wherein R'_{1 1} represents a hydrogen, an alkyl, an aryl, an alkenyl, an alkynyl or -(CH₂)_m-R₈, where m is 1-30 and R₈ represents a group permitted by the rules of valence. The terms "amine" and "amino" are art-recognized and include both unsubstituted and substituted amines, e.g. , a moiety that may be represented by the general formulas:

wherein R50, R51 and R52 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R61, or R50 and R51, taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R61 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In certain embodiments, only one of R50 or R51 may be a carbonyl, e.g., R50, R51 and the nitrogen together do not form an imide. In other embodiments, R50 and R51 (and optionally R52) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)_m-R61. Thus, the term "alkylamine" includes an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R50 and R51 is an alkyl group.

The term "acylamino" is art-recognized and includes a moiety that may be represented by the general formula:

wherein R50 is as defined above, and R54 represents a hydrogen, an alkyl, an alkenyl or -(CH₂)_m-R61, where m and R61 are as defined above.

The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined above. Certain embodiments of the amide in the present invention will not include amides which may be unstable.

The term "alkylthio" is art recognized and includes an alkyl group, as defined above, having a sulfur radical attached thereto. In certain embodiments, the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH₂)_m-R61, wherein m and R61 are defined above. Representative alkylthio groups include methylthio, ethylthio, and the like.

The term "carbonyl" is art recognized and includes such moieties as may be represented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and R55 represents a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R6 lor a pharmaceutically acceptable salt, R56 represents a hydrogen, an alkyl, an alkenyl or -(CH₂)_m-R61, where m and R61 are defined above. Where X50 is an oxygen and R55 is not hydrogen, the formula represents an "ester". Where X50 is an oxygen, and R55 is as first defined above, the moiety is referred to herein as a carboxyl group, and particularly when R55 is a hydrogen, the formula represents a "carboxylic acid". Where X50 is an oxygen, and R56 is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiocarbonyl" group. Where X50 is a sulfur and R55 or R56 is not hydrogen, the formula represents a "thioester." Where X50 is a sulfur and R55 is hydrogen, the formula represents a "thiocarboxylic acid." Where X50 is a sulfur and R56 is hydrogen, the formula represents a "thioformate." On the other hand, where X50 is a bond, and R55 is not hydrogen, the above formula represents a "ketone" group. Where X50 is a bond, and R55 is hydrogen, the above formula represents an "aldehyde" group. The terms "oxime" and "oxime ether" are art-recognized and refer to moieties that may be represented by the general formula:

wherein R75 is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, or -(CH₂)_m-R61. The moiety is an "oxime" when R is H; and it is an "oxime ether" when R is alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, or -(CH₂)_m-R61.

The terms "alkoxyl" or "alkoxy" are art recognized and include an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as may be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2)_m-R61, where m and R61 are described above. The term "sulfonate" is art recognized and includes a moiety that may be represented by the general formula: o

-OR57

O in which R57 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The term "sulfate" is art recognized and includes a moiety that may be represented by the general formula:

O O S OR57

O Il in which R57 is as defined above.

The term "sulfonamido" is art recognized and includes a moiety that may be represented by the general formula:

in which R50 and R56 are as defined above. The term "sulfamoyl" is art-recognized and includes a moiety that may be represented by the general formula:

in which R50 and R51 are as defined above.

The term "sulfonyl" is art recognized and includes a moiety that may be represented by the general formula:

in which R58 is one of the following: hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

The term "sulfoxido" is art recognized and includes a moiety that may be represented by the general formula:

/ R58 in which R58 is defined above.

The term "phosphoryl" is art-recognized and may in general be represented by the formula:

Q50

OR59 wherein Q50 represents S or O, and R59 represents hydrogen, a lower alkyl or an aryl.

When used to substitute, e.g., an alkyl, the phosphoryl group of the phosphorylalkyl may be represented by the general formulas:

Q50 Q50 Q51— p o Q51— P-OR59

OR59 OR59 wherein Q50 and R59, each independently, are defined above, and Q51 represents O, S or N. When Q50 is S, the phosphoryl moiety is a "phosphorothioate".

