ZA200101541B

ZA200101541B - Method for administering insulinotropic peptides.

Info

Publication number: ZA200101541B
Application number: ZA200101541A
Authority: ZA
Inventors: Benjamin Lee Hughes; Ronald Keith Wolff
Original assignee: Lilly Co Eli
Priority date: 1998-08-28
Filing date: 2001-02-23
Publication date: 2002-05-23

Description

. 1 b4 v WO 00/12116 PCT/US99/19348

METHOD FOR ADMINISTERING INSULINOTROPIC PEPTIDES

Field of the Invention

This invention relates to methods of treating humans suffering from diabetes and insulin resistance. In particular, the invention relates to the pulmonary delivery of glucagon-like peptide-1 (GLP-1) and analogs thereof for systemic absorption through the lungs to eliminate the need for administering anti-diabetic compounds by injection.

Background of the Invention

Glucagon-like peptide-1 was first identified in 1987 as a incretin hormone, a peptide secreted by the gut upon ingestion of food. Glucagon-like peptide-1 is secreted by the L-cells of the intestine after being proteolytically processed from the 160 amino acid precursor protein, preproglucagon. Cleavage of preproglucagon first yields glucagon-like peptide-1, a 37 amino acid peptide that is poorly active. A subsequent cleavage of the peptide bond between residues 6 and 7 yields biologically active glucagon-like peptide-1 referred to as GLP-1(7-37). It should be noted that this specification uses the nomenclature scheme that has developed around this hormone.

By convention in the art, the amino terminus of GLP-1(7-37) has been assigned number 7 and the carboxy terminus number 37. Approximately 80% of the GLP-1(7-37) that is synthesized is amidated at the C-terminal after removal of the terminal glycine residue in the L-cells. The biological effects and metabolic turnover of the free acid GLP-1(7-37), and the amide, GLP-1(7-36)NH , are indistinguishable. As used herein, these two naturally-occurring forms will be referred to collectively as GLP-1.

GLP-1 is known to stimulate insulin secretion (insulinotropic action) causing glucose uptake by cells which decreases serum glucose levels (see, e g., Mojsov, S.,

Int. J. Peptide Protein Research, 40:333-343 (1992)).

Numerous GLP-1 analogs and derivatives demonstrating insulinetropic action are known in the art. Also it has been demonstrated that the N-terminal histidine residue (His 7) is very important to insulinotropic activity of GLP-1 (Suzuki, S., et al. Diabetes Res. ; Clinical Practice 5 (Supp. 1):830 (1988).

Multiple authors have demonstrated the nexus between laboratory experimentation and mammalian, particularly human, insulinotropic responses to exogenous administration of GLP-1. See, e.g., Nauck, M.A., et al.,

Diabetologia, 36:741-744 (1993); Gutniak, M., et al., New

England J. of Medicine, 326 (20) :1316-1322 (1992) ; Nauck,

M.A., et al., J. Clin. Invest., 91:301-307 (1993); and

Thorens, B., et al., Diabetes, 42:1219-1225 (1993)].

GLP-1 based peptides hold great promise as alternatives to insulin therapy for patients with diabetes who have failed on sulfonylureas. GLP-1 has been studied intensively by academic investigators, and this research has established the following for patients with type II diabetes who have failed on sulfonylureas: 1) GLP-1 stimulates insulin secretion, but only during periods of hyperglycemia. The safety of GLP-1 compared to insulin is enhanced by this property of GLP-1 and by the observation that the amount of insulin secreted is

I proportional to the magnitude of the hyperglycemia. In addition, GLP-1 therapy will result in pancreatic release of insulin and first-pass insulin action at the liver. This results in lower circulating levels of insulin in the periphery compared to subcutaneous insulin injections. 2) GLP-1 suppresses glucagon secretion, and this, in addition to the delivery of insulin via the portal vein helps suppress the excessive hepatic glucose output in diabetic patients. 3) GLP-1 slows gastric emptying which is desirable in that it spreads nutrient absorption over a longer time period, decreasing the postprandial glucose peak. 4) Several reports have suggested that GLP-1 may enhance insulin sensitivity in peripheral tissues such as muscle and fat. 5) Finally, GLP-1 has been shown to be a potential regulator of appetite.

