ES2371148T3 - PP2-36, SELECTOR RECEIVER AGONIST Y4 FOR THERAPEUTIC INTERVENTIONS. - Google Patents

Abstract

The use of a Y agonist stops for the Y4 receptor on the Y1 and Y2 receptors, said Y receptor agonist being a pancreatic peptide PP2-36 with Sequential Identification Number: 10, in the preparation of a composition for (i) reducing the food intake, (ii) treatment of obesity or overweight, (iii) treatment of diarrhea or hypersecretion associated with small intestinal ileo-stomia or (iv) treatment of a condition in which obesity or overweight is considered a factor that contribute, selected from bulimia, bulimia nervosa, Syndrome X (metabolic syndrome), diabetes, type 2 diabetes mellitus or non-insulin-dependent diabetes mellitus (NIDDM), hyperglycemia, insulin resistance, glucose intolerance, cardiovascular disease, hypertension , arteriosclerosis, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, thromboembolic disease, hypercholesterolemia, hyperlipidemia, gallbladder disease, osteoarthritis, sleep apnea, polycystic ovarian syndrome, and breast, prostate or colon cancer.

Description

PP2-36, SELECTOR RECEIVER AGONIST Y4 FOR THERAPEUTIC INTERVENTIONS

FIELD OF THE INVENTION

The present invention relates to the pancreatic peptide PP2.36 with sequential identification number 10 that acts as a selective agonist of Y4 relative to Y1 and Y2 receptors, for use in the treatment of conditions that respond to receptor activation Y4, for example for the treatment of obesity and overweight, and conditions in which the above are considered contributing factors, diarrhea and intestinal hypersecretion.

BACKGROUND OF THE INVENTION

The family of PP-NPY fold peptides (Neuropeptide Y) (human sequence - sequential identification number No. 1), PYY (Peptide YY) (human sequence - sequential identification number No. 2), and PP (Pancreatic Polypeptide) (human sequence - sequence identification number No. 3), are Cterminally peptide-like peptides of 36 amino acids, homologous, naturally secreted, which are characterized by a common three-dimensional structure - the PP fold - which is surprisingly stable even in dilute aqueous solution and it is important for recognition by the receptor of the peptides.

Initially, the X-ray structure of the avian PP was characterized in great detail by means of X-ray crystallographic analysis at a resolution of 0.98 A and the unique structure got its name from this peptide (Blundell et al. 1981, Proc.Natl. Acad.Sci. USA 78: 4175-79; Glover et al. 1984, Eur.J. Biochem. 142: 379-85). Subsequently, the PP fold structure of other family members have been analyzed through NMR spectroscopic analysis. Both the X-ray and NMR analyzes are obviously performed in solid or high concentration conditions; However, detailed circular dichroism analysis suggests that NPY and PP adopt the PP fold structure even in aqueous solution, which is unusual for such a small peptide (Fuhlendorff et al. 1990 J. BioLChem. 265: 11706-12) . Importantly, the analysis of the proteolytic stability of the peptides and the fragments and analogs thereof clearly indicate that, for example, the full length PP1-36 is maintained in a folded configuration even in a dilute aqueous solution that protects it from degradation by certain enzymes that promptly and rapidly degrade analogs that cannot adopt the PP fold structure due to minor substitutions (Schwartz et al. 1990 Annals NY Acad. Sci. 611: 35-47).

The PP folding structure common to NPY, PYY and PP consists of 1) a helix similar to N-terminal polyproline (corresponding to residues 1 through 8 with Pro2, Pro5, and Pro8), followed by 2) a zone of change to beta type I (corresponding to residues 9 through 12) followed by 3) an amphiphilic alpha helix (residues 1330) that remains anti-parallel with the polyroline propeller at an angle of about 152 degrees between the axes of the helices, and 4) a C-terminal hexapeptide (residues 31-36). The folded structure is stabilized by hydrophobic interactions between the side chains of the amphiphilic alpha helix that closely inter-digest with the three hydrophobic proline residues (Schwartz et al 1990). In addition to the main residues in the receptor that recognize the C-terminal hexapeptide, there are the hydrophobic residues of the nucleus, which stabilize the PP folding structure, which are conserved throughout the family of PP folding peptides. Figure 1A shows the NPY sequence, with residues that are conserved between NPY, PYY and PP shown as white text on a black background. Figure 1 also illustrates the elements of the PP fold structure described above. The C-terminal hexapeptide, which is important for the recognition of the receptor is unstructured, but the PP fold provides a stable scaffold, which presents the C-terminal hexapeptide to the receptors (illustrated in Fig. 1 B), which is dependent or independent, in varying degrees, on parts of the N-term of the peptides. NMR spectroscopic analysis has shown that the C-distant and N-terminal parts of, for example, NPY, are quite mobile, indicating that the PP fold is constantly in danger of being "decompressed" from the free terminal end.

NPY is a broad spectrum neuropeptide with multiple actions in various parts of both the central and peripheral nervous systems acting through a number of different receptor subtypes, mainly: Y1, Y2, Y4 and Y5. The main NPY receptors are the Y1 receptor, which is generally the post-synaptic receptor that carries the “action” of NPY neurons and the Y2 receptor, which is generally a pre-synaptic, inhibitory receptor. This is also the case in the hypothalamus, where NPY neurons - which also express the melanocortin receptor antagonist / reverse agonist PRD (peptide related to dasyproctae) - act as the primary "sensory" neurons in the curved nucleus stimulatory branch. Thus, in this the "sensor core" for appetite control and energy display, NPY / PRD neurons together with POMC / CART inhibitory neurons monitor the hormonal and nutritional status of the body as soon as these neurons are the target of both long-term regulators such as leptin and insulin and short-term regulators such as ghrelin and PYY (see below). The stimulatory NPY / PRD neurons project, for example, to the paraventricular nucleus - also the hypothalamus - where its post-synaptic object receptors are believed to be Y1 and Y5 receptors. NPY is the most potent compound known in relation to increased food intake, as rodents under NPY intracerebroventricular injection (ICV) will eat until they literally explode. The PRD of NPY / PRD neurons acts as an antagonist primarily on type 4 melanocortin receptors (MC-4) and block the action of POMC-derived peptides - primarily aMSH - on this receptor. As soon as the MC4 receptor signal acts as a food intake inhibitor, the PRD action is - exactly the same as the NPY action - a stimulus signal for food intake (that is, an inhibition of an inhibition). In NPY / PRD neurons are inhibitory Y2 receptors - pre-synaptic - which are the object of both locally released NPY in addition to a target for the intestine PYY hormone - another PP fold peptide.

The PYY is released during a meal - in proportion to the caloric content of the food - from whole-endocrine cells in the extreme small intestine and colon, to act both on the periphery, on functions of the GI tract and centrally as a signal of satiety Peripherally, it is believed that PYY acts as an inhibitor - an "illicit rupture" - on, for example, the motility of the GI tract, gastric acid and exocrine pancreatic secretion. Centrally, it is believed that PYY acts primarily on inhibitory, pre-synaptic Y2 receptors on NPY / PRD neurons in the curved nucleus, believed to be accessed from the blood (Batterham et al. 2002 Nature 418: 650- 4). The peptide is released as PYY1-36, but a fraction - approximately 50% - circulates as PYY3-36, which is a product of degradation by dipeptidylpeptidase-IV, an enzyme that removes a dipeptide from the N-terminus of a peptide , provided that a Pro or Ala is in position two, as in all three peptides of the PP-PP, PYY and NPY fold (Eberlein et al., 1989 Peptides 10: 797-803). Thus, the PYY in circulation is a mixture of PYY1-36, which acts on both the Y1 and Y2 receptors (also as Y4 and Y5 with different affinities), and PYY3-36 - which has fewer affinities for the Y1, Y4 receptors and Y5 than for the Y2 receiver.