The term "phosphoramidite" is art recognized and includes moieties represented by the general formulas:

O O Q51—P¹ o- Q51 — P-OR59

R50 R51 R50 R51 wherein Q51, R50, R51 and R59 are as defined above. The term "phosphonamidite" is art recognized and includes moieties represented by the general formulas: R60 R60 Q51 P O Q51 P— OR59

R50 R51 R50 R51 wherein Q51, R50, R51 and R59 are as defined above, and R60 represents a lower alkyl or an aryl.

The term "selenoalkyl" is art-recognized and refers to an alkyl group having a substituted seleno group attached thereto. Exemplary "selenoethers" which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and - Se-(CH₂)_m-R61, m and R61 being defined above.

The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms, represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and methanesulfonyl, respectively. A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.

Certain compounds contained in compositions of the present invention may exist in particular geometric or stereoisomeric forms. In addition, compounds of the present invention may also be optically active. The present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)- isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention. If, for instance, a particular enantiomer of compound of the present invention is desired, it may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers. Alternatively, where the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. The term "substituted" is also contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described herein above. The permissible substituents may be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds. Analogous substitutions may be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

The definition of each expression, e.g. alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure unless otherwise indicated expressly or by the context.

For purposes of the invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (ed. Parker, S., 1985), McGraw-Hill, San Francisco, incorporated herein by reference). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

The term "ED₅₀" is art-recognized. In certain embodiments, ED50 means the dose of an agent, which produces 50% of its maximum response or effect, or alternatively, the dose which produces a pre-determined response in 50% of test subjects or preparations. Compounds of the Invention Certain aspects of the present invention relate to synthetic peptide analogues of native peptides or peptidic fragments thereof, which are incretin hormones, such as those selected from the group consisting of secretin, insulin, somatostatin, gastric inhibitory peptide (GIP), vasoactive intestinal peptide (VIP), PACAP, glicentin, glucagon, glucagon- like peptide 1 (GLP-I), glucagon-like peptide 2 (GLP-2), and GLP-I (7-36) amide. Certain embodiments of the present invention contemplate modification of native

GLP-I or peptidic fragments thereof. Native GLP- 1(7-36) amide has the all-L amino acid sequence depicted in Scheme 1. Scheme 1 Native GLP -1(7-36) amide, all L-amino acids [W-terminus] H⁷ A E G T¹¹ F T¹³ S D V S S Y L E G Q A A K E F I²⁹ A W L V K G R³⁶ [C- terminus] Retro-Inverso Modifications

One embodiment of the present invention contemplates a retro-inverso modification of the native GLP- 1(7-36) amide model, whereby the use of complementary D-amino acid enantiomers constitutes an inversion of the chirality of the amino acid residues in the native sequence (inverso modification), and whereby said D-amino acids are attached in a peptide chain such that the sequence of residues in the resulting analogue is exactly opposite of that in the native GLP-I peptide (retro modification), furnishing a stable synthetic GLP-I analogue, consistent with >90% sequence homology with native GLP-I (7-36) amide (see Figures 1 and 2).

Following the same design strategy, a further embodiment of the present invention makes use of complementarily diastereoisomeric D-allo amino acids as a conservative substitution for the two threonine and one isoleucine residues of the model sequence in the preparation of a variant peptide retro-inverso GLP-I analogue, giving a stable synthetic GLP-I analogue, consistent with >90% sequence homology with native GLP-l(7-36) amide (Figure 2).

Termini Modifications As described previously, the biological activity of native GLP-I is curtailed by rapid removal of the N-terminal dipeptide by DPP-IV. It is envisaged that terminus modifications could increase resistance to DPP-IV degradation, thus leading to prolonged duration of action in vivo, and/or optionally enhance receptor interactions.

