Analogues of glp-1

The present invention relates to a glucagon-type peptide analogue peptide, a pharmaceutically acceptable salt thereof, the use of this compound for the treatment of mammals, and useful pharmaceutical compositions containing this analogue.

Glucagon (7-36) (GLP-1) peptide-1 amide (SEQ ED NO: 1) is synthesized in intestinal L cells as a result of tissue specific post-translational processing of glucagon precursor preproglucagon (Vamdell, JM, et al., J. Histochem Cytochem, 1985: 33: 1080-6) and is released into the bloodstream in response to food. The plasma concentration of GLP-1 increases from a constant level of approximately 15 pmoles / 1 to a maximum level of 40 pmoles / 1. It has been proven that for a given increase in plasma glucose, the increase in serum insulin is about three times higher when glucose is administered orally compared to intravascular administration (Kreymann, B., et al., Lancet 1987: 2, 1300-4). This food-related increase in insulin release also known as endocrine effect is primarily humoral and it is now known that GLP-1 is physiologically the most important incretin in humans. In addition to insulinotropic effects, GLP-1 inhibits glucagon secretion by delaying gastric emptying (Wettergren A., et al., Dig Dis Sci 1993: 38: 665-73) and may increase peripheral glucose availability (D'Alessio, DA et al., J. Clin Invest 1994: 93: 2293-6).

In 1994, therapeutic properties of GLP-1 were suggested in line with the observation that a single subcutaneous (s / c) GLP-1 dose can completely normalize glucose levels in patients with non-insulin-dependent diabetes mellitus (NIDDM) (Gutniak, MK, et al., Diabetes Care 1994: 17: 1039-1044). This effect is thought to be due to both increased insulin release and reduced glucagon secretion. It has also been shown that the GLP-1 intravenous ingot delays gastric emptying in patients with NIDDM (Williams, B., et al., J. Clin Endo Metab 1996: 81: 327-32). Unlike sulfonylurea derivatives, the insulinotropic effect of GLP-1 is dependent on serum glucose (Holz, GG 4ty., Et al., Nature 1993: 361: 362-5). Thus, the loss of GLP-1 dependent insulin release at low plasma glucose protects against acute hypoglycemia. This combination and combination of actions give GLP-1 effective, unique, therapeutic advantages over other factors currently used in the treatment of NIDDM.

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Numerous studies have shown that administration of GLP-1 to healthy individuals is effective at affecting glycemic levels as well as insulin and glucagon levels (Orskov, C, Diabetologia 35: 701-711, 1992; Hoist, JJ, et al., Potential of GLP-1 in diabetes management in Glucagon III, Handbook of Experimental Pharmacology, Lefevbre PJ, published by Berlin, Springer Verlag, 1996, p. 311-326) these effects are glucose dependent (Kreymann, B., et al., Lancet ii: 13001304, 1987; Weir, GC, et al., Diabetes 38: 338-342, 1989). In addition, GLP-1 is also effective in patients with diabetes (Gutniak, M., N. Engl J Med 226: 1316-1322, 1992; Nathan, DM, et al., Diabetes Care 15: 270-276, 1992), causing normalization of glucose levels in patients with type 2 diabetes (Nauck, MA, et al., Diagbetologia 36: 741744, 1993) and improving glycemic control in type 1 patients (Creutzfeldt, WO, et al., Diabetes Care 19: 580-586, 1996) extending the possibility of its use as a therapeutic agent.

However, GLP-1 is metabolically unstable with a serum half life (i.e. / 2) of only 1-2 min in vivo. Also exogenously administered GLP-1 is rapidly degraded (Deacon, CF, et al., Diabetes 44: 1126-1131, 1995). This metabolic instability limits the therapeutic potential of natural GLP-1. Therefore, there is a need to develop GLP-1 analogues that are more active or more metabolically stable than natural GLP-1.

The subject of the invention is a compound of:

(Aib⁸’³⁵) hGLP-1 (7-36) NH2 (SEQ ID NO: 2), or a pharmaceutically acceptable salt thereof.