PP is a hormone, which is released from endocrine cells of the pancreatic islets, almost exclusively governed by vagal cholinergic stimuli especially obtained by food intake (Schwartz 1983 Gastroenterology 85: 1411-25). PP has various effects on the gastrointestinal tract, but none of these is observed in isolated cells and organs, and all of them appear to be dependent on an intact vagal nerve supply (Schwartz 1983 Gastroenterology 85: 1411-25). Accordingly, PP receptors, which are called Y4 receptors, are located in the brain stem with a strong manifestation in vagal motor neurons - whose activation results in the effects of PP as a satiety hormone (Whitecomb et al. 1990 Am.J. Physiol. 259: G687-9 1, Larsen & Kristensen 1997 Brain Res. Mol.Brain Res 48: 1-6). It should be noted that the PP of the blood has access to this area of the brain when the blood brain barrier is "porous" in this area where various hormones are perceived from the periphery. Recently, it has been discussed that part of the effect of PP on food intake occurs through an action on neurons - especially POMC / CART neurons in the curved nucleus (Batterham et al. 2004 Abstract 3.3 International NPY Symposium in Coimbra, Portugal). The PP acts through Y4 receptors for which it has a sub-nanomolar affinity as opposed to PYY and NPY that have nanomolar affinity for this receptor (Michel et al. 1998 Pharmacol. Rev. 50: 143-150). The PP also has an appreciable affinity for the Y5 receptor, but it is not of probable physiological importance in relation to the circulating PP due both to the lack of access to the cells in the central nervous system, where this receptor is especially expressed and due to the relatively low affinity for PP.

PP fold peptide receptors

There are four well-defined types of PP fold peptide receptors in man: Y1, Y2, Y4 and Y5, all of which recognize NPY1-36 and PYY1-36 with similar affinity. On one occasion, a type of Y3 receptor was suggested, which might prefer NPY over PYY, but today this is not accepted as a true receptor subtype (Michel et al. 1998 Pharmacol. Rev. 50: 143-150). A Y6 receptor subtype has been cloned, which is expressed in man in a truncated form that lacks TM-VII, in addition to the tail of the receptor and, consequently, at least on itself it does not appear to form a functional receptor molecule.

Y1 receptors - affinity studies suggest that Y1 links equally well with NPY and PYY, but not substantially with PP. The affinity for Y1 depends on the identities of both final sequences of the PP fold molecule (NPY / PYY) - for example, Tyr1 and Pro2 residues are essential - and it depends on the peptide ends being presented in exactly the right way. At the C-terminal end, where the side chains of several of the residues are essential, the Y1 receptor - such as the Y5 and Y4 receptors but not the Y2 receptor - tolerates certain substitutions at position 34 (usually a Gln) - such as Pro (Fuhlendorff et al. 1990 J. Biol. Chem. 265: 11706-12, Schwartz et al. 1990 Annals NY Acad.Sci. 61: 35-47). Such structure-function studies have been reported that refer to the requirements of Y1 and Y2 receptors (Beck-Sickinger et al. 1994 Eur.J. Biochem. 225: 947-58; Beck-Sickinger and Jung 1995 Biopolymers 37 : 123-42; S611 et al. 2001 Eur.J. Biochem. 268: 2828-37).

Y2 receptors - affinity studies suggest that Y2 binds equally well with NPY and PYY, but not substantially with PP. The receiver especially demands the C-terminal end of the PP fold (NPY / PYY). Thus, long C-terminal fragments - up to for example NPY13-36 (the complete alpha helix plus the C-terminal hexapeptide) - are recognized with a relatively high affinity, that is, up to ten-fold peptide affinity in full extension (Scheikh et al. 1989 FEBS Lett. 245: 209-14, Scheikh et al. 1989 J.Biol.Chem. 264: 6648-54). Consequently, various N-terminal deletions, which eliminate the binding to the Y1 receptor, still preserve some degree of binding to the Y2 receptor. However, the affinity of the C-terminal fragments is reduced approximately tenfold compared to NPY / PYY even for relatively long fragments. The Gln residue at position 34 of NPY and PYY is enormously important for binding recognition of the Y2 receptor (Schwartz et al. 1990 Annals NY Acad.Sci. 611: 35-47).

Y4 receptors - affinity studies suggest that Y4 binds PP with subnanomolar affinity corresponding to the concentrations found in plasma where NPY and PYY are recognized with much lower affinity. Such studies suggest that the Y4 receptor is highly dependent on the C-terminal end of the PP-fold peptides, and that relatively short N-terminal deletions disable link affinities. Such studies of structure activity related to the Y4 receptor have been reported (Gehlert et al. 1996 Mol. Pharmacol. 50: 112-18; Walker et al. 1997 Peptides 18: 609-12).

Y5 receptors - affinity studies suggest that Y5 binds equally well with NPY and PYY, and also binds PP with lower affinity, which, however, is below the normal circulation levels of this hormone. PYY3-36 is also well recognized by the Y5 receptor, however this receptor is expressed, to a high degree, in the Central Nervous System where such a peptide cannot gain access to the receptor directly when administered at the periphery.

The PP fold peptides and their analogues have been suggested for use in the treatment of obesity and related diseases, including, for example, Prader Willi syndrome, based on the demonstrated effects of certain of these peptides in animal models and in man and in the fact that obese people have low baseline levels of PP and PYY in addition to low nutritional responses of these peptides (Holst JJ et al. 1983 Int.J.Obes. 7: 529-38; Batterham et al. 1990 Nature). It has been known since the mid-1970s that PP could affect food intake in rodents. In 1993 it was reported that the infusion of PP in morbidly obese patients with Prader Willi syndrome decreased food intake (Berntson et al. 1993 Peptides 14: 497-503). Recently, this effect of PP was confirmed by infusion of PP in normal human subjects where long-term appetite suppression and reduced food intake were observed in 24 hours (Batterham et al. 2003, Clin. Endocrinol. Metab. 88: 3989-92).

However, native PP fold peptides are not optimal for use as biopharmaceuticals, for example, they are susceptible to degradation by various proteases such as DPP-IV. Thus, natural peptides are not optimized for protein stability as soon as they are produced to act, normally, for a relatively short time as a neuropeptide or hormone.