The use of "protecting groups" as described herein may also be advantageous in this regard (see definitions). Further examples of N-terminal modifications include glycation (e.g., N-glucitol), N-pyroglutamyl, N-acetyl, N-methylation (N-Me, α-Me), desamination, and substitution with imidazole-lactic acid.

Much less attention has focused on elaboration at the C-terminus. As described above, the GLP-I derivative CJC-1131 was engineered with a reactive C-terminus chemical linker that allows covalent bonding to endogenous serum albumin (vide supra).

Certain embodiments of the present invention provide for synthetic peptide analogues that may be optionally derivatized at the terminal residues, independently for each occurrence. One embodiment of the present invention considers a retro-inverso modified GLP-I analogue, as described above, which is optionally derivatized, whereby the corresponding C-terminal histidine carboxylate is replaced with a geminal-amino D- histidine residue to afford a stable synthetic GLP-I analogue, consistent with >90% sequence homology with native GLP-l(7-36) amide (Figure 2).

In certain embodiments of the present invention, the sequence of the retro-inverso modified GLP-I analogue can be extended by a number of amino acid residues, e.g., whereby the corresponding N-terminus is extended by up to about nine amino acids to furnish a variant peptide, hi certain embodiments, said variant peptide may be a retro- inverso modified GLP-I analogue as described above, wherein the corresponding N- terminus has been extended further with nine amino acid residues corresponding to those found in the C-terminal sequence of native exendin-4: P S S G A P P P S. According to U.S. Patent No. 6,583,111 (incorporated by reference) to DiMarchi et al., as many as about 14 substitutions can be made for amino acid residues along the native GLP-I sequence to afford variant peptide GLP-I compounds. In certain embodiments, aspects of the present invention provide for substitution of as many as about 14 residues in the sequence of a retro-inverso modified GLP-I analogue.

As used herein, the terminology "GLP-I compound," be it the native sequence, synthetic versions and variants thereof, analogues and derivatives thereof, including those of the present invention, also includes pharmaceutically acceptable salts of said compounds described, and in accordance with the detailed definitions herein. A GLP-I compound of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4- dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1 -sulfonate, naphthalene-2- sulfonate, mandelate, and the like. Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.

In the formulas representing selected specific embodiments of the present invention, the amino- and carboxy-terminal groups, although often not specifically shown, may be understood to be in the form they would assume at physiological pH values, unless otherwise specified. Thus, the N-terminal-H₂⁺ and C-terminal-CT at physiological pH may be understood to be present, though not necessarily specified and shown, either in specific examples or in generic formulas. The foregoing describes the status of the termini at neutral pH; it is understood, of course, that the acid addition salts or the basic salts of the peptides are also included within the scope of the invention. At high pH, basic salts of the C-terminus and carboxyl- containing side chains may be formed from nontoxic pharmaceutically acceptable bases, and suitable counter-ions include, for example, Na⁺, K⁺, Ca²⁺, and the like. Suitable pharmaceutically acceptable nontoxic organic cations can also be used as counter ions. In addition, as set forth herein, the peptides may be prepared as the corresponding amides. Suitable acid addition salts with respect to the N-terminus or amino group-containing side chains include the salts formed from inorganic acids such as hydrochloric, sulfuric, or phosphoric acid and those formed from organic acids such as acetic, citric, or other pharmaceutically acceptable nontoxic acids.

One aspect of the invention relates to a retro-inverso analogue of a peptide or a fragment thereof, or a pharmaceutically acceptable salt of the peptide or fragment, selected from the group consisting of secretin, insulin, somatostatin, gastric inhibitory peptide (GIP), vasoactive intestinal peptide (VIP), PACAP, glicentin, glucagon, glucagon-like peptide 1 (GLP-I), glucagon-like peptide 2 (GLP -2), and GLP-I (7-36) amide, wherein said analogue has at least 80% sequence homology to the peptide or fragment thereof; and said analogue comprises D-amino acids assembled in reversed order along the peptide chain.

