ES2526672T3 - New uses of oxytocin-type molecules and related methods - Google Patents

Abstract

A derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist for use in the treatment of a disorder selected from obesity and insulin resistance, wherein the derivative of oxytocin is selected from 4 -treonine-1-hydroxideaminooxytocin, 4-serine, isoleucine-oxytocin-8, 9-deamidooxitocin, 7-D-proline-oxytocin, (2, 4-diisoleucine) -oxytocin, 1-deamino-1-monocarba-E12-Tir (OMe)] - OT (dCOMOT), carbetocin (SEQ ID NO .: 2: Butiril-Tir (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2), 4-threonine, 7-glycine -oxytocin (TG-OT), (1-butanoic acid-2- (O-methyl-L-tyrosine) -1-carbaoxitocina, Ile-conopresina, atosiban, oxipresina, deamino-6-carba-oxytoxino (dC60), desmopressina and 1-deamino-oxytocin where the disulfide bridge between residues 1 and 6 is replaced by a thioether, wherein said oxytocin, derived from oxytocin or oxytocin agonist is to be administered periferally.

Description

DESCRIPTION

New uses of oxytocin type molecules and related methods.

Scope of the invention 5

The present invention relates to the substances and compositions thereof in the treatment of obesity and / or insulin resistance. In particular, the invention relates to the substances and compositions thereof useful in the treatment of metabolic syndrome.

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Background of the invention

According to a world estimate of the World Health Organization (WHO) for 2005, at least 400 million adults have excess body weight and most of them suffer from pathologies related to this overweight. WHO estimates that by 2015, 2300 million adults will be overweight and that 15.7 billion adults will be obese. Obesity is associated with increased plasma circulation levels of free fatty acids and triglycerides that contribute to insulin resistance in peripheral tissues such as skeletal muscle. In obesity, adipose tissues enlarge and have an altered profile of hormone secretion, such as adipokines such as leptin and adiponectin, which play a role in the control of body weight, insulin sensitivity, inflammation, angiogenesis and metabolism of 20 lipids, compared to healthy individuals.

It is known that this excess body weight and in particular adiposity, is directly related to a reduction in insulin sensitivity, generally compensated by an additional stimulation of pancreatic insulin secretion to prevent an increase in glucose levels in the blood. Therefore, increased adiposity, especially in the abdominal region, is known to be a strong risk factor for the development of insulin resistance that can then progress in type 2 diabetes. The increased peripheral resistance to Insulin, defined as a less effective tissue response to insulin in terms of glucose uptake and inhibition of hepatic glucose production, first leads to β-cells having an initial attempt to cancel the growing demand for insulin and then give as a result an alteration in its functional role and a decrease in its quantity, a dysfunction that leads to hyperglycemia and that characterizes type 2 diabetes.

In addition, obesity is one of the main components of the metabolic syndrome, characterized as a cluster of syndromes including central obesity in addition to at least two other selected conditions of high serum triglycerides, low levels of high density cholesterol (HDL) , high blood pressure and high fasting glucose due to insulin resistance. Patients with metabolic syndrome are twice as likely to die and three times more likely to have a heart attack or heart attack compared to people without the syndrome. Additionally, people with metabolic syndrome have a fivefold risk of developing type 2 diabetes. About 60% of patients with metabolic syndrome 40 will progress to type II diabetes, 50% will develop cardiovascular disease, more than 35% will suffer an acute myocardial infarction and up to 20% will suffer a stroke. It is estimated that about 20-25% of the world's adult population has metabolic syndrome.

In addition, obesity is also known to be a strong risk factor for the development of other disorders, such as some types of cancer. Four. Five

Therefore, there is a huge wasteland and economic needs for the development of new treatments in order to manage obesity and / or insulin resistance. There is also a need for the development of treatments for the metabolic syndrome because there is currently no treatment available for this multi-component syndrome and this syndrome is the source of additional disorders such as severe cardiovascular and diabetic complications.

Oxytocin (OT) is a neuro hypophyseal hormone nonapeptide (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly (SEQ ID. Number: 1)) (cysteine-tyrosine-isoleucine-glutamine-asparagine-cysteine - proline-leucine-glycine) synthesized both centrally and peripherally and that exerts various physiological effects. Within the central nervous system 55 (CNS), the OT gene is expressed in the hypothalamic paraventricular neurons (PVN) and the supraoptic nucleus (SON). The magnocellular OT neurons in these nuclei project to the neurohypophysis and are the largest source of systemically released OT, while the parvocellular OT neurons of the PVN project centrally. OT is synthesized periferally in several organs, such as the ovary, the testicles, the thymus, the kidney and the heart. So far, a single receptor OT (OTR) has been cloned, which is expressed in several tissues, including adipose tissue. According to such a wide distribution in its production and joining centers, it has been shown that OT is involved in several central and peripheral processes (Gimpl et al., 2001, Physiol. Rev., 81, 629-683). OT is currently used for the stimulation of uterine contraction in order to induce labor, for the control of postpartum hemorrhage after delivery of the placenta and for the stimulation of lactation (Pitocin®, Parke-Davis, Morris Plains , NJ and Syntocinon®, Novartis Pharmaceuticals, East Hanover, NJ). 65

The administration of OT has also been reported to increase the female sexual response and to be beneficial in the treatment of male sexual dysfunction (Pfaus, 2009, J. Sex. Med. Jun., 6, 1506-33).

Muchmore et al., 1981, THE JOURNAL OF BIOLOGICAL CHEMISTRY î 0 JAN î 98î LNKDPUBMED, 256 (1), 365-372 refers to a hypothetical existence of two distinct classes of OT receiver to explain the observed dual action mechanism of the OT, which has been completely abandoned since then.

Hanif et al., 1982, MOLECULAR PHARMACOLOGY, 22 (2) 381-388, discloses in vitro induction by insulin resistance oxytocin in isolated rat adipocytes.

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Fabian et al., 1977, ACTA UNIVERSITATIS CAROLINAE. MEDICA MONOGRAPHIA, No. 77 Pt 1, 141-145 shows a reduction of non-esterified plasma fatty acids (AGNE) in the presence of oxytocin.

WO 2008/008357 refers to methods that use satiety factors for the treatment of disorders associated with an undesirable level of satiety factor. fifteen

CAMERINO, 2009, OBESITY, 17 (5), 980-984 refers to a genetically engineered mouse (knock out mouse) using the OT (OT KO) (Oxt - / -).

KATSUHIKO ET AL., New aspects of oxytocin receptor function revealed by knockout mice: partnersexual behavior 20 and control of energy balance, Section V (New aspects of oxytocin receptor function revealed by knockout mice: partnersexual behavior and energy balance control, Section V,) PROGRESS IN BRAIN RESEARCH, ELSEVIER AMSTERDAM, NL, 170, 79-9 describes an OT receiving KO mouse (OTR KO) (Oxtr - / -).

Summary of the invention

The present invention relates to the unexpected finding that peripheral administration of OT in a model of obesity induced by a high-fat diet induces a dose-dependent decrease in body weight gain due to improved lipid metabolism. , in particular the increase in lipolysis and β-oxidation of fatty acids in adipose tissue, accompanied by improvements in glucose intolerance and insulin resistance, regardless of food intake.

A first aspect of the invention provides a derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist for repression or treatment of a disorder selected from obesity and insulin resistance, wherein said oxytocin, Oxytocin derivative or oxytocin agonist is to be administered peripherally.