For the treatment of conditions that respond to Y4 receptor modulation, such as obesity and intestinal hypersecretion, it would therefore be desirable to use PP fold peptides or PP fold peptide mimics, which acted as agonists and were specific for the Y4 receiver sought as an object, and which stably preserve elements of the PP fold structure important for the receptor link. In particular, it would be highly desirable to use such agents that are selective for the Y4 receptor over the Y1 and Y2 receptors. This is especially important, as expected from the activation of the Y1 receptor that potentially causes unwanted renal and cardiovascular side effects such as vasoconstriction and natriuresis. In addition, activation of the Y2 receptor can also cause side effects. Although it is not yet clear what the really efficient angiogenic Y-receptor profile is, Y2 agonists such as HPY3-35 can induce revascularization in, for example, models of ischemic hind limbs, that is, when administered in high doses. with constant exposure, such as when released from inoperative pellets (Zukowska Z et al. Trends Cardiovasc Med. 2003, 13: 86-92). The angiogenic response to NPY is reduced in animals knocked out with the Y2 receptor; however, the response to this NPY agonist of the broad spectrum Y is, in fact, not eliminated and both Y2 and Y5 receptors are over-regulated in ischemic vessels (Lee et al. J.Clin.Invest. 2003, 111: 1853-62). However, a PP-fold peptide or a PP-fold peptide mimic could, through activation of the Y2 receptor, cause side effects such as worsening retinopathy, for example, in diabetic patients and could potentially help in neo- vascularization associated with the growth of certain cancers. Thus, the use of the efficient and selective Y4 receptor on the Y1 and Y2 receptor agonists would be particularly useful in diseases and conditions susceptible to Y4 receptor activation.

Some common terms used in this report

Affinity: The affinity of a peptide to a specific receptor is given for example as with IC50 value or a Ki or Kd value, which in a specific non-limiting example is determined in an assay, as in a competition binding assay. The IC50 value corresponds to the concentration of the peptide that displaces - for the given relevant receptor - radioactive bond used in an amount much less than the Kd for 50% radioactive bond.

Appetite: A natural desire or craving for food. An increased appetite generally leads to increased eating behavior.

Appetite suppressants: Compounds that decrease the desire for food.

Link: A specific interaction between two molecules, so that both molecules interact. The binding of a receptor can be specific and selective, such that one molecule is preferentially linked in comparison to another molecule. The specific link can be identified by a dissociation constant (Kd). This value is dependent on the selectivity of the analyzed compound. For example, a compound with a Kd that is less than 10 nM is generally considered an excellent drug candidate. However, a compound that has a low affinity, but is selective for the particular receptor, may also be a good drug candidate.

Body Mass Index (BMI): A mathematical formula for measuring body mass, also sometimes called Quetelet Index. BMI is calculated by dividing weight (in kilograms) by height2 (in meters). The current standards for both men and women accepted as "normal" are a BMI of around 20 kg / m2. In one embodiment, a BMI of more than 25 kg / m2 can be used to identify an obese subject. Grade I obesity corresponds to a BMI of 25 kg / m2. A Grade II obesity corresponds to a BMI of 3040 kg / m2; and a Grade III of obesity corresponds to a BMI greater than 40 kg / m2 (Jequier 1987 Ain. J Clin. Nutr.

45: 1035-47). An ideal body weight will vary between species and individuals based on weight, body constitution, bone structure and sex.

Caloric intake or calorie intake: The number of calories (energy) consumed by an individual. In the present context, this term is identical to the term "energy intake."

Cosmetic treatment: the term is intended to designate a treatment not intended for medical purposes, but to improve the well-being of a subject, for example, in relation to the appearance of a subject. The term includes the treatment of a subject who wishes to reduce his weight without necessarily being overweight or obese.

Food intake: The amount of food consumed by an individual. Food intake can be measured by volume or weight. Its meaning includes i) food intake as a total amount of food consumed by an individual, and ii) food intake is the amount of protein, fat, carbohydrates, cholesterol, vitamins, minerals or any other food component of the individual . Accordingly, the term food intake used in the present context is similar to the term "energy intake."

Normal Daily Diet: The average dietary intake of an individual of a given species. A normal daily diet can be expressed in terms of caloric intake, protein intake, carbohydrate intake, and / or fat intake. A normal daily diet in humans generally comprises the following: from about 2,000, from about 2,400 or from about 2,800 to significantly more calories. Additionally, a normal daily diet in humans generally includes about 12 g to about 45 g of protein, about 120 g to about 610 g of carbohydrate, and about 11 g to about 90 g of fat. A hypocaloric diet would be no more than about 85%, and preferably no more than about 70% of the normal caloric intake of a human individual. In animals, caloric and nutrient requirements vary depending on the species and size of the animal. For example, in cats, the total caloric intake per kg, in addition to the percentage distribution of protein, carbohydrates and fats varies with the cat's age and reproductive status.

Obesity: A condition in which excess body fat can put a person at risk for their health (see Barlow and Dietz, Pediatrics 102: E29, 1998; National Institutes of Health, National Heart, Lung and Blood Institute (NHLBI) , Obes. Res. 6 (suppl. 2): 51 S209S, 1998). Excess body fat is the result of an imbalance in energy intake and energy consumption. In one embodiment, the Body Mass Index (BMI) is used to analyze obesity. In one embodiment, a BMI of about 22 kg / m2 (that is, about 10% above normal value) and up to about 30 kg / m2 is estimated to be overweight, especially around 25.0 kg / m2 and up to 30 kg / m2. It should be noted that subjects who have a BMI that is only slightly higher than the normal value (for example, from about 22 to about 25 kg / m2), very often have a desire to lose weight although this It may be only for cosmetic reasons.

Potency: The in vitro potency of a compound is defined in terms of EC50 values, that is, the concentration that leads to 50% of the maximum effect obtainable as determined in and for the relevant given recipient signing the assay.

Subject: The subject can be any subject, including both human and veterinary mammalian subjects. Thus, the subject may be a human, or it may be a non-human primate, a farm animal such as pig, cattle, sheep and poultry, an animal for sports or pets such as dogs, cats, horses, hamsters and rodents .

Therapeutically effective amount: A dose sufficient to prevent, treat or improve a specific condition or disease and / or relieve specific signs or symptoms of a specific condition or disease. The term includes a dose that is sufficient or prevents the advance or causes the regression of a disorder, or from which it is able to relieve a sign or symptom of a disorder, or from which it is able to achieve a desired result. In embodiments relating to cosmetic treatment or overpressure or obesity treatment, a therapeutic effect of a receptor agonist is an amount sufficient to inhibit or stop weight gain, or an amount sufficient to decrease appetite, or an amount sufficient to reduce energy. or food intake or increase energy consumption. The term "cosmetically effective amount" is intended to designate a dose that is sufficient for the subject being treated to achieve a desired effect.

DETAILED DESCRIPTION OF THE INVENTION

In its broadest aspect, the present invention provides the use of a Y receptor agonist that is selective for the Y4 receptor on the Y1 and Y2 receptors, said Y receptor agonist being the pancreatic peptide PP2-36 with sequential identification number: 10, in the preparation of a composition to (i) reduce food intake, (ii) treatment of obesity or overweight, (iii) treatment of diarrhea or hypersecretion associated with small intestinal ileostomy or (iv) treatment of a condition in which obesity or overweight is a contributory factor, selected from bulimia, bulimia nervosa, Syndrome X (metabolic syndrome), diabetes, type 2 diabetes mellitus or non-insulin-dependent diabetes mellitus (NIDDM), hyperglycemia, insulin resistance, glucose intolerance , cardiovascular disease, hypertension, arteriosclerosis, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, disease d thromboembolic, hypercholesterolemia, hyperlipidemia, gallbladder disease, osteoarthritis, sleep apnea, polycystic ovarian syndrome, and breast, prostate or colon cancer. PP2-36 (Sequential identification number: 10), is referenced in Bard et al., J.Biol.Chem. (1995) 270: 26762-26765 and Walker et al. Peptides vol 18, no. 4, 609-612 (1997). However, no reference discloses the use of the peptide for the purposes of this invention.

Gehlert et al. Molecular Pharmacology 199607 US, Vol. 50, no. 1, July 1996 (1996-07), pp. 112-118, includes receiver link data related to bovine PP truncation.

