MXPA99011320A - Methods for treating obesity - Google Patents

Abstract

Se describen métodos para tratar la obesidad, los cuales comprenden la administración de una cantidad terapéuticamente efectiva de una amilina o un agonista de amilina solos o en conjunto con otro agente para el alivio de la obesidad;se describen además métodos para reducir el aumento de peso inducido por insulina, los cuales comprenden la administración de una cantidad terapéuticamente efectiva de una amilina o un agonista de amilina.

Description

METHODS TO TREAT OBESITY This application is a continuation in part of the US patent application Serial No. 08 / 870,762, filed on June 6, 1997, the contents of which are incorporated herein by this reference, in its entirety.

FIELD OF THE INVENTION The present invention relates to methods for treating obesity. More particularly, the invention relates to the use of an amylin or an amylin agonist to treat obesity.

BACKGROUND THE AMILINA The structure and biology of amylin had been previously summarized. See, for example, Rink and co-authors, Trends in Pharmaceutical Sciences, 14: 113-118 (1993); Gaeta and Rink, ed. Chem. Res., 3: 483-490 (1994); and Pittner and co-authors, J. Cell. Bim., 55S: 19-28 (1994). Amylin is a protein hormone of 37 amino acids. It was isolated, purified and chemically characterized as the main component of amyloid deposits in the islets of the pancreas of human type 2 diabetics, deceased (Cooper and co-authors, Proc. Nati, Acad. Sci. USA, 84: 8628-8632 (1987)). The amylin molecule has two important post-translational modifications: the C-terminus is amidated (ie, the 37th amino acid residue is tyrosinamide) and the cysteines at positions 2 and 7 are entangled to form an N-terminal loop (via a cystine residue). The sequence of the open reading frame of the human amylin gene shows the presence of the proteolytic cleavage signal of the dibasic amino acid Lys-Arg, before the N-terminal codon for Lys, and the Gly, before the proteolytic signal Lys-Arg, in the C-terminal position, a typical sequence for amidation by PAM protein amidating enzyme (Cooper and co-authors, Bim. Biophys. Acta, 1014: 247-258 (1989)). Amylin is described and claimed in U.S. Patent No. 5,367,052, issued November 22, 1994. In type 1 diabetes, amylin has been shown to be deficient and replacement combined with insulin has been proposed as the preferred treatment with respect to insulin alone, in all forms of diabetes. The use of amylin and other amylin agonists for the treatment of diabetes mellitus is the subject of US Patent No. 5,175,145, issued December 29, 1992. Pharmaceutical compositions containing amylin and amylin plus insulin are described and claimed in U.S. Patent No. 5,124,314, issued June 23, 1992.

It has been said that the action of amylin excess mimics the key aspects of type 2 diabetes, and amylin blockade has been proposed as a novel therapeutic strategy. It has been described in US Patent No. 5,266,561, issued November 30, 1993, that amylin causes reduction in the basal, and insulin-stimulated, uptake of labeled glucose in skeletal muscle glycogen. It was also described that the latter effect was shared by the peptide related to the calcitonin gene (CGRP, acronym for its English designation: Calcitonin Gene Related Peptide) (see also Leighton and Cooper, Nature, 335: 632-635 (1988)). ). It is also reported that amylin reduces glucose uptake, stimulated by insulin, in the skeletal muscle and reduces the glycogen content (Young et al., Amer. J. Physiol., 259: 45746-1 (1990)). The treatment of type 2 diabetes and insulin resistance, with amylin antagonists, is described. The sequence of amylin has an approximate homology of 50% with the CGRP; also with 37 amino acid proteins that are widely diffused neurotransmitters, with many potent biological actions, including vasodilation. Amylin and CGRPs share the 2 Cys-7Cys disulfide bridge and a C-terminal amino acid amide residue, both of which are essential for full biological activity (Cooper and co-authors, Proc. Nati, Acad. Sci. USA, 857763-7766 ( 1988)). It is reported that amylin can be a member of a family of related peptides that includes CGRP, insulin, insulin-like growth factors, and relaxins, and they share a common genetic inheritance (Cooper and co-authors, Prog. Growth Factor Research, 1: 99-105 (1989)). Amine is synthesized primarily in beta pancreatic cells and is secreted in response to nutrient stimuli, such as glucose and arginine. Studies with tumor lines in beta cells, cloned (Moore and co-authors, Biochem Biophys, Res. Commun., 179 (1) (1991)), have shown that nutrient secretagogues, such as glucose and arginine, stimulate the release of amylin, as well as insulin. The molar ratio of amylin: insulin of the secreted proteins varies between preparations from about 0.01 to 0.4, but does not seem to vary much with acute stimuli in any preparation. However, during prolonged stimulation by high amount of glucose, the amylin: insulin ratio may increase progressively (Gedulin and co-authors, Biochem Biophys., Res. Commun., 180 (1): 782-789 (1991)). Thus, amylin and insulin are not always secreted in a constant ratio. It has been discovered and reported that certain actions of amylin are similar to some non-metabolic actions of CGRP and calcitonin; however, the metabolic actions of amylin, discovered during investigations of this newly identified protein, seem to reflect its primary biological role. At least some of these metabolic actions are imitated by CGRP; however, at doses that are remarkably vasodilatory (see, for example, Leighton and co-authors, Nature, 335: 632-635 (1988)); Molina and co-authors, Diabetes, 39: 260-265 (1990)). The first discovered action of amylin was the reduction of glucose-to-glycogen incorporation, stimulated by insulin, in the skeletal muscle of rats (Leighton and co-authors, Nature, 335: 632-635 (1988)); the muscle became "insulin resistant". Subsequent work with the rat soleus muscle ex vivo and in vitro has indicated that amylin reduces the glycogen synthase activity, promotes the conversion of glycogen phosphorylase from the inactive form "b" to the active form "a", promotes the net loss of glycogen (in the presence or absence of insulin), increases glucose 6-phosphate levels and may increase lactate output (see, for example, Deems and coauthors, Biochem. Biophys. Res. Co / 77m? < 77., 181 (1): 116-120 (1991)); Young and coauthors, FEBS Letts., 281 (1, 2): 149-151 (1991)). It appears that amylin does not affect glucose transport per se (eg, Pittner and co-authors, FEBS, Letts., 365 (1): 98-100 (1995)). Studies of dose response ratios of amylin and insulin show that amylin acts as a noncompetitive or functional antagonist of insulin in skeletal muscle (Young et al., Am. J. Physiol., 263 (2): E274-E281 (1992)). There is no evidence that amylin interferes with the binding of insulin to its receptors, or with the subsequent activation of insulin receptor tyrosine kinase (Follett et al., Clinical Research, 39 (1): 39A (1991)); Koopmans and co-authors, Diabetologia, 34: 218-224 (1991)).

