EA045828B1 - METAP2 INHIBITORS AND METHODS FOR RESTORING INSULIN SENSITIVITY - Google Patents

Description

Cross reference to related applications

This application claims the benefit of U.S. Provisional Patent Application No. 61/810,468, filed April 10, 2013, and U.S. Provisional Patent Application No. 61/925,918, filed January 10, 2014. The contents of each of these applications are incorporated by reference herein. in their entirety.

BACKGROUND OF THE INVENTION

Obesity is a chronic disease and is of particular health importance in modern society. According to the Centers for Disease Control and Prevention (CDC), the United States is in the midst of an obesity epidemic. In the United States, approximately 65% of adults are overweight, 30% of adults are obese, and more than 5 million adults are classified as morbidly obese. More than ten million are close to this level and are at risk of obesity-related health problems.

Current treatments for obesity include standard diet and exercise, very low calorie diets, behavioral therapy, pharmacotherapy including appetite suppressants, thermogenic drugs, food absorption inhibitors, mechanical devices such as jaw wires, waist belts and inflatable balloons, and surgery. However, these existing treatments are not very effective. Energy-restrictive diets are problematic and usually unsuccessful, and medical treatments are only moderately effective for long-term weight control. In most cases, toxicity and side effects have hampered the development of potential drug candidates for weight loss in patients. Metabolic syndrome (Sutherland, et al., Metabolic Syndrome and Related Disorders 2:82-104 (2004); Esposito, et al., Nutr.

Metab. Cardiovasc. Dis. 14:228-232 (2004)) refers to obesity and is characterized by a group of metabolic risk factors, including: 1) abdominal obesity (excess adipose tissue in and around the abdominal cavity); 2) atherogenic dyslipidemia (high triglycerides, low HDL (high density lipoprotein) cholesterol and high LDL (low density lipoprotein) cholesterol); 3) high blood pressure; 4) insulin resistance or glucose intolerance; 5) a prothrombotic state (eg, high fibrinogen or plasminogen activator inhibitor-1 in the blood) and 6) a pro-inflammatory state (eg, increased levels of CRP in the blood). Metabolic syndrome is becoming increasingly common in developed countries and is closely associated with the risk of coronary heart disease (Malik, et al., Circulation 110:1245-1250 (2004); Irabarren, et al., J. Am. Coll. Cardiol. 48 :1800-1807 (2006)).

Cardiometabolic syndrome includes obesity-related metabolic disorders and atherosclerosis.

Cardiometabolic disorders also promote arterial and valvular calcification, which can lead to the devastating clinical complications of acute myocardial infarction and aortic stenosis. In addition, diabetes causes chronic kidney disease, which also leads to cardiovascular ectopic calcification and acute myocardial infarction. Collectively, several major components of the cardiometabolic syndrome, developed through interrelated mechanisms, reinforce each other through local or systemic inflammation. Additionally, patient non-adherence to prescribed medication poses a huge challenge to the global healthcare community. In the US alone, avoidable medical costs were estimated at $300 billion in 2009. With the expiration of top-selling drugs draining pipelines and increasing payer cost containment, closing this gap is the shortage is best for pharmaceutical companies. Accordingly, new compounds and methods that induce and/or enhance weight loss and treat obesity and metabolic syndrome are needed. The present invention is aimed at addressing these needs.

The essence of the invention

The present invention relates to the use of a compound for improving or restoring insulin sensitivity in a patient in need thereof, where the patient is overweight or obese and wherein the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of

- 1 045828

The present invention also relates to the use of a compound of the formula

- 2 045828 r_ H 1 hn'kd z'^o

I

L ^R 6 J _n or a pharmaceutically acceptable salt thereof for improving or restoring insulin sensitivity in a patient in need thereof, where the patient is overweight or obese; and where in each case, regardless

R4 represents H or C1- _C6 alkyl;

R5 is H or C1- _C6 alkyl;

R6 is C2-C6 hydroxyalkyl;

Z represents -NH-AA ₁ -AA ₂ -AA ₃ -AA ₄ -AA ₅ -AA ₆ -C(O)-QXYC(O)-W;

AA ₁ is glycine, alanine or H2N (CH ₂ )mCO ₂ H, where m is 2, 3, 4 or 5;

AA ₂ represents a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, threonine, valine, tryptophan or tyrosine;

AA3 is a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, threonine, valine, tryptophan or tyrosine;

AA ₄ is a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, threonine, valine, tryptophan or tyrosine;

AA ₅ is a bond or glycine, valine, tyrosine, tryptophan, phenylalanine, methionine, leucine, isoleucine or asparagine;

AA ₆ represents a bond or alanine, asparagine, citrulline, glutamine, glycine, leucine, methionine, phenylalanine, series, threonine, tryptophan, tyrosine, valine or H ₂ N(CH ₂ ) _m CO ₂ H, where m is 2.3 , 4 or 5;

Q-X-Y represents

W represents o

x is equal to a number in the range from 1 to 450;

y is equal to a number in the range from 1 to 30;

n is equal to a number in the range from 1 to 50; and the compound has a molecular weight of less than 60 kDa.

Unless otherwise indicated, all technical and scientific terms used in the context have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. As used herein, the singular number also includes the plural unless the context clearly requires otherwise. Although methods and substances similar or equivalent to those described in the context may be used in the practice or testing of the present invention, suitable methods and substances are described below. All publications, patent applications, patents and other references cited in the context are incorporated by reference. References cited in context are not presumed to be prior art for the claimed invention. In the event of a conflict, this application, including the definitions, may be governed by. In addition, the substances, methods and examples are illustrative only and are not intended to be limiting.

Brief description of drawings

Fig. 1 is a graph showing the change in body weight over time after administration of the compounds of the present invention.

Fig. 2 is a graph showing average weekly food intake after administration of the compounds of the present invention.

Fig. 3 is a graph showing the relationship between total fat mass and body weight after administration of the compounds of the present invention.

Fi. 4 is a graph showing blood glucose levels after administering a sugar load to mice during a treatment period study using the compounds of the present invention.

Fig. 5 is a graph showing the change in body weight over time after administration of various doses of a compound of the present invention according to a dosage regimen

- 3 045828 stva q4d.

Fig. 6 is a graph showing changes in total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol at the end of a 32-day study as a function of the dose level of the compounds of the present invention administered.

Fig. 7 is a graph showing the change in body weight over time after administration of various doses of the compound of the present invention to rats under drug regimen q7d.

Fig. 8 is a graph showing the change in body weight of rats treated with one dose of various test agents.

Fig. 9 is a graph showing plasma concentration of a compound of the present invention over time based on administration of two different compounds.

Fig. 10 is a graph showing the change in body weight over time after administration of various compounds of the present invention.