PP2-36 is selective for the Y4 receptor over the Y1 and Y2 receptors. In the present context, this condition is met if the agonist has an IC50 value that is at least 10-fold lower for Y4 than for Y1 and Y2 receptors when measured in the affinity test described here. With respect to the power, PP2-36 has an EC50 value at least 10-fold lower for the Y4 receiver than for the Y1 and Y2 receivers when measured in the power test described here.

PP2-36 is a highly selective Y4 receptor agonist with improved stability compared to PP1

36. PP is normally a substrate for DPP-IV degradation as soon as it has a Pro in position 2. Thus, by eliminating the first PP residue a peptide is generated where the PP folding structure is not compromised because residue 1 (Ala1) does not make any interaction with the anti-parallel helix (this is guarded by Pro2) and because PP2-36 does not like the wild type peptide as a substrate for DPP-IV. Importantly, the Y4 receptor is not dependent on the presence of a residue in position 1. Thus, PP2-36 is both a more stable peptide and is a selective Y4 receptor agonist that has an improved selectivity window towards the Y1 receptor.

Receptor recognition profile - PP2-36 links with an affinity to the Y4 receptor, IC50 = 0.64 nM, which is similar to the affinity of PP, IC50 = 0.41. The affinity of PP2-36 on the Y1 and Y2 receptors is> 1000 nM (Table 1). PP2-36 acts as an agonist on the Y4 receptor with a power of 0.64 nM (EC50) which is also very similar to the power of PP (EC50 = 0.85 nM) as measured in the inositol phosphate motion test carried out in COS-7 cells transfected as described in values> 1000 nM. However, on the Y1 receiver, PP2-36 has a power that is 3-4 folds lower than that of the PP values, EC50 being 297 against 83 nM (Table 2). Thus, PP2-36 is a selective Y4 agonist with> 300 fold window both the Y1 and Y2 receptor as determined in a functional in vitro assay.

Protein stability - PP2-36 has the advantage compared to PP1-36 which is more stable and, consequently, a better biopharmaceutical agent as it is not a substrate for DPP-IV, as PP is due to the presence of a Pro residue in position 2- and insofar as the novel Pro N-terminal residue in PP2-36 prevents against degradation by most other amino peptides. These stability properties are especially useful in the context where a prolonged presence of the peptide in body fluids is obtained through various means such as motif modifications of the peptide as such or by means of various forms of pharmaceutical procedures such as sustained release formulations. .

Effect in vivo on acute food intake - either a saline solution or PP2-36 was administered by individual subcutaneous injections to groups of 8 mice at doses of 1 and 10 µg per animal after 16 hours of fasting. Figure 2 shows the cumulative dietary intake of the mice for 8 hours. The selective Y4 receptor agonist, PP2-36 inhibited food intake during the first 20 minutes after the initiation of the food at approximately 50% of the amount swallowed by the group of animals receiving saline. The suppressive effect of 50% on food intake was also observed when measured during the first full hour. The effect of 1 ug and 10 ug of PP2-36 was quite similar. The suppressive effect of PP2-36 was sustained for the entire period of 8 hours after individual subcutaneous injection (Fig 2). Thus, PP2-36, which is highly selective for Y4 on Y2 and Y1 receptors, is an efficient suppressor of food intake in mice as previously demonstrated for PP (Asakawa et al. 1999 Peptides 20: 1445-48. Asakawa 2003 Gastroenterology 124: 1325-36). It has been previously shown that this effect of PP is also observed in normal human subjects (Batterham et al. 2003 Clin.Endocrinol.Metab. 88: 3989-92) in addition to morbidly obese patients (Berntson et al. 1993 Peptides 14: 497-503).

Clinical Indications

PP2-36 is of value in the treatment of conditions that respond to the activation of Y4 receptors. Such conditions include those for which the regulation of energy intake or energy metabolism is indicated.

Diseases or conditions in which the regulation of energy intake or energy metabolism is indicated include obesity and overweight, and conditions in which obesity and overweight are considered as contributing factors, such as bulimia, bulimia nervosa, Syndrome X ( metabolic syndrome), diabetes, type 2 diabetes mellitus or Dependent Non-Insulin Diabetes Mellitus (NIDDM), hyperglycemia, insulin resistance, glucose intolerance, cardiovascular disease, hypertension, atherosclerosis, coronary artery disease, myocardial infarction, disease peripheral vascular, stroke, thromboembolic disease, hypercholesterolemia, hyperlipidemia, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, and breast, prostate or colon cancer.

PP2-36 is also of importance in the treatment of diarrhea or hypersecretion of intestinal stomy.

1.Obesity and overweight

It was suggested already in the seventies that the PP could be involved in the control of food intake. Recently, much evidence has been accumulated from studies on rodents that show very clearly that PP is, in fact, a powerful and efficient anorexigenic peptide when administered peripherally (Asakawa et al. Peptides 1999, 20; 1445-8; Katsuura et al Peptides 2002, 23: 323-9; Asakawa et al. Gastroenterology 2003, 124: 1325-36). As soon as the PP has no effect on appetite, food intake, etc. In animals knocked out with Y4, the PP is likely to act through the Y4 receptor to reduce appetite and food intake (Batterham et al. 2004 abstract S3.3 of the International Symposium on NPY in Coimbra [Portugal]. The PP also it has an effect on the intake of food in obese animals with an induced diet.PP receptors have been found in the brainstem in the last zone and in vague motor neurons where the blood brain barrier is not efficient and where circulating hormones such as PP can access neurons, so it is very likely that Y4 receptors in the NTS in the brainstem are a main objective through which PP acts to suppress appetite and food intake, however, recent evidence they also point to the possibility that the PP can also act through Y receptors in the conceivable arcuate nucleus in POMC neurons and perhaps also in NPY / AgRP (Batterham et al. Coim bra NPY meeting abstract S3.3). Low levels of PP are found in obese subjects, especially in Prader-Willi syndrome (Zipf et al.

J.C.E.M. 1981, 52: 1264-6, Holst et al. 1983, Int.J.Obes. 7: 529-38, Glaser et al. Horm Metab 1988, 20: 288-92) and high levels of PP are found in patients with anorexia nervosa. Importantly, infusion of PP in man decreases appetite and food intake up to 24 hours (Batterham et al. JCEM 2003, 88: 3989-92). Thus, the effect of PP on food intake was observed after the levels of PP in the circulation had returned to normal levels. Such long-lasting effects on appetite, etc., are notoriously known from other compounds, for example, also in the ICV injection of AgRP. Importantly, the infusion of PP has been shown to decrease food intake in morbidly obese patients with Prader Willi syndrome (Berntson et al 1993 Peptides 14: 497-503).

Hence, PP2-36 is suitable for use in a subject, such as a mammal, including a human, in order to regulate energy intake. Accordingly, PP2-36 can be used to alter energy intake, food intake, and energy consumption. The administration of PP2-36 results in a decrease in the amount, either in the total weight or the total volume or the calorie content of the food and / or a decrease in the intake of a food component, such as a decrease in the ingestion of lipids, carbohydrates, cholesterol or proteins. Appetite can be reduced by the administration of a therapeutically effective amount of PP2-36. Appetite can be measured by any means known to a person skilled in the art.