It is believed that amylin acts by means of the receptors present in the membranes of the plasma. The studies of amylin and CGRP and the effect of selective antagonists suggest that amylin acts through its own receptor (Beaumont and co-authors, Br. J. Pharmacol., 115 (5): 713-715 (1995); co-authors, FEBS Letts., 219: 195-198 (1991 b)), against the conclusion of other investigators that amylin acts primarily on CGRP receptors (eg, Chantry and co-authors, Biochem. J., 2772: 139 -143 (1991)); Galeazaa and coauthors, Peptides, 12: 585-591 (1991)); Zhu and co-authors, Biochem. Biophys. Res. Commun., 177 (2): 771-776 (1991)). Amylin receptors and their use in methods for screening and analyzing amylin agonist and antagonist compounds are described in U.S. Patent No. 5,264,372, issued November 23, 1993. Although amylin has remarkable effects on the metabolism of the amylin. liver fuel in vivo, there is no general agreement as to what actions of amylin are seen in isolated hepatocytes or in perfused liver. The available data do not support the idea that amylin promotes hepatic glycogenolysis, that is, it does not act as glucagon (for example, Stephens and co-authors, Diabetes, 40: 395-400 (1991); Gomez-Foix and co-authors , Biochem J., 276: 607-610 (1991)). It has been suggested that amylin can act on the liver to promote the conversion of lactate to glycogen and to increase the amount of glucose capable of being released by glucagon (see Roden and co-authors, Diabetologia, 35: 116-120 (1992)). In this way, amylin could act as an anabolic participant of insulin in the liver, in contrast to its catabolic action in the muscle. In fat cells, contrary to their action in muscle, amylin has no detectable actions on glucose uptake, stimulated by insulin, the incorporation of glucose into triglycerides, the production of CO2 (Cooper and coauthors, Proc. Nati. Acad. Sci., 85: 7763-7766 (1988)), lipolysis stimulated by epinephrine, or inhibition of insulin lipolysis (Lupien and Young Diabetes Nutrition and Metabolism - Clinical and Experimental, volume 6 (1), pages 1318 ( February 1993)). In this way, amylin exerts tissue-specific effects, with direct action on the skeletal muscle, notable indirect effects (through substrate supply) and perhaps direct effects on the liver, while the adipocytes appear to be "blinded" by the presence or absence of amylin. It has been reported that amylin may have remarkable effects on insulin secretion. Experiments in intact rats (Young and coauthors, Mol Cell Endocrinol., 85: R1-R5 (1992)) indicate that amylin inhibits insulin secretion. However, other investigators have not been able to detect the effects of amylin on isolated beta cells, on isolated islets, or on the whole animal (see Broderick and co-authors, Biochem, Biophys, Res. Commun., 177: 932-938). (1991)). Amylin or amylin agonists potently inhibit gastric emptying in rats (Young and co-authors, Diabetologia, 38/6): 642-648 (1995)); in dogs (Brown and co-authors, Diabetes 4 (Supplement 1): 172A (1994); and in humans (Macdonald and co-authors, Diabetologia38 (Supplement 1): A32 (abstract 118) (1995)). It is reported that gastric emptying is accelerated in type 1 diabetes diabetic rats, deficient in amylin (Young and co-authors, Diabetologia, supra, Nowak and co-authors, J. Lab. Clin. Med., 123 (1): 110-6 (1994)) and in rats treated with the selective amylin antagonist AC187 (Gedulin and co-authors, DiabetologiaZQ (Supplement 1): A244 (1995).) It seems that the effect of amylin on Gastric emptying is physiological (operative at concentrations to which it normally circulates) .The non-metabolic actions of amine include vasodilatory effects that can be mediated by interaction with vascular receptors of CGRP.It is reported that living tests suggest that amylin at least it is about 100 to 1000 times less potent than CGRP as a vasodilator (Brain and co-authors, Eur. J. Pharmacol., 183: 2221 (1990); Wang and coauthors, FEBS Letts., 291: 195-198 (1991)). The effect of amylin on regional hemodynamic actions, including renal blood flow, in conscious rats has been reported (Gardiner and co-authors, Diabetes, 40: 948-951 (1991)). The authors noted that the infusion of rat amylin was associated with greater renal vasodilation and less mesenteric vasoconstriction than that seen with infusion of human alpha-CGRP. They concluded that by promoting renal hyperemia to a greater extent than alpha-CGRP, rat amylin could provoke less marked stimulation of the renin-angiotensin system and, thus, less secondary vasoconstriction, mediated by angiotensin II. However, it was also noted that, during co-infusion of human 37CGRP and rat amylin, renal and mesenteric vasoconstrictions were not masked, presumably due to the vasoconstricting effects lacking in opposition, of angiotensin II, and that this discovery is similar to that seen during coinfusion of human A-CGRP and human alpha-8"37CGRP (id. at 951). It has also been reported that amylin has effects on both osteoclasts isolated, when it caused cell inactivity, and in vivo, when it was reported that plasma calcium fell to 20% in rats, rabbits and humans, with the wrong de Paget (see, for example, Zaidi and co-authors, Trends in Endrocinal and Metab., 4: 255-259 (1993)). From the available data, it seems that amylin is 10 to 30 times less potent than human calcitonin, for these actions. Interestingly, it was reported that amylin seemed to increase the production of osteoclast cAMP, but that it did not increase cytosolic Ca2, whereas calcitonin causes both results (Alam et al., Biochem. Biophys. Res. Commun., 179 (1 ): 134-139 (1991)) It was suggested, although not established, that calcitonin can act through two types of receptor and that amylin can interact with one of them, and it has also been discovered that, surprisingly in view of its Renal and other vasodilatory properties, described above, amylin significantly increases plasma resin activity in intact rats, when administered subcutaneously in a manner that prevents any alteration in blood pressure.This last point is important because blood pressure Low is a strong stimulus for the release of renin Amylin antagonists, such as amylin receptor antagonists for CGRP and / or the receptors Calcitonin, can be used to block the elevation of plasma renin activity, evoked by amylin. The use of amylin antagonists to treat disorders related to renin is described and claimed in U.S. Patent No. 5,376,638, issued Decem27, 1994. In normal humans, fasting amylin levels have been reported. at 10 pM and post-prandial or post-glucose levels, from 5 to 20 pM (eg, Koda and co-authors, The Lancet, 339: 1179-1180 (1992)). In obese, insulin-resistant individuals, amylin levels after food may rise further, reaching up to about 50 pM. In comparison, the values for insulin in fasting and after eating food (post-prandial) are 20 to 50 pM and 100 to 300 pM, respectively, in healthy people, with levels perhaps 3 to 4 times higher in people resistant to insulin. In type 1 diabetes, in which beta cells are destroyed, amylin levels are at or below detection levels, and do not rise in response to glucose (Koda and coauthors, The Lancet, 339 : 1179: 1180 (1992)). In normal mice and rats, basal amylin levels of 30 to 100 pM have been reported, while values up to 600 pM have been measured in certain diabetic, insulin-resistant strains of rodents (eg, Huang and co-authors, Hypertension, 19: 1- 101-1-109 (1991) Injected into the brain or peripherally administeredIt has been reported that amylin suppresses food intake, for example, Chance and co-authors, Brain Res., 607: 352-354 (1991) and Chance and co-authors, Brain Res., 607: 185-188 (1993), an action shared with CGRP and with calcitonin. The effective concentrations in cells that measured this action are unknown. The use of amylin and amylin agonists for the treatment of anorexia is described and claimed in U.S. Patent No. 5,656,590, issued August 12, 1997. Compositions that include a cholecystokinin agonist and an amylin agonist or a molecule Hybrid for use in reducing food intake or controlling appetite or body weight, are described and claimed in U.S. Patent No. 5,739,106, issued April 14, 1998.

THE OBESITY Obesity is a chronic disease that is highly prevalent in modern society and is associated not only with a social stigma, but also with a diminished life expectancy and numerous medical problems, including adverse psychological development, reproductive disorders, such as polycystic diseases of the ovaries, dermatological disorders, such as infections, varicose veins, Acanthosis nigrícans and eczema; exercise intolerance, diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, osteoarthritis, orthopedic damage, thromboembolic diseases, cancer and coronary heart disease. Rissanen and co-authors, British Medical Journal, 301: 835-837 (1990). Obesity, and especially obesity of the upper body, is a very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25 percent of the US population and 27 percent of the Canadian population are overweight. Kuczmarski, Amer. J. of Clin. Nut., 55: 495S-502S (1992); Reeder and co-authors, Can. Med. Ass. J., 23: 226-233 (1992). Obesity of the body is the strongest known risk factor for type II diabetes mellitus, and is a strong risk factor for cardiovascular diseases and cancer as well. Recent estimates of the medical cost of obesity show a result of 150 billion dollars worldwide. The problem has become so serious that the Ministry of Health has taken the initiative to combat the increasing fatigue that it deprives in American society. Much of this pathology induced by obesity can be attributed to a strong association with dyslipidemia, hypertension and insulin resistance. Many studies have shown that reducing obesity through diet and exercise dramatically reduces these risk factors. Unfortunately, these treatments are largely unsatisfactory with a failure rate that reaches 95%. That failure may be due to the fact that the condition is strongly associated with genetically inherited factors, which contribute to an increased appetite, preference for high calorie foods, reduced physical activity and increased lipogen metabolism. This indicates that women who inherit these genetic tendencies are likely to become obese, regardless of their efforts to combat the condition. Therefore, a new pharmacological agent is needed that can correct this disadvantage of adiposity and allow the doctor to successfully treat obese patients, despite their genetic inheritance. Existing therapies for obesity include diets and normal exercises, very low calorie diets, behavioral therapy, pharmacotherapy that involves appetite suppressants, thermogenic drugs, food absorption inhibitors, mechanical devices, such as mandibular braces, waist-forming cords and balloons, as well as surgery. Jung and Chong, Clinical Endocrinology, 35: 11-20 (1991); Bray, Am. J. Clin. Nutr., 55: 538S-544 (1992). It has been reported that fasting modified with additional protein is effective in reducing weight in adolescents. Lee and co-authors, Clin. Pediatr., 31: 234-236 (April 1991). Caloric restriction as a treatment for obesity causes catabolism of the body's protein storage and produces a negative nitrogen balance. Therefore, diets supplemented with proteins have become popular as a means to decrease nitrogen loss during calorie restriction. Because these diets produce only modest nitrogen consumption, a more effective way to maintain lean body mass and protein storage is necessary.

