MXPA99001873A - Use of glp-1 or analogs in treatment of myocardial infarction - Google Patents

Abstract

This invention provides a method of reducing mortality and morbidity after myocardial infarction. GLP-1, a GLP-1 analog, or a GLP-1 derivative, is administered at a dose effective to normalize blood glucose.

Description

USE OF GLP-1 OR ANALOGS OF THE SAME IN THE TREATMENT OF MYOCARDIAL INFARCTION BACKGROUND OF THE INVENTION 1. Field of Invention This invention relates to a method for reducing mortality and morbidity after myocardial infarction in diabetic patients. 2. Background information. Morbidity and mortality from cardiovascular disease is higher in patients who manifest diabetes or impaired glucose tolerance, compared to patients without these disorders. Diabetics represent up to 24% of the total number of patients admitted to coronary care units for suspected infarction, while these constitute only approximately 5% of the general population [Malmber and Rydén; Fuller J.H., Di abe t. I tab. 1 9: 96-99 (1993)]. The mortality of diabetic patients in hospital with myocardial infarction is twice that of non-diabetics [Hamsten A., et al., J. In t. Med. 736: 1-3 (1994) Malmberg K. and Rydén L., Eur. Heart J. 9: 256-264 (1988)]. Diabetics REF .: 29518 experience more morbidity and die more frequently in the post-acute recovery phase, mainly due to fatal reinfarction and congestive heart failure [Mal berg and Rydén; Stone P. et al., J. Am. Coi l. Cardi ol. 14: 49-57 (1989); Karlson B.W. and collaborators, Di abe t. Med. 10 (5): 449-54 (1993); Barbash G.I. and collaborators, J. Am. Coll. Cardi ol. 22: 707-713 (1993)]. The difference in mortality and morbidity between diabetics and non-diabetics after myocardial infarction persists, despite the reduction in the incidence of morbidity and mortality after acute myocardial infarction [Granger C.B. and collaborators, J. Am. Coll. Cardi ol. , 21 (4): 920-5 (1993); Grines C. et al., N. Engl. J. Med. 328: 673-679 (1993)]. The factors responsible for the poor prognosis among diabetic patients with acute myocardial infarction, can act before, during or after the acute event. These include diffuse coronary atheromatosis, with more advanced and widespread coronary artery disease, which, together with possible diabetic cardiomyopathy, may contribute to a high prevalence of congestive heart failure. The autonomic neuropathy with deteriorated perception of pain and variability of the remaining cardiac rhythm, increased, may also be of importance. A coronary thrombus is an essential part of an evolving infarction, and principally, it has been found that platelet activity, coagulation and fibrinolytic functions are disturbed in diabetic patients [Davi G. et al., New Engl. J. Med., 322: 1769-1774 (1990)]. The exaggerated metabolic disturbances in diabetics can play an important role. Myocardial infarction causes a reduction in circulating insulin, a dramatic increase in adrenergic tone, and the release of stress or tension hormones, such as cortisone, catecholamines, and glucagon, which together increase hyperglycemia and stimulate lipolysis. The free fatty acids released further deteriorate the myocardium by various mechanisms, and excessive oxidation of free fatty acids can possibly damage the nonischemic parts of the myocardium [Rodrigues B. et al., Cardi ova scul ar Research, 26 (10) : 913-922 (1992)]. Palliative measures are necessary to normalize blood glucose in diabetics to control the metabolic cascade that exacerbates infarct damage. In a recent trial, the improved metabolic care of diabetic patients during acute myocardial infarction, including the carefully verified infusion of insulin and glucose, and the strong post-acute regulation of blood glucose by treatment with multiple dose insulin , subcutaneous, decreased mortality during the year after myocardial infarction by 30%, compared with a control group of diabetics who did not receive insulin treatment, unless considered clinically necessary [Malmberg, K. et al., J. Am. Coll ege Cardi olgy, 26: 57-65 (1995)]. Infusion with insulin, however, creates the potential for hypoglycemia, which is defined as blood glucose below 0.3 mM. Hypoglycemia increases the risk of ventricular arrhythmia and is a dangerous consequence of insulin infusion. An algorithm for insulin infusion for diabetics with myocardial infarction was developed to prevent hypoglycemia [Hendra, T.J. and collaborators, Di abe t es Res. Cl in. Pra c t. 16: 213-220 (1992)]. However, 21% of patients developed hypoglycaemia under this algorithm. In another study of glucose control after myocardial infarction, 18% of patients developed hypoglycemia when infused with insulin and glucose [Malmberg, K.A. and collaborators, Di abe t es Care, 17: 1007-1014 (1994)]. Infusion with insulin also requires frequent periodic verification of blood glucose levels, so that the onset of hypoglycaemia can be