Meal-time use of GLP-1 based peptides offers several advantages over insulin therapy. Insulin therapy requires blood glucose monitoring, which is both expensive and painful. The glucose-dependency of GLP-1 provides an enhanced therapeutic window in comparison to insulin, and should minimize the need to monitor blood glucose. Weight gain also can be a problem with intensive insulin therapy, particularly in the obese type II diabetic patients.

The therapeutic potential for native GLP-1 is further increased if one considers its use in patients with type I diabetes. A number of studies have demonstrated the effectiveness of native GLP-1 in the treatment of insulin dependent diabetes mellitus. Similar to patients with type

IT diabetes, GLP-1 is effective in reducing fasting hyperglycemia through its glucagonostatic properties.

Additional studies have indicated that GLP-1 also reduces postprandial glycemic excursions in type I patients, most likely through a delay in gastric emptying. These observations indicate that GLP-1 may be useful as a treatment in type I and type II patients.

To date administration of clinically proven peptide hormones and as well as GLP-1 has generally been accomplished by subcutaneous injection which is both inconvenient and unattractive. Therefore, many investigators have studied alternate routes for administering peptide hormones such as oral, rectal, transdermal, and nasal routes. Thus far, however, these routes of administration have not resulted in clinically proven peptide hormone therapy.

It has been known for a number of years that some proteins can be absorbed from the lung. For example, insulin administered by inhalation aerosol to the lung was first reported by Gaensslen in 1925. Despite the fact that a number of human and animal studies have shown that some insulin formulations can be absorbed through the lungs, pulmonary delivery of peptide hormones has not been vigorously pursued because of very low bioavailability.

Larger proteins, such as cytokines and growth factors which are generally larger than 150 amino acid residues, are often readily absorbed by the cells lining the alveolar regions of the lung. Pulmonary absorption of smaller proteins is however much less predictable; though insulin (51 residues), calcitonin (32 residues) and parathyroid hormone v WO 00/12116 PCT/US99/19348 (34 residues) have been reported to be systemically absorbed through the pulmonary route. See US Patent No: 5,607,915, herein incorporated by reference. Despite systemic absorption by the lung of some small protein hormones, the pharmacodynamics associated with pulmonary delivery of peptides is unpredictable.

Thus, there is a need to provide a reliable pulmonary method of delivering GLP-1 and related analogs because it would offer patients an attractive, non-invasive alternative to insulin. This need is particularly true since insulin has a very narrow therapeutic index while GLP- 1 treatment offers a way to normalize blood glucose only in response to hyperglycemic conditions without the threat of hypoglycemia.

Not all protein hormones can be efficiently absorbed through the lungs, and there are many factors that affect it. Absorption of proteins in the lung is largely dependent on the physical characteristics of the protein.

Thus, even though pulmonary delivery of some protein hormones has been observed, the physical properties and short length of GLP-1 and some related peptides made it unclear whether such peptides could be effectively delivered through the pulmonary route.

Efficient pulmonary delivery is dependent on the ability to deliver the protein to the deep lung alveolar epithelium. Protein particles that lodge in the upper airway epithelium are not absorbed to a significant extent because the overlying mucus functions to trap, and then clear debris by mucociliary transport up the airway. This mechanism is also a major contributor to low bicavailability. The extent to which proteins are not absorbed and instead eliminated by these routes depends on their solubility, their size, and other largely uncharacterized mechanisms.

Even when a peptide hormone can be reproducibly delivered to the deep lung alveolar epithelium, it is difficult to predict whether it will be rapidly absorbed and transported to the blood. Absorption values for some proteins delivered through the lungs have been calculated and range from fifteen minutes for parathyroid hormone (1- 34) to 48 hours for glycosylated al-antitrypsin. Moreover a variety of endogenous peptidases exist in the lung which can degrade peptides prior to absorption. Thus, the longer it takes for a peptide particle to dissolve and be absorbed, the greater the chance for enzymatic inactivation. Thus, because of the small size of GLP-1 and its inherent susceptibility to certain enzymes, it was most surprising to find that an aerosolized GLP-1 analog could be reproducibly and effectively delivered through the lungs.