In certain embodiments, the present invention relates to the aforementioned retro- inverse analogue, wherein said analogue has at least 90% sequence homology to the peptide or fragment thereof.

In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said analogue has at least 95% sequence homology to the peptide or fragment thereof. In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said analogue has at least 99% sequence homology to the peptide or fragment thereof.

In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said peptide is GLP-I (7-36) amide. In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said analogue is independently derivatized at one or both of the terminal residues. In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein the C-terminal histidine residue of said analogue has been replaced with a geminal-amino histidine analogue.

In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said analogue comprises D-allo amino acids.

In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein all D-threonines and D-isoleucines are allo amino acids. hi certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said analogue comprises O-allo amino acids. In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein all D-threonines and D-isoleucines are allo amino acids.

In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein the peptide sequence is extended by 1 to about 10 additional amino acid residues. In certain embodiments, the present invention relates to the aforementioned retro- inverso analogue, wherein said additional amino acid residues are the sequence P S S G A P P P S. Methods of the Invention

Said GLP-I compounds may be used to treat subjects with a wide variety of diseases and conditions. It is believed that GLP-I compounds, including those of the present invention, exert their biological effects by acting at a receptor referred to as the "GLP-I receptor" (see U.S. Pat. No. 5,670,360 to Thorrens). Subjects with diseases and/or conditions that respond favorably to GLP-I receptor stimulation, or to the administration of GLP-I compounds, can therefore be treated with the GLP-I compounds of the present invention. These subjects are said to "be in need of treatment with GLP-I compounds" or "in need of GLP-I receptor stimulation." Included are subjects with non-insulin dependent diabetes, insulin dependent diabetes, stroke (see WO 00/16797 by Efendic), myocardial infarction (see WO 98/08531 by Efendic), obesity (see WO 98/19698 by Efendic), catabolic changes after surgery (see U.S. Pat. No. 6,006,753 to Efendic), functional dyspepsia and irritable bowel syndrome (see WO 99/64060 by Efendic). Also included are subjects requiring prophylactic treatment with a GLP-I compound, e.g., subjects at risk for developing non-insulin dependent diabetes (see WO 00/07617). Subjects with impaired glucose tolerance or impaired fasting glucose, subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects with a partial pancreatectomy, subjects having one or more parents with non-insulin dependent diabetes, subjects who have had gestational diabetes and subjects who have had acute or chronic pancreatitis are at risk for developing non-insulin dependent diabetes. Accordingly, in certain embodiments, the present relates to a method of treating

Type II diabetes, hypertension, cardiovascular disease, periodontal disease, retinopathy, glaucoma, renal disease, neuropathy, ketoacidosis, Type I diabetes, stroke, myocardial infarction, obesity, catabolic changes after surgery, functional dyspepsia, irritable bowel syndrome, impaired glucose tolerance, impaired fasting glucose, or partial pancreatectomy comprising administering to a mammal in need thereof a therapeutically effective amount of a composition including one or more analogues according to any one of the aforementioned compounds of the invention and any of the attendant definitions, as detailed above. In certain embodiments, the present invention relates to said method, wherein the mammal is a primate, bovine, ovine, equine, porcine, rodent, feline, or canine. In certain embodiments, the present invention relates to said method, wherein the mammal is a human.

Another aspect of the present invention relates to a method of treating Type II diabetes, comprising administering to a mammal in need thereof a therapeutically effective amount of a retro-inverso analogue according to the present invention. In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has at least 90% sequence homology to the peptide or fragment thereof.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has at least 95% sequence homology to the peptide or fragment thereof.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has at least 99% sequence homology to the peptide or fragment thereof.