detected and remedied as soon as possible. In patients who receive infusion with insulin in the study cited [Malmberg, 1994], blood glucose was measured at least every second hour, and consequently the infusion rate was adjusted. Thus, the safety and efficacy of insulin-glucose infusion therapy for patients with myocardial infarction depends on quick and easy access to blood glucose data. Such an intense need to periodically check blood glucose places a heavy burden on health care professionals, and increases the inconvenience and cost of treatment. As a result, intensive care cardiac units often do not allocate resources to optimize blood glucose levels in diabetics with acute myocardial infarction, as can be obtained by intravenous administration of insulin. Considering the risks and burdens inherent in insulin infusion, an alternative procedure is needed to manage blood glucose during acute myocardial infarction in diabetics. The incretin hormone, glucagon-like peptide 1, abbreviated as GLP-1, is processed from proglucagon in the intestine and improves nutrient-induced insulin release [Krcymann B. and colleagues Lance t 2: 1300-1303 (1987)]. Various truncated forms of GLP-1 which stimulate insulin secretion (insulinotropic action) and the formation of cyclic AMP (cAMP) are known [see, for example, Mojsov, S., Int. J. Peptide Protein Research, 40: 333-343 (1992)]. A relationship has been established between various in vitro laboratory experiments and the insulinotropic responses of mammals, especially humans, to the exogenous administration of GLP-1, GLP-1 amide (7-36) and GLP-1 acid (7). -37) [see, for example, Nauc, MA and collaborators, Di abe t ol ogi a, 36: 741-744 (1993); Gutniak, M. et al., New Engl and J. of Medi cine, 326 (20): 1316-1322 (1992); Nauck, M.A. and collaborators, J. Clin. In ves t. , 91: 301-307 (1993); and Thorens, B. et al., Di abe t es, 42: 1219-1225 (1993)]. The amide GLP-1 (7-36) exerts a pronounced anti-diabetic effect in insulin-dependent diabetics by stimulating insulin sensitivity and by improving the glucose-induced release of insulin at physiological concentrations [Gutniak M. et al., New Engl and J. Med. 326: 1316-1322 (1992)]. When administered to noninsulin-dependent diabetics, the GLP-1 (7-36) amide stimulates insulin release, decreases glucagon secretion, inhibits gastric emptying and improves glucose utilization [? Auck, 1993; Gutniak, 1992; Auck, 1993]. The use of GLP-1 type molecules for the prolonged therapy of diabetes has been obstructed because the serum half-life of such peptides is very short. For example, GLP-1 (7-37) has a serum half-life of only 3 to 5 minutes. The GLP-1 amide (7-36) has a half-life of approximately 50 minutes when administered subcutaneously. Thus, these GLP-1 molecules should be administered as a continuous infusion to achieve a prolonged effect [Gutniak M. et al., Di abe t es Care 17: 1039-1044 (1994)]. In the present invention, the short half-life of GLP-1 and the consequent need for continuous administration are not disadvantages because the patient is typically encaged in a cardiac intensive care unit, where the fluids are continuously administered from parenteral way.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for reducing mortality and morbidity after myocardial infarction, which comprises administering a compound from the group consisting of GLP-1., GLP-1 analogs, GLP-1 derivatives and the pharmaceutically acceptable salts thereof, at an effective dose to normalize the blood glucose, to a patient in need thereof. The present invention provides the benefits of the reduction of mortality and morbidity after myocardial infarction, observed in the combined treatment with glucose and insulin in diabetics during acute myocardial infarction, but without the inconvenience and expensive requirement of verification frequent periodic blood glucose, interpretation of blood glucose results, and adjustment of insulin dosing rate, and without the ever present risk of hypoglycaemia accompanying insulin infusion.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph showing the effect of continuous infusion of the GLP-1 amide (7-36) on the average blood glucose concentration (mM) (-B-) in five patients with NIDDM during the night. The graph also describes the effect of continuous insulin infusion on the average blood glucose concentration (--O--) in the same five patients with NIDDM, but on a different night. Figure 2 is a graph showing the effect of GLP-1 amide infusion (7-36) on the average blood glucose concentration (mM) (- M-) in five patients with NIDDM when infused during the day , for three hours starting at the beginning of each of the three meals. The graph also describes the effect of subcutaneous insulin injection on the average blood glucose concentration (-O -) in the same five patients with NIDDM, but on a different day, and with the injection shortly before each meal.