Summary of the Invention

The present invention relates to a method for administering a glucagon-like peptide-1 molecule comprising, administering an effective amount of the peptide to a patient in need thereof by pulmonary delivery. The present invention also relates to a method for treating diabetes comprising, administering an effective dose of a glucagon- like peptide-1 to a patient in need thereof by pulmonary delivery. Another aspect of the invention relates to a method for treating hyperglycemia comprising, administering an effective dose of a glucagon-like peptide-1 to a patient

} r 1 ¥ : -7- in need thereof by pulmonary delivery. Preferably, the glucagon-like peptide-1 molecule is delivered by inhalation and to the lower airway of the patient.

The glucagon-like peptide-1 can be delivered in a carrier, as a solution or suspension, or as a dry powder, using any of a variety of devices suitable for administration by inhalation. Preferably, the glucagon-like peptide-1 is delivered in a particle size effective for reaching the lower airways of the lung.

Detailed Description of the Invention

The term "GLP-1" refers to human glucagon-like peptide-1 whose sequences and structures are known in the art. See US Patent No. 5,120,712, herein incorporated by reference. As previously discussed, there are two native forms of human GLP-1, GLP-1(7-37) and GLP-1(7-36)NH, which will be distinguished only when necessary.

The term "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) and GLP-1(7-35), GLP-1(7-36), Val -GLP-1(7-37), Gln'-

GLP-1(7-37), D-Gln -GLP-1(7-37), Thr -Lys -GLP-1(7-37), and

Lys -GLP-1(7-37). Preferred GLP-1 analogs are GLP-1(7-34) and GLP-1(7-35), which are disclosed in U.S. Patent No: 5,118,666, herein incorporated by reference.

The term "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, a-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. Modifications at amino acid side groups include, without limitation, acylation of lysine e-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, and deamidation of glutamine or asparagine. Modifications of the terminal amino include, without limitation, the des- amino, N-lower alkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, may be protected by protective groups known to the ordinarily-skilled protein chemist. The a- carbon of an amino acid may be mono- or di-methylated.

The term "GLP-1 molecule" means GLP-1, GLP-1 analog, or GLP-1 derivative.

Another preferred group of GLP-1 analogs is defined by the formula:

R,-X-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu- ¥-Gly-Gln-Ala-Ala-Lys-Z-Phe-Ile-Ala-Trp-Leu-Val-Lys-

Gly-Arg-R, (SEQ ID NO:1) and the pharmaceutically-acceptable salts thereof, wherein:

R, is selected from the group consisting of L-histidine, D- histidine, desamino-histidine, 2-amino-histidine, b-~hydroxy- histidine, homohistidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine; X is selected from the group consisting of Ala, Gly, Val, Thr, Ile, and alpha-methyl-Ala;

. . ‘

Y is selected from the group consisting of Glu, Gln, Ala,

Thr, Ser, and Gly; 2 is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R, is selected from the group consisting of NH2, and Gly-OH; providing that when

R, is His, X is Ala, Y is Glu, and Z is Glu, R, must be NH,

Yet another preferred group of compounds consistent with the present invention is disclosed in WO 91/11457 (U.S. Patent No. 5,545,618, herein incorporated by reference) and consists essentially of GLP-1(7-34), GLP-1(7- 35), GLP-1(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

D-arginine for arginine at position 36; (b) substitution of an oxidation-resistant amino acid for tryptophan at position 31; (c) substitution of at least one of: tyrosine for valine at position 16; lysine for serine at position 18; aspartic acid for glutamic acid at position 21; serine for glycine at position 22; arginine for glutamine at position 23; arginine for alanine at position 24; and glutamine for lysine at position 26; and

Tr (d) substitution of at least one of: glycine, serine, or cysteine for alanine at position 8; aspartic acid, glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glutamic acid at position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine for glycine at position 10; and glutamic acid for aspartic acid at position 15; and (e) substitution of glycine, serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine, or the D- or N-acylated or alkylated form of histidine for histidine at position 7; wherein, in the substitutions is (a), (b), (d), and (e), the substituted amino acids can optionally be in the D-form and the amino acids substituted at position 7 can optionally be in the N-acylated or N-alkylated form.