In certain embodiments, the present invention relates to the aforementioned method, wherein said peptide is GLP-I (7-36) amide.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue is independently derivatized at one or both of the terminal residues. In certain embodiments, the present invention relates to the aforementioned method, wherein the C-terminal histidine residue of said analogue has been replaced with a geminal- amino histidine analogue. hi certain embodiments, the present invention relates to the aforementioned method, wherein said analogue comprises O-allo amino acids.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has only allo amino acids at positions corresponding to D-threonine and D-isoleucine. hi certain embodiments, the present invention relates to the aforementioned method, wherein said analogue is extended by 1 to about 10 additional amino acid residues.

In certain embodiments, the present invention relates to the aforementioned method, wherein said additional amino acid residues of said analogue are the sequence P S S G A P P P S.

In certain embodiments, the present invention relates to the aforementioned method, wherein the mammal is a primate, bovine, ovine, equine, porcine, rodent, feline, or canine.

In certain embodiments, the present invention relates to the aforementioned method, wherein the mammal is a human.

Yet another aspect of the present invention relates to a method of treating hypertension, cardiovascular disease, periodontal disease, retinopathy, glaucoma, renal disease, neuropathy, ketoacidosis, Type I diabetes, stroke, myocardial infarction, obesity, catabolic changes after surgery, functional dyspepsia, irritable bowel syndrome, impaired glucose tolerance, impaired fasting glucose, or partial pancreatectomy, comprising administering to a mammal in need thereof a therapeutically effective amount of a retro- inverso analogue according to the present invention. In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has at least 90% sequence homology to the peptide or fragment thereof.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has at least 99% sequence homology to the peptide or fragment thereof. In certain embodiments, the present invention relates to the aforementioned method, wherein said peptide is GLP-I (7-36) amide.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue is independently derivatized at one or both of the terminal residues. In certain embodiments, the present invention relates to the aforementioned method, wherein the C-terminal histidine residue of said analogue has been replaced with a geminal- amino histidine analogue.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue comprises O-allo amino acids. In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue has only allo amino acids at positions corresponding to D-threonine and D-isoleucine.

In certain embodiments, the present invention relates to the aforementioned method, wherein said analogue is extended by 1 to about 10 additional amino acid residues. In certain embodiments, the present invention relates to the aforementioned method, wherein said additional amino acid residues of said analogue are the sequence P S S G A P P P S.

In certain embodiments, the present invention relates to the aforementioned method, wherein the mammal is a primate, bovine, ovine, equine, porcine, rodent, feline, or canine. In certain embodiments, the present invention relates to the aforementioned method, wherein the mammal is a human. Pharmaceutical Compositions

In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the compounds described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; (8) nasally; (9) pulmonary; or (10) intrathecally.

The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.

The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

As set out above, certain embodiments of the present compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable acids. The term "pharmaceutically-acceptable salts" in this respect, refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed during subsequent purification. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. ScL 66:1-19).

The pharmaceutically acceptable salts of the subject compounds include the conventional nontoxic salts or quaternary ammonium salts of the compounds, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically-acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically-acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of compounds of the present invention. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically- acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 percent to about 30 percent. In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present invention. Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. A compound of the present invention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules, trouches and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxamer and sodium lauryl sulfate; (7) wetting agents, such as, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof; (10) coloring agents; and (11) controlled-release agents, such as crospovidone or ethyl cellulose. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients. Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption, such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This result may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

The preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.

The phrases "parenteral administration" and "administered parenterally" as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration. Compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically- acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular compound of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. Generally, oral, intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 100 mg per kilogram of body weight per day. If desired, the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms, dosing is one administration per day.

While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals. In another aspect, the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of one or more of the subject compounds, as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, or mucous membranes; or (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually or buccally; (6) ocularly; (7) transdermally; or (8) nasally.

The term "treatment" is intended to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals, such as equines, cattle, swine and sheep; and poultry and pets in general.