Additionally, the treatment of obesity would improve if said regimen would also result in the accelerated loss of body fat. Various techniques for such treatment include those discussed by Weintraub and Bray, Med. Clinics N. Amer., 73: 237 (1989).; Bray, Nutrition Reviews, 49:33 (1991). Considering the high prevalence of obesity in today's society and the serious consequences associated with it, which are discussed above, any therapeutic drug potentially useful for reducing the weight of obese people could have a profound beneficial effect on their health. There is a need for a drug that reduces the total body weight of the obese subjects to their ideal body weight and helps maintain the reduced weight level.

BRIEF DESCRIPTION OF THE INVENTION It has now been discovered, surprisingly, that amylin and amylin agonists, for example, the amylin agonist analogue 25,28,29pro_n amj | na (also called "pramlintide" and formerly known as "AC-'137" ) can be used to treat obesity in humans.

The present invention is directed to novel methods for treating or preventing obesity in humans, which comprises administering an amylin or an amylin agonist, for example, the amylin agonist analogue 25,28'29 Pro-h-amylin. The amylin or the amylin agonist can be administered alone or together with another agent to alleviate obesity. In one aspect, the invention is directed to a method for treating obesity in a human subject, comprising administering to the subject an effective amount of an amylin, or said amylin agonist. By "treating" is meant the management and care of a patient for the purpose of combating the disease, condition or disorder, and includes the administration of an amylin or an amylin agonist to prevent the onset of symptoms or complications, the relief of symptoms or complications, or elimination of the disease condition or disorder. Treating obesity, therefore, includes the inhibition of weight gain and the induction of weight loss in patients who need it. Additionally, treating obesity means including weight control for cosmetic purposes in humans, that is, controlling body weight to improve physical appearance. It is understood that the term "amylin" includes compounds as defined in U.S. Patent No. 5,234,906, issued August 10, 1993, for "Hyperglycemic compositions", the content of which is incorporated herein by this reference. For example, it includes the human peptide hormone called amylin and secreted by beta cells of the pancreas, and species variations thereof. "Amylin agonist" is also a term known in the art, and refers to a compound that duplicates or mimics the effects of amylin. An amylin agonist can be a peptide or a non-peptide compound, and includes amylin agonist analogues. The term "amylin agonist analog" is understood as referring to derivatives of an amylin that are currently believed to act as amylin agonists, normally, by virtue of binding to, or otherwise interacting, either directly or indirectly with an amylin receptor or another or other receptors, with which amylin itself can interact to elicit a biological response. Useful amylin agonist analogs include those identified in an international patent application WPI Accession No. 93-182488 / 22, entitled "New amylin agonist peptides, used to treat and prevent hypoglycemia and diabetes mellitus", whose content is also incorporated herein by this reference. In a preferred embodiment, the amylin agonist is an analogue of amylin agonist, preferably 25,28,29Pro-h-amylin. 25'2829pro-h-amiline and other amylin agonist analogs are described and claimed in U.S. Patent No. 5,686,411, issued November 11, 1997, the contents of which are also incorporated herein by this reference. In another aspect, the present invention is directed to novel methods for reducing insulin-induced weight gain in human subjects who are taking insulin by administering a therapeutically effective amount of an amylin or an amylin agonist. In one embodiment, the subject has diabetes mellitus, for example, type 1 or type 2 diabetes mellitus. In a preferred embodiment, the amylin agonist is ,28,29pro_h_am¡ | na_ DETAILED DESCRIPTION OF THE INVENTION The study described in Example 1 showed that administration of the amylin agonist 25,28,29Pro-h-amylin (pramlintide) to diabetics using insulin (type 2) resulted in a decrease in body weight after 4 weeks, which reached statistical significance within two dose groups: 60 μg TID and 60 μg QID. The study described in Example 2 showed that the administration of pramlintide (30 μg or 60 μg QID) to type 1 diabetics resulted in a statistically significant decrease in body weight, compared to a placebo, at 13, 26 and 52 weeks. The study described in Example 3 showed that administration of pramlintide (30, 75 or 150 μg TID) to patients with type 2 diabetes, requiring insulin, resulted in a statistically significant decrease in body weight, in comparison with placebo at 13, 26 and 52 weeks. These results are in total contrast to insulin therapy alone in patients with type 1 or type 2 diabetes, which is usually associated with weight gain.