Because the enzyme, dipeptidyl-peptidase IV (DPP

IV), may be responsible for the observed rapid in vivo inactivation of administered GLP-1, [see, e.g., Mentlein,

R., et al., Bur. J. Biochem., 214:829-835 (1993)], administration of GLP-1 analogs and derivatives that are protected from the activity of DPP IV is preferred, and the administration of Gly -GLP-1(7-36)NH, Val'-GLP-1(7-37)OH, a- methyl-Ala -GLP-1(7-36)NHz, and Gly -Gln’ -GLP-1(7-37)OH, or pharmaceutically-acceptable salts thereof, is more preferred.

The use in the present invention of a molecule claimed in U.S. Patent No. 5,188,666, herein incorporated by reference, is preferred. 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-X (SEQ ID NO:2) wherein X is selected from the group consisting of Lys and

Lys-Gly; and a derivative of said peptide, wherein said peptide is selected from the group consisting of: a pharmaceutically-acceptable acid addition salt of said peptide; a pharmaceutically-acceptable carboxylate salt of sald peptide; a pharmaceutically-acceptable lower alkylester of said peptide; and a pharmaceutically-acceptable amide of said peptide selected from the group consisting of amide, lower alkyl amide, and lower dialkyl amide.

Another preferred group of molecules for use in the present invention consists of compounds disclosed in

U.S. Patent No. 5,512,543, herein incorporated by reference, having the general formula:

R R'-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-

Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala-Trp-

Leu-Val-Lys-Gly-Arg-R’ rR’ (SEQ ID NO:3) and pharmaceutically-acceptable salts thereof, wherein R is selected from the group consisting of 4-imidazopropionyl, 4- imidazoacetyl, or 4-imidazo-a, a dimethyl-acetyl; R is selected from the group consisting of C.-C , unbranched acyl, or is absent; R’ is selected from the group consisting of

Gly-OH or NH; and, Xaa is Lys or Arg, may be used in present invention.

More preferred compounds of SEQ ID NO:3 for use in the present invention are those in which Xaa is Arg and

R is C.-C, unbranched acyl.

Highly preferred compounds of SEQ ID NO:3 for use in the present invention are those in which Xaa is Arg,

Ris C,-C,, unbranched acyl, and R’ is Gly-OH.

More highly preferred compounds of SEQ ID NO:3 for use in the present invention are those in which Xaa is

Arg, R is C,-C,, unbranched acyl, R is Gly-OH, and R is 4- imidazopropionyl.

The most preferred compound of SEQ ID NO:3 for use in the present invention is that in which Xaa is Arg, R is

Cg unbranched acyl, Ris Gly-OH, and R' is 4- imidazopropionyl.

The use of val’-GLP-1 (7-37) OH or a ~ pharmaceutically-acceptable salt thereof, as claimed in US

Patent Number 5,705,483, herein incorporated by reference, in the present invention is highly preferred.

Methods for preparing the GLP-1, GLP-1 analogs, or

GLP-1 derivatives useful in the present invention are well- known in the art and are easily within the grasp of ordinarily skilled protein chemists or biochemists. The amino acid portion of the active compound used in the - present invention, or a precursor thereto, can be made either by solid-phase synthetic chemistry, purification of

GLP-1 molecules from natural sources, or recombinant DNA

, : . v WO 00/12116 PCT/US99/19348 technology. Routine synthetic organic techniques enable the alkylation and acylation of the GLP-1 derivatives.

The term “GLP-1 related compound” refers to any compound falling within the GLP-1, GLP-1 analog, or GLP-1 derivative definition.