The invention also relates to a pharmaceutical composition.

The essence of the invention is that it contains an effective amount of the compound (Aib^S'³⁵) HGLP-1 (7-36) Nib. a pharmaceutically acceptable salt and a pharmaceutically acceptable carrier or solvent for this compound.

The invention furthermore relates to the use of an effective amount of a compound (Aib^8,35) hGLP-1 (7-36) NH2, or a pharmaceutically acceptable salt thereof for the treatment of diseases susceptible to agonization of the GLP-1 receptor in a patient in need thereof.

The invention also relates to the use of a compound (Aib * '³⁵) hGLP-1 (7-36) NH2 for the treatment of diseases selected from the group consisting of type I diabetes, type II diabetes, obesity, glucagonoma, respiratory secretory diseases, metabolic diseases, arthritis, osteoporosis, central nervous system disease, recurrence of narrowing, neurodegenerative disease, renal failure, myocardial infarction, nephrotic syndrome, pulmonary edema, liver cirrhosis, hypertension and diseases in which it is desirable to limit food intake in a patient in need thereof, comprising administering to said patient an effective amount of a compound (A ^ / óhGLP-ipGóNHb, or a pharmaceutically acceptable salt thereof. The use of the compound in the manufacture of a medicament for the treatment of type I diabetes and type II diabetes is preferred.

With the exception of N-terminal amino acids, the amino acid abbreviations (e.g. Ala) in the disclosure correspond to the formula -NH-CH (R) -CO-, where R is the amino acid side chain (e.g., OH3 in Ala). For the N-terminal amino acid, the abbreviation corresponds to the structure (r2r3) -N-CH (R) -CO-, where R is the amino acid side chain, A and b is α-aminoisobutyric acid.

o ii

The peptide (Aib ') hGLP-1 (7-36) NH 2 can be prepared by standard solid phase peptide synthesis. See, e.g., Stewart, JM et al., Solid Phase Synthesis (Pierce Chemical Co., II Edition 1984).

In the synthesis of the GLP-1 analogue, (Aib3^,3⁵) hGLP-1 (7-36) NH2 according to the invention, the attachment time is 2 hours. for successive base-by-base attachment reactions.

The peptide of the invention may be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, salts formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic or pamoic acid), inorganic acids (e.g. hydrochloric acid, sulfuric or phosphoric acid) and polymeric acids (e.g. tannic acid, carboxymethyl cellulose, polylactic, polyglycolic or polylactic acid glycol copolymers). A typical method for preparing the peptide salts of the invention is well known and can be carried out by conventional salt exchange methods. Accordingly, the TFA salt of the peptide of the invention (the TFA salt obtained by purifying the peptide by preparative HPLC, eluting with a buffer solution of 4).

189 664 drilling TFA) can be converted into another salt, such as acetate, by dissolving the peptide in a small amount of 0.25 N acetic acid aqueous solution.

The resulting solution is applied to a semi-preparative HPLC column (Zorbax, 300 SB, CB). The column is eluted (1) for 0.5 hour. 0.1 N aqueous ammonium acetate solution (2) for 0.5 hour 0.25 N acetic acid aqueous solution and (3) a linear gradient (20% to 100% solution B over 30 min) at a flow rate of 4 ml / min (solution A is 0.25 N acetic acid in water, solution B is 0 , 25 N acetic acid in acetonitrile / water, 80:20). Peptide-containing fractions were collected and dried by lyophilization.

As is well known to those skilled in the art, the potential use of GLP-1 is broad and diverse (see, Todd, JF, et al., Clinical Science, 1998, 95, pp. 325-329; and Todd, JF in the European Journal of Clinical Investigation , 1997, 27, pp. 533-536). Thus, administration of the compounds of the invention to produce an agonist effect may have the same effect as administration of GLP-1 alone. Various applications of GLP-1 can be summarized as treatment for: type I diabetes, type II diabetes, obesity, glucagononemia, respiratory secretory diseases, metabolic diseases, arthritis, osteoporosis, central nervous system diseases, recurrence of narrowing, neurodegenerative disease, renal failure, myocardial infarction, nephrotic syndrome, pulmonary edema, liver cirrhosis, hypertension and diseases in which it is desirable to limit food intake. The GLP-1 analog of the invention, which causes an agonist effect in a patient, can be used in the treatment of: hyperglycaemia and disturbed syndrome associated with gastroectomy or removal of the small intestine.