Fig. 11 is a graph showing changes in body weight in male Levin rats fed a high fat diet following administration of various compounds of the present invention.

Fig. 12 is a graph showing the effect of fumagillol as a function of body weight loss in DIO rats after administration of various compounds of the present invention.

Fig. 13 is a graph showing changes in insulin levels in male Levin DIO rats fed a high fat diet following administration of various compounds of the present invention.

Fig. 14 is a graph showing insulin levels during an oral glucose load (OGTT) after administration of various compounds of the present invention.

Fig. 15 is a graph showing the decrease in blood glucose levels in DIO rats during oral sugar loading (OGTT) after administration of various compounds of the present invention.

Fig. 16 is a graph showing the HOMA-ir product during OGTT in DIO rats after administration of various compounds of the present invention.

Fig. 17 is a graph showing weekly feed intake after administration of various compounds of the present invention.

Fig. 18 is a graph showing changes in leptin levels from baseline following administration of various compounds of the present invention.

Fig. 19 is a graph showing changes in body weight in DIO mice after administration of various compounds of the present invention under drug regimen Q4D and Q8D.

Fig. 20 is a graph showing body weight loss after administration of CKD-732 under the Q2D and Q4D drug regimen.

Fig. 21 is a graph showing reductions in feed intake following administration of various compounds of the present invention.

Fig. 22 is a graph showing significantly reduced insulin levels during ipGTT in male C57B16 mice fed a high fat diet for 25 weeks following administration of various compounds of the present invention.

Fig. 23 is a graph showing the AUC (area under the curve) of insulin during sugar loading in male DIO mice after administration of various compounds of the present invention.

Fig. 24 is a graph showing blood glucose levels in male C57B16 mice fed a high fat diet following administration of various compounds of the present invention.

Fi. 25 is a graph showing the HOMA product during ipGTT in C57B16 mice fed a high fat diet following administration of various compounds of the present invention.

Detailed Description of the Invention

Compounds of the present invention

The present invention relates to the use of a compound for improving or restoring insulin sensitivity in a patient in need thereof, where the patient is overweight or obese and wherein the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of

- 4 045828

The present invention also relates to the use of a compound of the formula

- 5 045828

-'x

HN^^O Z^^O

I

L Re Jn or a pharmaceutically acceptable salt thereof for improving or restoring insulin sensitivity in a patient in need thereof, where the patient is overweight or obese; and where in each case, regardless

R4 represents H or C1-C6alkyl;

R5 represents H or C1-C6alkyl;

R6 is _C2 -C6hydroxyalkyl;

Z represents -NH-AA ₁ -AA ₂ -AA ₃ -AA ₄ -AA ₅ -AA ₆ -C(O)-QXYC(O)-W;

AA1 is glycine, alanine or H2N(CH ₂ )mCO ₂ H, where m is 2, 3, 4 or 5;

AA ₂ represents a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, threonine, valine, tryptophan or tyrosine;

AA ₃ is a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, threonine, valine, tryptophan or tyrosine;

AA ₄ is a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, series, threonine, valine, tryptophan or tyrosine;

AA5 is a linkage or glycine, valine, tyrosine, tryptophan, phenylalanine, methionine, leucine, isoleucine or asparagine;

AA ₆ represents a bond or alanine, asparagine, citrulline, glutamine, glycine, leucine, methionine, phenylalanine, series, threonine, tryptophan, tyrosine, valine or H ₂ N(CH ₂ ) _m CO ₂ H, where m is 2.3 , 4 or 5;

Q-X-Y represents

W represents

x is equal to a number in the range from 1 to 450;

y is equal to a number in the range from 1 to 30;

n is equal to a number in the range from 1 to 50; and the compound has a molecular weight of less than 60 kDa.

In some embodiments, R4 is methyl. In some embodiments, R5 is methyl.

In some embodiments, R6 is 2-hydroxypropyl.

In some embodiments, AA1 is glycine, AA2, _AA3 , AA4, AA5, and AA6 are linkages.

In some embodiments, AA1 is glycine, AA2, AA ₃ , AA4 and AA5 are bonds, and AA ₆ is glycine.

In other embodiments, the ratio AA1 is glycine, AA2 is a bond, AA3 is a bond, AA4 is glycine, AA5 is leucine, and AA ₆ is glycine. In some embodiments, AA ₁ is glycine, AA ₂ is a bond, AA ₃ is a bond, AA ₄ is glycine, AA ₅ is valine, and AA ₆ is glycine. In some embodiments, AA ₁ is glycine, AA ₂ is a bond, AA ₃ is a bond, AA4 is glycine, AA5 is phenylalanine, and AA6 is glycine. In some embodiments, AA1, AA4, AA5, and AA6 are each glycine, AA ₂ is a bond, and AA ₃ is a bond.

In some embodiments, the x to y ratio is in the range of 20:1 to 4:1, preferably 11:1.

In some embodiments, the patient's body mass index (BMI) is between 25 and 29.9 kg/m ² ; 30 kg/ ^m2 or more; 35 kg/ ^m2 or more; or 40 kg/ ^m2 or more.

In some embodiments, the patient has at least one underlying health care

- 6 045828 obesity-induced or obesity-related hypertension, selected from the group consisting of diabetes, impaired glucose tolerance, impaired fasting glucose, elevated plasma insulin concentrations, insulin resistance syndrome, hyperlipidemia, dyslipidemia, hypertension, hyperuricemia, gout , coronary artery disease, myocardial infarction disease, angina pectoris, sleep apnea, obstructive sleep apnea, Pickwick syndrome, fatty liver, cerebral infarction, stroke, cerebral vascular thrombosis, respiratory complications, gallbladder disease, kidney disease, gastrointestinal tract esophageal reflux, stress urinary incontinence, atherosclerosis, heart disease, abnormal heart rhythms, cardiac arrhythmia, transient ischemic attack, orthopedic disorders, osteoarthritis, arthritis deformans, lumbago, menstrual disorders, endocrinopathies, hormonal imbalances and infertility.

In some embodiments, the compound is further used to treat, reduce or improve one or more cardiometabolic risk factors in the subject, and the cardiometabolic risk factors are selected from plasma triglyceride levels, LDL cholesterol levels, C-reactive protein (CRP) levels, systolic blood pressure , diastolic blood pressure, abdominal tissue adiposity, fasting glucose levels, fasting insulin levels, fibrinogen levels, plasminogen activator inhibitor-1 levels, and leptin levels.

In some embodiments, the compound is administered in a therapeutically effective amount that is 0.0001 to 5 mg/kg body weight per day or 0.001 to 1 mg/kg body weight per day.

In some embodiments, the compound is administered 1 to 5 times per week, preferably every two weeks, more preferably the q4d dosing schedule, or most preferably the q7d dosing schedule.