A second aspect of the invention relates to a use of a selected oxytocin derivative, an oxytocin derivative and an oxytocin agonist for the preparation of a pharmaceutical preparation for the treatment of a selected disorder of obesity and resistance to insulin, wherein said oxytocin, derived from oxytocin or oxytocin agonist is to be administered peripherally. 40

A third aspect according to the invention relates to a pharmaceutical formulation comprising a derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist combined with at least one co-agent useful in treating a disease or a Selected disorder of obesity and insulin resistance and at least one pharmaceutically acceptable carrier. Four. Five

Description of the figures

Figure 1 shows the effect of central administration of OT on body weight gain in an obesity-induced rat model using a high-fat diet as described in Example 50 1. A: Cumulative weight gain body during the 14-day treatment period (weeks 5 to 7 of high-fat diet, 45%) of (●) rat controls with saline infusion and (◊) with OT infusion (1.6 nmol / d ). B: Delta weight gain and C: cumulative food intake during the 14-day treatment period of rat controls with the infusion of saline (black bars) and with the infusion of OT (1.6 nmol / d) (white bars). D: food efficiency (body weight gain ratio in relation to cumulative food intake during the experimental period of 14 days).

Figure 2 shows the effect of central administration of OT on lipid metabolism in a rat model induced to obesity by a high-fat diet as described in Example 1. A: The expression of messenger ribonucleic acid MRNA of the enzymes involved in the absorption of triglycerides, lipogenesis and lipolysis in epididymal white adipose tissue (TABe) of the controls of 60 rats with saline infusion (black bars) and with infusion of OT (1.6 nmol / d) (white bars). B: expression of PPAR-alpha mRNA and enzymes involved in β-oxidation of fatty acids in epididymal white adipose tissue (TABe) of rats with saline infusion controls (black bars) and with OT infusion - (1.6 nmol / d) (white bars).

Figure 3 shows the effect of central administration of OT against insulin resistance 65 induced by a high fat diet as described in Example 1. A: glucose delta and B:

image 1

Delta insulin levels measured during the Intraperitoneal TTG Glucose Tolerance Test (1.5 g / kg) before treatment (A) (3 weeks of high fat diet, 45%; n = 16 rats), at the end of treatment (7 weeks of high fat diet, 45%; n = 6 for each treatment) for (●) controls of rats with infusion of saline solution and (◊) with infusion of OT (1, 6 nmol / d). C: Glucose infusion ratio (RIG) during hyperinsulinemic euglycemic clamps in rats with obesity induced by high fat diet (DAG) with saline infusion (black bar) and with OT infusion (white bar) ( infusion of intracerebroventricular OT (icv) for 14 days (1.6 nmol / d)). D and E: Glucose-stimulated insulin utilization rate measured during hyperinsulinemic euglycemic clamps in different types of muscles (D): red quadriceps (QR) white quadriceps (QB), red gastrocnemius (GR), white gastrocnemius (GB), soleus (S), tibial (Tib) (14 days of infusion of OT icv (1, 6 nmol / d)) and in different types of deposits 10 of adipose tissue (E): inguinal white adipose tissue (TABi) and epididymis ( TABe) of controls of rats with obesity induced by high fat diet (DAG) with saline infusion (black bars) and with infusion of OT (white bars).

Figures 1-3: Values are means ± ESM (standard error of the mean) of 6 to 7 animals per group. * P <0.05, ** P <0.01 compared to controls. fifteen

Figure 4 shows the direct effect of OT on lipid metabolism in adipocytes in vitro as described in Example 2. A: The mRNA expression of enzymes related to lipid metabolism in 3T3 differentiated adipocytes -L1 after 24 h of incubation with saline solution (black bars) or 5 µM of OT (white bars) (three independent experiments performed on different series of cultured adipocytes 3T3 L1) B: The content of oleyletanolamide (OAS) in adipocytes 20 differentiated 3T3 L1 after 24 h of incubation with saline solution (black bar) or 5 µM of OT (white bar) (two independent experiments performed on different series of 3T3 L1 cultured adipocytes).

Figure 5 shows the effect of peripheral administration of OT in an obesity-induced rat model by a high-fat diet as described in Example 3. A: Accumulated body weight gain during the treatment period 14 days (week 5 to 7 of the high-fat diet, 25 45%) of (●) controls of rats with infusion of saline, (◊) and with infusion of OT (50 nmol / d) and controls (A ) of the pair-fed feeding (AP). B: Cumulative food intake during the 14-day experiment period of rat controls with saline infusion (black bar) and with OT infusion (50 nmol / d, white bar) and AP controls (gray bars). C: The parameters of body composition (fat and lean masses) during a 10-day treatment period (D10) of 30 controls of rats infused with saline solution (black bar), and infusion with OT (50 nmol / d, white bar) and AP controls (gray bar). D: The parameters of delta body composition between the beginning (D0) and the end (D10) of a 10-day treatment period. E: The ratio of different weights of fat pads (inguinal white adipose tissue (TABi); epididymal white adipose tissue (TABe) on the body weight of rats controls with infusion of saline solution (black bar), and with 35 infusion of OT (50 nmol / d, white bar) and AP controls (gray bars) Values are means ± ESM (standard error of the mean) of 7 to 8 animals per group. * P <0.05 compared with controls F: Photographs of newly dissected epididymal white adipose tissue (TABe) and fat pad of mesenteric white adipose tissue (TABm) of rat controls with saline infusion, with OT infusion (50 nmol / d ) and AP controls 40

Figure 6 shows the effect of peripheral administration of OT on insulin resistance induced by high fat diet (DAG) as described in Example 4. A: The homeostasis model of insulin resistance (MHO-RI) of rat controls with saline infusion (black bar), with OT infusion (50 nmol / day) (white bar) and paired feeding controls (AP) (gray bar). B: The glucose infusion rate (RIG) during hyperinsulinemic euglycemic clamps in rats with obesity induced by high fat diet with saline infusion (black bar), with OT infusion (50 nmol / day) ( white bar) and AP controls (gray bars). Values are means ± ESM (Standard Error of the Average) of 7 to 9 animals per group. * P <0.05 compared to controls.

Figure 7 shows the effect of central (and) peripheral OT (A) administration on plasma glycerol levels in a high-fat diet-induced obesity rat model as described in the Examples. 1 and 5. A: Plasma glycerol levels in rat controls with saline infusion (black bar) and OT infusion (1.6 nmol / day) (white bar). B: Plasma glycerol levels in rat controls with saline infusion (black bar) and infusion with OT (50 nmol / day) (white bar) and AP controls (gray bars). Values are means ± ESM (Standard Error of the Average) of 6 to 8 animals per group. 55

Figure 8 shows the effect of peripheral administration of carbetocin in an obesity rat model induced by a high-fat diet as described in Example 6. A: The cumulative body weight gain during the treatment period of 14 days (week 5 to 7 of high-fat diet, 45%) of (●) controls of rats with saline infusion rats (Δ) infusion of carbetocin (5 nmol / d) and associated controls (A) of feeding paired (AP), (◊) rats infused with 60 carbetocin (50 nmol / d) and associated AP controls (♦). B: Accumulated food intake during the 14-day experiment period of controls rats with saline infusion (black bar), rats with carbetocin infusion (5 nmol / d, white bar) and associated AP controls (bar gray), infusion of carbetocin in rats (50 nmol / d, white striped bar) and with associated AP controls (gray striped bar). C: Percent changes in body fat during a 10-day treatment period in controls of 65 rats with saline infusion (black bar), with carbetocin infusion (5 nmol / d, white bar) and

with associated AP controls (gray bar), with carbetocin infusion (50 nmol / d, white striped bar) and associated AP controls (gray striped bar). Values are mean ± ESM (Standard Error of the Average) of 7 to 8 animals per group * P <0.05 compared to controls.