For example, reduced appetite can be assessed by a psychological evaluation. In such an embodiment, administration of the receptor agonist results in a change in hunger, satiety and / or perceived fullness. Hunger can be assessed by any means known to the person skilled in the art. In one embodiment, hunger is assessed using psychological trials, such as by evaluating feelings of hunger and sensory perception using, for example, a questionnaire.

PP2-35 can be used to decrease the motility of the upper GI tract such as by decreasing gastric emptying. From PP, the prototype of agonist Y4, its ability to reduce gastric emptying is known. A therapeutically effective amount of PP2-36 can be administered to the subject, thereby decreasing the motility of the GI tract. It is well known that compounds that decrease gastric emptying will have a beneficial effect in also decreasing food intake as soon as the subject feels fuller or satisfied.

PP2-36 can be used to alter the energy metabolism in a subject. Energy is burned in all physiological processes. The body can alter the energy consumption index directly, by modulating the efficiency of these processes, or by changing the number or nature of procedures that are taking place. For example, during ingestion, the body expends energy by moving food through the intestine, and digesting food, and, inside cells, the efficiency of cellular metabolism can be altered to produce more or less heat. In a subsequent embodiment, a procedure is disclosed here for any and all manipulations of the arc circuit described in this application, which alters, in a coordinated and reciprocal manner, the energy consumption. Energy consumption is a result of metabolism, cellular, protein synthesis, metabolic index, and calorie utilization. Thus, peripheral administration of PP2-36 results in increased energy consumption, and decreased efficiency of calorie utilization.

PP2-36 can be used for weight control and treatment, reduction or prevention of obesity, especially any or more of the following: prevention and reduction of weight gain; induction and promotion of weight loss; and obesity reduction as measured by the Body Mass Index. As mentioned above, the invention also relates to the use of PP2-36 to control any or more of appetite, satiety and hunger, especially any or more of the following: appetite reduction, suppression and inhibition; induction, increase, reinforcement and promotion of satiety and feelings of satiety; and reduction, inhibition and suppression of hunger and feelings of hunger. The disclosure also relates to the use of PP236 in maintaining any or more of a desired body weight, a desired Body Mass Index, a desired appearance and good health.

PP2-36 can be used for the treatment and / or prevention of eating disorders, appetite disorders, overweight, bulimia, bulimia nervosa, Syndrome X (metabolic syndrome) or complications or risks associated with them, including diabetes, diabetes Type 2 mellitus or Dependent Non-Insulin Mellitus Diabetes (NIDDM), hyperglycemia, insulin resistance, glucose intolerance, cardiovascular disease, hypertension, arteriosclerosis, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, thromboembolic disease , hypercholesterolemia, hyperlipidemia, gallbladder disease, osteoarthritis, sleep apnea, reproductive disorders such as polycystic ovarian syndrome, and breast, prostate or colon cancer, the procedure comprising administration to a subject such as a mammal, including a human , an effective dose of PP2-36, as described here.

2. Intestinal hypersecretion

PP is known to have a strong anti-secretory effect on both the small and large intestines and this appears to be mediated by Y4 receptors located in epithelial cells (Cox & Tough 2001 Br.J.Pharmacol. 135: 1505 -12). It was shown in vivo that peripheral administration of PYY - another PP-fold peptide that activates Y1 and Y2 receptors - can cause a lasting reduction in intestinal secretion induced by vasoactive intestinal polypeptide in human subjects with ileostomies (Playford et al 1990 Lancet 335 : 1555-57). It was concluded that the PYY could be a therapeutic agent against diarrhea. However, for example the natriuretic and hypertensive effects of the combined Y1 and Y2 agonists, NPY and PYY peptides have prevented this. Such side effects will not be relevant for PP2-36. Thus, PP2-36 is especially useful for the treatment or protection against hypersecretion of the GI tract including various forms of diarrhea whether or not caused by hypersecretion as a suppression of intestinal secretion will eliminate either the cause of the diarrhea or the symptoms. An especially interesting indication is the hyper-secretion observed in patients with ileostomy, who often lose large amounts of fluid. PP2-36 is especially useful for the treatment or protection against hypersecretion associated with ileostomy of the small intestine.

Additional comments regarding the administration of PP2-36 for the treatment or prevention of obesity and related diseases

During a meal, a wide variety of gastrointestinal hormones and neurotransmitter systems are activated in a carefully coordinated, sequential and superimposed way. In addition, food components influence not only the secretion of GI hormones and the activity of various afferent neuronal pathways, but these food components also influence various hormones and central nervous system centers directly after being absorbed. Thus, the regulation of food intake and energy consumption is a highly complex and multi-faceted process. In view of this, it is surprising that certain hormones such as PP, in fact, can substantially affect the system when administered in a manner that results in, for example, only 3-4 times the plasma levels that are reached during a meal.

Administration of the PP2-36 agonist has, mainly, and apparently, the desired effect if the compound is administered on an empty stomach in an effective dose as described. If the agonist is supplied in a situation where the varied hormonal and neuronal systems are active due to the presence of food components in the GI tract or the expectation of a meal, the effect is not seen or a minor effect is observed. Thus, the selective Y4 agonist is preferably administered on an empty stomach in an effective dose either subcutaneously, nasally or through other means as described herein. In the present context, the term "fasting" means that the subject has not eaten any food or drink within at least the last 2 hours before administration of the Y2 receptor agonist, such as, for example, within, at least , the last three hours, within at least the last four hours, within at least the last five hours, within at least the last six hours, within at least the last seven hours, within at least the last eight hours , within at least the last nine hours, within at least the last ten hours, within at least the last eleven hours or within at least the last twelve hours before dosing.

In a subgroup of the population, PP2-36 may not have the intended action due to genetic variations such as polymorphisms of the Y4 receptor gene. Loss of function by mutations in these receptors can probably be associated with obesity. Thus, in a preferred embodiment of the invention, an analysis of the Y4 gene of the subject to be treated is carried out in order to investigate the polymorphisms / mutations in these genes and the identification of such polymorphisms. An optimal treatment of the subjects can be performed based on such analysis. For example, only subjects with normal genotype or polymorphisms, which do not affect the function of the Y4 agonist, should be treated with PP2-36. Another possibility is to increase the dose of PP2-36 in subjects expressing a receptor with abnormalities in order to ensure an optimal effect of the drug. In the case where the obesity of a subject is caused by an abnormality in Y4 receptor function, it could be argued that treatment with - for example - large doses of PP2-36 is a form of replacement therapy - provided that at least some of the relevant functions of the recipient have been left out - for example in heterozygous patients.

An acute analysis can be carried out where PP2-36 is administered to ensure that it has the intended effect on the subject to be treated before a chronic treatment is initiated. Through these means, it is ensured that only subjects that are susceptible to treatment with PP2-36 are treated with the compound.

PP2-36 can be combined in the treatment of obesity, diabetes and diseases related to the use of various other drugs aimed at appetite and energy consumption. This includes, but is not limited to, drugs such as GI tract lipase inhibitors, neurotransmitter reuptake inhibitors, cannabis receptor antagonists, and inverse agonists, in addition to other types of neurotransmitters - including but not limited to receptors. 5HT - and / or hormones - including but not limited to receptor agonists or GLP-1 antagonists, MC4, MC3. Due to the fact that the selective Y4 agonist aims at a homeostatic regulatory mechanism in the communication between the GI tract and the Central Nervous System - that is, the Y4 receptors that are normally the object of satiety by the PP hormone of the pancreas - It is especially beneficial to combine treatment with PP2-36 with treatment with a drug that has as its object a central, hedonic mechanism in the regulation of appetite and energy consumption, such as CB1 receptors, for example being part of the reward system.