In some embodiments, the claimed compound is administered parenterally or subcutaneously.

In some embodiments, the claimed compound may be provided in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier.

In some embodiments, Z is

In a preferred embodiment, Z is o

H I/ oV \ ^Gly-Phe-Leu-N^J^

N

In some embodiments, the diabetes is non-insulin-dependent diabetes mellitus type II. In some embodiments, the sleep apnea is obstructive sleep apnea. In some embodiments, the gallbladder disease is gallstone disease.

For the purposes of the present invention, chemical elements are designated according to the periodic table of the elements, version, CAS, Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover.

The term alkyl refers to a fully saturated branched or straight carbon chain radical having the number of carbon atoms indicated, or up to 30 carbon atoms if the number of atoms is not specified. For example, the term lower alkyl refers to alkyl having from 1 to 10 carbon atoms, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl and octyl, and alkyls that are positional isomers of these alkyls. Alkyl containing from 10 to 30 carbon atoms includes decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl and tetracosyl. In some variants

- 7 045828 implementations of a straight chain or branched alkyl have 30 or fewer carbon atoms in its main chain (eg C1-C30 for straight chains, C ₃ -C ₃₀ for branched chains) and more preferably 20 or less. Likewise, some cycloalkyls have 3-10 carbon atoms in their ring structure and may have 5, 6, or 7 carbon atoms in their ring structure.

Unless the number of carbon atoms is otherwise indicated, the term lower alkyl as used in the context means an alkyl group as defined above but having from one to ten carbon atoms or from one to six carbon atoms in the main chain, such as methyl, ethyl, n- propyl, isopropyl, nbutyl, isobutyl, sec-butyl and tert-butyl. Likewise, lower alkenyl and lower alkynyl have similar chain lengths. Throughout the application, some alkyl groups are lower alkyls. In some embodiments, the substituent referred to in the context as alkyl is lower alkyl.

The term amino acid is intended to include all compounds, whether natural or synthetic, that contain both an amino and an acid functional group, including analogs and derivatives of amino acids. In some embodiments, the amino acids contemplated by the present invention are naturally occurring amino acids found in proteins, or naturally occurring anabolic or catabolic products of such amino acids, which contain amino groups and carboxyl groups. Natural amino acids are designated in all cases by generally accepted three-letter and/or one-letter abbreviations corresponding to the trivial name of the amino acid according to the following list. Such abbreviations are recognized in the peptide field and are recommended by the IUPAC-IUB commission for biochemical nomenclature.

The term amino acid residue means an amino acid. In general, the abbreviations used in the present context to designate natural amino acids are based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). For example, Met, Ile, Leu, Ala and Gly represent methionine, isoleucine, leucine, alanine and glycine residues, respectively. The term residue means a radical obtained from the corresponding α-amino acid by eliminating the OH part of the carboxyl group and the H atom of the α-amino group.

The term amino acid side chain means that portion of an amino acid residue other than the main chain, as defined by KD Kopple, Peptides and Amino Acids, W.A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and 33; examples of such common amino acid side chains are CH ₂ CH ₂ SCH ₃ (methionine side chain), -CH ₂ (CH ₃ )-CH ₂ CH ₃ (isoleucine side chain), CH ₂ CH (CH ₃ ) ₂ (leucine side chain) or H- (glycine side chain). These side chains are pendants to the α carbon atom of the main chain.

The term peptide, as used herein, refers to a sequence of amino acid residues joined together by peptide bonds or modified peptide bonds. The term peptide is intended to include peptide analogs, peptide derivatives, peptidomimetics, and peptide variants. It should be understood that the term peptide includes peptides of any length. Peptide sequences presented in context are written according to the common convention whereby the N-terminal amino acid is on the left and the Terminal amino acid is on the right (e.g., H ₂ N-AA ₁ -AA ₂ -AA ₃ -AA ₄ -AA ₅ -AA ₆ -CO ₂ H).

Synthesis of the compounds of the invention

The synthetic methods of this invention may be suitable for different functional groups, so different substituted starting compounds can be used. Such methods generally provide the desired final compound at or near the end of the overall production process, although in some cases further conversion of the compound to its pharmaceutically acceptable salt may be desirable.

The compounds of the present invention can be prepared in a variety of ways using commercially available starting materials, compounds known in the literature, or from readily prepared intermediates, using standard synthetic methods and procedures that are either known to those skilled in the art or will be apparent to those skilled in the art. in the light of the material presented in the context. Standard synthetic methods and procedures for the preparation of organic molecules and functional group transformations or treatments can be obtained from the relevant scientific literature or from standard textbooks in the field. Although not limited to any one or more sources, classic textbooks such as Smith, M.B., March, J. March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, ^5th edition, John Wiley & Sons: New York, 2001 ; and Greene, T.W., Wuts, P.G.M., Protective Groups in Organic Synthesis, ^3rd edition, John Wiley & Sons: New York, 1999, incorporated herein by reference, are the applicable and recognized reference textbooks of organic synthesis known to those skilled in the art. . The following descriptions of synthetic methods are intended to illustrate, but not to limit, the general procedures for preparing the compounds of the present invention.

- 8 045828

The compounds of the present invention can be conveniently prepared by various methods known to those skilled in the art. The compounds of the present invention can be prepared according to the schemes and examples presented in the context, from commercially available starting materials or starting materials that can be prepared using procedures described in the literature. The compounds of the present invention and their synthesis are further described in PCT Publications Nos. W0 2011/150088 and WO 2011/150022. Each of these publications is incorporated by reference in its entirety for all purposes.

Pharmaceutical compositions

The present invention also relates to the use of the claimed compounds or their pharmaceutically acceptable salts in the form of a pharmaceutical composition containing a pharmaceutically acceptable carrier or excipient.

The term pharmaceutically acceptable excipient or pharmaceutically acceptable carrier, as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, the standard reference text in the field. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.

Pharmaceutically acceptable carriers include solid carriers such as lactose, calcium sulfate, sucrose, talc, gelatin, agar, pectin, gum acacia, magnesium stearate, stearic acid and the like. Representative liquid carriers include syrup, peanut butter, olive oil, water, and the like. Likewise, the carrier or diluent may include a time-delay agent known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methyl methacrylate, and the like. Other fillers, excipients, flavoring agents and other additives, such as those known in the art, can also be included in the pharmaceutical composition of the present invention. Liposomes and non-aqueous excipients such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of a conventional vehicle or agent in the compositions of the invention is contemplated, excluding any conventional vehicle or agent that is incompatible with the active compound. Additional active compounds may also be included in the compositions. In some embodiments, the pharmaceutical composition contains DMSO.