The term "preservative" refers to a compound added to a pharmaceutical formulation to act as an anti-microbial agent. A parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product. Among preservatives known in the art as being effective and acceptable in parenteral formulations are benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. See, e.g., Wallhauser, K.,

Develop. Biol. Standard, 24: 9-28 (Basel, S. Krager, 1974).

The term "buffer" or "pharmaceutically acceptable buffer" refers to a compound that is known to be safe for use in protein formulations and that has the effect of controlling the pH of the formulation at the pH desired for the formulation. Pharmaceutically acceptable buffers for controlling pH at a moderately acid pH to a moderately basic pH include, for example, such compounds as phosphate, acetate, citrate, TRIS, arginine, or histidine.

The term "isotonicity agent" refers to a compound that is tolerated physiologically and imparts a suitable tonicity to a formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations.

’ ' + ‘ . -14- 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-GIn-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-

Val-X (SEQ ID NO:2) wherein X is selected from the group consisting of Lys and

Lys-Gly; and a derivative of said peptide, wherein said peptide is selected from the group consisting of: a pharmaceutically-acceptable acid addition salt of said peptide; a pharmaceutically-acceptable carboxylate salt of said peptide; a pharmaceutically-acceptable lower alkylester of said peptide; and a pharmaceutically-acceptable amide of said peptide selected from the group consisting of amide, lower alkyl amide, and lower dialkyl amide.

U.S. Patent No. 5,512,549, herein incorporated by reference, having the general formula:

R'-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-

Leu-Glu-Gly-Gln-Ala-Ala-Xaa-Glu-Phe-Ile-Ala-Trp- bea-val-iys-oly-arg-® (SEQ ID NO:3) 1 * and pharmaceutically-acceptable salts thereof, wherein R is selected from the group consisting of 4-imidazopropionyl, 4-

Claims

h Y PCT/US99/19348 CLAIMS:

1. A method of administering a glucagon-like peptide-1(GLP-1) molecule comprising, administering an effective amount of a GLP-1 molecule selected from the group consisting of GLP-1, GLP-1 analogs, or GLP-1 derivatives to a subject by pulmonary means.

2. The method of Claim 1, wherein the GLP-1 molecule is delivered to lower airways of the subject.

3. The method of Claim 2, wherein the GLP-1 molecule is deposited in the alveoli.

4. The method of Claim 1, wherein the GLP-1 molecule is inhaled through. the mouth of the subject.

5. The method of Claim 1, wherein the GLP-1 molecule is administered as a pharmaceutical formulation comprising the GLP-1 molecule in a pharmaceutically acceptable carrier.

6. The method of Claim 5, wherein the formulation is selected from the group consisting of a solution in an aqueous medium and a suspension in a non-aqueous medium.

7. The method of Claim 6, wherein the formulation is administered as an aerosol.

8. The method of Claim 5, wherein the formulation is in the form of a dry powder.

9S. The method of Claim 5, wherein the GLP-1 molecule has a particle size of less than about 10 microns MMAD. AMENDED SHEET

PCT/US99/19348

10. The method of Claim 39, wherein the GLP-1 molecule has a particle size of about 1 to about 5 microns MMAD.

11. The method of Claim 10, wherein the GLP-1 molecule has a particle size of about 2 tO about 3 microns MMAD.

12. The method of Claim 1, wherein at least about 10% of the GLP-1 molecule delivered is deposited in the lung.

13. The method of Claim 1, wherein the GLP-1 molecule is delivered from an inhalation device suitable for pulmonary administration and capable of depositing the GLP-1 molecule in the lungs of the subject.

14. The method of Claim 13, wherein the device is selected from the group consisting of a nebulizer, a metered-dose inhaler, a dry powder inhaler, and a sprayer.

15. The method of Claim 14, wherein the device is a dry powder inhaler. :

16. The method of Claim 1, wherein the GLP-1 molecule is selected from the group consisting of GLP-1 analogs and GLP-1 derivatives.