Figure 9 depicts the primer sequences used for quantitative (real-time) PCR as described in Example 1. 5

Detailed description

The term "oxytocin derivative" as mentioned herein, includes peptide fragments, peptide analogs and chemically modified derivatives or salts of peptides, natural or synthetic, therapeutic or prophylactically active, presenting activity similar to the activity or substantially retaining the activity of the parental molecule of oxytocin. For example, oxytocin derivatives include oxytocin or oxytocin peptide analogs that are chemically modified, for example, by amidation of the carboxyl terminal (-NH2), the use of amino acids D in the peptide sequence, the incorporation of small fractions non-peptidyl, as well as the modification of the amino acids themselves (ie, alkylation or esterification of the side chain R groups) 15 (peptidomimetics). In particular, the term oxytocin derivative includes "oxytocin analogs" which means polypeptides substantially homologous to native human oxytocin, respectively, but which have a different amino acid sequence different from that of native human oxytocin due to one or more deletions. , insertions or substitutions. Substantially, homologs means a variant amino acid sequence, that is, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, so 20 minus 97%, at least 98% or at least 99% identical to native amino acid sequences, as set forth above. The percentage identity of two amino acids or two nucleic acid sequences can be determined by visual inspection and / or mathematical calculation or more easily by comparing the sequence information by a known computer program used for comparing the sequence such as the package. Clustal version 1.83. Oxytocin analogs can encompass a sequence that has at least one conservatively substituted amino acid, which means that a residue of a given amino acid is substituted by a residue that has similar physicochemical characteristics. Oxytocin derivatives may include, but are not limited to those described in WO 2008/042452 or EP 1928484, the contents of which are incorporated herein in their entirety by reference, such as 4-threonine-1 -hydroxideaminooxytocin, 4-serine, 8-isoleucine-oxytocin, 9-deamidooxytocin, 7-D-30 proline-oxytocin and its deamino analogue, (2, 4-diisoleucine) -oxytocin, deamino analogue oxytocin, a long active oxytocin analog, 1-deamino-1-monocarba-E12-Tir (OMe)] - OT (dCOMOT), carbetocin (SEQ ID NO: 2: Butiril-i (Me) -lle-Gln-Asn-Cys-Pro-Leu- Gly-NH2), 4-threonine, 7-glycine-oxytocin (TG-OT), (1-butanoic acid-2- (O-methyl-L-tyrosine) -1-carbaoxytocin, Ile-conopressin, atosiban, oxyipresin, deamino-6-carba-oxytoxino (dC60), desmopressin and 1-deamino-oxytocin in which the disulfide bridge between residues 1 and 6 is replaced by a thioether.

The term "oxytocin agonist" includes substances that are capable of interacting with oxytocin receptors (OTR) and / or mimicking the biological activity of oxytocin, particularly through its interaction with the OTR: For example, Oxytocin agonists include those described in WO 2007/050353, WO 2005/023812, WO 2004/072083 and WO 03/016316, the contents of which are incorporated herein by reference in their entirety. Oxytocin agonists include peptides, peptidomimetics and small molecules. In particular, they include WAY-267,464 (4- (3,5-dihydroxybenzyl) -N- (2-methyl-4 - [(1-methyl-4,10-dihydropyrazolo [3,4-b] [1,5] benzodiazepin-5 (1H) -yl) carbonyl] benzyl) piperazine-1-carboxamide), decomoton and 4, - (3,5-Dihydroxybenzyl) -piperazinea-1-carboxylic acid 2-methyl-4- (3-methyl- 4,10-dihydro-3H-2,3,4,9-tetra-aza-benzo [f] azulene-9-carbonyl) -dipeptide.

The term "peptidomimetics" is defined as a peptide analog that contains non-peptide structural elements, in which the peptide is able to mimic or agonize the biological action (s) of a natural parental peptide.

Other useful forms of oxytocin derivatives, oxytocin analogs or oxytocin agonists in the context of the invention include other pharmaceutically acceptable active salts of said compounds, as well as active isomers, enantiomers, polymorphs, solvates, hydrates and / or pro medicines of said compounds. fifty

The term "pharmaceutically acceptable" refers to a carrier composed of a material that is not biologically or in any other undesirable way.

The term "carrier" refers to any component present in a pharmaceutical formulation that is different from the active agents and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents. , preservatives 55 and the like.

The term "obesity" includes a disease or disorder that includes having a high amount of body fat. In general, obese humans have a body mass index (BMI) (body weight of an individual divided by the square of their height) of about 30 kg / m2 or greater. Obesity is known to be accompanied by greater risks for several comorbidities such as type 2 diabetes, hypertension, cardiovascular disease and arthritis.

The term "insulin resistance" refers to elevated blood glucose levels (fasting glucose levels of 110-126 mg / dL; glucose levels of more than 140 mg / dL at 2 hours after the administration of 75 g of glucose).

The term "metabolic syndrome" refers to a group of syndromes including central obesity (excess of 65 fat in the abdomen measured at waist circumference, usually equal to or greater than 80 cm in women

and equal to or greater than 90 cm in males) in addition to at least two other selected conditions of dyslipidemia (in particular high serum triglycerides, typically equal to or greater than 150 mg / dL and low levels of high density cholesterol ( HDL) normally less than 40 mg / dL in men and 50 mg / dL in women), high blood pressure (BP), normally equal to or greater than 130 mm Hg for systolic BP and equal to or greater than 85 mm Hg for Diastolic PA or insulin resistance. 5

As used herein, "treatment" and "treating" and the like generally mean obtaining a desired pharmacological and physiological effect. The effect may be prophylactic in terms of preventing or partially preventing a disease, symptom or condition thereof or it may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a mammal, especially a human being and includes: (a) prevention of the disease that occurs in a subject that may be pre-disposed to the disease, but that has not yet been diagnosed as having it; (b) inhibition of the disease, that is, stopping its development; or relief of the disease, that is, causing the regression of the disease and / or its symptoms or conditions in such a way that the damage is improved or repaired.

In particular, the treatment of obesity includes normalizing body weight or improving the body with overweight and in particular the central fat deposit. The treatment of obesity also includes a reduction in the risk of co-morbidities related to obesity mentioned above.

In particular, the treatment of insulin resistance is comprised of normalizing or improving a disability of normal biological responses to both exogenous or endogenous insulin, typically through the control of blood glucose levels or the production of insulin. The treatment of insulin resistance comprises the prevention or delay of the progression of the syndrome towards type 2 diabetes.

In particular, the treatment of metabolic syndrome comprises normalizing or improving at least one of the components of the syndrome, regardless of weight loss. In a particular aspect, the treatment of metabolic syndrome comprises the prevention of the development of type 2 diabetes.

The term "subject" as used herein refers to mammals. For example, mammals contemplated in the present invention include humans, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents and the like.

The term "efficacy" of a treatment according to the invention can be measured based on changes in the course of the disease before a use according to the invention. For example, the effectiveness of a treatment according to the invention can be measured by a decrease in central obesity (i.e. a decrease in waist circumference, a decrease in fat mass) or a decrease in insulin resistance. using the oral glucose tolerance test (OTTG), fasting glucose test (TGA) or hyperinsulinemic euglycemic clamp. For another example, the effectiveness of obesity treatment comprises a reduction in plasma triglycerides and the like.

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The method of preparation

Oxytocin, oxytocin derivatives, oxytocin agonists and oxytocin analogs are commercially available (e.g. NeoMPS, Strasbourg, France) or can be easily prepared by the qualified person, as described, for example , in US 2,938. 891 and US 3,076,797 and tested for biological activity according to known methods. All peptide analogs or derivatives of oxytocin described and / or contemplated herein can be prepared by chemical synthesis using either automated synthetic technologies or manual solid phase synthetic technologies or by using recombinant molecular techniques, techniques that are generally known in the art.

 Four. Five

The compositions

The invention provides pharmaceutical or therapeutic agents such as the compositions and methods for the treatment of a patient, preferably a mammalian patient and more preferably a human patient suffering from a medical disorder and in particular a disorder selected from obesity and resistance to 50 insulin

In a particular embodiment, the invention provides pharmaceutical or therapeutic agents such as compositions and methods for treating a patient, preferably a mammalian patient and more preferably a human patient suffering from a medical disorder, wherein the disorder is the syndrome. metabolic. 55

In a particular embodiment, the invention provides pharmaceutical or therapeutic agents such as compositions and methods for the treatment of a patient, preferably a mammalian patient and more preferably a human patient suffering from a medical disorder, wherein the disease is the type 2 diabetes

In a particular embodiment, the invention provides a pharmaceutical formulation according to the invention for use as a medicament.