Accordingly, the present invention includes within its scope pharmaceutical compositions wherein the active ingredient is compound (Aib^8,35) hGLP-1 (7-36) NH2 (SEQ ID NO: 2) combined with a pharmaceutically acceptable carrier.

The doses of the active ingredient according to the invention may vary; however, it is necessary that the amount of active ingredient is such that a useful dosage form can be obtained. The dose chosen depends on the desired therapeutic effect, route of administration and duration of treatment. In general, the dose of the active substance according to the invention is in the range of 1x10 '⁷up to 200 mg / kg / day, preferably 1x 10⁴ up to 100 mg / kg / day, which can be given as a single dose or divided into multiple doses.

Relationship (Aib8³⁵) hGLP-1 (7-36NHl2 (SEQ ID NO: 2) can be administered orally, parenterally (e.g. intramuscularly, intraperitoneally, intravascularly, or by subcutaneous injection or implant), into the nose, vagina, anus, under the tongue or locally and may be in a form with pharmaceutically acceptable carriers to match the dosage form to the route of administration.

Solid dosage forms for oral administration include capsules, pills, powders or granules. In such a solid form, the active compound is provided in admixture with at least one inert pharmaceutically acceptable carrier, such as sucrose, lactose or starch. Such dosage form may also include, as normal practice, additional substances other than inert diluents, e.g. wetting agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coating.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions and syrups, elixirs containing unreactive solvents commonly used by scientists, such as water. In addition to inert solvents, the composition may also contain additives such as wetting, emulsifying and suspending agents, and sweeteners, flavors and aromas.

Preparations according to the invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or carriers are polypropylene glycol, vegetable oils such as corn oil and olive oil, gelatin and injectable organic esters such as ethyl oleate. Such dosage forms may also contain additives such as preserving, wetting, emulsifying and dispersing agents. They can be sterilized, for example by filtration through a bacterial-retaining filter, inclusion of sterilizing agents in the composition, or by heating the composition. They may also be prepared in the form of a sterile solid, which may be dissolved immediately before use in water or some other sterile injectable medium.

Compositions for rectal or vaginal administration are preferably suppositories, which may contain, in addition to the active substance, fillers such as coconut butter or suppository wax.

Compositions for nasal or sublingual administration are also prepared with common additives well known in the art.

In addition, the compound of the invention may be administered in a sustained release form as described in the patents and patent applications set out below. In US Patent No. 5,672,659, sustained release compositions containing a biologically active agent and polyester are provided. U.S. Patent No. 5,595,760 describes sustained release compositions containing a biologically active agent in the form of a gel. In US Patent Application No. 08 / 929.363 filed on September 9, 1997, a sustained release polymer composition comprising a biologically active agent and chitosan is provided. In US Patent Application No. 08 / 740,778, filed November 1, 1996, a sustained release polymer composition comprising a biologically active agent and cyclodextrin is provided. In the US patent application No. 09 / 015,394 filed January 29, 1998, depicted adsorption-based sustained release compositions containing a biologically active agent. In US Patent Application No. 09/121, 653, filed June 23, 1998, a method of making microparticles containing a therapeutic agent such as a peptide in an oil-in-water process is described. In the US patent application No. 09 / 131,472 filed on August 10, 1998, complexes containing a therapeutic agent such as peptide and phosphorylated polymer are shown. In US Patent Application No. 09 / 184,413 filed on November 2, 1998, therapeutic complexes containing a therapeutic agent such as a peptide and a polymer carrying a non-polymerizable lactone are presented. The above patents are incorporated by reference.

Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art. By citation, all publications, patent applications, patents and other literature are included.