The term pharmaceutically acceptable salts refers to relatively non-toxic addition salts of a compound(s) with inorganic and organic acids. These salts can be prepared in situ during the final isolation and purification of the compound(s) or separately by reacting the purified compound(s) in free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Репрезентативные соли включают в себя гидробромидные, гидрохлоридные, сульфатные, бисульфатные, фосфатные, нитратные, ацетатные, валератные, олеатные, пальмитатные, стеаратные, лауратные, бензоатные, лактатные, фосфатные, тозилатные, цитратные, малеатные, фумаратные, сукцинатные, тартратные, нафтилатные, мезилатные , glucoheptonate, lactobionate and lauryl sulfonate salts and the like. Representative alkali or alkaline earth metal salts include lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Representative organic amines useful for preparing base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, etc. (See, for example, Berge et al. (1977) Pharmaceutical Salts, J. Pharm. Sci. 66:1-19).

The expression pharmaceutically acceptable is used in the context to refer to ligands, substances, compositions and/or dosage forms that, within the limits of medical judgment, are suitable for use in contact with human and animal tissues, are substantially non-pyrogenic, and do not exhibit excessive toxicity, irritation, allergic reactions or are not associated with other problems or complications and have an acceptable benefit/risk ratio.

The present invention relates to the use of a compound or a pharmaceutically acceptable salt thereof for improving or restoring insulin sensitivity in a patient in need thereof where the patient is overweight or obese.

Obesity and overweight refer to excess fat in a subject relative to lean body mass. The accumulation of excess fat is associated with an increase in the size (hypertrophy or steatosis) as well as the number (hyperplasia) of fat tissue cells. Obesity can be due to any cause, regardless of whether it is genetic (eg, Prader-Willi syndrome) or caused by external conditions (environmental). Obesity is variously measured in terms of absolute mass, weight:height ratio, degree of excess body fat, distribution of visceral and subcutaneous fat, and social and aesthetic norms. A common measure of body fat is body mass index (BMI). BMI refers to the ratio of body weight (expressed in kilograms)

- 9 045828 max) to the square of height (expressed in meters). Body mass index can be accurately calculated using the formula: SI units: BMI = mass (kg)/(height ² (m ² ) or US units: BMI = (weight (lb) * 703)/(height ² (in ² ).

As described in the context, overweight refers to a condition in which an otherwise healthy adult has a BMI of 25 to 29.9 kg/m ² . As used herein, the term obese or obese refers to a condition in which an otherwise healthy adult has a BMI of 30 kg/m ² or more. Obesity has several subcategories. An adult who has a BMI of 35 kg/m ² or more is called a morbidly obese person or a morbidly obese person. An adult who has a BMI >40-44.9 kg/m ² and/or an adult who has a body mass index of 35 kg/m ² or more and at least one obesity-related health condition is called a person suffering from morbidity. obese, or morbidly obese person. An adult who has a BMI of 45 kg/m ² or more is called an extremely obese person or an extremely obese person. For children, definitions of overweight and obesity take into account age and gender influences on body fat mass.

Different countries may define obesity and overweight using different BMIs. This means that the term obesity has definitions in all countries. For example, increased risks associated with obesity occur with lower body mass index (BMI) in Asia. In Asian countries, including Japan, obesity refers to a condition in which a subject with at least one obesity-induced or obesity-related comorbidity that requires weight loss or the symptoms of which can be improved by weight loss has a BMI greater than or equal to 25.0 kg/ ^m2 . Ethnic residents of South and Central America tend to be classified closer to Asian than European or North American.

BMI does not account for the fact that excess adipose tissue may occur selectively in different parts of the body, and the development of adipose tissue may be more harmful to health in some parts of the body than in other parts of the body. For example, central obesity, commonly associated with an apple-shaped body, is the result of excess adiposity, especially in the abdominal area, including belly fat and visceral fat, and carries a higher risk of comorbidity than peripheral obesity, which is usually associated with a piri-shaped body. body, resulting from excess fat, especially on the thighs. Waist/hip ratio (WHR) measurements can be used as an indicator of central obesity. The minimum WHR value characteristic of central obesity has been established in various ways, and adults with central obesity typically have a WHR of approximately 0.85 or greater for women and approximately 0.9 or greater for men.

Disease assessment is carried out using standard methods known in the art, for example, monitoring the appropriate marker(s). For example, the following markers can be monitored to assess obesity: body weight, BMI, body composition studies, body fat distribution, central fat distribution, food or calorie intake, behavioral measures of hunger and satiety, metabolic rate, and obesity-related comorbidities.

Methods for determining whether a subject is overweight or obese, which calculate the ratio of excess adipose tissue to lean body mass, may involve determining the subject's body type. Body type can be determined by measuring the thickness of subcutaneous fat in several places of the body, such as the abdominal region, subscapular region, arms, buttocks and thighs. These measurements are then used to estimate total body fat with a margin of error of approximately four percent. Another method is bioelectrical impedance analysis (BIA), which uses the resistance of electrical flow as it passes through the body to estimate body fat. Another method uses a large tank of water to measure the buoyancy of a body. Increased body fat will make the body more buoyant, while higher muscle mass will cause a tendency to sink. Another method is dual-energy fan-beam X-ray absorptiometry (DEXA). DEXA allows non-invasive determination of body composition, especially total body fat and/or regional fat mass. MRI can also be used to non-invasively determine body composition.

A subject in need of treatment according to the present invention may also have (ie have been diagnosed with or have) at least one obesity-induced or obesity-related comorbidity, i.e. diseases and other adverse conditions associated with overweight or obesity, or aggravated or provoked by overweight or obesity.

Obesity-induced or obesity-related comorbidities The obesity-induced or obesity-related comorbidity is selected from the group consisting of diabetes, impaired glucose tolerance, impaired fasting glucose, elevated

- 10 045828 plasma insulin concentrations, insulin resistance syndrome, hyperlipidemia, dyslipidemia, hypertension, hyperuricemia, gout, coronary artery disease, myocardial infarction disease, angina pectoris, sleep apnea, obstructive sleep apnea, Pickwick's syndrome, fatty liver, infarction brain, stroke, cerebral thrombosis, respiratory complications, gallbladder disease, kidney disease, gastroesophageal reflux, stress urinary incontinence, atherosclerosis, heart disease, abnormal heart rhythms, cardiac arrhythmia, transient ischemic attack, orthopedic disorders, osteoarthritis, arthritis deformans, lumbago, menstrual disorders, endocrinopathy, hormonal imbalances and infertility.

The present invention also relates to the use of a compound for the treatment, reduction or improvement of one or more cardiometabolic risk factors in said patient, and the cardiometabolic risk factors are selected from plasma triglyceride levels, LDL cholesterol levels, C-reactive protein (CRP) levels, systolic blood pressure, diastolic blood pressure, abdominal tissue adiposity, fasting glucose levels, fasting insulin levels, fibrinogen levels, plasminogen activator inhibitor-1 levels, and leptin levels.