The amylin agonist analogs useful in this invention include the amylin agonist analogs described and claimed in the above-noted US Patent No. 5,686,411. Amine agonists include the amylin agonist analogs, as follows: 1. An amylin agonist analog having the amino acid sequence: ^ ArX-Asn-Thr ^ Ala-Thr-Y-Ala-Thr- ^ GIn-Arg -Leu-B Asn- 15Phe-Leu-CrD Ei- ^ FrG Asn-H GIy- ^ Pro- -Leu-Pro-Jr ^ Thr-K Val-Gly-Ser-35Asn-Thr-Tyr-Z; in which: Ai is Lys, Ala, Ser or hydrogen; Bi is Ala, Ser or Thr; C -i is Val, Leu or lie; D -i is His or Arg; Ei is Ser or Thr; F ^ is Ser, Thr, Gln or Asn; G 1 is Asn, Gln or His; H 1 is Phe, Leu or Tyr; 11 is lie, Val, Ala or Leu; J 1 is Ser, Pro or Thr; X and Y are independently selected residues having side chains that are chemically linked together to form an intramolecular linkage, wherein the intramolecular linkage comprises a disulfide bond, a lactam bond or a thioether bond; and Z is amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy; and on the condition that, when Ai is Lys, B-? is Ala, Ci is Val, D-i is Arg, Ei is Ser, F-? is Ser, Gi is Asn, Hi is Leu, is Val, Ji is Pro and Ki is Asn, then one or more of Ai to Ki is a D-amino acid and Z is selected from the group consisting of alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy. 2. An amylin agonist analog having the amino acid sequence: 1A? -X-Asn-Thr-5Ala-Thr-Y-Ala-Thr-10Gln-Arg-Leu-B Asn-5Phe-Leu-d-DrEr ^ FrGrAsn-HrGIy- ^ Pro- -Leu-JrPro- ^ Thr-KrVal-Gly-Ser-35Asn-Thr-Tyr-Z; wherein: Ai is Lys, Ala, Ser or hydrogen; Bi is Ala, Ser or Thr; Ci is Val, Leu or lie; Di es His or Arg; E 1 is Ser or Thr; Fi is Ser, Thr, Gln or Asn; G 1 is Asn, Gln or His Hi is Phe, Leu or Tyr; l-i is lie, Val, Ala or Leu; J -i is Ser, Pro, Leu, lie or Thr; Ki is Asn, Asp or Gln; X and Y are independently selected residues having side chains that are chemically linked together to form an intramolecular linkage; wherein said intramolecular linkage comprises a disulfide bond, a lactam bond or thioether; and Z is amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy; and provided that when: (a). Ai is Lys, B-? is Ala, Ci is Val, Di is Arg, Ei is Ser, Fi is Ser, Gi is Asn, Hi is Leu, Li is Val, J 1 is Pro and Ki is Asn; or (b). Ai is Lys, Bi is Ala, Ci is Val, D-i is His, Ei is Ser, F-? is Asn, G 1 is Asn, Hi is Leu, li is Val, Ji is Ser and Ki is Asn; then one or more of Ai to Ki is a D-amino acid and Z is selected from the group consisting of alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy. 3. An amylin agonist analogue having the amino acid sequence: 1A? -X-Asn-Thr-5Ala-Thr-Y-Ala-Thr-10Gln-Arg-Leu-B? -Asn-15Phe-Leu-d -Di-Er ^ Fi-G Asn-HrGIy- ^ lrJrLeu-Pro-Pro- ^ Thr-KrVal-GIy-Ser-35Asn-Thr-Tyr-Z; wherein: Ai is Lys, Ala, Ser or hydrogen; Bi is Ala, Sero Thr; Ci is Val, Leu or lie; Di es His or Arg; Ei is Ser or Thr; Fi is Ser, Thr, Gln or Asn; Gi is Asn, Gln or His; Ji is lie, Val, Ala or Leu; Ki is Asn, Asp or Gln; X and Y are independently selected residues having side chains that are chemically linked together to form an intramolecular linkage; wherein the intramolecular linkage comprises a disulfide linkage, a lactam linkage or thioether; and Z is amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy; and provided that when Ai is Lys, Bi is Ala, Ci is Val, Di is Arg, Ei is Ser, Fi is Ser, d is Asn, Hi is Leu, li is Pro, Ji is Val and Ki is Asn; then one or more of Ai to Ki is a D-amino acid and Z is selected from the group consisting of alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy. 4. An amylin agonist analog having the amino acid sequence: 1Ai-X-Asn-Thr-5Ala-Thr-Y-Ala-Thr-10Gln-Arg-Leu-B? -Asn-15Phe-Ñeu-C? -D? -E? -20F? -G? -Asn-H? -Gly-25Pro-l? -Leu-Pro-Pro-30Thr-J? -Val-Gly-Ser-35Asn-Thr-Tyr-Z; wherein: Ai is Lys, Ala, Ser or hydrogen; Bi is Ala, Ser or Thr; Ci is Val, Leu or He; Di es His or Arg; Ei is Ser or Thr; Gi is Asn, Gln or His; Hi is Phe, Leu or Tyr; h is lie, Val, Ala or Leu; Ji is Asn, Asp or Gln; X and Y are independently selected residues, having side chains that are chemically linked together to form an intramolecular linkage; wherein said intramolecular linkage comprises a disulfide bond, a lactam bond or thioether; and Z is amino, alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy; and provided that when Ai is Lys, Bi is Ala, Ci is Val, Di is Arg, Ei is Ser, Fi is Ser, Gi is Asn, Hi is Leu, h is Val and Ji is Asn; then one or more of Ai to Ki is a D-amino acid and Z is selected from the group consisting of alkylamino, dialkylamino, cycloalkylamino, arylamino, aralkylamino, alkyloxy, aryloxy or aralkyloxy. Preferred amylin agonist analogues include 25,28,29Pro-h-amylin, 18Arg25'28'29Pro-h-amylin and 18Arg25-28Pro-h-amylin. The activity as amylin agonists can be confirmed and quantified by performing various selection analyzes, including analysis of receptor binding of the acupuncture nucleus, described later in Example 7, followed by soleus muscle analysis described later in Example 8; a gastric emptying analysis described below in Example 9 or 10, or by the ability to induce hypocalcemia or reduce postprandial hyperglycemia in mammals, as described herein. The receptor binding assay, a competence analysis, which measures the ability of the compounds to bind amylin receptors attached to the membrane, is described and claimed in U.S. Patent No. 5,264,372, issued November 23, 1993, whose description is incorporated herein by this reference. The analysis of receptor binding is also described in the following example 7. A preferred source of the membrane preparations used in the analysis is the basal antecerebra, which comprises membranes of the accumbent nucleus and the surrounding regions. The compounds that are subjected to analysis compete for the binding to these receptor preparations, with Bolton-Hunter rat 125l-amylin. The competition curves are analyzed by computer, in which the binding quantity (B) is plotted as a function of the logarithm of the ligand concentration; using non-linear regression analysis to a 4-parameter logistic equation (Inplot program, GraphPAD Software, San Diego, CA, EU A), or DeLean's ALLFIT program and co-authors (ALLFIT, version 2.7 (NIH, Bethesda MD 20892)) . Munson and Rodbard, Anal. Biochem., 107: 220-239 (1980).

Analyzes of the biological activity of amylin agonists in the soleus muscle can be performed using the previously described methods (Leighton, B and Cooper, Nature, 335: 632-635 (1988); Cooper and co-authors, Proc. Nati. Sci. USA, 85: 7763-7766 (1988), in which the amylin agonist activity can be determined by measuring the inhibition of insulin-stimulated glycogen synthesis The analysis of the soleus muscle in Example 8 is also described. The methods for measuring the gastric emptying regime are described, for example, in Young and coauthors, Diabetologia, 38 (6): 642-648 (1995) .In a phenol red method, which is described in Example 9 which follows, conscious rats receive by feeding an acolyte gel containing methylcellulose and a phenol red indicator.Two minutes after administration, the animals are anesthetized using halothane, the stomach is exposed and pinched in the pyloric and lower esophageal sphincters; It is removed and opened to an alkaline solution. The stomach content of the intensity of the phenol red present in the alkaline solution can be derived, it is measured by absorbance at the wavelength of 560 nm. In a tritiated glucose method, which is described in the example 10 that follows, is fed conscious rats with tritiated glucose in water. The rats are gently restricted by the tail, whose tip is anesthetized using lidocaine. Tritium is collected from the plasma separated from the caudal blood at various points of time and detected in a beta counter. Test compounds are usually administered approximately one minute before administration. The effects of amylin or amylin agonists on body weight can be identified, evaluated or selected to use the methods described in the following examples 1-3, or other methods known in the art or equivalents, to determine the effects on body weight. Preferred amylin agonist compounds exhibit activity in the receptor binding assay, in the order of less than about 1 to 5 nM, preferably, less than about 1 nM and, more preferably, less than about 50 pM. In the analysis of the soleus muscle, the preferred amylin agonist compounds show EC50 values of the order of less than about 1 to 10 micromolar. In gastric emptying analyzes, preferred agonist compounds show ED 50 values of the order of less than 100 μg / rat. Amylin and amylin peptide agonists can be prepared using common and current solid phase peptide synthesis techniques, and preferably an automatic or semi-automatic peptide synthesizer. Typically, by using those techniques, an amino acid protected with alpha-N-carbamoyl, and an amino acid attached to the growing peptide chain, are coupled in a resin, at room temperature, in an inert solvent such as dimethylformamide, N-methylpyrrolidinone or methylene chloride, in the presence of coupling agents, such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole, in the presence of a base such as diisopropiiethiamine. The aifa-2N-carbamoyl protecting group is removed from the resulting peptide-resin, using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction is repeated with the next desired N-protected amino acid, which is to be added to the peptide chain. Suitable N-protecting groups are well known in the art, with terbutoxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc) being preferred. Solvents, amino acid derivatives and 4-methylbenzhydrylamine resin, used in the peptide synthesizer, can be purchased from Applied Biosystems Inc. (Foster City, CA, E.U.A.). The following amino acids can be purchased with protected side chain, from Applied Biosystems, Inc .: Boc-Arg (Mts), Fmoc-Arg (Pmc), Boc-Thr (Bzl), Fmoc-Thr (t-Bu), Ser (Bz1), Fmoc-Serit-Bu), Boc-Tyr (BrZ), Fmoc-Tyr (t-Bu), Boc-Lys (CI-Z), Fmoc-Lys (Boc), Boc-Glu (Bzl) , Fmoc-Glu (t-Bu), Fmoc-His (Trt), Fmoc-Asn (Trt), and Fmoc-G? N (Trt). Boc-His (BOM) from Applied Biosystems, Inc. or from Bachem Inc. (Torrance, CA, E. U. A.) can be purchased. Anisole, methyl sulfide, phenol, ethanedithiol and thioanisole can be obtained from Aldrich Chemical Company (Milwaukee, Wl, E. U. A.); Air Products and Chemicals (Allentown, PA, E. U. A.) provide HF. Ethyl ether, acetic acid and methanol can be purchased from Fisher Scientific (Pittsburgh, PA). Peptide synthesis in solid phase can be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems, Inc., Foster City, CA, USA), using the NMP / HOBt system (option 1) and Tboc chemistry or Fmoc (see the Applied Biosystems User's Manual for the ABI 430A peptide synthesizer, version 1.3B, July 1, 1988, section 6, pages 49-70, Applied Biosystems, Inc., Foster City, CA, USA) , with cap.