The full names of the abbreviations used here are as follows: Boo for t-butyloxycarbonyl, HF for hydrogen fluoride, Fm for formyl, Xan for xantyl, Bzl for benzyl, Tos for tosyl, DNP for 2,4-dinitrophenyl, DMF for dimethylformamide, DCM for dichloromethane, HBTU for 2- (1H -Benzotriazol-1-yl) -1,1,3,3-tetramethyl uronium hexafluorophosphate, DIEA for diisopropylethylcarboxylamine, HOAc for acetic acid, TFA for trifluoroacetic acid, 2CIZ for 2-chlorobenzyloxycarbonyl, 2BrZ for 2-bromobenzyl cyclohexyl, Fmoc for 9-fluorenylmethoxycarbonyl, HOBt for N-hydroxybenzotriazole and PAM resin for 4-hydroxymethylphenylacetamidomethyl resin.

The following examples describe a method of synthesis for preparing the peptide of the invention, which method is well known to those skilled in the art and testing the biological activity of the peptide of the invention. Other methods are also known to those skilled in the art. The examples are provided to illustrate and not intend to limit in any way the scope of the present invention.

Boc-PAla-OH, Boc-D-Arg (Tos) -OH and Boc-D-Asp (OcHex) were provided from Nova Biochem, San Diego, California. Boc-Aun-OH was provided by Bachem, King of Prussia, PA. Boc-Ava-OH and Boc-Ado-OH were supplied from Chem -mpex International, Wood Dale, IL. Boc-Nal-OH was supplied from Synthetech, Inc. Albany, OR.

Example I (Aib⁸’³⁵) HGLP-1 (7-36) NH₂

The title compound was synthesized using the Applied Biosystems peptide synthesizer (Foster City, CA) model 430A modified to accelerate solid phase synthesis. See Schnolzer, et al., Int. J. Peptide Protein Res., 90: 180 (1992). A 4-methylbenzhydrylamine (MBHA) resin (Peninsula, Belmont, CA) with a 0.91 mmol / g substitution was used. The amino acid Boc (Bachem, CA, Torrance, CA; Nova Biochem., LaJolla, CA) was used

189 664 with the following side chain protecting substituents: Boc-Ala-OH, Boc-Arg (Tos) -OH, Boc-Asp (OCHeX) OH, Boc-Tyr (2BrZ) -OH, Boc-His (DNP) -OH, Boc -VaI-OH, Boc-Leu-OH, Boc-Gly-OH, Boc-Gln-OH, Boc-IIe-OH, Boc-Lys (2CIZ) -OH, Boc-Thr (Bzl) -OH, Boc-Ser (Bzl) -OH, Boc-Phe-OH, Boc-Aib-OH, Boc-Glu (OcHex) -OH and Boc-Trp (Fm) -OH. The synthesis was carried out on a 0.20 mmol scale. Boc groups were removed by treatment with 100% TFA for 2x1 min. Boc amino acids (2.5 mmol) were pre-activated with HBTU (2.0 mmol) and DIEA (1.0 mL) in 4 m DMF and bound without prior neutralization of the peptide-resin TFA salt. The setting time was 5 min. except for Boc-Aib-OH residues and subsequent Boc-Lys (2CIZ) -OH and Boc-His (DNP) -OH residues for which the attachment time was 2 hours.

At the end of the polypeptide chain synthesis, the resin was treated with a solution of 20% mercaptoethanol / 10% DIEA in DMF for 2x30 min. to remove the DNP group in the His side chain. The Boc group at the N-terminus was removed by treatment with 100% TFA for 2x2 min. After neutralizing the peptide resin with 10% DIEA in DMF (1x1 min), the formyl group of the Trp side chain was removed by treatment with a solution of 15% ethanolamine / 15% water / 70% DMF for 2x30 min. The peptide resin was washed with DMF and DCM and dried under reduced pressure. The final cut was made by mixing the peptide resin in 10 ml HF containing 1 ml anisole and dithiothreitol (24 mg) at 0 ° C for 75 min. HF was removed by flowing nitrogen. The residue was washed with ether (6x10 mL) and extracted with 4N HOAc (6x10 mL).