The term patient or subject, when used in context, can mean either a human or a non-human subject. In some embodiments, the subject is a vertebrate animal. In some embodiments, the vertebrate animal is a mammal. The term mammals also includes, but is not limited to, farm animals, sporting animals, primates (including humans), horses, dogs, cats, mice and rats. In some embodiments, the mammal is a human.

As used herein, the expression subject in need means a subject who is overweight or obese (who may or may not have one or more comorbidities), or a subject who has an increased risk of being overweight or obese relative to the population in in general. In some aspects, the subject in need thereof is an obese patient having a BMI of 30 kg/m ² or more. In some aspects of the invention, the subject who needs this is a subject who is overweight or obese, or has an increased risk of being overweight or developing obesity, compared to the general population who is not overweight or who has not been diagnosed with a disease, selected from the group consisting of cancer, hyperproliferative disorder, retinal neovascularization due to macular degeneration, psoriasis and pyogenic granuloma, rheumatoid, immune and degenerative arthritis.

The term prophylactic or therapeutic treatment is a recognized term in the art and includes the administration of one or more subject compositions to the host. If it is administered before the clinical manifestation of an undesirable condition (e.g., a disease or other undesirable condition in the host animal), then the treatment is prophylactic (i.e., it protects the owner from developing the undesirable condition), whereas if it is administered after the manifestation of the undesirable condition, the treatment is therapeutic (i.e. it is intended to reduce, alleviate or stabilize the intensity of the symptoms of an existing unwanted condition or its side effects).

As used herein, the term treatment is an approach to achieve beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following results: improvement, reduction in severity, reduction of one or more symptoms associated with the disease. In obesity, beneficial or desired clinical outcomes include any one or more of the following: weight loss or maintenance; regulating (including reducing) food or caloric intake; increasing metabolic rate or inhibiting decrease in metabolic rate and improving, reducing the severity and/or attenuating any of the disorders associated with obesity, such as diabetes mellitus, non-insulin-dependent diabetes mellitus, hyperglycemia, low glucose tolerance, insulin resistance, lipid disorder, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, abdominal obesity, eating disorder, metabolic syndrome, hypertension, osteoarthritis, myocardial infarction, fatty liver, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver degeneration (NAFLD), stroke and other associated diseases; improving the quality of life of obese people and/or extending life expectancy.

An individual at risk for obesity may or may not have a detectable disease and may or may not have said detectable disease prior to treatment with the methods described in the context. The expression at risk means that an individual has one or more so-called risk factors, which are measurable parameters that correlate with the development of obesity. An individual who has one or more of these risk factors has a higher likelihood of becoming obese than an individual without these risk factor(s). These risk factors include, but are not limited to, age, diet, physical inactivity, metabolic

- 11 045828 syndrome, family history of obesity, ethnicity, hereditary syndromes, history of previous disease (eg, eating disorders, metabolic syndrome and obesity), presence of previous disease (eg, overweight). For example, to put it another way, a healthy individual with a BMI of 25.0 to less than 30.0 kg/ ^m2 or an individual with at least one comorbidity and a BMI of 25.0 to less than 27.0 kg/ ^m2 is at risk of developing obesity.

The development of obesity means the onset and/or progression of a disease in an individual (which may be another embodiment of the invention). The development of obesity can be detected using standard clinical methods described in context. However, development also refers to the progression of the disease, which may initially be undetected. For the purposes of the present invention, progression refers to the biological course of a pathological condition, in this case determined by assessing height and weight to calculate BMI, measuring waist circumference, identifying comorbidities, and the onset and/or worsening of complications of obesity, such as atherosclerosis, type II diabetes , polycystic ovarian disease, cardiovascular disease, osteoarthritis, dermatological disorders, hypertension, insulin resistance, hypercholesterolemia, hypertriglyceridemia and cholelithiasis. Various such diagnostic tests are well known in the art. The term development includes emergence, repetition and beginning of development. As used herein, the term onset or occurrence of obesity includes the onset of first occurrence and/or recurrence.

As used herein, the expression weight management or weight improvement refers to the reduction or maintenance of an individual's body weight (compared to the pre-treatment weight level). In some embodiments, body weight is typically maintained within a normal range. Body weight can be reduced by reducing calorie intake and/or reducing body fat accumulation. In some embodiments, the subject's body weight is reduced by at least about 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% compared to the pre-treatment level.

As used herein, food regulation refers to reducing or maintaining an individual's food intake (compared to pre-treatment levels). In some embodiments, food intake is typically maintained within normal limits. In some embodiments, the individual's food intake is reduced by approximately 3%, 4%, 5%, 10%, 20%, 30%, 40%, or 50% compared to pre-treatment levels.

The term therapeutically effective amount of a compound, when used in treatment, refers to the amount of a compound in a formulation that, when administered as part of a desired dosage regimen (in a mammal, preferably a human), reduces a symptom, improves a condition, or delays or prevents the onset of disease states according to clinically acceptable standards for the disorder or condition being treated, or for a cosmetic purpose, for example, with an acceptable benefit/risk ratio applicable to any drug treatment. The term therapeutically effective amount is synonymous with the term effective dose.

As used herein, the term effective dose or effective amount of a drug, compound or pharmaceutical composition is an amount sufficient to achieve medicinal or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, reducing severity or delaying the onset of disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes present during development of the disease. For therapeutic use, beneficial or desired results include clinical results such as a decrease in the intensity, duration or frequency of attacks of the disease and a decrease in the intensity of one or more symptoms resulting from the disease (biochemical, histological and/or behavioral symptoms), including its complications and intermediate pathological phenotypes present during the development of a disease, improving the quality of life of people suffering from such a disease, reducing the dose of other drugs required to treat that disease, increasing the effect of another drug and/or slowing the progression of the disease in patients. An effective dose can be administered in one or more administrations. For purposes of the present invention, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment, either directly or indirectly. As understood from the clinical context, an effective dose of a drug, compound or pharmaceutical composition may or may not be obtained in combination with another drug, compound or pharmaceutical composition. Thus, an effective dose can be considered in the context of the administration of one or more therapeutic agents, and one agent can be considered to be administered in an effective amount if the desired result can be achieved or is achieved in combination with one

- 12 045828 or several other agents. For example, an effective amount of a compound of the present invention for treating obesity is an amount sufficient to treat or reduce the intensity of one or more symptoms associated with obesity. The term effective amount means an amount sufficient to produce one or more of the following results (which may also be consistent with various embodiments of the present invention): weight reduction, weight restoration or weight control, reduction in intake, restoration of normal food intake, or regulating food intake, increasing metabolic rate, reducing one or more symptoms resulting from obesity-related diseases, improving the quality of life of obese people, and/or prolonging life expectancy.