The pharmaceutical compositions of the invention may contain at least one substance selected from oxytocin, an oxytocin derivative and an oxytocin agonist according to the invention in any manner described herein. The compositions of this invention may be composed in addition to one or more 65

Additional pharmaceutically acceptable ingredients such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavorings, adjuvants and the like.

The compositions according to the invention, together with an adjuvant conventionally employed, a carrier, a diluent or an excipient can be made in the form of the pharmaceutical compositions and can be used as solids, such as tablets or filled capsules or liquids such as solutions, suspensions, 5 ointments, emulsions, elixirs or capsules filled therewith, films or gels, all for oral use or in the form of sterile parenteral injectable solutions (including subcutaneous) used by continuous injection or infusion. If desired, sustained release compositions, for example sustained release gels, films, and transdermal patches, can be readily prepared. The compositions can also be formulated as a dry product for reconstitution with water or other suitable vehicle before use. Compositions 10 of this invention such as liquid formulations, including, but not limited to aqueous or oily suspensions, solutions, emulsions, syrups and elixirs may be used as drops, for use in an injection, such as A spray or impregnated in a nasal buffer.

Injectable compositions are usually based on sterile injectable saline or phosphate buffer saline or other injectable carriers known in the art. Such pharmaceutical compositions and their unit dosage forms may include ingredients in conventional proportions, with or without additional active compounds or principles and such unit dosage form forms may contain any suitable effective amount of the active ingredient in line with the range of daily dose planned to be used. According to a particular embodiment, the compositions according to the invention are injectable or inhalable. Such liquid preparations may contain additives including, but not limited to, 20 suspending agents, emulsifying agents, non-aqueous vehicles and preservatives. Suspension agents include, but are not limited to sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel and fats. Hydrogenated edibles. Emulsifying agents include, but are not limited to lecithin, sorbitan monooleate and acacia. Preservatives include, but are not limited to methyl or propyl p-hydroxybenzoate and sorbic acid. Dispersing or wetting agents include but are not limited to poly (ethylene glycol), glycerol, bovine serum albumin, Tween®, Span®. Other materials as well as the process techniques for the formulation and the like are set forth in Part 5 of Remington's Pharmaceutical Sciences, 21st Edition, 2005, University of the Sciences in Philadelphia, Lippincott Williams & Wilkins, which is incorporated herein as reference. 30

The compositions of this invention can also be formulated as a depot preparation, which can be administered by implantation or by intramuscular injection. The compositions may be formulated with suitable polymeric or hydrophobic materials (such as an emulsion in an acceptable oil, for example), with ion exchange resins or as sparingly soluble derivatives (such as a poorly soluble salt, for example). 35

The solid compositions of this invention may be in the form of tablets or tablets formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to binding agents, fillers, lubricants, disintegrants and wetting agents. Binding agents include, but are not limited to syrup, acacia, gelatin, sorbitol, gum tragacanth, starch mucilage and polyvinylpyrrolidone. Fillers 40 include, but are not limited to lactose, sugar, microcrystalline cellulose, corn starch, calcium phosphate and sorbitol. Lubricants include, but are not limited to magnesium stearate, stearic acid, talc, polyethylene glycol and silica. Disintegrants include, but are not limited to potato starch and sodium starch glycolate. Wetting agents include, but are not limited to sodium lauryl sulfate.

The tablets may be coated according to methods well known in the art. Four. Five

The compositions of this invention may also be formulated for nasal administration which may be in a form including, but not limited to a nasal spray powder or liquid, a suspension, nose drops, a gel, a film or ointment, through of a tube or catheter, by means of a syringe, by packtail, by swab (a small flat absorbent pad), by a nasal buffer or by submucosal infusion. Nasal administration of the medicament can be carried out as described in patent documents 50 EP 1928484 or WO 2008/042452, for example by using devices, including, but not limited to, unit dose containers, sprinklers pump drippers, tightening bottles, aerosols without preservatives and without air, nebulizers, metered dose inhalers and pressurized metered dose inhalers. In the art, devices for nasal administration of medications are known and there are several that are commercially available. Aerosols can be dispensed by using a pressurized package or a nebulizer and a suitable propellant including, but not limited to dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, hydrocarbons, compressed air, nitrogen or carbon dioxide. An aerosol system requires that the propellant be inert in relation to the pharmaceutical composition. In the case of a pressurized aerosol, the dosing unit can be controlled by providing a valve in order to administer a precisely measured amount. Powdered nasal sprays 60 may be formulated in the form of microspheres dispensed by a nasal insufflator device (device for blowing a gas, dust or vapor into a body cavity) or a pressurized aerosol canister.

The compounds of this invention can also be administered in forms of sustained release or sustained release systems for the administration of medicaments (WO 2004/078147). A description of representative sustained release materials can also be found in materials incorporated in Remington’s Pharmaceutical Sciences.

The modes of administration

The compositions of this invention can be administered in any form including intravenous injection, oral route, mucosal administration (delivery to the mucosal surfaces of the nose, nasal passages, nasal cavity, mucosal surfaces of the oral cavity including the gum, the floor of the oral cavity, the cheeks, the lips, the tongue, the teeth; and to the mucous surfaces of or around the eye including the conjunctiva, the lacrimal gland, the naso lacrimal ducts, the mucosa of the upper or lower eyelid and the eye), intranasal administration (i.e., by aerosol, by drops, by powder, by gel, by film, inhalant or other means), dermal or transdermal administration (i.e. delivery to the skin including the face, neck, scalp, body or combinations thereof) for example by intradermal or subcutaneous injection combinations thereof.

The combination

 fifteen

According to the invention, oxytocin, an oxytocin derivative and an oxytocin agonist and the pharmaceutical formulations thereof can be administered alone or in combination with a cooperative agent useful in the treatment of obesity and / or resistance. to insulin, such as substances useful for treatment, stabilization, prevention and / or delay in obesity and / or insulin resistance selected from the classes of biguanides, glitazones, sulfonylureas, inhibitors of dipeptidyl 20 peptidase-4 (DPP-4) or glucagon type 1 agonists (GLP-1), as described in De Fronzo, 2010, Am. J. Med., 3 (1), S38-48 .

The invention encompasses the administration of a selected derivative of oxytocin, an oxytocin derivative and an oxytocin agonist and the pharmaceutical formulations thereof to an individual before, simultaneously or sequentially with other therapeutic regimens or co-agents useful in the treatment. of obesity and insulin resistance (i.e. multiple drug regimens), in a therapeutically effective amount. A derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist or pharmaceutical formulations thereof that is administered simultaneously with said co-agents can be administered in the same or in different composition (s) and by the same or by different route (s) of administration. 30

According to one embodiment, a pharmaceutical formulation is provided comprising a derivative selected from oxytocin, an oxytocin derivative and an oxytocin agonist combined with at least one co-agent useful in the treatment of a selected disease or disorder. of obesity and insulin resistance and at least one pharmaceutically acceptable carrier. 35

According to another embodiment, a pharmaceutical formulation according to the invention is provided wherein the derivative is oxytocin.

The dose administered to an individual, as a single or multiple doses, will vary depending on a variety of 40 factors, including the pharmacokinetic properties, conditions and characteristics of the patient (sex, age, body weight, health, size), degree of symptoms, concurrent treatments, frequency of treatment and the desired effect.

In a particular embodiment, an effective amount of oxytocin, an oxytocin derivative or an oxytocin agonist will depend on the form and composition that is used for administration. Typically, the effective amount of oxytocin, an oxytocin derivative or an oxytocin agonist administered transmucosally or transdermally is generally less than the dosage used when the agent is delivered by other routes (eg, oral, intravenous, intramuscular or subcutaneous).

 fifty

In a particular embodiment, the doses used for the administration of oxytocin, an oxytocin derivative or an oxytocin agonist include, but are not limited to an effective amount within the dose range of 10 to about 1,500 µg. , usually from about 20 to about 1,000 µg, such as about 25 to about 750 µg.