DETAILED DESCRIPTION OF THE INVENTION "GLP-1" means GLP-1 (7-37). By custom in the art, the amino terminus of GLP-1 (7-37) has been assigned with the number 7 and the carboxyl terminus with the number 37. The amino acid sequence of GLP-1 (7-37) is well known in the technique, but is presented later for the convenience of the reader.

NH2-Hi s7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu-Gly-Gln-Ala-Al a25-Lys-Glu-Phe- I e-Ala30- Trp-Leu-Val-Lys-Gly35-Arg-Gly37-COOH (SEQ ID NO.1) A "GLP-1 analog" is defined as a molecule that has one or more substitutions, deletions, inversions or additions of amino acids compared to GLP-1. GLP-1 analogs known in the art include, for example, GLP-1 (7-34) and GLP-1 (7-35), GLP-1 (7-36), Gln9-GLP-1 (7- 37), D-Gln9-GLP-1 (7-37), Thr16-Lys18-GLP-1 (7-37) and Lys18-GLP-1 (7-37). Preferred GLP-1 analogs are GLP-1 (7-34) and GLP-1 (7-35), which are described in US Pat. No. 5,118,666, incorporated by reference herein, and also GLP-1. (7-36), which are biologically processed forms of GLP-1 that have insulinotropic properties. Other GLP-1 analogs are described in U.S. Patent No. 5,545,618, which is incorporated by reference herein. A "GLP-1 derivative" is defined as a molecule having the amino acid sequence of GLP-1 or a GLP-1 analog, but which additionally has chemical modification of one or more of its amino acid side groups, the atoms of carbon, the terminal amino group or the terminal carboxylic acid group. A chemical modification includes, but is not limited to, the addition of chemical portions, creating new bonds, and eliminating chemical portions. Modifications in the amino acid side groups include, without limitation, the acylation of the e-amino groups of the usine, the N-alkylation of the arginine, histidine or usine, the alkylation of the carboxylic, glutamic or aspartic acid groups, and the deamidation of glutamine or asparagine. Modifications of the terminal amino group include, without limitation, modifications such as des-amino, N-lower alkyl, N-di-lower alkyl and N-acyl. Modifications of the terminal carboxyl group include, without limitation, modifications such as amide, lower dialkyl amide, dialkyl amide, and lower alkyl ester. The lower alkyl is alkyl of 1 to 4 carbon atoms. In addition, one or more side groups or terminal groups may be protected by protective groups known to the chemist of ordinary protein experience. The carbon a of an amino acid can be mono- or dimethylated. A preferred group of GLP-1 analogs and derivatives for use in the present invention is composed of molecules of the formula: Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20- and -Gly- Gln-Ala-Ala25-Lys-Z -Phe-Ile-Ala30- Trp-Leu-Val-Lys-Gly 3J5 -Arg-R2 (SEQ ID NO.2) and pharmaceutically acceptable salts thereof, wherein: Ri is selected from the group consisting of L-histidine, D-histidine, desamino-histidine, 2-amino-histidine, ß-hydroxy-histidine, ho ohistidina, alpha-fluoromethyl -histidine, and alpha-methyl-histidine; X is selected from the group consisting of Ala, Gly, Val, Thr, lie and alpha-methyl-Ala; And it is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; Z is selected from the group consisting of Glu, Gln, Ala, Thr, Ser, and Gly; and R2 is selected from the group consisting of NH2, and Gly-OH; with the proviso that the compound has an isoelectric point in the range of about 6.0 to about 9.0 and with the additional proviso that when Ri is His, X is Ala, Y is Glu, and Z is Glu, R2 must be NH2. The numerous analogues and derivatives of GLP-1 having an isoelectric point in this range have been described and include, for example: GLP-1 (7-36) NH2 Gly8-GLP-1 (7-36) NH2 Gln9-GLP -1 (7-37) D-Gln9-GLP-1 (7-37) acetyl-Lys9-GLP-I (7-37) Thr9-GLP-1 (7-37) D-Thr9-GLP-1 (7 -37) Asn9-GLP-1 (7-37) D-Asn9-GLP-1 (7-37) Ser22-Arg23-Arg24-Gln26-GLP-1 (7-37) Thr16-Lys18-GLP-1 (7 -37) Lys18-GLP-1 (7-37) Arg23-GLP-1 (7-37) Arg24-GLP-1 (7-37), and the like [see, for example, W091 / 11457]. Another preferred group of active compounds for use in the present invention is described in International Patent WO 91/11457, and consists essentially of GLP-1 (7-34), GLP-1 (7-35) -, GLP-1 (7-36), or GLP-1 (7-37), or the amide form thereof, and pharmaceutically acceptable salts thereof, having at least one modification selected from the group consisting of: a) substitution 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 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; power plant by serine in position 18; aspartic acid by glutamic acid in position 21; serine by glycine in position 22; arginine for glutamine in position 23; arginine by alanine in position 24; and glutamine by Usina in position 26; 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 by glutamic acid in position 9; serine, cysteine, threonine, asparagine, glutamine, tyrosine, alanine, valine, isoleucine, leucine, methionine, or phenylalanine by glycine in position 10; and glutamic acid by aspartic acid in the 15-position; 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; where, in the substitutions of (a), (b), (d) and (e), the substituted amino acids may be optionally in the D form and the amino acids substituted in the 7 position may 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 the administered GLP-1 [see, for example, Mentlein, R. et al., Eur. J. Bi ochem. , 214: 829-835 (1993)], administration of GLP-1 analogs and derivatives that are protected from DPP IV activity, is preferred, and administration of Gly8-GLP-1 (7-36) NH2, Val8-GLP-1 (7-37) OH, α-methyl-Ala8-GLP-I (7-36) NH2, and Gly8-Gln21-GLP-1 (7-37) OH, or the pharmaceutically acceptable salts thereof, is more preferred . The use in the present invention of a molecule claimed in US Pat. No. ,188,666, which is expressly incorporated by reference herein, is preferred. Such a molecule is selected from the group consisting of a peptide having the amino acid sequence: NH2-His7-Ala-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20-Glu- Gly-Gln-Ala-Ala25-Lys-Glu-Phe-Ile-Ala30- Trp-Leu-Val-X (SEQ ID No. 3) wherein X is selected from the group consisting of Lys and Lys-Gly; and a derivative of said peptide, wherein the peptide is selected from the group consisting of: a pharmaceutically acceptable acid addition salt of the peptide; a pharmaceutically acceptable carboxylate salt d-e said peptide; a pharmaceutically acceptable lower alkyl ester 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 the compounds, claimed in U.S. Patent No. 5,512,549, which is expressly incorporated by reference herein, of the general formula: R ^ Ala-Glu-Gly10- Thr-Phe-Thr-Ser-Asp15-Val-Ser-Ser-Tyr-Leu20- Glu-Gly-Gln-Ala-Ala25-Xaa-Glu-Phe-IIe-Ala30- Trp-Leu-Val-Lys-Gly35- Arg-R3 I R2 (SEQ ID No. 4) and the pharmaceutically acceptable salts thereof, wherein R1 is selected from the group consisting of 4-imidazopropionyl, 4-imidazoacetyl, or 4-imidazo-a, a-dimethyl- acetyl; R2 is selected from the group consisting of unbranched acyl of 6 to 10 carbon atoms, or absent; R3 is selected from the group consisting of Gly-OH or NH2; and Xaa is Lys or Arg, which can be used in the present invention. The most preferred compounds of SEQ ID NO. 4 for use in the present invention are those in which Xaa is Arg and R2 is unbranched acyl of 6 to 10 carbon atoms. The highly preferred compounds of the SEQ ID NO. 4 for use in the present invention are those in which Xaa is Arg, R 2 is unbranched acyl of 6 to 10 carbon atoms, and R 3 is Gly-OH The most highly preferred compounds of SEQ ID NO. 4 for use in the present invention, are those in which Xaa is Arg, R2 is unbranched acyl of 6 to 10 carbon atoms, R3 is Gly-OH, and R1 is 4-imidazopropionyl. The most preferred compound of SEQ ID DO NOT. 4 for use in the present invention is one in which Xaa is Arg, R2 is unbranched acyl 8 carbon atoms, R3 is Gly-OH, and R1 is 4-imidazopropionyl. The use in the present invention of a molecule claimed in U.S. Patent No. 5,120,712, which is expressly incorporated by reference herein, is highly preferred.