17. The method of Claim 16, wherein the GLP-1 molecule is a GLP-1 analog.

18. The method of Claim 17, wherein the GLP-1 analog is selected from the group consisting of val8-GLp- 1(7-37)0H, Gly%-GLP-1(7-37)0H, and AspS-GLP-1(7-37)OH. AMENDED SHEET

PCT/US99/19348

19. The method of Claim 18, wherein the GLP-1 analog is val®-GLP-1({7-37)CH.

20. The method of Claim 18, wherein the GLP-1 analog is Gly®-GLP-1(7-37)CH.

21. Use of a GLP-1 molecule in the manufacture of a medicament for treating diabetes by administering an effective dose of said medicament to a subject by pulmonary delivery.

22. Use according to Claim 21, wherein the GLP-1 molecule is administered as a pharmaceutical formulation comprising the GLP-1 molecule in a pharmaceutically acceptable carrier. :

23. Use according to Claim 21, wherein the GLP-1 molecule is val®-GLP-1(7-37)CH.

24. Use according to Claim 21, wherein the GLP-1 molecule is Gly8-GLP-1(7-37) OH.

25. Use according to Claim 21, wherein the GLP-1 molecule is delivered from an inhalation device suitable for pulmonary administration and capable of depositing the GLP-1 molecule in the lungs of the subject.

26. Use according to Claim 25, wherein the device is a sprayer or a dry powder inhaler.

27. Use according to Claim 25, wherein an actuation of the device administers about 40 ug to about 4,000 ug of the GLP-1 molecule. AMENDED SHEET

PCT/US99/19348

28. Use according to Claim 25, wherein an actuation of the device administers about 80 ug tc about 2,000 ug of the GLP-1 molecule.

29. Use according to Claim 25, wherein an actuation of the device administers about 160 pg to about 1,000 ug of the GLP-1 molecule.

30. Use according to Claim 25, wherein an actuation of the device administers about 320 pg to about 500 ug of the GLP-1 molecule.

31. Use of a GLP-1 molecule in the manufacture of a medicament for treating hyperglycemia by administering an effective dose of said medicament to a subject by pulmonary means.

32. Use according to Claim 31, wherein the GLP-1 molecule is administered as a pharmaceutical formulation comprising the GLP-1 molecule in a pharmaceutically acceptable carrier.

33. Use according to Claim 31, wherein the GLP-1 molecule is val3-GLP-1(7-37)OCH.

34. Use according to Claim 31, wherein the GLP-1 molecule is Gly®-GLP-1 (7-37) OH.

35. Use according to Claim 31, wherein the GLP-1 molecule is delivered from an inhalation device suitable for pulmonary administration and capable of depositing the GLP-1 molecule in the lungs of the subject. AMENDED SHEET

PCT/US99/19348

36. Use according to Claim 35, wherein the device is selected from the group consisting cf a sprayer and a dry powder inhaler.

37. Use according to Claim 35, wherein an actuation of the device administers about 40 ug to about 4,000 ug of the GLP-1 molecule.

38. Use according to Claim 35, wherein an actuation of the device administers about 80 ug to about 2,000ug of the GLP-1 molecule.

39. Use according to Claim 35, wherein an actuation of the device administers about 160 ug to about 1,000 ug of the GLP-1 molecule.

40. Use according to Claim 35, wherein an actuation of the device administers about 320 ug to about 500 pg of the GLP-1 molecule.

41. A substance or composition for use in a method of treating diabetes, said substance or composition comprising a GLP-1 molecule, and said method comprising administering an effective dose of said substance or composition to a subject by pulmonary delivery.

42. A substance or composition for use in a method of treatment according to Claim 41, wherein the GLP-1 molecule is administered as a pharmaceutical formulation comprising the GLP-1 molecule in a pharmaceutically acceptable carrier.

43. A substance or composition for use in a method of treatment according to Claim 41, wherein the GLP-1 molecule is val®-GLP-1(7-37)CH. AMENDED SHEET

PCT/US99/19348

44. A substance or composition for use in a method of treatment acccrding to Claim 41, wherein the GLP-1 molecule is Gly3-GLP-1(7-37)OH.