Dose

The therapeutically effective amount of PP2-36 will depend on the age, weight and condition of the subject being treated, the severity and type of the condition or disease being treated, the form of administration and the strength of the composition that is applied. .

For example, a therapeutically effective amount of PP2-36 can vary from about 0.01 µg per kilogram (kg) of body weight to about 1 g per kg of body weight, such as about 1 µg to about 5 mg per kg of body weight, or about 5 µg to about 1 mg per kg body weight. In another embodiment, PP2-36 is administered to a subject at 0.5 to 135 picomoles (pmol) per kilogram of body weight,

or about 72 pmol per kg of body weight.

In a specific, non-limiting example, about 5 to about 50 nmol are administered as a subcutaneous injection, such as about 2 to about 20 nmol, or about 1.0 nmol is administered as a subcutaneous injection. The exact dose is readily determined by one skilled in the art based on the potency of the specific compound (such as the receptor agonist) used, the age, weight, sex and physiological condition of the subject. The dose of an agonist can be a molar equivalent of the therapeutically effective dose of PYY3-36.

The amounts can be divided into one or several doses for daily administration, every second day, weekly, every two weeks, monthly or with any other suitable frequency. Normally, administration is once or twice daily.

Administration Procedures

PP2-36 in addition to pharmaceutical compositions according to the invention can be administered by any route, including the enteral route (for example, oral administration) or parenteral route. In a specific embodiment, the parenteral route is preferred and includes intravenous, intra-articular, intraperitoneal, subcutaneous, intramuscular, intrasternal and infusion injection, in addition to administration by sublingual, transdermal, topical, transmucosal route, including the nasal route, by inhalation, by example, pulmonary inhalation. In specific embodiments, the subcutaneous and / or nasal route of administration is preferred.

When administered centrally, the natural Y4 selective PP peptide can, as NPY and PYY do when ICV has been administered, induce eating (probably due to the activation of central receptors that circulating hormones or compounds of normally do not reach peripherally administered peptides). Thus, in cases where increased appetite, it is preferred that PP2-36 be administered peripherally.

The PP2-36 may be administered as such dispersed in a suitable vehicle or may be administered in the form of a suitable pharmaceutical composition. Such compositions are also within the scope of the invention. In the following, suitable pharmaceutical compositions are described.

Pharmaceutical compositions

PP2-36 for use in medicine is normally presented in the form of a pharmaceutical composition comprising PP2-36 in conjunction with one or more physiologically or pharmaceutically acceptable excipients.

PP2-36 can be administered to an animal including a mammal such as, for example, a human, by any suitable route of administration such as, for example, the oral, buccal, nasal, ocular, pulmonary, topical, transdermal, vaginal route. , rectal, ocular, parenteral (including but not limited to subcutaneous, intramuscular, and intravenous, see above), at a dose that is effective for individual purposes. A person skilled in the art will know how to choose a suitable route of administration. As mentioned above, the parenteral route of administration is preferred. In a specific embodiment, the receptor agonist is administered subcutaneously and / or nasally. It is well known in the state of the art that subcutaneous injections can be easily self-administered.

A composition suitable for a specific route of administration is easily determined by a healthcare professional for each individual patient. Various pharmaceutically acceptable carriers and their formulation are described in standardized formulation treaties, for example, Remington’s Pharmaceutical Sciences, by E.W. Martin.

Pharmaceutical compositions comprising PP2-36 may be in the form of a solid, semi-solid or liquid composition. For parenteral use, the composition normally has a liquid form or a semi-solid or solid form for implantation.

Liquid compositions, which are sterile solutions or dispersions can be used by, for example, intravenous, intramuscular, intrathecal, epidural, intraperitoneal or subcutaneous injection or by infusion. PP2-36 can also be prepared as a sterile solid composition, which can be dissolved or dispersed before or at the time of administration using, for example, distilled water, saline solution or other suitable sterile injectable medium.

The liquid form of the composition may be a solution, an emulsion containing nano-emulsions, a suspension, a dispersion, a liposomal composition, a mixture, a spray, or an aerosol (the last two types are especially relevant for nasal administration) .

Suitable media for solutions or dispersions are usually based on pharmaceutically acceptable water or solvents, such as an oil (for example, sesame or peanut oil) or an organic solvent such as, for example, propanol or isopropanol. A composition according to the invention may comprise further pharmaceutically acceptable excipients, such as, for example, pH adjusting agents, osmotically active agents, for example, in order to adjust the isotonicity of the composition to physiologically acceptable levels, viscosity adjusting agents , suspending agents, emulsifiers, stabilizers, preservatives, antioxidants, etc. A preferred medium is water.

Compositions for nasal administration may also contain suitable non-irritating carriers, such as, for example, polyethylene glycols, glycofurol, etc., in addition to absorption enhancers well known to those skilled in the art (for example, with reference to Remington's Pharmaceutical Sciences).

For parenteral administration, in one embodiment, PP2-36 can generally be formulated by mixing it in the desired degree of purity, in an injectable unit dosage form (solution, suspension

or emulsion), with a pharmaceutically acceptable carrier or excipient, that is, one that is not toxic to receptors at the dosages and concentrations used and is compatible with other ingredients of the composition.

Generally, the formulations are prepared by contacting PP2-36 uniformly and intimately with liquid carriers or subtly divided solid carriers or both. Then, if necessary, the product is shaped in the desired formulation. Preferably, the carrier is a parenteral carrier, more preferably a solution that is isotonic with the recipient's blood. Examples of such carriers include water, saline, Ringer's solution, and dextrose solution. Non-aqueous carriers such as fixed oils and ethyl oleate are also useful here, in addition to liposomes. Due to the amphipathic nature of the peptides described herein, other suitable forms also include micellar formulations, liposomes and other types of formulations comprising one or more suitable lipids, such as, for example, phospholipids and the like.

Preferably, they are suspended in an aqueous carrier, for example, in a buffered isotonic solution at a pH of about 8.0, preferably at a pH of about 3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5. Useful buffer substances include acetate, citrate, phosphate, borate, carbonate, such as, for example, sodium citrate-citric acid and sodium phosphate phosphoric acid buffers, and sodium acetate / acetic acid.

The compositions may be designed for controlled or prolonged release of the receptor agonist after administration in order to obtain a less frequent administration regimen. Normally, a dosing regimen including 1-2 daily administrations is considered adequate, but other administration regimes are also included within the scope of the present invention, such as, for example, more frequency and less frequency. In order to achieve a prolonged release of PP2-36, a suitable carrier, including, for example, lipids or oils, can be used in order to form a reservoir in the administration area from which PP2-36 is slowly released into the circulatory system, or an implant can be used. Suitable compositions in this regard include liposomes and biodegradable particles to which PP2-36 has been incorporated.

In these situations, where solid compositions are required, the solid composition may be in the form of tablets such as, for example, conventional tablets, effervescent tablets, coated tablets, molten tablets or sublingual tablets, pellets, powders, granules, granules, material in particles, solid dispersions or solid solutions.

A semi-solid form of the composition may be a chewing gum, an ointment, a cream, a liniment, a paste, a gel or a hydrogel.

Other suitable dosage forms of the pharmaceutical compositions according to the invention may be vagitories, suppositories, strips, patches, tablets, capsules, sacks, troches, devices, etc.