Such a molecule is selected from the group consisting of a peptide having the amino acid sequence: NH2-His7-ALa-Glu-Gly10-Thr-Phe-Thr-Ser-Asp15-VaL-Ser-Ser-Tyr-Lau20-Glu- Gly-Gln-Ala-Ala ^ -Lys-Glu-Phe-Ile-Ala30- Trp-Leu-Val-Lys-Gly35-Arg-Gly37-C00H1 (SEQ ID NO: 1) and a derivative of said peptide: wherein the 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 alkyl ester 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. The use of GLP-1 (7-36.} .ANG. Amide or a pharmaceutically acceptable salt thereof, in the present invention is more highly preferred.The amino acid sequence of the GLP-1 amide (7-36) is : NH2His7-Ala-Glu-Gly10-Thr-Phe-Thr-S-Asp15-Val-S-Se-Tyr-Leu20-. Glu-Gly-Gln-Al a-Al a25-Lys-Glu-Phe-XI e-Al a30- Trp-Le? Ir-Val-Lys-Gly35-Arg-NH2 (SEQ ID NO.5) The methods to prepare the. active compound used in the present invention, namely GLP-1, an analog of G.LP-1, or a GLP-1 derivative used in the present invention, are well known, and are described in the Patents North American Nos. 5,118.66, 5,120,712, and ,523,549, which are incorporated by reference herein. The amino acid portion of the active compound used in the present invention, or a precursor thereof, is made either by 1) synthetic chemistry in solid phase; 2). the purification of GLP molecules from natural sources; or 3) recombinant DNA technology. The chemical synthesis in solid phase of the polypeptides is well known in. the technique and can. be found in general texts in the area such as Dugas, H. and Penney, C, Bi oorgani c Chemi s try, Springer-Verlag, New York (1981), pp. 54-92, Merrifield, J.M., Chem. Soc, 85: 2149 (1962), and Stewart and Young, Soli d Phase - Pepti by Syn thesi s, Freeman, San Francisco (1969) p. 24-66 .. For example, the amino acid portion can be synthesized by solid-phase methodology using a 43 OA peptide synthesizer (PE-Applied Biosystems, Inc., 850 Lincoln Center drive, Foster City, CA 94404) and cycles of synthesis supplied by PE-Applied Biosystems. BOC-amino acids and other reagents are commercially available from PE-Applied Biosystems and other chemical supply companies. The sequential Boc chemistry using the double-pair protocols is applied to the initial p-methyl-benzhydril-amine resins, for the production of C-terminal carboxamides. For the production of the C-terminal acids, the corresponding PAM resin is used. Asn, Gln, and Arg are coupled using the preformed hydroxybenzotriazole esters. The following side chain protective groups can be used: Arg, Tosyl Asp, cyclohexyl Glu, cyclohexyl Ser, Benzyl Thr, Benzyl Tyr, 4-bromocarbobenz.oxi Boc deprotection can be achieved with trifluoroacetic acid in sodium chloride. methylene. After completion of the synthesis the peptides can be de-protected and cleaved from the resin with anhydrous hydrogen fluoride (HF) containing 10% .meta-cresol ... The cleavage of the a of the protecting groups of the side chain and the peptide from the resin is carried out at -5 ° C to 5 ° C, preferably on ice for 60 minutes. After the removal of the HF, the gone peptide / resin is washed with ether, and the peptide is extracted with ascetic acid .gl a ci ai y. is lio.fiii.z_a. Techniques well known to those of ordinary skill in the art in recombinant DNA technology can be used to prepare the compound. active. used. in the .. present., invention. In fact, recombinant DNA methods can be preferred due to the higher yield. The basic steps in the production rec_u__m.bin.ante are: a) isolation of a sequence of natural ADJSf that codes for, a molecule of GLP-1, or the construction of a coding sequence of synthetic or semi-synthetic DNA for a molecule of GLP-1, b) the placement of the coding sequence within an expression vector, in a manner suitable for the expression of proteins already. either alone or as a fusion protein, c) the transformation of an appropriate prokaryotic or eukaryotic host cell, with the expression vector, d) the cultivation of the transformed host cell under conditions that will allow the expression of a GLP molecule -L *. Y. e) the recovery and purification of the GL.P-1 molecule recombinantly produced. As previously stated, the coding sequences can be completely synthetic or the result of modifications to the DNA encoding the larger, active glucagon.