 55

In another particular embodiment, the doses used for the administration of oxytocin, an oxytocin derivative or an oxytocin agonist include the doses for nasal administration as described in EP 1928484 or WO 2008/042452.

60 patients

In one embodiment, the patients according to the invention are the patients suffering from a selected disorder of obesity and insulin resistance.

In a further embodiment, the patients according to the invention are patients suffering from obesity.

In another additional embodiment, patients according to the invention are suffering from insulin resistance.

In another additional embodiment, patients are suffering from metabolic syndrome.

In another additional embodiment, patients are suffering from type 2 diabetes.

The use according to the invention

 5

In another embodiment of the invention there is provided a use of a derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist for the preparation of a pharmaceutical composition for the repression or treatment of obesity and / or insulin resistance , wherein the oxytocin derivative is selected from 4-threonine-1-hydroxideaminooxytocin, 4-serine, 8-isoleucine-oxytocin, 9-deamidooxytocin, 7-D-proline-oxytocin, (2,4-diisoleucine) -oxytocin , 1-deamino-1-monocarba-E12-Tir (OMe)] - OT (dCOMOT), carbetocin (SEQ ID NO .: 2: Butiril-10 Tir (Me) -Ile-Gln-Asn-Cys-Pro-Leu -Gly-NH2), 4-threonine, 7-glycine-oxytocin (TG-OT), (1- butanoic acid-2- (O-methyl-L-tyrosine) -I-carbaoxytocin, Ile-conopressin, atosiban , oxyprine, deamino-6-carba-oxytoxin (dC60), desmopressin and 1-deamino-oxytocin in which the disulfide bridge between residues 1 and 6 is replaced by a thioether, wherein said oxytocin, derived from oxytocin or agonist of oxytocin is to be administered peripherally. fifteen

In a further embodiment, a use of a derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist is provided for the preparation of a pharmaceutical composition for the prevention or treatment of metabolic syndrome. *

 twenty

In a further embodiment, a use of a derivative selected from oxytocin, an oxytocin derivative and an oxytocin agonist is provided for the preparation of a pharmaceutical composition for the prevention or treatment of type 2 diabetes, wherein The oxytocin derivative is selected from 4-threonine-1-hydroxideaminooxytocin, 4-serine, isoleucine-oxytocin-8, 9-deamidooxytocin, 7-D-proline-oxytocin, (2, 4-diisoleucine) -oxytocin, 1- deamino-1-monocarba-E12-Tyr (OMe)] - OT (dCOMOT), carbetocin (SEQ ID NO .: 2: Butiril-25 Tir (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly- NH2), 4-threonine, 7-glycineoxytocin (TG-OT), (1-butanoic acid-2- (O-methyl-L-tyrosine) -I-carbaoxitocin, Ile-conopressin, atosiban, oxyprine, deamino- 6-carba-oxytoxino (dC60), desmopressin and 1-deamino-oxytocin in which the disulfide bridge between residues 1 and 6 is replaced by a thioether, wherein said oxytocin, derived from oxytocin or oxytocin agonist is for be administered peripherally. 30

In another embodiment, a compound, a use or a method according to the invention is provided, in which the disorder is obesity.

In another embodiment, a compound, a use or a method according to the invention is provided, wherein the disorder is insulin resistance.

In another embodiment, a compound, a use or a method according to the invention is provided, in which the derivative is oxytocin.

 40

In another embodiment, a compound, a use or a method according to the invention is provided, in which the derivative is an oxytocin analog.

In another embodiment, a compound, a use or a method according to the invention is provided, in which the oxytocin analog is carbetocin. Four. Five

In another embodiment, a compound, a use or a method according to the invention is provided, in which the derivative is an oxytocin agonist.

The compounds and compositions according to the invention may be useful in the suppression or treatment of obesity and / or insulin resistance. In a particular embodiment, the compounds and compositions according to the invention may be useful in the repression or treatment of metabolic syndrome. In another particular embodiment, the compounds and compositions according to the invention may be useful in the repression or prevention or treatment of type 2 diabetes.

 55

The examples illustrating the invention will be described hereinafter in a more detailed manner and by reference to the embodiments depicted in the Figures.

EXAMPLES

 60

The following abbreviations refer respectively to the following definitions:

Acadm (medium chain acyl-CoA dehydrogenase), Acox1 (acyl-CoA oxidase 1) TAM (brown adipose tissue), Ci (Curie), d (day), h (hour), µg (microgram), mg (milligram) , min (minute), mM (millimolar), nm (nanometer), ACACA (acetyl-coenzyme A carboxylase alpha), AEA (Anandamide), PC (body weight), cDNA 65 (complementary DNA), DGAT1 (diacylglycerol O-Acyltransferase counterpart 1), Ehhadh (enoil-CoA

hydratase / 3-hydroxyacyl-CoA dehydrogenase), ELISA (Enzyme-linked immunosorbent assay), TABe (white adipose tissue of the epididymis), SAG (fatty acid synthase), RIG (glucose infusion rate), GR (red gastrocnemius) , GB (white gastrocnemius), DAG (high fat diet), LHS (sensitive hormone lipase), TABi (inguinal white adipose tissue), Lpl (lipoprotein lipase), M-MLV-RT (reverse transcriptase of murine leukemia virus of Moloney), AGNE (non-esterified fatty acids), OEA 5 (Oleoylethanolamide), OT (oxytocin), PCR (polymerase chain reaction), PEA (Palmitoylethanolamide), PNPLA2 (phospholipase domain similar to patatin containing 2), QR ( red quadriceps) QB (white quadriceps), RNA (ribonucleic acid), TI (reverse transcriptase), S (Soleo), SCD1 (stearoyl-coenzyme A 1 desaturase), TG (triglycerides), TIB (tibial), UCP3 (protein 3 decoupled), 2-AG (2-arachidonyl glycerol).

 10

Example 1: Positive impact of central or peripheral OT infusion on body weight gain, lipid metabolism and insulin resistance

The beneficial metabolic effects of OT administration in the following obesity model induced by a high fat diet in rats were evaluated. fifteen

Animals and diets

Wistar male rats (300-325 g) were purchased from Charles River (L'Arbresle, France) and housed in individual cages under controlled temperature conditions (22 ° C) and lighting (light on: 7: 00-19: 00 h). 20 They had free access to water and a 45% high-fat diet (DAG) (metabolized energy, 4.56 kcal / g) (R / M Ssniff® EF according to D12451 (I) mod., Ssniff Spezialdiäten GmbH, Soest , Germany) for seven weeks. Body weight, food intake and water intake were recorded daily (8: 30-9: 30 h). All procedures were approved by the Ethics Committee of our University and were in accordance with the Swiss guidelines for animal experimentation. 25

Intra ventricular brain infusion (i.c.v.)

In order to study the role of OT on metabolic homeostasis, the Wistar male rats above were taken to obese during 7 weeks of a 45% high-fat diet (DAG) and were infused intra ventricularly (30 cv) well brain with OT (1.6 nmol / d) or with a saline solution during the last 2 weeks of the experiment.

After three weeks of feeding with a high-fat diet (DAG), the rats underwent surgical procedures. They were anesthetized with an intramuscular injection of ketamine-xylasin (Ketalar @ -35 Rompun @, Parke-Davis and Bayer, Leverkusen, Switzerland) of 40 and 9 mg / kg, respectively and implanted with a cannula in the right lateral cerebral ventricle fixed in the skull with dental cement, as described previously (Rohner-Jeanrenaud et al., 1989, 124 of Endocrinology, 733-9). After a week of recovery, the beverage response to the injection i.c.v. of Angiotensin II (5 ng / µl) (Novabiochem, Laüfelfingen, Switzerland) in order to confirm the correct placement of the cannula. At the end of the fifth week of feeding the high-fat diet, osmotic mini-pumps (Alzet®, model 2001, Alza Corporation, Cupertino, CA) that administered oxytocin (NeoMPS®, Strasbourg, France) or its subcutaneously were implanted vehicle (NaCl 0.9%) and connected to the cannula of the icv via a polyethylene catheter, as described above (Rohner-Jeanrenaud et al., 1989, above). The OT was administered at a dose of 1.6 nmol / day or 16 nmol / day / rat for 14 days. A group of rats with vehicle infusion was fed in a paired manner to the amount of food consumed by the animals infused with the OT in order to assess the effects of the OT regardless of changes in food intake. The paired diet consisted of giving one third of the daily amount of food in the morning and the remaining two thirds just before the onset of darkness. Rats were sacrificed using isoflurane anesthesia (Halocarbon Laboratories, River Edge, NJ) and rapid decapitation between 9:00 and 13:00 h. Blood samples were collected in tubes coated with AEDT 50 (ethyldiaminotetraacetic acid), centrifuged and plasma stored at -20 ° C. The tissues were quickly removed, frozen on a vacuum table (freeze-clamped) and stored at -80 ° C.