The Boc-peptide resins can be divided with HF (-5 ° C at 0 ° C, one hour). The peptide can be extracted from the resin by alternating water and acetic acid, and the filtrates are lyophilized. The Fmoc-peptide resins can be divided according to common and current methods (Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pages 6-12). The peptides can also be assembled using an Advanced Chem Tech synthesizer (model MPS 350, Louisville, KY, E. U. A.). The peptides can be purified by RP-HPLC (preparatory and analytical) using the Waters Delta Prep 3000 system. A preparatory column of C4, C8 or C18 can be used (10 μ, 2.2 x 25 cm, Vydac, Hesperia, CA, USA) ), to isolate the peptides; and the purity can be determined using an analytical column of C4, C8 or C18 (5 μ, 0.46 x 25 cm, Vydac). Solvents (A = 0.1% TFA / water and B = 0.1% TFA / CH3CN) can be supplied to the analytical column at a flow rate of 1.0 ml / min and to the preparatory column at 15 ml / min. The amino acid analysis can be carried out in the Waters Pico Tag system, and it can be processed using the Maximum program. The peptides can be hydrolysed by hydrolysis with acid in the vapor phase (115 ° C, 20-24 hours). Derivatives of hydrolysates can be formed and can be analyzed by common methods (Cohen and coauthors, The Pico Tag Method: A Manual of Advanced Techniques for Amino Acid Analysis, pages 11-52, Millipore Corporation, Milford, MA (1989 )). You can carry out analysis by bombarding with fast atoms, by M-Scan, Incorporated (West Chester, PA, E. U. A.). The mass calibration can be performed using cesium iodide or cesium / glycerol iodide. The ionization analysis can be carried out by plasma desorption, using a flight detection time, in an Applied Biosystems Bio-lon 20 mass spectrometer. The peptide compounds useful in the invention can also be prepared using recombinant DNA techniques. , using methods now known in the art. See, for example, Sambrook and coauthors, Molecular Cloning: A Laboratory Manual, 2a. edition, Cold Spring Harbor (1989). Non-peptide compounds, useful in the present invention, can be prepared by methods known in the art. The compounds referred to above can form salts with various inorganic and organic acids and bases. These salts include salts prepared with organic and inorganic acids, for example, HCl, HBr, H2SO4, H3PO4, trifluoroacetic acid, acetic acid, formic acid, methanesulfonic acid, toluenesulfonic acid, maleic acid, fumaric acid and camphor sulfonic acid. Salts prepared with bases include: ammonium salts, alkali metal salts, for example, sodium and potassium salts and alkaline earth salts, for example, calcium and magnesium salts. Acetate, hydrochloride and trifluoroacetate salts are preferred. The acetate salts are preferred over the others. The salts can be formed by conventional means, for example, by reacting the free acid or the base form of the product with one or more equivalents of the appropriate base or acid in a solvent or in a medium in which the salt is insoluble. , or in a solvent such as water, which is then removed under vacuum or by freeze drying, or by changing the ions of an existing salt by another, in a suitable ion exchange resin. The compositions useful in the invention may conveniently be provided in the form of formulations suitable for parenteral (including intravenous, intramuscular and subcutaneous) or nasal or oral administration. The proper administration format can best be determined by a practicing physician, individually for each patient. Suitable pharmaceutically acceptable carriers and their formulation are described in common formulating treaties, for example, in Remington's Pharmaceutical Sciences, by E. W. Martin. See also Wang, Y. J. and Hanson, M.A., Parenteral Formulations of Proteins and Peptides: Stability and Stabilizers, Journal of Parenteral Science and Technology, technical report No. 10, supplement 42: 2S (1988). The compounds provided as parenteral compositions for injection or for infusion, in an inert oil, suitably a vegetable oil, such as sesame, peanut, olive oil) or other acceptable carrier may be suspended, for example. It is preferred to suspend them in an aqueous carrier, for example, in an isotonic buffer, at a pH of about 5.6 to 7.4. These compositions can be sterilized by conventional sterilization techniques, or they can be filtered to sterilize them. The compositions may contain pharmaceutically acceptable auxiliary substances, which are necessary to approximate physiological conditions, such as pH regulating agents. Useful regulators include, for example, sodium acetate / acetic acid regulators. A slow-release "build-up" form of preparation can be used, so that therapeutically effective amounts of the preparation are delivered to the bloodstream for many hours or days after the transdermal injection or delivery. It is preferred that these parenteral dosage forms be prepared in accordance with the patent application of the same successor, entitled "Parenteral liquid formulations for amylin agonist peptides", Serial No. 60 / 035,140, filed on January 8, 1997, and in U.S. Patent Application No. 09 / 005,262, filed January 8, 1998, which is incorporated herein by this reference; and include approximately 0.01 to 0.5% (w / v), respectively, of an amylin or an amylin agonist, in an aqueous system together with about 0.02 to 0.5% (w / v) of an acetate, phosphate, citrate regulator or glutamate, to obtain a pH of the final composition of about 3.0 to 6.0 (more preferably, 3.0 to 5.5), as well as about 1.0 to 10% (w / v) of a carbohydrate or a polyhydric alcohol toner, in a continuous aqueous phase. Also present is about 0.005 to 1.0% (w / v) of antimicrobial preservative, selected from the group consisting of m-cresol, benzyl alcohol, methyl-, ethyl-, propyl- and butyl-parabens, and phenol; in the preferred product formulation, intended to allow multiple doses to be administered to the patient. A stabilizer is not necessary. A sufficient amount of water for injection is used to obtain the desired concentration of the solution. Sodium chloride as well as other excipients may also be present, if desired. However, said excipients must maintain the general stability of amylin or the amylin agonist peptide. The liquid formulations should be substantially isotonic, ie, within ± 20% of the isotonicity and, preferably, within 10% of the isotonicity. It is highly preferable that in the formulation of amylin or amylin agonist for parenteral administration, the polyhydric alcohol be mannitol, the regulator be acetate regulator, the preservative be about 0.1 to 0.3% (w / v) m-cresol and the pH is approximately 3.7 to 4.3. The desired isotonicity can be achieved using sodium chloride or other pharmaceutically acceptable salts. If desired, the solutions of the above compositions can be thickened with a thickening agent, such as methylcellulose. They can be prepared in emulsified form, either from water to oil or from oil to water. Any of a wide variety of pharmaceutically acceptable emulsifying agents can be used, including, for example: acacia powder, a nonionic surfactant (such as Tween) or an ionic surfactant (such as sulfates or sulfonates of polyether alkali alcohol, for example, a Triton). The compositions useful in the invention are prepared by mixing the ingredients following generally accepted procedures. For example, the components can simply be mixed in a mixer or other common and ordinary device to produce a concentrated mixture which can then be adjusted to the final concentration and viscosity by adding water or a thickener, and possibly a regulator to control the pH , or an additional solute to control tonicity. For use by the physician, the compositions will be provided in unit dosage form containing an amount of an amylin or an amylin agonist, for example, an analogous amylin agonist compound, which is effective in single or multiple doses, to control Obesity at the selected level. Therapeutically effective amounts of an amylin or an amylin agonist, such as an amylin agonist analog, for use in the control of obesity, are those that decrease body weight. As will be recognized by those skilled in the art, an effective amount of therapeutic agent will vary with many factors, including the age and weight of the patient, the physical condition of the patient, the action to be obtained, and other factors. Individual, divided or continuous analgesic doses of the compounds, for example, including 25.28'29Pro-h-amylin, 18Arg-25,28'29Pro-h-amylin and 19Arg-25,28Pro-h-amylin will typically be the approximate scale of 0.01 to 5 mg / day, preferably about 0.05 to 2 mg / day and, more preferably, about 0.1 to 1 mg / day, for a 70 kg patient, administered as a single dose, as divided doses or as continuous doses. The exact dose that will be administered is determined by the attending physician, and depends on numerous factors including those noted above. Administration should begin at the first sign of obesity. The administration can be effected by injection or infusion, preferably intravenous, subcutaneous or intramuscular. Orally active compounds can be taken orally; however, the doses should be increased from 5 to 10 times. In general, when treating or preventing obesity, the compounds of this invention can be administered to patients in need of such treatment at dose scales similar to those given hereinbefore; however, the compounds can be administered more frequently, for example, once, twice or three times a day, or continuously. It is preferred that doses of peptide agonists, e.g., pramlintide, be administered subcutaneously at doses of 30-300 μg, given one to four times a day and, more preferably, doses of 30 to 120 μg, administered from two to four times per day. To assist in understanding the present invention, the following examples are included, which describe the results of a series of experiments. The studies that refer to this invention, of course, should not be taken as a specific limitation for the present invention, and it is considered that those variations of the invention, now known or later developed, which are within the knowledge of the experts in the art. matter, fall within the scope of the invention as described herein and is subsequently claimed.