A DNA sequence encoding the preproglucagon is presented in Lund et al., Proc. Na ti. Aca d. Sci. U. S A 79: 345-349 (1982) and can be used as starting material in the semisynthetic production -of the compounds of the present invention, by altering the native sequence to achieve the desired results .. Synthetic genes, transcription and translation in vi tro a ± n vivo of which gives. As a result the production of a GLP-1 molecule can be constructed by techniques well known in the art. Due to the natural degeneracy of the genetic code, one skilled in the art will recognize that a considerable but defined number of DNA sequences can be constructed, all of which code for GLP-1 molecules. The methodology of synthetic gene construction is well known in the art. See Brown et al. (1979) Me thods in Enzymol ogy, Academic Press, N.Y.,. Vol. 68, pages 109-151. The DNA sequence is designed from the desired amino acid sequence using the genetic code, which is easily ascertained by the biologist of ordinary experience in the art. Once designed, the sequence itself can be generated using the conventional apparatus for DNA synthesis such as the Model 380A or 38OB DNA synthesizers (PE-Applied Biosystems, Inc., 85 W. Lincoln Center Drive, Foster City, CA 94404) . To express the amino acid portion of a compound used in the present invention, the synthetic DNA sequence, engineered, is inserted into any of the many appropriate recombinant DNA expression vectors through the use of restriction endonucleases. appropriate. See in general Maniatis et al. (1989) Mol ecul ar Cloning; TO Labora Tory Manual, Cold Springs Harbor Laboratory Press, N.Y., Vol. 1-3. The restriction endonuclease cleavage sites are genetically engineered at either end of the DNA encoding the GLP-1 molecule to facilitate the isolation of, and integration into, the amplification and expression vectors well known in the art. . The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector used. The restriction sites are chosen to appropriately orient the coding sequence with the control sequences, thereby achieving the appropriate reading in structure and the expression of. the. protein of interest. The coding sequence must be placed to be in proper reading structure with the promoter and ribosome binding site of the expression vector, which are functional in the host cell in which the protein is to be expressed. To achieve efficient transcription of the synthetic gene, it must be operably associated with a promoter-operator region. Therefore, the promoter-operator region of the synthetic gene is placed in the same sequential orientation with respect to the codon. of start T.G d l. gene ... synthetic. A variety of are well known in the art. expression vectors useful for transforming prokaryotic and eukaryotic cells. See TJie Prome.ga Biol ogi.cal Research Producer ts Ca tal ogue (1992) (Pro ega Corp., 2800 Woods Hollow Road, Madison, Wl, 53711-5399); and The Strata Gene Clinging Sys t ems Ca tal ogue (1992) (Stratagene Corp., 11011 North, Torxe.and Pines Road, La Jolla, CA, 92037). Also, the Patent North American No. 4,710,473. describes the transformation vectors of circular DNA plasmids, useful for the expression of exogenous genes in E. col i at high levels. These plasmids are useful as transformation vectors in recombinant DNA procedures and a) confer on the plasmid the ability for autonomous replication in a host cell; b) control the autonomous replication of the plasmid in relation to the temperature at which the host cell cultures are maintained; c) stabilize the maintenance of the plasmid in the host cell populations; d) direct the synthesis of a protein product, indicator of the maintenance of the plasmid in a population of host cells; e) provide the restriction, in series, restriction sites unique to the plasmid; and f) terminate mRNA transcription. These circular DNA plasmids are useful as vectors in procedures, of recombinant DNA to ensure high levels of expression of exogenous genes.