The dosage of the compound of the present invention can be determined empirically for individuals to whom the compound is administered one or more times. The compounds of the present invention are administered to individuals in incremental doses. To assess the effectiveness of a compound of the present invention, markers of pathological conditions can be monitored. It will be apparent to one skilled in the art that the dosage will vary depending on the individual, the stage of the disease (eg, the stage of obesity), and the past and concurrent treatments used.

The toxicity and therapeutic efficacy of the compounds of the present invention can be determined by standard pharmaceutical procedures using experimental animals. Toxic doses can be defined as the maximum tolerated dose (MTD) or alternatively the LD50 (dose lethal to 50% of the population). Effective doses can be defined as the ED50 (the dose therapeutically effective in 50% of the population) or the dose required to produce some average magnitude of change in an animal (e.g., the dose required to produce an average reduction in systolic blood pressure of 10 mmHg in a group of subjects) .

Ideally effective and toxic doses can be determined for individuals of the same species. However, if they are determined for different species, allometric scaling can be used to convert the effective or toxic dose to a different species. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. When comparing mice to rats, the generally accepted scaling factor (proportionality) of 2 is usually used; it is estimated that the rat dose is half that of mice. Thus, if the toxic dose for a rat is 100 mg/kg and the effective dose for a mouse is 1 mg/kg, the therapeutic index for the rat can be calculated as the effective dose for the rat being 1 mg/kg/2 or 0.5 mg/kg , and the therapeutic index is 200. The FDA defines a drug as having a narrow therapeutic range if (a) there is less than a 2-fold difference between the mean lethal dose and the mean effective dose or (b) there is less than a 2-fold difference between the minimal toxic and minimum effective blood concentrations.

Although it is possible to use compounds of the present invention that exhibit toxic side effects, care should be taken to develop a delivery system that specifically targets such compounds of the present invention to the site of the affected tissue to minimize possible damage to uninfected cells and thereby reduce side effects.

Data obtained from animal studies can be used to determine dosage ranges for use in humans. The dose of such compounds of the present invention is preferably within a range of circulating concentrations that includes a range of effective doses with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration used. For compounds of the present invention with M.M. less than 1000, the therapeutically effective dose can be estimated initially from cell culture assays, although animal models may provide more accurate dose estimates for conjugates that require cleavage of the linker to release the active moiety. Such information can be used to more accurately determine doses applicable to humans. It is well known in the art that conjugation with a polymer dilutes (ie reduces) the activity of the active moiety (the polymer is the diluent). This is illustrated by the mouse anticancer drug dosing model shown in the following table.

Parent drug Drug dose (mg/kg) Conjugate Conjugate dose (mg/kg) TNP-470 30 (qod) XMT-1107 800 Docetaxil 12 (Q4d) Opaxio 480 SRT-11 20 (q2d) EZN-2208 145 (q2d) Doxorubicin 5 (q4d) RK1 62 (q7d) Carboplatin 60 (Qd) AR-5356 2200

- 13 045828

Thus, it is quite clear that conjugation with a polymer increases clinical doses, while the therapeutic index does not improve. This is illustrated by the human dosing model for anticancer drugs shown in the following table.

Parent drug Drug dose (mg/kg) Conjugate Conjugate dose (mg/ ^m2 ) TNP-470 180 XMT-1107 tbd Paclitaxil 175 Opaxio 473 SRT-11 125 EZN-2208 260 Doxorubicin 78 RK1 280 Oxaliplatin 85 AR-5346 6400

The polymer conjugate and modified compounds of the present invention surprisingly provide superior efficacy and lower toxicity compared to the unconjugated and/or unmodified parent drug/active moiety.

For example, the fumagillol conjugates and modified fumagillol derivatives of the present invention are surprisingly superior to small molecule fumagillol because they provide enhanced weight loss in DIO mice at equivalent molar doses. The compounds of the present invention can be used at lower molar doses and with less frequent dosing to provide equivalent weight loss. Lower molar doses and reduced dosing frequency reduce systemic drug exposure and systemic drug toxicity. In addition, the fumagillol conjugates and modified fumagillol derivatives of the present invention provide the following effects similar to those of fumagillol small molecules: preferential fat loss in DIO mice and reduced food intake.

Traditional polymer conjugates attenuate activity, increase doses by 5-20 times and provide a small change in therapeutic index (<2x). In contrast, the polymer conjugates of the compounds of the present invention surprisingly and unexpectedly provide an increased therapeutic index (an order of magnitude improvement) and demonstrate increased activity at a reduced dose.

In the methods of the present invention, the polymer conjugates of the conjugated compounds of the present invention unexpectedly demonstrate less frequent dosing (e.g., q4d, dosing every fourth day, q7d, dosing every seventh day, q8d, dosing every eighth day), dosing, which are less than at least 84 mol.% fumagillol equivalent, decreased AUC (area under the curve) in non-target compartments, while the therapeutic index is increased (> 10x).

In another embodiment, the present context provides effective doses, for example, a daily dose of a compound of the present invention. For example, methods are provided herein that involve administering dosages of a compound of the present invention that are effective for reducing body weight. For example, the intended dosage of a compound of the present invention in the methods described in the context may include dosing, regardless of body weight, about 200 mg/day, about 80 mg/day, about 40 mg/day, about 20 mg/day, approximately 10 mg/day, approximately 5 mg/day, approximately 3 mg/day, approximately 2 mg/day, approximately 1 mg/day, approximately 0.5 mg/day, approximately 0.2 mg/day, approximately 0.05 mg/day, approximately 0.01 mg/day or approximately 0.001 mg/day.

An effective amount of a drug for weight loss in a patient may also be a body weight or surface area based dose of from about 0.0001 to about 5 mg/kg body weight per day. For example, the intended dose may be from about 0.001 to 5 mg/kg body weight per day, from about 0.001 to 1 mg/kg body weight per day, from about 0.001 to 0.1 mg/kg body weight per day, from about 0.001 up to 0.010 mg/kg body weight per day or approximately 0.007 mg/kg body weight per day.

The compounds of the present invention can be administered in an amount sufficient to reduce a patient's body weight from about 0.5 kg/week to about 1 kg/week (or from about 0.5% body weight per week to about 1% body weight per week). In some embodiments, weekly weight loss occurs throughout the duration of treatment.

Administration of a compound of the present invention according to the method of the present invention may be continuous or intermittent, depending, for example, on the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to skilled practitioners. Administration of a compound of the present invention may be substantially continuous over a preselected period of time or may be sequential dosing at specified intervals.