EXAMPLE 1 MEASUREMENT OF BODY WEIGHT: A 4-WEEK STUDY IN DIABETICS OF TYPE 2, WHICH REQUIRES INSULIN Participants in the study were men and women 25 to 78 years of age, with a history of type II diabetes mellitus, which requires treatment with insulin for at least six months before the prescreening visit. The patients had a body weight that did not vary more than 45% of the desirable weight before admission to the study (based on the Metropolitan Life tables). The study employed methods described in Thompson and co-authors, Diabetes 46: 632-636 (1997). After a placebo-controlled period, patients were randomly assigned to receive placebo or one of three dose regimens of 25.28'29 Pro-h-amylin (pramlintide) for four weeks: 30 μg QID (before breakfast; lunch, lunch and snack), 60 μg TID (before breakfast, lunch and dinner) or 60 μg QID (before breakfast, lunch, lunch and snack). During the entire period with the study drug, patients self-administered four injections of the study drug daily, within 15 minutes of each food, and afternoon snacks. During the double-blind period, pramlintide, 60 μg or placebo administered before evening snack was randomly distributed to patients. Both pramlintide and placebo were administered as separate injections into the subcutaneous tissue of the anterior abdominal wall; the specific site was alternated after each injection. The patient was instructed to stay on his usual diet, insulin, and exercise regimens throughout the study, unless the investigator gave instructions to the contrary, and to abstain from alcoholic beverages before all clinical visits. As shown in Table I, there was a statistically significant weight reduction of the basic weight line, at week 4 within the 60 μg groups of pramlintide TID (meaning = -0.89 kg, p = 0.0056) and 60 μg of pramlintide QID (meaning = -0.72 kg, p) 0.0014). With the Hochberg adjustment for multiple comparisons, there was no statistically significant change in body weight with respect to the baseline, at week 4, in any of the three pramlintide groups, compared to the placebo group. Thus, administration of pramlintide with continuous insulin use improved glycemic control, with a decrease in body weight that reached statistical significance within the groups of 60 μg ID and QID. This decrease is in sharp contrast to the weight gain usually associated with improved glucose control achieved with insulin alone, in patients with type 2 diabetes.

TABLE I WEIGHT OF BODY. CHANGE OF BASIC LINE IN WEEK 4 * Student t test (within the comparison with the group of drug under study). Two-way ANOVA (comparison with placebo) with the Hochberg adjustment. NS = not statistically important; NAP = not applicable.

EXAMPLE 2 MEASUREMENT OF THE BODY WEIGHT: A 52-WEEK STUDY IN TYPE 1 DIABETICS This study was a double-blind, placebo-controlled, multiple-center parallel group study with a potential dose escalation. Participants in the study were men and women between the ages of 16 and 70 years, with type 1 diabetes mellitus. Four subcutaneous injections of 30 μg of pramlintide or placebo were self-administered daily, one before each meal and one snack before about sleeping. Certain patients (those in the pramlintide arm who had a HbAlc reduction from the baseline of less than 1.0% after 13 weeks of treatment) were randomly reassigned at 20 weeks with 30 μg or 60 μg QID for the remainder of the study. . The patients in this study were treated with the study medication, formulated at pH 4.0, at a concentration that allowed the injection of 0.1 ml per dose. Four hundred and seventy-seven patients received at least one dose of the study medication (pramlintide or placebo). Of the 477 patients randomly distributed and dosed, 341 completed the 52-week study. Patients treated with pramlintide experienced a clinically significant and statistically significant decrease in body weight, compared to placebo, at 13, 26 and 52 weeks (table II). The maximum difference was observed at 26 weeks and at 52 weeks (decrease of at least 1.2 kg compared with placebo, at each time point). Weight loss occurred particularly in those patients who had a baseline body mass index (BMI) of at least 17.0 kg / m2, which indicates the maximum benefit among those obese patients (Table III). Patients who took pramlintide within the subgroup of patients with baseline HbAlc levels of at least 8.0% and stable with insulin, experienced a mean decrease in body weight, compared with placebo, at all three time points (Table IV). This observation is consistent with the well-known effect of insulin, of facilitating the increase in body weight. Thus, it seems that pramlintide reduces insulin-induced weight gain. Normally distributed data was analyzed, using two-way variation analysis. In the cases in which the data were not found to follow a normal distribution, nonparametric methods (Kruskal-Wallis test) were used, based on ranges. In these cases, the Hodges-Lehman estimator is presented for the difference with respect to the placebo, instead of the mean.

TABLE II CHANGES IN THE WEIGHT OF THE BODY FROM THE LINE PESOS ** Kruskal-Wallis test *** Two-way ANOVA * Statistically significant difference compared to placebo.

PICTURE BODY WEIGHT: CHANGES FROM THE BASIC LINE FOR PATIENTS WITH BASIC LINE BMI > 27.0 KG / M2 O < 27.0 KG / M2. WEIGHTS AT WEEKS 13, 26 AND 52 TABLE IV BODY WEIGHT: CHANGES FROM BASIC LINE.- PATIENTS ON HBA1C > 8.0%, INSULIN WITHIN ± 10% OF BASIC LINE PESOS IN WEEKS 13, 26 AND 52 Two-way ANOVA ^ Statistically significant difference compared with placebo.

EXAMPLE 3 WEIGHT MEASUREMENT OF THE BODY: A 52-WEEK STUDY IN TYPE 2 DIABETICS, WHICH REQUIRES INSULIN This study was a double-blind, placebo-controlled, parallel-dose, multiple-center dose variation study. The participants in the study are men and women between the ages of 18 and 75, with type 2 diabetes mellitus, who require insulin. Three subcutaneous injections of pramlintide (30, 75 or 150 μg TID) or placebo (TID), one before each feed, were self-administered daily for 52 weeks. The patients of this study were treated with the study medication, formulated at pH 4.7, at a concentration that required the injection of 0.3 ml per dose. The period of double blind treatment was preceded by a period of induction with placebo, individually blind, from 3 to 10 days. Of the 539 patients randomly assigned and randomly dosed, 381 completed the 52-week study. Patients treated with any of the three doses of pramlintide experienced a clinically significant and statistically significant decrease in body weight, compared with placebo, at 13, 26 and 52 weeks (Table V). The maximum difference was observed with respect to placebo at 26 weeks and at 52 weeks (decreases of 2.3 and 2.7 kg, compared with placebo, at these points of time). The weight of the patients treated with placebo increased with respect to the baseline at all three time points, in contrast to weight decreases in the three pramlintide groups, at all points of time. Weight loss occurred in patients who had a baseline body mass index (BMI) of at least 27.0 kg / m2 as well as those who had a baseline BMI of less than 27.0 kg / m2 (Table VI) . Patients with Pramlintide in all three groups, with levels of Baseline HbAlc of at least 8.0% and stable with insulin, experienced a decrease in body weight, compared to placebo at all points of time (Table VII). The magnitude of the response in general was comparable with that observed for all patients, suggesting an effect independent of changes in insulin dose. Normally distributed data were analyzed using two-way variation analysis (with the Hochberg adjustment to the Bonferroni procedure for multiple comparisons). In the cases in which the data were not found to follow a normal distribution, nonparametric methods (Kruskal-Wallis test), based on ranges, were used. In these cases, the Hodges-Lehman estimator is presented for the difference from the placebo, instead of the mean.

TABLE V BODY WEIGHT: CHANGES FROM BASIC LINE PESOS IN WEEKS 13, 26 AND 52 ** Kruskal-Wallis test with Hochberg adjustment for multiple comparisons, versus placebo. * Statistically significant difference compared to placebo.