Having constructed an expression vector for the amino acid portion of a compound used in the present invention, the next step is to place the vector within an appropriate cell and thereby construct a recombinant host cell, useful for expression of the polypeptide. Techniques for transforming cells with recombinant DNA vectors are well known in the art, and can be found in general references such as Maniatis et al. Supra. The host cells can be constructed already e.ea from eukaryotic or prokaryotic cells. Prokaryotic host cells generally produce the protein at higher rates, and they are. easier to grow. Proteins expressed in high level bacterial expression systems are typically added in granules or in inclusion bodies, which contain high levels of overexpressed protein. Such protein aggregates should typically be recovered, solubilized, denatured and refolded using techniques well known in the art. See Krenger et al. (1990) in Pro t ein Folding, Gierasch and King, eds., Pages 136-142, American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C.; and U.S. Patent No. 4,923,967. Alterations to a precursor amino acid sequence of GLP-1 or the GLP-1 analog to produce a GL_P-1 analog deduced or GLP-1 derivative are carried out by well-known methods: chemical modification, enzymatic modification or combination of chemical modification and enzym-C_a ,. of pr ___ mr .__. res of GLP-1 .. The s. Techniques of classical methods in solution phase and semi-synthetic methods, can also be useful for the preparation of the GLP-1 molecules used in the present invention. The methods for preparing the GJL.P-1 molecules of the present invention are well known to the chemist of ordinary peptide experience. The addition of an acyl group to the group. epsilon-amino of Lys34 can be achieved using any of a variety of methods known in the art. technique .. See Bi oconj uga t e Chem. "Chemical Modifications of Proteins: History and Applications" 'pages 1, 2-12 (1990) and Hashimoto et al., Pha rma ceuti cal Res. 6 (2): 171-176 (1989). For example, an N-hydroxy-succinimide ester of octanoic acid can be added to the lysyl-epsilon-amine using 50% acetonitrile in borate buffer. ELI peptide can be acylated either before or after the im.idaz.ol group is added. In addition, if the peptide is prepared recombinantly, acylation is possible before enzymatic cleavage. Also, the plant in the GLP-1 derivative can be acylated as shown in International Patent W096-29342, which is incorporated by reference in the present. The existence and preparation of a plurality of functional analogues and derivatives of amide molecules of GLP-1 (7-36) and GLP-1 (7-37) deprotected, and partially protected, natural have been described in the art. and non-natural, [see for example, US Pat. Nos. 5,120,712 and 5,118,666, which are incorporated by reference herein, and Orskov, C. et al., J. Bi ol. Chem. , 264 (22): 12826-12829 (1989) and W091 / 11457 (Burckley, D. I. et al., Published August 8, 1991)]. Optionally, the amino- and carboxyl-terminal amino acid residues of the GLP-1 derivatives can be protected or, optionally, only one of the ends is protected. Reactions for the formation and removal of such protecting groups are described in standard works including, for example, "Protective Groups in Organic Chemistry," Plenum Press, London and New York (1973); Green, T.H., "Protective Groups in Organic Synthesis ", Wiley, New York (1981); and "The Peptides", Vol. I, Schroder and Lübke, Academic Press London and New York: (1965). Representative amino protecting groups include, for example, formyl, acetyl, isopropyl, butoxycarbonyl, fluorenylmethoxycarbonyl, carbobenzyloxy, and the like. Representative carboxyl protecting groups include, for example, benzyl ester, methyl ester, ethyl ester, t-butyl ester, p-nitrofenyl ester, and the like. The carboxyl-terminal lower alkyl ester GLP-1 derivatives, used in the present invention, are prepared by reaction of the desired alkanol of 1 to 4 carbon atoms with the desired polypeptide in the presence of a catalytic acid such as hydrochloric acid. Appropriate conditions for such formation of the alkyl ester include a reaction temperature of about 50 ° C and a. reaction time from about 1 hour to about 3 hours. Similarly, the alkyl ester derivatives of the Asp and / or Glu residues can be formed. The preparation of a carboxamide derivative of a compound used in the present invention is formed, for example, as described in Stewart, JM et al., Soli d Pha se Pepti by Syn thesi s, Pierce Chemical Company Press, 1984. use in the present invention a pharmaceutically acceptable salt form of GLP-1, a GLP-1 analog or a GLP-1 derivative. The acids commonly used to form the 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 salts of sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monoacid phosphate, diacid phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate. , caproate, heptanoate, propiolate, axalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butin-1,4-dioate, exin-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate , sulfonate, xylene sulfonate, phenylacetate, phenylprapionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propansul fonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred acid addition salts are those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and, especially, hydrochloric acid. The base addition salts include those derived from inorganic bases, such as the ammonium or alkali metal or alkaline earth metal hydroxides, carbonates, bicarbonates and the like. Such bases useful in the preparation of the salts of this invention, thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate and the like. Saline forms are particularly preferred. A GLP-1, GLP-1 analog or GLP-1 derivative used in the present invention can be formulated with one or more excipients before use in the present invention. For example, the active compound used in the present invention can be complexed with a divalent metal cation