Subcutaneous Infusion (s.c.)

 55

Another group of Wistar male rats from above were taken to obese as described above and were infused subcutaneously with saline or with OT (NeoMPS®, Strasbourg, France) (50 nmol / day / rat) for 14 days.

Glucose tolerance tests 60

The impact of OT-induced changes in peripheral lipid metabolism on glucose tolerance and insulin sensitivity was determined by glucose tolerance tests (TTG). Glucose (75 mg / kg body weight) was administered intraperitoneally 4 hours after food withdrawal. Glucose levels were measured using a glucose meter (One Touch II; LifeScan) from tail blood samples of 0, 15, 30, 60, 65 90 and 120 minutes after glucose injection.

Euglycemic-hyperinsulinemic clamps

The global glucose and specific tissue utilization rates were measured by performing euglycemic-hyperinsulinemic clamps associated with the 2-deoxyglucose labeled technique as described previously (Vettor et al., 1994, Diabetology, 37, 1202 -1208). Obese rats induced by a high-fat diet were fasted overnight and anesthetized intraperitoneally with Nembutal (75 mg / kg sodium pentobarbital; Abbott Laboratories, Chicago, IL). Total glucose utilization was measured during the hyperinsulinemic euglycemic clamps, as described previously (Vettor et al., 1994, above). At the end of the clamp, the utilization rate of the individual tissues of glucose stimulated by 10 insulin in vivo was determined by injecting a single bolus of the 2-deoxy-D- [1-3H] glucose label (30 µCi; Amersham Biosciences UK, Little Chalfont, United Kingdom), as described above (Vettor et al., 1994, above). The rats were sacrificed by rapid decapitation and the tissues were quickly removed, frozen on a vacuum table (freeze-clamped) and stored at -80 ° C. Measurement of the tissue concentration of 2-deoxy-d- [1-3H ] glucose-6-phosphate allowed calculation (Vettor et al., 1994, above) of the in vivo glucose utilization rate of individual tissues and was expressed in ng / mg.min.

Plasma measurements

In order to determine the effect of OT on peripheral metabolism, glucose, insulin, leptin, AGL, glycerol and TG levels were determined. Plasma glucose was measured by the glucose oxidase method (Glu, Roche Diagnostics GmbH, Rotkreuz, Switzerland). Plasma non-esterified fatty acids (AGNE) and triglyceride levels (TG) were determined using enzymatic AGNE C and TG PAP150 commercial kits from Wako Chemicals GmbH (Neuss, Germany) and Biomérieux (Marcy l'Etoile, France), respectively . Plasma levels of OT were determined by an ELISA, as previously described (Pequeux et al., 2001, 25 Scand. J. Clin. Lab. Invest., 61, 407-415). Plasma glycerol levels were measured using the Free Glycerol Reagent (Sigma). The central infusion of OT promoted increasing levels of OT in plasma, suggesting a direct potential effect on adipose tissue.

Tissue processing and R T-PCR in real time 30

In order to delineate the mechanisms responsible for the effect on peripheral metabolism, the expression of the enzymes involved in lipid metabolism in adipose tissues (white epididymal adipose tissues and brown interscapular) was analyzed. Total RNAs were extracted from the frozen tissue using one-step extraction with the Trizol reagent (Sigma-Aldrich, Buchs, Switzerland). The integrity of the RNA was evaluated by 1% agarose gel electrophoresis and the concentration was determined by spectrophotometry. An amount of 2.5 µg of total RNA was used for RT (reverse transcriptase) using random hexamers (Microsynth, Geneva, Switzerland), dNTPs (deoxynucleotides) (Promega Corporation, Madison, WI, United States), RNasin as an inhibitor of RiboNuclease (Promega Corporation) and the Moloney murine leukemia virus reverse transcriptase enzyme kit (RT-MLV-M) (Invitrogen, Basel, Switzerland). For quantitative PCR (PCRc), gene amplification was performed from cDNA of 6.25 or 12.5 ng, using the SYBR® green PCR Master Mix (Applied Biosystems, Warrington, United Kingdom) and an ABI7500 machine (Applied Biosystems, Foster City, CA). All primers were designed (Figure 9, Table 1) with PrimerExpress software (http://phym.unige.ch/) and used in a concentration of 200 to 300 nM for PCRc. The results were normalized to the expression levels of the constitutive gene, ribosomal protein S29 (RPS29). Four. Five

Role of OT administration in body weight gain regardless of changes in food intake

The chronic infusion i.c.v. of OT decreased body weight gain and resulted in a 50-fold decrease in cumulative body weight gain of more than fifty percent compared to the effect of saline (Figures 1A and 1B; P <0.05) , without any effect on food intake (Fig. 1). This resulted in a marked decrease in food efficiency in rats treated with OT compared to controls of saline infusion, an index calculated as the ratio of body weight gain in relation to food intake accumulated during 14-day experimental period (Figure 1D; P <0.05). 55

Role of OT administration in lipid metabolism

All the measured parameters of peripheral metabolism (i.e. plasma glucose, insulin, leptin and AGL levels) remained unchanged by the central administration of OT, compared with saline infusion, except 60 concentrations of plasma TGs that were decreased, as shown in Table 2 below.

In addition, both administrations of central OT (Fig. 7A) and peripheral (Fig. 7B) increased plasma glycerol levels, supporting that chronic administration of OT increases lipolysis.

 5

In white epididymal adipose tissue (TABe), considered as an intra-abdominal fat deposit, the central infusion of OT promoted an increase in the expression of Lpl mRNA, an enzyme responsible for the absorption of circulating TG (Fig. 2A; P <0.01). Central infusion of OT did not modify the mRNA expression of enzymes involved in lipogenesis and storage of TG [ie, acetyl-coenzyme A carboxylase alpha (Acaca, also known as ACC-alpha), fatty acid synthase (SAG) and the homologous diacylglycerol O-acyltransferase 10 (DGAT1)] (Figure 2A), while increasing that of two enzymes involved in lipolysis, especially the patatin-like phospholipase domain containing 2 (PLAPN2) (Figure 2A; P <0.05) and sensitive hormone lipase (LHS) (Fig. 2A; P <0.01). The stimulating effect of OT on LHS was also detected at the protein level.

As an improved intracellular availability of AGLs in adipocytes could be expected as a result of such increased absorption of TG and lipolysis in rats infused with OT (1.6 nmol / d) compared to controls. , the observation of the current unchanged levels of plasma AGLs in rats infused with OT (Table 2) suggests an endogenous use of these substrates induced by the administration of OT.

Measurement of mRNA expression of enzymes involved in the real-time β-oxidation of fatty acids RT-PCR as described above showed that the infusion of OT increased the TABe expression of acyl-20 CoA oxidase 1 ( Acoxl, Figure 2B, P <0.05), of enoyl-CoA hydratase / 3-hydroxyacyl-CoA dehydrogenase (Ehhadh, Figure 2B, P <0.05) and of medium chain acyl-CoA dehydrogenase (Acadm, also known as MCAD, Figure 2B, P <0.05) and the expression of the detectable protein of fatty acids, decoupling protein 3 (Ucp3, Figure 2B, P <0.05) has also improved. For the purpose of comparison, the mRNA expression of most of the genes cited above in interscapular brown adipose tissue (TAMi) was also analyzed and it was observed that infusion of OT significantly increased the expression of the main enzymes involved in the β-oxidation of fatty acids, as well as that of PPAR-alpha in this tissue. In contrast, the central infusion of OT did not change the expression of the genes involved in the β-oxidation of fatty acids in the liver and skeletal muscle.