TABLE VI BODY WEIGHT: CHANGES FROM THE BASIC LINE FOR PATIENTS WITH BASIC LINE BMI GREATER OR EQUAL TO 27.0 KG / M2 OR LESS THAN 27.0 KG / M2.- WEIGHTS TO WEEKS 13. 26 AND 52 TABLE VII BODY WEIGHT: CHANGES FROM THE BASIC LINE.- PATIENTS WITH HBA GREATER THAN OR EQUAL TO 8.0%. INSULIN WITHIN ± 10% OF BASIC LINE PESOS. AT WEEKS 13. 26 AND 52 ** Kruskal-Wailis test with Hochberg adjustment for multiple comparisons against placebo. *** Two-way ANOVA with Hochberg adjustment for multiple comparisons against placebo. * Statistically significant difference, compared with placebo.

EXAMPLE 4 PREPARATION OF 25 829PRO-H-AMILINE The solid phase synthesis of 25.28'29Pro-h-amylin was carried out using methylbenzhydrylamine anchor / binding resin, and side chain protection with Na-Boc / benzyl, by common methods and peptide synthesis streams. The 2, r - [disulfurojamilin-MBHA-resin was obtained by treatment of cysteines protected with mAb, with thallium trifluoroacetate (III) in trifluoroacetic acid. After obtaining the cyclization, the resin and the side chain protecting groups were divided with liquid HF in the presence of dimethyl sulphide and anisole. The 25,28,29Pro-h-amyllan was purified by Reverse phase HPLC, preparatory. The peptide was found to be homogeneous to analytical HPLC and capillary electrophoresis and the structure was confirmed by amino acid analysis and sequence analysis. The product gave the desired mass. FAB mass spectrum: (M + H) + = 3,949.

EXAMPLE 5 PREPARATION OF 18Arq252829Pro-h-AMILINE The solid phase synthesis of 18Arg25,28,29Pro-h-amylin was carried out, using an attachment of methylbenzhydril-amine-bonding resin and side chain protection of Na-Boc / benzyl, by common peptide synthesis methods and currents The 2'7- [disulfide] amyl-MBHA-resin was obtained by treatment of cysteines protected with mAb, with thallium trifluoroacetate (III) in trifluoroacetic acid. After cyclization, the resin was obtained and the side chain protective groups were divided with liquid HF in the presence of dimethyl sulfide and anisole. The i8Arg25,28.29pro.h_ami | ina was purified by preparatory HpLC, in reverse phase. The peptide was found to be homogeneous by analytical HPLC and capillary electrophoresis, and the structure was confirmed by amino acid analysis and sequence analysis. The product gave the desired mass ion. FAB mass spectrum: (M + H) + = 3,971.

EXAMPLE 6 PREPARATION OF 18Ara252829Pro-h-AMILINE Solid phase synthesis of 18Arg 25.28Pro-h-amylin was carried out, using methylbenzhydrylamine-binding resin anchor, and side chain protection with Na-Boc / benzyl, by common methods and currents of peptide synthesis. 2.7- [disulfide] amilin-MBHA-resin was obtained, by treatment of cysteine protected with mAb, with thallium (III) trifluoroacetate in trifluoroacetic acid. After cyclization, the resin was obtained and the side chain protective groups were divided with liquid HF in the presence of dimethyl sulfide and anisole. The 18Arg25.28Pro-h-amiline, by preparatory reverse phase HPLC. The peptide was found to be homogeneous by analytical HPLC and capillary electrophoresis, and the structure was confirmed by amino acid analysis and sequence analysis. The product gave the desired mass ion.

FAB mass spectrum: (M + H) + = 3,959.

EXAMPLE 7 ANALYSIS OF UNION TO RECEIVER The evaluation of the binding of the compounds to amylin receptors was carried out in the following manner: 125 I-rat amylin (Bolton Hunter, marked on N-terminal lysine) was purchased from Amersham Corporation (Arlington Heights), IL, E. U. A.). The specific activities at the time of use varied from 1950 to 2000 Ci / mmol. Unlabelled peptides were obtained from BACHEM Inc. (Torrance, CA, E.U.A.) and Peninsula Laboratories (Belmont, CA, E.U.A.). Sprague-Dawley male rats, weighing 200-250 grams, were sacrificed by decapitation. The brains were excised to phosphate-regulated cold saline (PBS). Cuts were made of the ventral surface, rostral with respect to the hypothalamus, joined laterally by the olfactory tracts, and extending at an angle of 45 ° medially from these tracts. This basal antecerebro tissue, containing the accumbent nucleus and the surrounding regions, was weighed and homogenized in 20 mM of ice-cold HEPES regulator (20 mM HEPES acid, pH adjusted to 7.4 with NaOH at 23 ° C). The membranes were washed three times in a new regulator, centrifuging for 15 minutes at 48,000 x g. The final membrane pellet was resuspended in 20 mM of HEPES regulator containing 0.2 mM phenylmethylsulfonyl fluoride (PMSF).

To measure the binding to 125 I-amylin, membranes were incubated with 4 mg of original wet tissue weight, with 125-1-amylin at 12-16 pM in 20 mM regulator HEPES, containing 0.5 mg / ml bacitracin, 0.5 mg / ml. ml of bovine serum albumin and 0.2 mM of PMSF. Solutions were incubated for 60 minutes at 23 ° C. Incubations were terminated by filtration through GF / B glass fiber filters (Whatman Inc., Clifton, NJ, USA), which had previously been soaked for four hours in 0.3% polyethylenimine, in order to reduce non-union. specific for radiolabelled peptides. The filters were washed immediately before filtration with 5 ml of cold PBS and immediately after filtration with 15 ml of cold PBS. The filters were removed and the radioactivity was determined in a gamma counter, at a count efficiency of 77%. Competition curves were generated by measuring the binding in the presence of 10"12 to 10" 6 M unlabelled test compound, and analyzed by non-linear regression, using a 4-parameter logistic equation (Inplot program, GraphPAD Software, San Diego , CA, USA). In this analysis, purified human amylin binds to its receptor at a Cl50 measured at around 50 pM. The results for the test compounds are indicated in Table VIII, which shows that each of the compounds has important receptor binding activity.

EXAMPLE 8 MUSCLE ANALYSIS "SOLEUS" The determination of the amylin agonist activity of the compounds was depleted, using soleus muscle analysis, as follows. Male Harlan Sprague-Dawley rats, of approximately 200 g of mass, were used in order to maintain the soleus muscle mass divided, at less than 40 mg. The animals were fasted for 4 hours before sacrificing by decapitation. The skin of the lower limb was detached, which was pinned with bugs on a cork board. The Achilles tendon was cut just above the calcareous bone and the m was reflected outwards. gastrocnemius from the posterior aspect of the tibia. The soleus muscle, a smooth, small muscle, 15-20 mm long, 0.5 mm thick, was then completely dislodged from the bone surface of m. gastrocnemius, and cleaned of the perimysium using fine scissors and forceps. Next, the soleus muscle was divided into equal parts, using a blade passed anteriorly, through the belly of the muscle, to obtain a total of four muscle strips of each animal. After dissecting the animal's muscle, it was kept for a short period in physiological saline. It was not necessary to keep the muscle in tension, as this had no demonstrable effects on the incorporation of radioglucose into glycogen.