by well-known methods. Such metal cations include, for example, Zn ++, Mn ++, Fe ++, Co + +, Cd ++, Ni + +, and the like. Optionally, the active compound used in the present invention can be combined with a pharmaceutically acceptable buffer, and the pH adjusted to provide acceptable stability, and an acceptable pH for parenteral administration. Optionally, one or more pharmaceutically acceptable antimicrobial agents can be added. Meta-cresol and phenol are preferred antimicrobial agents, pharmaceutically acceptable. One or more pharmaceutically acceptable salts can be added to adjust the ionic strength or tonicity. One or more excipients may be added to further adjust the isotonicity of the formulation. Glycerin is an example of an isotonicity-adjusting excipient. Administration can be by any known route, which is effective by the doctor of ordinary experience. Parenteral administration is preferred. By parenteral administration, it is commonly understood in the medical literature to inject a dosage form into the body by means of a sterile syringe or some other mechanical device such as an infusion pump. Parenteral routes include intravenous, intramuscular, subcutaneous, intraperitoneal, intraspinal, intrathecal, intracerebroventricular, intravascular, subarachnoid, and epidural routes. Routes for intravenous, intramuscular and subcutaneous administration of the compounds used in the present invention are more preferred. Routes for intravenous and subcutaneous administration of the compounds used in the invention are even more highly preferred. present invention. For parenteral administration, an active compound used in the present invention is preferably combined with distilled water to an appropriate pH. Additional pharmaceutical methods can be used to control the duration of action. Controlled release preparations can be made by using polymers to complex with or absorb the active compound used in the present invention. The prolonged duration can be obtained by the selection of appropriate macromolecules, for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene vinyl acetate, methylcellulose, carboxymethylcellulose, or protamine sulfate, and by the selection of the concentration of macromolecules, as well as the methods of incorporation, in order to prolong the release. Another possible method for extending the duration of action by controlled release preparations is to incorporate an active compound used in the present invention into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or vinylacetate copolymers. ethylene. Alternatively, instead of incorporating a compound within these polymeric particles, it is possible to trap a compound used in the present invention in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose microcapsules, or gelatin, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, or in macroemulsions. Such teachings are. describe in Remington r s Pharmaceuti cal Sci ences (1980). A diagnosis, of "myocardial infarction" is one that involves medical judgment, and typically relies on the finding of at least two of the following indications symptoms: 1) pain, of. breast of. At least 15 minutes long; 2) at least two values of serum creatine kinase and serum creatine kinase B, at least two standard deviations above the normal range, 10 to 16 hours after the onset of symptoms; 3) two or more levels of lactate dehydrogenase that are at least two standard deviations above the normal range within 48-72 hours after onset of symptoms, including an isoenzyme pattern typical of myocardial infarction; and 4) development of new Q waves and / or initial ST elevation followed by inversion of the T wave in at least two of the 12 standard ECG guidelines. The acute phase of myocardial infarction occurs during the first 72 hours after the onset of symptoms or the indications described above. The treatment that is the objective of this invention occurs during the acute phase of myocardial infarction, that is, in acute myocardial infarction. A patient in need of the compounds used in the present invention is one who is in the acute phase of myocardial infarction, and who is also unable to self-regulate blood glucose. A patient is unable to perform self-regulation if that patient: 1) was previously diagnosed with insulin-dependent diabetes (IDDM) or non-insulin-dependent diabetes (NIDDM), according to the definitions of the National Diabetes Data Group (National Diabetes Data Group [Di abe t es, 28: 10.39-1057 (1979) 1); 2) has a blood glucose level greater than 11 mmol / l, even without. a previous diagnosis of diabetes; or 3) has an abnormal tolerance to glucose. The dose of GLP-1, of the analogue of G.LP-1 or of the GLP-1 derivative, effective to normalize a patient's blood glucose level, will depend on a number of factors, among which are included, without. limitation, sex, weight and age of the patient, the severity of the disability, to regulate blood glucose, the underlying causes of the inability to regulate blood glucose * either that the glucos.au other source of carbohydrate is simultaneously administered, the route of administration and bioavailability, persistence in the body, formulation * and potency. Where administration is continuous, an appropriate dosage rate or rate is between 0.25 and 6 pmol / kg body weight / minute, preferably from about 0.5 to about 1.2 picomole / kg / minute. Where administration is intermittent, the dose per administration should take into account the interval between doses, the bioavailability of GLP-1, the GLP-1 analog or the GLP-1 derivative, and the level necessary to affect normal blood glucose. . It is within the ordinary practitioner's experience to titrate the dose and the rate or rate of administration of GL.P-1, the GLP-1 analog or the GLP-1 derivative to achieve the desired clinical result. The present invention will be more readily understood by reference to the specific examples, which are provided to illustrate, not to limit, the present invention.