In addition, the central infusion of OT promoted a marked increase in the expression of stearoyl-30 coenzyme A desaturase 1 mRNA (SCD1) that converts unsaturated fatty acids from palmitic acid (16: 1) and stearic acid (18: 0 ) to unsaturated mono fatty acids of palmitoleic acid (16: 1) and oleic acid (18: 1), respectively (Figure 2A; P <0.05). It is known that these resulting fatty acids can be incorporated into the membrane as phospholipids or stored as TG. Since no change was observed in the mRNA expression of the enzymes involved in the storage of TGs in TABe of animals with the infusion of OT (Figure 2A), an increase induced by OT in synthesis and synthesis could have occurred. incorporation as phospholipids (PL) in the membrane of palmitoleic or oleic acids. Oleoylethanolamide (OAS), a member of the endocannabinoid family, results from the division of N-oleoyl phosphatidylethanolamine (NOPE) by N-acyl phosphatidylethanolamine phospholipase D (NAPA-PLD), resulting from the transfer of oleic acid from the position sn-1 phosphatidylcholine (PC) to phosphatidylethanolamine-free amine (PE). It is observed that this chronic infusion i.c.v. 40 of the OT increases the content of the OAS in the TABe compared to the effect of the saline solution, while not modifying the content in the TABe of other endocannabinoids, such as the saturated analogue of the OEA,

Palmitoylethanolamide (PEA) derived from palmitoleic acid, anandamide (AEA) or 2-arachidonylglycerol (2-AG), nor does it alter the plasma levels of the OAS and other endocannabinoids (Table 2).

In order to verify the hypothesis that central infusion of OT modulates peripheral lipid metabolism by promoting OAS synthesis in adipose tissue, obese rats induced by high-fat diet were infused i.c.v. for 14 days with a tenfold higher dose of OT (16 nmol / d). At this dose, the central infusion 5 of the OT decreases the accumulated food intake compared to the controls of the i.c.v. infusion. of saline solution. Therefore, in order to determine the metabolic effects caused by the OT regardless of changes in food intake, a second control group with saline solution was infused that was fed pair (AP) in the amount of food consumed by the rats treated with OT. Body weight gain of infused rats i.c.v. with the OT it was significantly lower than in the 10 saline infusion control group, an effect that was independent of changes in food intake, as was not observed in the AP control group. The stimulating effect of this dose of OT on the SCD1 mRNA expression and the OAS content was more marked than that of the lowest dose (5.6 and 2 times versus 4.2 and 1.3 times, respectively), being oblivious to changes in food intake, further supporting that this central action of the OT on the metabolism of lipids in adipose tissue implies the synthesis of the OAS promoted by the OT.

According to the results obtained with the tenfold lower dose, the infusion of 16 nmol / d of the OT did not modify PPAR-alpha of the mRNA of the TABe, but resulted in an independent increase in food intake in the expression of PPAR-alpha target genes (ie Acadm, Acox1, Ehhadh, Ucp3). The infusion of OT 20 significantly increased the expression of PPAR-alpha mRNA and its target genes in TAM. A potential effect of OT on the activity of TAM, including the increase in thermogenesis, cannot be excluded.

Role of OT administration in insulin resistance induced by high fat diet

 25

Two glucose tolerance tests (TTG) were performed, one first after 3 weeks of the high fat diet (DAG) (ie, before the respective saline and OT treatments) and a second at the end of the week 2 with infusion of saline or OT (i.e., after 7 weeks of DAG). After 7 weeks of DAG, both rats infused with saline solution and OT exhibited a weakening of glucose tolerance compared to the TTG performed after 3 weeks of DAG feeding (Figure 3A; P <0.05). 30 Such deterioration was accompanied by insulin secretion in the saline infusion group (Figure 3B; P <0.05), which implies the occurrence of decreased insulin sensitivity while this was not observed in the animals infused with OT, suggesting a protective role for OT against glucose intolerance and insulin resistance induced by DAG. Experiments of hyperinsulinemic euglycemic clamps confirmed that infusion with OT promoted significant increases in the infusion rate of 35 glucose (RIG) (Figure 3; P <0.05) and in the rate of glucose utilization in skeletal muscles red, such as the red quadriceps (Figure 3D; P <0.01), the red gastrocnemius (Figure 3D; P <0.05) and the soleus (Fig. 3D; P <0.05) compared to the group of the Saline solution. An increase in glucose uptake in inguinal TAB (Figure 3E; P <0.05) and in epididymal TAB (Fig. 3E; P <0.05), was also observed in rats centrally infused with OT. All together, these results support that central chronic infusion of OT 40 decreases body weight gain in obese rats induced with high fat diet (DAG), regardless of changes in food intake, improves resistance to DAG-induced insulin (improved glucose tolerance, together with a significant decrease in insulin secretion during TTG, that is, improved insulin sensitivity, increased RIG and increased glucose utilization rate in skeletal muscles red, TABi and TABe), decreases plasma triglyceride levels and increases the expression of Lp1 in the white adipose tissue of the epididymis (TABe), an enzyme responsible for the absorption of TGs, as well as the enzymes involved in lipolysis and in the β oxidation of fatty acids. This effect selectively searches for white adipose tissue such as central chronic infusion of OT has no effect on lipid metabolism in skeletal muscle or liver. In addition, the increase caused by the OT in the content of OAS in the TABe and the increase in genes (for example, Acadm, Acox1, Ehhadh, Ucp3) that encode the 50 enzymes involved in the β-oxidation of fatty acid which are genes PPAR-alpha objective (Jeong et al., 2009. Exp. Mol. Med., 41, 397-405) is supporting an effect of OT on the enhanced activity of PPAR-alpha.

Example 2: Direct effect of OT on lipid metabolism in adipocytes in vitro

 55

The in vitro effect of OT on lipid metabolism in differentiated 3T3-L1 adipocytes was tested as follows. The Murine 3T3-L1 fibroblasts were differentiated to a pre-adipocyte phenotype as described previously (Olson et al., 1997, Mol. Cell. Biol, 2425-2435). In summary, the cells were cultured in a modified Dulbecco eagle medium (DMEM) with 4.5 g / l glucose supplemented with 10% reduced heat inactivated calf serum (SBR) at 37 ° C / 8% CO2. The cells were induced to differentiate, two days after the confluence, by sequential incubations with: 4.5 g / l DMEM glucose, 10% fetal bovine serum (SFB) containing 1 µg / ml insulin, 0.25 µM dexamethasone and 0.5 mM isobutylmethylxantane (IBMX) for 4 days; 4.5 g / l DMEM glucose, 10% SFB containing only insulin for an additional 4 days; 4.5 g / l DMEM glucose, SFB 10% for 3-4 days. Differentiation was considered achieved when more than 90% of the cells show an accumulation of large lipid droplets in their cytoplasm. 65

OT increased mRNA expression of all genes related to lipid metabolism that were affected by the central infusion of OT. This comprised a stimulatory effect of OT on PPAR-alpha (Figure 4A). Furthermore, even though the effect of OT on SCD1 mRNA expression was less pronounced than that obtained in centrally infused rats, exposure of 3T3-L1 adipocytes to OT resulted in an increased OEA content (Figure 4B). In order to further support that the OAS could be a mediator of the effects induced by OT on lipid metabolism in adipose tissue, 3T3-L1 adipocytes were incubated in the presence of OAS and the OAS was shown to induce the expression of PPAR-alpha mRNA and most of the PPAR-alpha-related genes (Acadm, Acox1, Ehhadh, Ucp3).