The compound of the invention can be administered as such or in admixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutical effects of the first administered one is not entirely disappeared when the subsequent is administered. Micelles Microemulsification technology improves bioavailability of some lipophilic (water insoluble) pharmaceutical agents. Examples include Trimetrine (Dordunoo, S. K., et al., Drug Development and Industrial Pharmacy, 17(12), 1685-1713, 1991 and REV 5901 (Sheen, P. C, et al., JPharm Sci 80(7), 712-714, 1991). Among other things, microemulsification provides enhanced bioavailability by preferentially directing absorption to the lymphatic system instead of the circulatory system, which thereby bypasses the liver, and prevents destruction of the compounds in the hepatobiliary circulation. While all suitable amphiphilic carriers are contemplated, the presently carriers are generally those that have Generally-Recognized-as-Safe (GRAS) status, and that can both solubilize the compound of the present invention and microemulsify it at a later stage when the solution comes into a contact with a complex water phase (such as one found in human gastro-intestinal tract). Usually, amphiphilic ingredients that satisfy these requirements have HLB (hydrophilic to lipophilic balance) values of 2-20, and their structures contain straight chain aliphatic radicals in the range of C-6 to C-20. Examples are polyethylene- glycolized fatty glycerides and polyethylene glycols.

Commercially available amphiphilic carriers are particularly contemplated, including Gelucire-series, Labrafil, Labrasol, or Lauro glycol (all manufactured and distributed by Gattefosse Corporation, Saint Priest, France), PEG-mono-oleate, PEG-di- oleate, PEG-mono-laurate and di-laurate, Lecithin, Polysorbate 80, etc (produced and distributed by a number of companies in USA and worldwide). EXEMPLIFICATION The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. GLP-J Receptor Agonist GLP-I receptors and the signal transduction cascade initiated by ligand binding to the GLP-I receptor are described in U.S. Patent No. 6,074,875 (incorporated by reference) to Thorens. The GLP-I receptor is a membrane protein with seven trans-membrane domains, coupled to heterotrimeric G-proteins that link activation of the receptor by ligand binding to production of intracellular secondary messengers, especially cyclic adenosine monophosphate (cAMP). The GLP-I analogue resulting from retro-inverso modification and C-terminal gem-amino derivatization was tested for efficacy in activating the GLP-I receptor. Materials and Methods

The methods used in these experiments have been described in Beinborn et al. (2001) J. Biol. Chem. 276:37787. Cell Culture and Transfection

COS-7 cells (l*10⁶ cells/10 cm dish) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with heat-inactivated fetal bovine serum (10%, v/v), 26mM sodium bicarbonate, and penicillin G sodium/streptomycin sulfate (100 units/ml and 100 μg/ml, respectively). After an overnight incubation in a humidified atmosphere with 5 % CO₂, cells were transiently transfected using the diethylaminoethyl-dextran method, adding 5 μg cDNA encoding the wild type human GLP-I receptor. Control cells (no receptor expression) were transfected with the empty vector, pcDNAl . Measurement of cAMP Formation

Twenty-four hours after transfection, COS-7 cells containing the wild type and mutant receptors were seeded on 24-well plates (100,000 cells/well) and allowed to attach for an additional 24 hours. Cells were incubated with indicated peptide concentrations at room temperature for one hour in Dulbecco's modified Eagle's medium. The medium was supplemented with 1% bovine serum albumin (BSA), ImM isobutyl-methylxanthine, 0.4 μM Pro-Boro-Pro, and 25 mM HEPES, pH 7.4. After removal of the supernatant, cells were incubated with 0.1N HCl and lysed by freeze-thawing in liquid nitrogen. After neutralization, cAMP levels were determined by radioimmunoassay (acetylation method) using a FlashPlate^® kit (Perkin Elmer Life Sciences). Plate-bound radioactivity was measured using a Packard Topcount^® proximity scintillation counter. Data Analysis

Data were normalized to the maximal response obtained with a saturating concentration of GLP-I (10 μM, = 100%). GraphPad Prism software version 3.0 (GraphPad, San Diego, CA) was used to calculate agonist concentration-response curves. Results