The dosage form can be designed to release PP2-36 freely or in a controlled manner, for example, with respect to tablets by suitable coatings.

The pharmaceutical composition may comprise a therapeutically effective amount of PP2-36.

The content of PP2-36 in a pharmaceutical composition of the invention is, for example, about

0.1 to about 100% by weight of the pharmaceutical composition.

The pharmaceutical compositions can be prepared by any of the methods known to a pharmaceutical formulation expert.

In pharmaceutical compositions, PP2-36 is normally combined with a pharmaceutical excipient, this is a therapeutically inert substance or carrier.

The carrier can take a wide variety of forms depending on the desired dosage and route of administration.

Pharmaceutically acceptable excipients may be, for example, putties, hardeners, disintegration stimulants, diluents, glidants, solvents, emulsifying agents, suspending agents, stabilizers, strengthening agents, aromas, colors, pH adjusting agents, retarding agents, humidifying agents, surface active agents, preservatives, antioxidants, etc. Details can be found in pharmaceutical manuals, such as Remington’s Pharmaceutical Sciences or the Pharmaceutical Exicipient Handbook.

Synthesis

PP2-36 can be synthesized by solid phase peptide synthesis, using either an automatic peptide synthesizer, or traditional bank synthesis. The solid support can be, for example, chlorotrityl (CI) or Wang resin (OH), both of which are commercially available. The active groups of these resins react immediately with the carboxyl group of an amino acid N-Fmoc, consequently covalently binding it to the

polymer. The resin-bound amine can be deprotected by exposure to piperidine. A second N-protected amino acid can then be coupled to the amino acid-resin. These phases are repeated until the desired sequence is obtained. At the end of the synthesis, the resin-bound protected peptide can be deprotected and separated from the resin with trifluoroacetic acid (ATF). Examples of reagents that facilitate the coupling of new amino acids to the chain of amino acids bonded to the resin are: tetra-methylurionium hexafluorophosphate (HATU), hexafluorophosphate O- (1H-benzotriazola-1-yl) -N, N, N ', N '-tetramethyluronium (HBTU), tetrafluorborate O- (1Hbenzotriazola-1-yl) -N, N, N', N'-tetramethyluronium (TBTU), 1H-hydroxybenzotriazole (HOBt).

Peptide synthesis by solution chemistry rather than solid phase chemistry is also feasible.

PP2-36 was made by solid phase synthesis, on PALPeg-PS resin, (amide resin (amide resin Applied Bioscience, Warrington, UK GEN913401), using Fmoc chemistry with an excess of 5x reagent. The coupling was carried out by HCTU everywhere, DMF being the solvent.Fmoc removal was carried out with 20% piperidine in DMF, 10-15 minutes, however, the peptide could simply also be synthesized by various other standardized methods of peptide synthesis such as tBOC chemistry and solution chemistry instead of solid state, etc. The synthesis is illustrated by the following description, but PP2-36 was made by similar procedures:

Illustrative Synthesis of [Cis2, D-Cis27] PP (SEQUENTIAL IDENTIFICATION Nº: 4)

In the protection of general lateral group there was normalized Fmoc except in:

Arg = Fmoc Arg (Pbf) –OH

Asn, Gln = Fmoc Asn (Trt) -OH

Thr, Ser, Asp, Glu, Tir = tButil

Ala-Ser 22-23 = Fmoc AlaSer pseudoproline

In the case of [Cis2, D-Cis27] PP, the following special protection groups were used in order to obtain selective deprotection:

Cis 27 = Fmoc DLis (Trt) –OH

The peptide was synthesized by solid phase synthesis, on PAL Peg-PS resin (a resin that will generate the biologically important carboxyamide group, by division), using Fmoc chemistry with excess 5 fold molar reagent. The coupling was carried out. by HCTU everywhere, using DMF as a solvent. Fmoc removal after each coupling phase was carried out with 20% piperidine in DMF for 10-15 minutes. The coupling was checked after each stage by quantitative ninhydrin test. In certain cases, double couplings could be carried out.

The resin was subsequently divided into different batches to generate the peptide.

Structure

Molecular formula Mw theoretical m / z m / z measured mass Reaction time minutes of in % purity Analytical Procedure

PP2-36

 C182H282N5 205352 4110.7 4109.2 18.1 96.2 C

[Cis2, DCis27] PP

C177H279N5 305354 4125.8 4124.4 17.9 98.7 C

HPLC Analytical Procedure C

 Column = Vydac C18 218TP54, 250 x 4.6 mm     Buffer A = 20 mL of MeCN and 2 mL of ATF in Water (Total Volume 2000 mL)     Shock Absorber B = 2 mL of ATF in Water (Total volume of 2000 mL)

   Gradient = 25% B to 75% over 27 minutes    Flow rate = 1.00 mL / min    Wavelength = 215 nm

Injection volume = 10 µL

Biological tests and results

I. IN VITRO TESTS TO DETERMINE THE AFFINITY AND POWER OF PEPTIDES

Affinity Assay of Human Y2 Receptor

The affinity of test compounds for the human Y2 receptor is determined in a competition binding analysis using 1251-PYY binding on COS-7 cells fleetingly transferred with the human Y2 receptor using a standardized calcium phosphate transfer method.

The transferred COS-7 cells are transferred to culture plates one day after transfer at a density of 40 x 103 cells per well looking for a 5-8% bond of the radioactive binder. Two days after transfer, competitive binding experiments are carried out for 3 hours at 4 ° C using 25 pM of 125l-PYY (Amersham, Little Chalfont, UK). Binding assays are carried out in 0.5 ml of a 50 mM Hepes buffer, pH 7.4, supplemented with 1 mM CaCl2, 5 mM MgCl2, and 0.1% (w / v) bovine serum albumin and 100 µg / ml of bacitracin. A non-specific link is determined as a link in the presence of 1 µM of unlabeled PYY. The cells are washed twice in 0.5 ml of frozen buffer and 0.5-1 ml of lithic buffer (8 M Urea, 2% NP40 in 3 M acetic acid) is added and the associated radioactivity is measured in a gamma counter. The determinations are made in duplicate. The fixed state link is reached with the radioactive linker under these conditions. EC50 values were calculated using standardized pharmacological data management programming, Prism 3.0 (graphPad software, San Diego, United States).

Affinity analysis of the human Y4 receptor

The protocol for the Y2 affinity analysis, except that human transformed COS-7 Y4 cells are used, the competence analysis uses 125l-PP, and PP is used for the determination of non-specific binding.

Affinity analysis of the human Y1 receptor

The protocol for Y2 affinity analysis, except that COS-7 Y1-transformed cells are used

human and are transferred to culture plates at a density of 1.5 x 106 cells per well, the analysis of

competition uses 125l-PP, and NPY is used for non-specific binding determination.

Affinity analysis of the human Y5 receptor

The protocol for Y2 affinity analysis, except that Y5-transformed COS-7 cells are used

human and are transferred to culture plates at a density of 5 x 105 cells per well, the analysis of

competition uses 125l-PP, and NPY is used for non-specific binding determination.

