reactions were then used as templates in a second round of PCR with just the outer primers, i.e. P0012 plus P0025, in order to join them together.
• the reaction conditions for both rounds of PCR were 1 x 94°C for 1 min, 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 finish.
• pREX0367 was then digested with Notl and Pvul (the latter to destroy the ampicillin resistance gene) and ligated into pSAC35 previously digested with Notl to create pREX0368 ( Figure 7).
• pREX0368 was transformed into the host Saccharomyces cerevsiae strain by electroporation and transformed colonies selected on the basis of leucine prototrophy on buffered minimal medium plates. After selection of single colonies, yeast transformants were stocked in 40% Trehalose and stored at -70°C. Expression was determined by growth in liquid minimal medium buffered to pH6.5 and analysis of supernatant by SDS-PAGE, western blot and ELISA.
• GLP-1/mTf pREXOlOO
• H-GLP-1 /mTf H-GLP-1 /mTf
• haegtftsdvssylegqaakefiawlvkgr (amino acids 1-30 of SEQ ID NO: 32) haegtftsdvssylegqaakefiawlvkgrg (SEQ ID NO: 32)
• the peptide sequence of GLP- 1(7-36) may be back translated into DNA and codon optimized for yeast:
• the primers were specifically designed to form 5' Xbal and 3' Kpnl sticky ends after annealing and to enable direct ligation into XballKp ⁇ l cut pREX0052, just 5' of the end of the leader sequence and at the N-terminus of mTf.
• other sticky ends may be engineered for ligations into other vectors.
• GLP-1 > h a e g t f t s d v s s y l
• This plasmid was then electroporated into the host Saccharomyces yeast strains and transformants selected for leucine protofrohy on minimal media plates. Expression was determined by growth in liquid minimal media and analysis of supernatant by SDS-PAGE, western blot, and ELISA.
• GLP-1/mTf and H-GLP-1/mTf were expressed and purfied from fermentation cultures, grown under standard conditions by cation exchange and anion exchange chromatography.
• Tissue culture plates (24-well) were seeded with CHO-GLPIR cells at a density of 1 x 10 5 cells per/well in RPMI/10% FBS medium one day prior to treatment. The next day the cells appeared uniformly distributed with an approximate confluency of 60-80 percent.
• KRB Krebs-Ringer buffer
• Dilutions of each test compound were prepared in KRB/IBMX and triplicate wells of CHO-GLPIR cells were treated with 0.15ml of test compound per well for exactly 50 minutes at 37°C. The treatment was halted by washing the cultures two times with ice-cold phosphate-buffered saline. Lysates were prepared by the addition of 0.2ml lysis buffer IB (Amersham Biosciences cAMP Biotrak EIA kit) for 10 minutes at room temperature, then lOO ⁇ l of each cell extract was then assayed to determine the cAMP concentration using the cAMP Biotrak Enzyme Immunoassay System (Amersham Biosciences) according to kit instructions.
• IB Amersham Biosciences cAMP Biotrak EIA kit
• H-GLP-1/mTf was found to be more resistant to DPP-IV than GLP-1/mTf
• Example 3 Modified GLP-1/mTf for the Treatment of Diabetes
• 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 treatment of these animals with GLP-1 induces insulin secretion and reduces blood glucose.
• H-GLP-1 or H-GLP-1 fused to fransferrin H-GLP-1/mTf.
• GTT Glucose Tolerance Test
• the blood glucose level of the untreated animals rises and slowly drops towards the base line while the animals which are injected with H-GLP-1 or H-GLP-1 /mTf show faster normalization of blood glucose level due to the insulinotropic effect of the GLP-1.
• modified H-GLP-1 or H-GLP-1/mTf is used to normalize the high fasting glucose of the Zucker rats without glucose adminisfration. While the blood glucose levels remain high in the untreated animals, a significant drop is seen in the H-GLP- 1 or modified H-GLP-1/mTf treated animals.
• Example 4 Modified Glucagon Having Dipeptidyl-Peptidase IV Protection
• This Example describes modified glucagon molecules protected from DPPIV activity.
• the following peptides are synthesized using standard solid phase Fmoc chemistry and purified by reverse phase HPLC using a C18 column and quantitated by absorbance at 220nm. The purified peptides are analyzed by mass spectrometry (MALDI-TOF):
• the peptides are pre-treated with DPP-IV as described above and then assayed for the ability to activate the glucagon receptor using a recombinant cell line expressing a cloned glucagon receptor.
• Example 5 Modified GIP Having Dipeptidyl-Peptidase IV Protection [00267] This Example provides modified GIP molecules protected from DPPIV activity.

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