Figure 3 depicts graphically data showing activation of the GLP-I receptor by a retro-inverso GLP-I analogue of the present invention. These results establish that the retro-inverso C-terminal gem-amino GLP-I analogue (as depicted in Figure 2) is a stable, biologically active GLP-I mimetic, demonstrating 100% agonist activity for the GLP-I receptor at 1.3 μM concentrations. These results are similar to those observed for Liraglutide, in that the required dosage is 1000-fold more than native GLP-I. In addition to stimulating insulin release, it may also be the case that the retro-inverso analogue of the present invention has an extended half-life in serum, i.e., that it is more stable toward degradation. INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications cited herein are hereby incorporated by reference. U.S. Patent 5,545,618 is expressly incorporated by reference in its entirety. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A retro-inverso analogue of a peptide or a fragment thereof, or a pharmaceutically acceptable salt of the peptide or fragment, selected from the group consisting of secretin, insulin, somatostatin, gastric inhibitory peptide (GIP), vasoactive intestinal peptide (VIP), PACAP, glicentin, glucagon, glucagon- like peptide 1 (GLP-I), glucagon-like peptide 2 (GLP-2), and GLP-I (7-36) amide, wherein said analogue has at least 80% sequence homology to the peptide or fragment thereof; and said analogue comprises D-amino acids assembled in reversed order along the peptide chain.

2. The retro-inverso analogue of claim 1, wherein said analogue has at least 90% sequence homology to the peptide or fragment thereof.

3. The retro-inverso analogue of claim 1, wherein said analogue has at least 95% sequence homology to the peptide or fragment thereof.

4. The retro-inverso analogue of claim 1, wherein said analogue has at least 99% sequence homology to the peptide or fragment thereof.

5. The retro-inverso analogue of any one of claims 1 -4, wherein said peptide is GLP- 1 (7-36) amide.

6. The retro-inverso analogue of claim 5, wherein said analogue is independently derivatized at one or both of the terminal residues.

7. The retro-inverso analogue of claim 5, wherein the C-terminal histidine residue of said analogue has been replaced with a geminal-amino histidine analogue.

8. The retro-inverso analogue of claim 5, 6, or 7, wherein said analogue comprises D- allo amino acids.

9. The retro-inverso analogue of claim 5, 6, or 7, wherein all D-threonines and D- isoleucines are allo amino acids.

10. The retro-inverso analogue of any one of claims 1 -9, wherein the peptide sequence is extended by 1 to about 10 additional amino acid residues.

11. The retro-inverso analogue of claim 10, wherein said additional amino acid residues are the sequence P S S G A P P P S.

12. A method of treating Type II diabetes, comprising administering to a mammal in need thereof a therapeutically effective amount of a retro-inverso analogue according to any one of claims 1-11.

13. The method of claim 12, wherein said retro-inverso analogue is according to any one of claims 5-11.

14. The method of claim 12, wherein said retro-inverso analogue is according to claim 9.

15. The method of any one of claims 12-14, wherein the mammal is a primate, bovine, ovine, equine, porcine, rodent, feline, or canine.

16. The method of any one of claims 12-14, wherein the mammal is a human.

17. A method of treating hypertension, cardiovascular disease, periodontal disease, retinopathy, glaucoma, renal disease, neuropathy, ketoacidosis, Type I diabetes, stroke, myocardial infarction, obesity, catabolic changes after surgery, functional dyspepsia, irritable bowel syndrome, impaired glucose tolerance, impaired fasting glucose, or partial pancreatectomy, comprising administering to a mammal in need thereof a therapeutically effective amount of a retro-inverso analogue according to any one of claims 1-11.

18. The method of claim 17, wherein said retro-inverso analogue is according to any one of claims 5-11.

19. The method of claim 17, wherein said retro-inverso analogue is according to claim 9.

20. The method of any one of claims 17-19, wherein the mammal is a primate, bovine, ovine, equine, porcine, rodent, feline, or canine.

21. The method of any one of claims 17-19, wherein the mammal is a human.