The results of analyzing NPY, PYY, PYY3-36, PP and PP2-36 in affinity affinity analyzes are given in Table 1:

Table 1

Agonist

 Y2 Y1 Y4

 IC50 Nm

SEM N IC50 nm SEM  N IC50 Nm SEM  n

NPY

0.30 0.07 4 2.3 0.3 4 26 5 4

PYY

0.22 0.02 2 16 one 2 30 8 2

PYY (3-36)

0.20 0.03 10 > 1000 2 343 42 4

PP

> 1000 4 > 1000 one 0.41 0.05 eleven

PP2-26

 > 1000 3 > 1000 2 0.64 0.04 4

5 Power analysis of the human Y2 receptor

The potency of the human Y2 receptor analysis compounds is determined by carrying out experiments that respond to doses in COS-7 cells transiently transfected with the human Y2 receptor in addition to a promiscuous G protein, Gqi5, which ensures that Y2 receptor pairs through a Gq path

10 which led to an increase in the volume of inositol phosphate.

Volume of Fofatidilinositol - One day after transfection, COS-7 cells are incubated for 24 hours with 5 µCi of [3 H] -myo-inositol (Amersham, PT6-271) in 1 ml of medium supplemented with 10% serum Fetal calf, 2 mM glutamine and 0.01 mg / ml gentamicin per well. The cells are washed twice in

15 buffer, 20 mM HEPES, pH 7.4, supplemented with 140 mM NaCl, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl2, 10 mM glucose, 0.05% (w / v) bovine serum; and are incubated in 0.5 ml of buffer supplemented with 10 mM LiCl at 37 ° C for 30 minutes. After stimulation with various concentrations of peptide for 45 minutes at 37 ° C, the cells are extracted with 10% frozen perchloric acid followed by incubation on ice for 30 min. The resulting floats are neutralized with KOH in HEPES buffer, and the

Phosphate [3H] -inositol generated is purified on Bio-Rad AG 1-X8 anion exchange resin and measured on a beta counter. The determinations are made in duplicates. EC50 values were calculated using standardized pharmacological data management programming, Prism 3.0 (graphPad Software, San Diego, United States).

25 Power analysis of the human Y4 receptor

The protocol is identical for the Y2 power analysis, except that COS-7 Y4transformed human cells are used.

30 Power analysis of the human Y1 receptor

The protocol is identical for the Y2 power analysis, except that COS-7 and transformed human COS-7 cells are used.

35 Power analysis of the Y5 Human receptor

The protocol is identical for the Y2 power analysis, except that COS-7 Y5transformed human cells are used.

40 The results of analyzing NPY, PYY, PYY3-36, PP and PP2-36 in affinity affinity analyzes are given in Table 2:

Table 2

Agonist

 Y2 Y1 Y4

 EC50 Nm

SEM N EC50 nm SEM  N EC50 Nm SEM  n

NPY

1.5 0.5  eleven  1.7 0.4 eleven 167 56 7

PYY

0.23 0.06 8 0.6 0.1 5 3. 4 5 6

PYY (3-36)

0.36 0.07 16 74 5 7 > 1000 8

PP

> 1000 8 83 13 5 0.64 0.07 17

PP2-26

 > 1000 4 297 Four. Five 3 0.88 0.12 5

II. IN VITRO ANALYSIS TO DETERMINE PROTEIN STABILITY

An important measurement for PP2-36 is protein stability, especially in terms of stability, for example, towards degradation by enzymes as soon as the peptide has been designed to have increased stability compared to, for example, PYY3-36 or even increased stability compared to PYY and PP of full length.

10 Stability of the PP-fold was determined as the stability of the peptide towards degradation by endopeptidases with adhesion, for example, in the loop zone, which is relatively flexible as described (O'Hare, M. & Schwartz, TW 1990 In Degradation of Bioactive Substances: Physiology and Pathophysiology. J. Henriksen, ed. CRC Press, Boca Raton, FI.). Like an enzyme

15 model, Asp-N endoproteinase (Pierce) is used. This enzyme adheres to the N-terminal side of the Asp residues, for example between residues 9 and 10 (Asp) in PP. The peptide is incubated with an effective dose of Asp-N endopeptidase as indicated by the manufacturer in 0.01 M Tris / HCl buffer, pH 7.5 at room temperature and samples were removed after several time periods of over 24 hours. The samples are analyzed by HPLC and the gradual degradation of the

20 peptide follows in time. The peptide is compared in relation to stability with PYY, PYY1336 and PP.

Stability towards aminopeptidases.- It was determined as described supra for endopeptidases but using aminopeptidase N and di-peptidylpeptidase IV instead. The 25 peptides are compared in relation to stability with PYY, PYY3-36 and PP.

III. IN VIVO STUDIES TO DETERMINE THE EFFECT OF PP2-36 ON THE APPETITE, FOOD INGESTION AND BODY WEIGHT

30 Effect of PP2-36 on acute food intake in mice

The ddy strain of mice, 34-37 grs. and 8-9 weeks old (Japan SLC, Shizuuoka, Japan). The mice were housed individually in a regulated environment (22 C, 55% humidity) in a 12-hour light-dark cycle with light at 7 AM. Food and water were available ad libitum except 35 just before the experiments (see below). The mice were accustomed to subcutaneous injections during the week prior to the start of the experiments. In the experiments, a mouse was used each time. Immediately before administration, the peptides were diluted in physiological saline and administered in 100 µL volume by subcutaneous administration. The results are expressed as average +/- SE. Variation analysis was used followed by Bonferroni analysis to estimate the

40 difference between groups.

The mice were deprived of food but with free access to water during the 16 hours prior to the analysis and the experiments were started at 10 AM the next day. A standardized diet (CLEA Japan, Inc., Tokyo, Japan) was used and food intake was measured by the subtraction of non-ingested food from

45 food previously measured after administration and checking of food spillage. Eight animals in each group receiving either saline solution, 1 µg PP2-36, or 10 µg of PP2-36 test compound.

The results for PP2-36 (Sequential Identification Number: 10) as a test compound are shown in Figure 2.

SEQUENCES NPY (Sequential Identification Number: 1)

PYY (Sequential identification number: 2)

Claims (4)

1. The use of a Y agonist stops for the Y4 receptor on the Y1 and Y2 receptors, 5 said receptor agonist Y being a pancreatic peptide PP2-36 with Sequential Identification Number: 10, in the preparation of a composition for (i) reducing food intake, (ii) obesity or overweight treatment, (iii) treatment of diarrhea or hypersecretion associated with small intestinal ileo-stomia or (iv) treatment of a condition in which obesity or overweight is considered a contributing factor, selected from bulimia, bulimia nervosa, Syndrome X (metabolic syndrome), diabetes Type 2 diabetes mellitus or diabetes mellitus no 10 insulin-dependent (NIDDM), hyperglycemia, insulin resistance, glucose intolerance, cardiovascular disease, hypertension, arteriosclerosis, coronary artery disease, myocardial infarction, peripheral vascular disease, stroke, thromboembolic disease, hypercholesterolemia, hyperlipidemia, gallbladder disease, osteoarthritis, sleep apnea, polycystic ovarian syndrome, and breast, prostate or colon cancer.

2.

The use of the pancreatic peptide PP2-36 with Sequential Identification Number: 10, in the preparation of a composition to reduce food intake.

3.

The use of a Y agonist stops for the Y4 receptor on the Y1 and Y2 receptors,

20 said receptor agonist Y being a pancreatic peptide PP2-36 with Sequential Identification Number: 10, in the preparation of a composition for the treatment of diarrhea or hypersecretion associated with small intestinal ileo-stomia. 4. The use claimed in any one of claims 1 to 3, wherein the composition is 25 adapts for administration to a patient subcutaneously, intramuscularly, intravenously, nasally, transdermally or orally. 40 5 10 5