The muscles were added to 50 ml Erlenmeyer flasks containing 10 ml of pre-gassed Krebs-Ringer bicarbonate regulator, containing (per liter): NaCl, 118.5 mmol (6.93 g) K KCl, . 94 mmol (443 mg); CaCl2, 2.54 mmol (282 mg); MgSO 4, 1.19 mmol (143 mg), KH 2 PO 4, 1.19 mmol (162 mg), NaHCO 3, 25 mmol (2.1 g), 5.5 mmol of glucose (1 g), and recombinant human insulin (Humulin-R, Eli Lilly, IN, USA) and the test compound, as detailed below. It was verified that the pH was at 37 ° C between 7.1 and 7.4. The muscles were assigned to different flasks, so that 4 pieces of muscle of each animal were evenly distributed among the different conditions of analysis. The incubation media was gassed, gently bubbling carbonaceous (95% O2, 5% CO2) onto the surface, while stirring continuously at 37 ° C in an oscillating water bath. After a half-hour "pre-incubation" period, 0.5 μCi of U-14C-glucose was added to each flask, which was incubated for another 60 minutes. Then each piece of muscle was quickly removed, stained and frozen in liquid nitrogen, weighed and stored for subsequent determination of 1 C-glycogen. The determination of 14C-glycogen was carried out in a 7 ml flash ampoule. Each sample of frozen muscle was placed in an ampoule and digested in 1 ml of 60% potassium hydroxide at 70 ° C for 45 minutes, under continuous agitation. The dissolved glycogen was precipitated on the ampule by the addition of 3 ml of absolute ethanol and cooled overnight at -20 ° C. The supernatant was gently aspirated, the glycogen was washed again with ethanol, the aspiration was dried and the precipitate was dried under vacuum. All ethanol is evaporated to prevent inactivation during flash counting. The remaining glycogen was re-dissolved in 1 ml of water and 4 ml of flash fluid and the count for carbon 14 was carried out. The rate of incorporation of glucose into the glycogen was obtained (expressed in μmol / g / hr) from the specific activity of 14C-glucose in the 5.5 mM glucose of the incubation medium, and the total carbon 14 counts remaining in the glycogen extracted from each muscle. The dose / response curves were adjusted to a 4-parameter logistic model, using a minimum-quadratic iterative routine (ALLF1T, v2.7, NIH, MD, E.U.A.) to derive the EC50. Since the EC50 is normally logarithmically distributed, the ± standard error of the logarithm is expressed. Pairwise comparisons were performed using base test routines t, from SYSTAT (Wilkinson, SYSTAT: the system for statistics, SYSTAT Inc., Evanston, IL, E. U. A. (1989)). Dose-response curves were generated with muscles added to media containing 7.1 nM (1000 μU / ml) of insulin and each test compound was added at final (nominal) concentrations of 0, 1, 3, 10, 30, 100 , 300 and 1000 nM. Each analysis also contained internal positive controls, which consisted of a single load of rat amylin stored, freeze-dried and stored at -70 ° C. Human amylin is known to be a hyperglycemic peptide, and EC50 measurements of amylin preparations in the soleus muscle analysis typically range from about 1-10 nM, although some commercial preparations having a purity of less than 90% have EC50 higher, due to the presence of contaminants, which results in less measured activity. The results for the test compounds are given in the HIV box.

TABLE VIII EXAMPLE 9 ANALYSIS OF GASTRIC EMPTYING TO RED FENOL Gastric emptying was measured using a modification (Plourde and coauthors, Life Sci., 53: 857-862 (1993)) of the original method of Scarpignato and coauthors (Arch. Int.Pharmacodyn. Ther., 246: 286-295 (1980 )). Briefly, conscious rats received by feeding, 1.5 ml of acoloric gel containing 1.5% methylcellulose (M-0262, Sigma Chemical Co., St. Louis, MO, E.U.A.) and 0.05% phenol red indicator. Twenty minutes after the administration, the rats were anesthetized using 5% halothane, the stomach was exposed and grasped with forceps in the pyloric and lower esophageal sphincters, using forceps for arteries; it was taken out and opened in an alkaline solution, which was constituted at a fixed volume. The stomach content of the phenol red intensity in the alkaline solution was derived, measured by absorbance at a wavelength of 560 nm. In most of the experiments the stomach was clear. In other experiments the gastric contents were centrifuged into particles, to clarify the solution to measure the absorbency. When the diluted gastric contents remained cloudy, the spectroscopic absorbance due to phenol red was derived as the difference between that present in alkaline diluent versus acetified diluent. In separate experiments in 7 rats both the stomach and the small intestine were removed and opened in an alkaline solution. The amount of phenol red that could be recovered from the upper gastrointestinal tract, within 29 minutes of administration, was 89 ± 4%; The dye that seemed to bind irretrievably to the luminal surface of the intestine, can be taken into account for the rest.

To compensate for this small loss, the percentage of stomach contents that remained after 20 minutes was expressed as a fraction of the gastric contents recovered from the control rats sacrificed immediately after administration in the same experiment. The percentage of contents of the remaining gastric emptying is equal to (absorbance at 20 minutes) / (absorbance at 0 minutes). We adjusted the dose-response curves for gastric emptying to a 4-parameter logistic model, using a quadratic minimum iterative routine (ALLFIT, v2.7, NIH, Bethesda, MD, USA) to derive the DE5o- Since the ED50 it is normally logarithmically distributed, the ± standard error of the logarithm is expressed. We carried out pairwise comparisons using one-way variation analysis and the Student-Newman-Keuls multiple comparison test (Instat v2.0, GraphPad Software, San Diego, CA, E.U.A), using P <; 0.05 as a level of significance. In dose-response studies, rat amylin (Bachem, Torrance, CA, USA), dissolved in 0.15M saline, was administered as a 0.1 ml subcutaneous bolus, at doses of 0.01, 0.1, 1, 10 or 100 μg, 5 minutes before foraging in Harran Sprague Dawley rats (non-diabetics) left on an empty stomach for 20 hours, and in diabetic BB rats, left on an empty stomach for 6 hours. When subcutaneous injections of amylin were administered 5 minutes before foraging, with phenol red indicator, there was a suppression of gastric emptying, dose dependent (data not shown). The suppression of gastric emptying was complete in normal HSD rats, to which 1 μg of amylin was administered and in diabetic rats given 10 μg (P = 0.22, 0.14). The ED50 for the inhibition of gastric emptying in normal rats was 0.43 μg (0.60 nmol / kg) ± 0.19 logarithmic units, and was 2.2 μ (2.3 nmol / kg) ± 0.18 logarithmic units in diabetic rats.

EXAMPLE 10 ANALYSIS OF GASTRIC EMPTYING WITH TRITIZED GLUCOSE Fastened rats were held by tail Sprague Dawley conscious, not fasted, and the tip of the tail was anesthetized using 2% lidocaine. Tritium was detected from the plasma separated from the caudal blood at 0, 15, 30, 60, 90 and 120 minutes after foraging, in a beta-counter. The rats were injected subcutaneously with 0.1 ml of saline containing 0, 0.1, 0.3, 1, 10 or 100 μg of rat amylin 1 minute before foraging (n = 8, 7, 5, 5, 5, respectively). After administering the rats previously injected with saline, tritiated glucose, the plasma tritium increased rapidly (t about 8 minutes) to an asymptote that slowly declined. Subcutaneous injection with amylin decreased and / or delayed the absorption of the label or label, depending on the dose. The plasma tritium activity was integrated for 30 minutes to obtain the areas under the curve drawn as a function of the dose of amine. The DE 0 derived from the logistic adjustment was 0.35 μg of amylin.

Claims (12)

NOVELTY OF THE INVENTION CLAIMS

1. The use of an amylin or an amylin agonist for the manufacture of a medicament for the treatment or prevention of obesity in a human subject.

2 - The use according to claim 1, wherein the amylin agonist is an amylin agonist analogue.

3. The use according to claim 2, wherein the amylin agonist analog is ^^ Pro-h-amylin.

4. The use according to claim 1, wherein the medicament containing the amylin or the amylin agonist is administered subcutaneously.

5. The use according to claim 4, wherein the medicament containing the amylin or the amylin agonist is administered from 1 to 4 times a day and provides from 30 mg to 300 mg / dose of amylin or the agonist of Amylin to the patient.

6. The use according to claim 5, wherein the medicament containing the amylin or the amylin agonist is administered three times a day and provides approximately 80 μg per dose of amylin or the amylin agonist to the patient.

7. The use according to claim 5, wherein the medicament containing the amylin or the amylin agonist is administered four times a day and provides approximately 60 μg per dose of amylin or the amylin agonist to the patient.

8. The use of amylin or an amylin agonist for the manufacture of a medicament for reducing weight gain, induced by insulin, in a human subject receiving insulin.

9. The use according to claim 8, wherein the amylin agonist is ^^ Pro-h-amylin.

10. The use according to claim 8, wherein the subject has diabetes mellitus.

11. The use according to claim 10, wherein the subject has type 1 diabetes mellitus.

12. The use according to claim 10, wherein the subject has type 2 diabetes. 13.- The use according to claim 6 or 7, wherein the amylin agonist is ^ Pro-amylin.