- 14 045828

For repeated administrations over several days or longer, depending on the condition, treatment is continued until desired suppression of disease symptoms or until sufficient therapeutic levels are achieved. For example, dosing is suggested between one and five times per week. In some embodiments, a compound of the present invention is administered approximately every fourth day. Other dosage regimens include a dosing schedule of 1 to 5 times per week, every three or four days, or less frequently. In some embodiments, a compound of the present invention is administered approximately once per week, once every two weeks, or approximately 1-4 times per month, depending on the duration of the drug response. An intermittent dosing regimen may be used, with alternating doses at intervals ranging from 2 days to 7 days or even 14 days. In some embodiments, treatment may begin with a daily dose and then progress to a weekly dose, even a monthly dose. The development of this therapy is easily monitored by conventional methods and assays or by measuring MetAP2, as described in US Pat. No. 6,548,477.

The frequency of administration can be determined and adjusted throughout the course of therapy. For example, the frequency of administration may be determined or adjusted depending on the type and severity of the disease being treated, whether the agent is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the agent, and the instructions of the attending physician. Typically, the clinician will administer a compound of the present invention until a dosage is reached that achieves the desired result.

Treatment can be continued for as long or as short a period of time as desired. A suitable treatment period may be, for example, at least about one week, at least about four weeks, at least about one month, at least about six months, at least about 1 year, at least about 2 years, or indefinitely. The treatment period can be limited when the desired result is achieved, for example, target weight loss. For example, when a loss of about 5% body weight, about 10% body weight, about 20% body weight, about 30% body weight or more has been achieved. The treatment regimen may include a correction phase, during which a compound of the present invention is administered in a dosage amount or at a dosing frequency sufficient to provide a reduction in excess obesity, followed by a maintenance phase, during which a lower dose of the compound is administered or a reduced dosing frequency is used, sufficient to prevent the recurrence of excess obesity.

The compounds or pharmaceutically acceptable salts thereof, or pharmaceutical compositions thereof, can be administered by any method known in the art. For example, the compounds or compositions of the present invention are administered by oral, intranasal, transdermal, topical, pulmonary, inhalation, buccal, sublingual, intraperitoneal, subcutaneous, intramuscular, intravenous, rectal, intrapleural, intrathecal, and parenteral routes. Administration may be systemic, for example, intravenous administration or localized administration. In some embodiments, the route of administration may be intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, intrapleural, intrauterine, rectal, vaginal, topical, etc. In some embodiments, the compound is administered subcutaneously.

In one aspect, the compounds of the present invention or pharmaceutically acceptable salts thereof are administered in a suitable dosage form or preparation prepared by combining a therapeutically effective amount (e.g., an effective level sufficient to achieve the desired therapeutic effect) of a compound of the present invention or pharmaceutically acceptable salts thereof, (as active ingredient) with standard pharmaceutical carriers or diluents according to conventional procedures (ie, procedures for preparing the pharmaceutical composition of the present invention). These procedures may include, as necessary, mixing, granulating and compressing or dissolving the ingredients to obtain the desired preparation.

Parenteral dosage forms can be prepared by any method known in the art. For example, sterile injectable aqueous or oily suspensions can be prepared according to a method known in the art using suitable dispersing or wetting agents and suspending agents.

Oral dosage forms such as capsules, tablets, pills, powders and granules can be prepared using any suitable method known in the art. For example, the compounds of the present invention can be mixed with intersolubility substances and compressed into tablets. Alternatively, the compositions of the invention are formulated into chewable tablets, crushable tablets, oral tablets, or oral rinses.

For pulmonary administration (eg, intrabronchial administration), the compounds of the present invention can be formulated with conventional excipients to form an inhalation-administered composition

- 15 045828 tion in the form of a fine powder or spray liquid. For ocular administration, the compounds of the present invention can be formulated with conventional excipients, for example, in the form of eye drops or an ocular implant. Excipients useful in eye drops include thickening or gelling agents to minimize loss due to lacrimation by increasing retention in the eye.

Liquid dosage forms for oral or other administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan fatty acid esters and mixtures thereof. Ophthalmic, oral or other systemically administered compositions may also include adjuvants, such as wetting agents and emulsifying and suspending agents, in addition to inert diluents.

Commercially available nebulizers, including jet nebulizers and ultrasonic nebulizers, are useful for administering liquid medications. Liquid drugs can be sprayed directly and lyophilized powder can be sprayed after reconstitution. Alternatively, the compounds of the present invention can be nebulized using a fluorocarbon formulation and a metered dose inhaler or inhaled as a lyophilized and milled powder.

Dosage forms for topical or transdermal administration of the pharmaceutical composition of the invention may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers or patches. The active substance is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers that may be required. For example, transdermal routes of administration are achieved using aqueous drops, aerosol, emulsion or cream.

Transdermal patches may have the added benefit of providing controlled delivery of active ingredients to the body. Such dosage forms can be prepared by dissolving or dispersing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound passing through the skin. The rate can be controlled either by the use of a rate control membrane or by dispersing the compound in a polymer matrix or gel.

Compositions for rectal or vaginal administration may be suppositories, which can be prepared by mixing the compounds of the present invention with suitable, non-irritating excipients or carriers, such as cocoa butter, polyethylene glycol or suppository wax, which are solid at ambient temperature but liquid at bodies and therefore melt in the rectum or vaginal cavity and release the active agent(s). Alternatively, the subject drugs can be administered by release from the lumen of the endoscope after insertion of the endoscope into the rectum of the subject.

One skilled in the art may refer to general reference texts for detailed descriptions of known methods discussed in the context, or equivalent methods. These texts include Ausubel et al. , Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed.), Cold Spring Harbor Press, Cold Spring Harbor. New York (2000); Coligan et al., Current Protocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., Current Protocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., The Pharmacological Basis of Therapeutics (1975), Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 18th edition (1990). These texts, of course, can also be referred to in the making or by an aspect of the invention.

Examples

The following are examples to further illustrate various features of the present invention. The examples also illustrate applicable methodology for practicing the invention. These examples do not limit the claimed invention.

General procedures

Tangential flow filtration (TFF) was used to purify the polymer products of the invention. TFF was performed using the Pall Minimate™ capsule and Minimate™ TFF system according to the manufacturer's instructions. For purification, either a Minimate TFF capsule with a 5kDa Omega membrane (5K) or a Minimate TFF capsule with a 10kDa Omega membrane cartridge (10K) was used. In all cases, the percolated portion was discharged and the retained portion was lyophilized, thereby obtaining a polymer product. The structures of the products were confirmed by 1H NMR, and small molecules were also characterized by MS. The polymer weights given in the examples were not adjusted for water content.

Carbamoylfumagillol and chloroacetylcarbamoylfumagillol can be prepared according to methods

- 16 045828 described in US patent No. 5166172 (Kishimoto, et al., which is incorporated by reference), p-nitrophenyl fumagyl-6-yl carbonate can be prepared according to published procedures. (See Han, S. et al. Biorg. Med. Chem. Lett. 2000, 10, 39-43). MA-GFLG-ONp can be prepared according to the methods described in US Pat. No. 5,258,453 (Kopecek et al., incorporated by reference).