The peptide mixture in the aqueous extract was purified using reverse phase high pressure chromatography (HPLC) using a VYDAC® Ci8 reverse phase column (Nest Group, Southborough, MA). The column was eluted with a linear gradient (20% to 50% solution B over 105 min) at a flow rate of 10 ml / min. (Solution A = containing 0.1% TFA water; Solution B = containing 0.1% TFA acetonitrile). Collected fractions were checked by analytical HPLC. Those that contained pure product were dried by lyophilization. 135 mg of a white solid with 98.6% purity was obtained as determined by HPLC analysis. Mass spectrometry analysis in an electron sprayer (MS (ES)) S showed a molecular weight of 3339.7 (consistent with the calculated molecular weight of 3339.7).

Example II

The compound of the invention was tested for GLP-1 binding activity according to the following procedure.

Cell culture.

RIN 5F islet cell cell adenoma (ATCC- # CRL-2058, American Type Culture Collection, Manassas, VA) cells expressing the GLP-1 receptor were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum and maintained about 37 ° C in a humidified atmosphere with 5% CO + / 95% air.

Radiolabeled ligand binding.

Membranes for radiolabeled ligand binding assay were prepared by homogenizing RIN cells in 20 ml ice-cold 50 mM Tris-HCl in Brinkman Polytron (Westbury, NY) (position 6, 15 sec.). Homogenates were washed twice by centrifugation (39,000 g / 10 min), final pellets were suspended in 50 mM Tris-HCl containing 2.5 mM MgCl2, 0.1 mg / ml bacitracin (Sigma Chemical, St. Louis, MO), and 0.1% BSA. For assay, samples (0.4 ml) were incubated with 0.05 nM (1²⁵I) GLP-1 (7-36) (SEQ ID NO: 5) (~ 2200 Ci / mmol, New England Nuclear, Boston, MA), with and without 0.05 ml of competitive unlabeled test peptide compound. After 100 min incubation (25 ° C), bound (1²5f) GLP-1 (7-36) (SEQ ID NO: 5) was separated from free by rapid filtration through Gf / C filters (Brandel, Gaithersburg, MD), which had previously been soaked in 0.5% polyethyleneimine. The filters were then washed three times in 5 ml ice-cold 50 mM Tris-HCl and the bound radioactivity remaining on the filter was measured by a gamma spectrophotometer (Wallac LKB, Gaithersburg, MD). Specific binding was defined as total binding (1²5f) GLP-1 (7-36) (SEQ ID NO: 5) minus bound in the presence of 1000 nM GLP-1 (7-36) (SEQ ID NO: 1) (Bachem, Torrence, CA).

189 664

LIST OF SEQUENCES <110> Dong, Zheng Xin <120> Analog GLP-1 <130> 00537-186002 <140> US 09 / 857,636 <141> 2001-06-07 <150> PCT / EP99 / 09660 <151> 1999- 12-07 <150> US 60 / 111.255 <151> 1998-12-07 <150> US 09 / 206.601 <151> 1998-12-07 <160> 5 <170> FastSEQ for Windows version 4.0 <210> 1 < 211> 30 <212> PRT <213> Homo sapiens <400> 1

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30 <210> 2 <211> 30 <212> PRT <213> Artificial sequence <220>

<223> Mutagen <221> VARIETY <222> 2.29 <223> Xaa = Aib (alpha-aminoisobutyric acid) <221> VARIETY <222>

<223> this sequence has an admitted end of C <400> 2

His Xaa Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Xaa Arg 20 22 30 <210> 3 <211> 31 <212> PRT <213> Homo sapiens <400> 413

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 '5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly 20 25 30

189 664 <210> 4 <211> 32 <212> PRT <213> Homo sapiens <400> 414

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Arg 20 25 30 <210> 5 <211> 30 <212> PRT <213> Artificial sequence <220>

<223> Mutagen <221> VARIETY <222> 13 <223> Xaa = Tyr radiolabelled 1251 <400> 415

His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Xaa Leu Glu Gly 1 5 10 15

Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg 20 25 30

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