45. A substance or composition for use in a method of treatment according to Claim 41, wherein the GLP-1 molecule is delivered from an inhalation device suitable for pulmonary administration and capable of depositing the GLP-1 molecule in the lungs of the subject.

46. A substance or composition for use in a method of treatment according to Claim 45, wherein the device is a sprayer or a dry powder inhaler.

47. A substance or composition for use in a method of treatment according to Claim 45, wherein an actuation of the device administers about 40 ug to about 4,000 ug of the GLP-1 molecule.

48. A substance or composition for use in a method of treatment according to Claim 45, wherein an actuation of the device administers about 80 pg to about 2,000 ug of the GLP-1 molecule.

49. A substance or composition for use in a method of treatment according to Claim 45, wherein an actuation of the device administers about 160 pg to about 1,000 ug of the GLP-1 molecule.

50. A substance or composition for use in a method of treatment according to Claim 45, wherein an actuation of the device administers about 320 pug to about 500 pg of the GLP-1 molecule. AMENDED SHEET

PCT/US99/19348

51. A substance or composition for use in a method for treating hyperglycemia, caid substance or composition comprising a GLP-1 molecule, and said method comprising administering an effective dose of said substance OT composition to a subject by pulmonary means.

52. A substance or composition for use in a method of treatment according to Claim 51, wherein the GLP-1 molecule is administered as a pharmaceutical formulation comprising the GLP-1 molecule in a pharmaceutically acceptable carrier.

53. A substance or composition for use in a method ’ of treatment according to Claim 51, wherein the GLP-1 molecule is val8-GLP-1(7-37) CH.

54. A substance or composition for use in a method of treatment according to Claim 51, wherein the GLP-1 molecule is Gly3-GLP-1(7-37)OH.

55. A substance or composition for use in a method of treatment according to Claim 51, wherein the GLP-1 molecule is delivered from an inhalation device suitable for pulmonary administration and capable of depositing the GLP-1 molecule in the lungs of the subject.

56. A substance or composition for use in a method of treatment according to Claim 55, wherein the device is selected from the group consisting of a sprayer and a dry powder inhaler.

57. A substance or composition for use in a method of treatment according to Claim 55, wherein an actuation of the device administers about 40 ug to about 4,000 ug of the GLP-1 molecule. AMENDED SHEET

PCT/USS99/19348 -4 1 -

58. A substance or composition for use in a method of treatment according to Claim E55, wherein an actuation of the device administers about 80 ug to about 2,000 ug of the GLP-1 molecule.

59. A substance or composition for use in a method of treatment according to Claim 55, wherein an actuation of the device administers about 160 ug to about 1,000 ug of the GLP-1 molecule.

60. A substance or composition for use in a method of treatment according to Claim 55, wherein an actuation of the device administers about 320 ug to about 500 ug of the GLP-1 molecule.

61. Use of a GLP-1 molecule selected from the group consisting of GLP-1, GLP-1 analogs, or GLP-1 derivatives in the manufacture of a preparation for use in a method of administering an effective amount of a GLP-1 molecule to a subject by pulmonary means.

62. A substance or composition for use in a method of administering a GLP-1 molecule, said substance or composition comprising a GLP-1 molecule selected from the group consisting of GLP-1, GLP-1 analogs, or GLP-1 derivatives, and said method comprising administering an effective amount of said substance Or composition to a © subject by pulmonary means.

63. A method according to Claim 1, substantially as herein described and illustrated.

64. Use according to Claim 21, Claim 31 or Claim 61, substantially as herein described and illustrated. AMENDED SHEET

PCT/US99/19348

65. A substance or composition for use in a method of treatment according tc claim 41 or Claim 51, substantially as herein described and illustrated.

66. A substance or composition for use in a method of administration according to Claim 62, substantially as herein described and illustrated.

67. A new method of administration; new use of a GLP-1 molecule or a GLP-1 molecule selected from the group consisting of GLP-1, GLP-1 analogs, or GLP-1 derivatives; or a substance or composition for a new use in a method of treatment or in a method of administration, substantially as herein described. AMENDED SHEET