Example 3: Positive impact of peripheral administration of OT on body weight gain and lipid metabolism

The beneficial metabolic effects of the administration of OT were evaluated in the same model of obesity in rats induced by the high-fat diet as in Example 1 and in PPAR-alpha knockout mice (Lee et al., 1995, Mol. Cell. Biol., 15 (6): 3012-22). Animals with obesity induced by the high-fat 15 DAG diet, saline or OT groups (see conditions in Example 1) were subcutaneously infused via mini pumps (Alzet®, as above). The OT was administered at a dose of 50 nmol / day / rat for 14 days.

The parameters of body composition 20

An EchoMRI-700 quantitative nuclear magnetic resonance (NMR) analyzer (Echo Medical Systems, Houston, TX) was used to measure total fat mass and lean body mass in rats at the beginning and end of treatment.

 25

At this dose, the administration of OT decreased food intake (Figure 5B; P <0.05) and body weight gain (Fig. 5A; P <0.05). The loss of body weight of rats treated with OT was in any case related to an anorexigenic effect of the hormone, as was not observed in the AP group (Figure 5A). The body composition analyzed by an NMR analyzer showed that the periferal administration of OT promoted a significant decrease in fat mass, while inducing an increase in lean body mass 30 percent, regardless of changes in food intake ( Figures 5C and D; P <0.05) after 10 days of treatment. In this way, a decrease in fat mass, independent of food intake, was also confirmed by examining and weighing different fatty tissue deposits (Figures 5E and F).

 35

PPAR-alpha in knockout and wild-type mice

OT was administered peripherally with 50 nmol / day for 3 days in PPAR-alpha knockout (KO) mice and their wild-type litter brothers. While peripheral infusion of OT induced a significant decrease in body weight gain of wild-type animals, it did not modify this parameter in 40 KO PPAR-alpha mice. Consistently, peripheral infusion of OT promoted an increase in mRNA expression of the PPAR-alpha target genes (Acadm, Acox1, Ehhadh, Ucp3) in wild-type mice, while having no effect on PPAR-alpha knockout mice .

Statistical Analysis 45

For examples 1-3, the results are expressed as means ± ESM The Levene test was used to check whether the equality of variance between groups (SPSS Inc., Chicago, IL). In order to evaluate the effects of the treatment, the comparison of the groups was performed using parametric tests (Student's t-test) and non-parametric tests (Mann-Whitney test) when normality tests and the same variance failed. The 50 statistical significance was established at * P <0.05, ** P <0.01.

These results support that the effects induced by OT on the lipid metabolism of adipose tissue are exerted by the direct activation of PPAR-α, stimulating the synthesis of endocannabinoid, oleoylethanolamide (OAS), in adipose tissue, thus stimulating lipolysis and oxidation of local fatty acids.

All observed metabolic effects support the benefit of OT administration and / or activation of the OT receptor pathway by OT-like molecules (e.g., derivatives of OT, OTR agonists or OT analogs). ) for the repression or treatment of obesity or insulin resistance. In particular, those 60 multiple component effects support the benefit of OT administration and / or activation of the OT receptor pathway by OT-like molecules for the treatment of a multi-component syndrome such as the syndrome. metabolic and in the prevention, repression, delay or treatment of disorders due to increased peripheral insulin resistance such as in type 2 diabetes.

 65

Claims (14)

1. A derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist for use in the treatment of a disorder selected from obesity and insulin resistance, wherein the derivative of oxytocin is selected from 4-threonine-1-hydroxideaminooxitocin, 4-serine, isoleucine-oxytocin-8, 9-5 deamidooxytocin, 7-D-proline-oxytocin, (2, 4-diisoleucine) -oxytocin, 1-deamino-1-monocarba- E12-Tir (OMe)] - OT (dCOMOT), carbetocin (SEQ ID NO .: 2: Butiril-Tir (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2), 4-threonine, 7-glycine-oxytocin (TG-OT), (1-butanoic acid-2- (O-methyl-L-tyrosine) -1-carbaoxitocina, Ile-conopresina, atosiban, oxipresina, deamino-6-carba-oxytoxino (dC60 ), desmopressin and 1-deamino-oxytocin where the disulfide bridge between residues 1 and 6 is replaced by a thioether, wherein said oxytocin, derived from oxytocin or agonist of oxytocin is to be administered periferally. 2. A derivative for use according to claim 1 wherein the disorder is insulin resistance. 3. A derivative for use according to claim 1 wherein the disorder is obesity. fifteen 4. A derivative of use according to any one of claims 1 to 3 wherein the disease is the metabolic syndrome. 5. A derivative for use according to claim 2 for the prevention of the development of diabetes of type 2. 6. A derivative for use according to any one of claims 1 to 5, wherein the derivative is an oxytocin analog.  25 7. A derivative for use according to any one of claims 1 to 5, wherein the derivative is oxytocin. 8. A derivative for use according to any one of claims 1 to 6, wherein the derivative is carbetocin.  30 9. A pharmaceutical formulation comprising a derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist, combined with at least one co-agent selected from the classes of biguanides and glitazones, sulfonylureas, inhibitors of dipeptidyl peptidase-4 (DPP-4) and glucagon type 1 agonists (GLP-1) and at least one pharmaceutically acceptable carrier, wherein the oxytocin derivative is selected from 4-threonine-1-hydroxideaminooxytocin, 4- serine, 8-isoleucine-oxytocin, 9-35 deamidooxytocin, 7-D-proline-oxytocin, (2, 4-diisoleucine) -oxytocin, 1-deamino-1-monocarba-E12-Tir (OMe)] - OT (dCOMOT ), carbetocin (SEC ID No.: 2: Butyryl-Tyr (Me) -Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2), 4-threonine, 7-glycine-oxytocin (TGOT), (1 -butanoic acid-2- (O-methyl-L-tyrosine) -1-carbaoxitocin, Ile-conopressin, atosiban, oxipresin, deamino-6-carba-oxytoxino (dC60), desmopressin and 1-deamino-oxytocin in where the disulfur bridge or between residues 1 and 6 it is replaced by a thioether. 40 10. A pharmaceutical formulation according to claim 9, wherein the derivative is an oxytocin analog. 11. A pharmaceutical formulation according to claim 9, wherein the derivative is oxytocin. Four. Five 12. A pharmaceutical formulation according to claims 9 or 10, wherein the derivative is carbetocin. 13. A pharmaceutical formulation according to any of claims 9 to 12 for use in the treatment of a disorder selected from obesity and insulin resistance, wherein the formulation is 50 to be administered periferally. 14. A derivative selected from oxytocin, a derivative of oxytocin and an oxytocin agonist for use in the treatment of a disorder selected from obesity and insulin resistance and, wherein said oxytocin, a derivative of Oxytocin and an oxytocin agonist is to be administered in combination with at least one co-agent selected from the classes of -biguanides and glitazones, sulfonylureas, dipeptidyl peptidase-4 (DPP-4) inhibitors and glucagon type agonists. 1 (GLP-1), wherein the oxytocin derivative is selected from 4-threonine-1-hydroxideaminooxytocin, 4-serine, isoleucine-oxytocin-8, 9-deamidooxytocin, 7-D-proline-oxytocin, (2, 4-diiso leucine) -oxytocin, 1-deamino-1-monocarba-E12-Tyr (OMe)] - OT (dCOMOT), carbetocin, (SEQ ID NO .: 2: Butiril-Tyr (Me) -lle-Gln- Asn-Cys-Pro-Leu-Gly-NH2), 4-threonine, 7-glycine-oxytocin (TG-OT), (1-butanoic acid-60 (O-methyl-L-tyrosine) -1-carbaoxitocina , Ile-conopressin, atosiban, oxyprine, deamino-6-carba-oxytoxino (dC60), desmopressin and 1-deamino-oxytocin where the disulfide bridge between residues 1 and 6 is replaced by a thioether, wherein said oxytocin, derived from oxytocin or the agonist of Oxytocin is to be administered peripherally.