Example 1 The amide of GL.P-1 (7-36) was administered by subcutaneous infusion at a rate or dose ratio of 1.2, picomole / kg / hour, for ten hours overnight, to five patients who had non-insulin diabetes -dependent (NIDDM.). As a control, insulin was continuously infused in the same five patients, but on a different day than in the amide infusion of GLP-1 (7-36). Insulin rate or rate of insulin was adjusted every two hours to achieve optimal control, and to avoid hypoglycemia. As demonstrated by the data in. Table 1, and in Figure 1, the subcutaneous infusion of the amide of GLP-1 (7-36) almost normalized glucose, blood without inducing hypoglycemia in any of the patients. The metabolic control with the amide of GLP-1 (7-36) was better than that achieved by insulin, and the average blood glucose level was lower for the treatment with GLP-1 amide (7-36) than for the control by a statistically significant amount at 23:00, 0:00 and 1:00.

Table 1. Average blood glucose levels for five patients with NIDDM infused continuously for ten hours overnight with the GLP-1 amide (7-36). In a control study with the same patients on a different day, insulin was administered by continuous infusion.

Infusion of insulin Infusion of GLP-1 (Control) Glucose Error Glucose Blood Error Standard Blood Standard Average Hour (mM) Average (mM) (mM) (mM) 21:00 7.5 0.45 6.9 0.68 22: 00 5.4 0.76 6.6 0.55 23: 00 4.1 0.16 5.9 0.98 0:00 4.4 0.23 5.6 0.90 1: 00 4.4 0.29 5.1 0.58 2: 00 4.8 0.34 5.2 0.58. 3:00 5.2 0.41 5.4 0.30 4: 00 5.4 0.41 5.7 0.25 :00 5.8 0.41 6.0 0.30 6: 00 6.0 0.45 6.1 Q.38 7: 00 6.2 0.45 6.1 0.33 Example 2 During the day, amide GL.P-1 (7-36) was infused in five patients with NIDDM for three hours during breakfast, lunch and dinner. The hours of infusion were from 7: 30-10: 30 (breakfast), 10: 30-1: 30 (lunch), and 4: 30-7: 30 (dinner), -as shown in Figure 2. In an experiment, control in the same five patients with UTDDM conducted on a different day, insulin was injected subcutaneously just before the start of meals, as indicated in Figure 2. While GLP-1 was infused, the excursions of post-prandial glucose observed with the. Injections with insulin were eliminated, and normal blood glucose levels were maintained. Immediately after finishing each infusion with GLJP-I amide (7-36), the blood glucose level increased significantly. No side effects of the amide of GLP-1 (7-36) were observed. These data indicate that the amide infusion of. GLP-1 (7-36) more effectively controls postprandial glucose levels than insulin injection, and that. Control is effective as long as the infusion with GLP-1 amide (7-36) is continued.

Table 2. Average blood glucose levels for five patients with NIDDM infused with the GLP-1 amide. (7-36) for three hours, starting at the beginning of each meal. In a control study with the same patients on a different day, insulin was administered by subcutaneous injection just before each meal. Meals started at 7:30, 10:30 and 4:30.

Infusion with GLP-1 Subcutaneous Insulin Injection Glucose Glucose Error Sanguine Error Standard Blood Standard Average Hour (mM) Average (mM) (mM) (mM) 7: 00 5.4 0.35 6.1 0.41 8: 00 4.9 0.38 7.0 0.51 9:00 5.7 0.59 9.1 0.74 0: 00 5.8 1.06 9.9 0.7. 1:00 8.1 0.94 8.Z 0.76 2: 00 9.4 0.59 6.5 0.74 3:00 7.2 1.18 .1 0.90 4: 00 5.3 1.21 8.1 0.91 5: 00 7.2 0.71 7.0 0.87 6: 00 10.4 0.26 7.2 0.57 7: 00 9.2 1.06 6.5 Q.59 8: 00 5.7 1.59 7.3 0.65 9: 00 6.6 0.94 6.1 0.59 Infusion with GLP-1 Subcutaneous Insulin Injection Glucose Glucose Error Sanguine Error Standard Blood Standard Hour Promise (M) Promise (M) (m) (M) 20: 00 8.3 0.71 6.0 0.41 9:00 PM 9.3 0.71 6.4 0.44 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, se. Claims as property what is contained in the following:

Claims (13)

1. A method for reducing mortality and morbidity after myocardial infarction, characterized in that it comprises administering to a patient in need thereof, a compound selected from the group consisting of GLP-1, GLP-1 analogs, GLP derivatives -1, and pharmaceutically acceptable salts thereof, at an effective dose to normalize blood glucose.

2. The method according to claim 1, characterized in that the compound is administered intravenously.

3. The method according to claim 1, characterized in that the compound is administered subcutaneously.

4. The method according to claims Z. or. 3, because the administration is continuous.

5. The method of compliance with claim 4, characterized in that the rate of administration of the compound is between 0.25 and 6 picomo1 / kg / minute.

6. The method according to claim 5 * characterized in that the rate of administration of the compound is between 0.5 and 2.4 pmal / kg ./minute.

7. The method according to claim 5, characterized in that the speed is between about 0.5 and about 1.2 pmol / kg / min.

8. The method according to claim 2, characterized in that the intravenous administration is intermittent.

9. The method according to claim 2, characterized in that the compound is administered intravenously and is also administered by another parenteral route.

10. The method of compliance with claim 9, characterized in that the other parenteral route is the subcutaneous route.

11. The method according to claim 1, characterized in that the compound is administered in the amide of GLP-1 (7-36), or a pharmaceutically acceptable salt thereof.

12. A method for reducing morbidity and mortality after myocardial infarction, characterized in that it comprises administering a compound that exerts insulinotropic activity by interacting with the same receptor, or receptors, with which GLP-1, the GLP-1 analogs, interact , and the GLP-1 derivatives, when exercising their insulinotropic activity.

13. A method for reducing morbidity and mortality after myocardial infarction * characterized in that it comprises administering a compound that increases insulin sensitivity by interacting with the same receptor, or receptors, with which GLP-1, analogues interact of GLP-1, and GLP-L derivatives to increase insulin sensitivity.