Synthesis of πpoly(HPMA-co-MA-GFLG-ONp)]

ONp polymer -GFL-NFr

ABOUT

Blend of hydroxypropyl methacrylamide (HPMA, 22.16 g, 155 mmol), N-methacryl-gly-phe-leu-gly-pnitrophenyl ether (MA-GFLG-ONp, 10.00 g, 17.19 mmol), AIBN (1.484 g , 9.037 mmol) and acetone (225 g) were degassed (frozen, pumped out, thawed, 4 cycles). The resulting reaction mixture was stirred at 50°C for 48 hours, then cooled to room temperature. The desired product was purified by trituration with acetone, then dried in vacuo to obtain 17.6 g of poly(HPMA-co-MA-GFLG-ONp) as a white solid. The structure was confirmed by 1H NMR and the product was shown to contain no significant impurities (eg p-nitrophenol). UV absorbance data indicated that the copolymer contained 0.47 mmol p-nitrophenyl ester per gram of polymer. The copolymer of this example was used in most of the following examples. A wide range of copolymers based on different monomers and/or monomer ratios can be prepared by this procedure by adjusting the stoichiometry and/or using different monomers.

Synthesis of poly(HPMA-co-MA-GFLG-OH)

Poly(HPMA-co-MA-GFLG-ONp) (700 mg) was added in portions to 0.1 M NaOH solution (11.3 ml) at 0°C. The yellow reaction mixture was stirred at 0°C for 0.5 h, then at room temperature for 4 h. Half of the solution was acidified with 0.1 M HCl to pH = 6. The aqueous phase was extracted with ethyl acetate to remove excess p-nitrophenol. The aqueous phase was lyophilized to obtain poly(HPMA-co-MA-GFLG-OH) as a colorless solid (360 mg).

Synthesis of poly(HPMA-co-MA-GG-ONp) o polymer -G-NH. JC ONp

A mixture of hydroxypropyl methacrylamide (HPMA, 82.5 g), N-methacryl-gly-gly p-nitrophenyl ether (MA-GG-ONp, 16.8 g), AIBN (5.7 g) and acetone (875 g) was bubbled through argon for 90 min. The resulting reaction mixture was stirred at 50°C for 48 hours, then cooled to room temperature. The desired product was purified by trituration with acetone, then dried in vacuo to obtain 69.3 g of poly(HPMA-co-MA-GG-ONp) as a white solid. The structure was confirmed by 1H NMR and the product was shown to be free of significant impurities (eg p-nitrophenol). The amount of p-nitrophenyl ether per gram of polymer can be determined by UV absorption. A wide range of copolymers based on different monomers and/or monomer ratios can be prepared by this procedure by adjusting the stoichiometry and/or using different monomers.

Synthesis of poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ N(Me)BOC) and general procedure A o polymer -GFL-NH N O

Oh Me t-Bu

A solution of poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6 ml) and H ₂ O (10 ml) was added dropwise over a 15-minute interval to the tert-butyl solution -N-(2-aminoethyl)-Nmethylcarbamate (0.20 g, 1.15 mmol) in water (20 ml) at 0°C. The reaction mixture was stirred at 0°C for 15 minutes, then warmed to room temperature and stirred for 12 hours. The solvents were evaporated under reduced pressure. The resulting residue was dissolved in water (50 ml), the pH was adjusted to approximately 8.0 with 0.1 M NaOH solution. The solution was filtered through a VacuCap filter, then purified with TFF (10K). The polymer containing solution was washed (as part of the TFF process) with 25 mM NaCl solution (800 ml) to remove p-nitrophenol, the pH of the solution was adjusted to approximately 4 with 0.1 M HCl and then washed (as part of the TFF process) with water (400 ml). The polymer solution was lyophilized to separate poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ N(Me)BOC) compound as a pale yellow solid (720 mg, 71%).

Synthesis of Fmoc-Phe-Gly-NH- (CH ₂ ) ₆ NH-Boc

To a solution of Fmoc-Phe-Gly-OH (0.66 g) in anhydrous THF (20 ml) at 0°C under N ₂ atmosphere was added N,N'-dicyclohexylcarbodiimide (0.307 g) and 1-hydroxybenzotriazole hydrate (0.201 g) . After stirring for 15 minutes, N-Boc-1,6-diaminohexane (0.322 g) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. Solids were collected by filtration and washed with EtOAc. The filtrate and washings were then concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (0 to 10% MeOH in CH ₂ Cl ₂ ) to give Fmoc-Phe-Gly-NH(CH ₂ ) ₆ NH-Boc as a white solid (0.9 g) . Synthesis of Fmoc-Phe-Gly-NH (CH ₂ ) ₆ NH ₂ -TFA

- 17 045828

Fmoc-Phe-Gly-NH-(CH ₂ )6NH-Boc (0.7 g) was dissolved in CH2C12 (4 ml) at 0°C under N2 atmosphere and then trifluoroacetic acid (TFA) (4 ml) was added. The reaction mixture was allowed to warm to room temperature and stirred for 2 hours under N2 atmosphere. The solvents were removed under reduced pressure and the residue was dried under high vacuum to obtain 0.71 g of Fmoc-Phe-Gly-NH(CH ₂ )6-NH ₂ -TFA. This crude product was used for preparation without further purification.

Synthesis of Fmoc-Phe-Gly-NH (CH ₂ ) ₆ NH-CO-fumagillol

At 0°C, to a solution of the compound Fmoc-Phe-Gly-NH (CH ₂ ) ₆ -NH ₂ -TFA (0.71 g) in anhydrous CH ₂ Cl ₂ (20 ml) and DMF (1 ml) under N2 atmosphere was added nitrophenyl fumagyl-6-yl carbonate (0.536 g). Diisopropylethylamine (DIPEA) (0.74 ml) was then added. The reaction mixture was allowed to warm to room temperature and then stirred overnight at the same temperature. The solvents were removed under reduced pressure and the resulting residue was dissolved in EtOAc (70 ml). The EtOAc solution was washed with water and brine. The ethyl acetate solution was then dried over MgSO ₄ , filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (0 to 10% MeOH in CH ₂ Cl ₂ ), yielding Fmoc-Phe-Gly-NH-(CH ₂ ) ₆ NH-COfumagillol as an off-white solid (0.81 g) .

Synthesis of H-Phe-Gly-NH (CH ₂ ) ₆ NH-CO-fumagillol

At 0°C, to a solution of Fmoc-Phe-Gly-NH (CH ₂ ) ₆ NH-CO-fumagillol (0.80 g) in anhydrous CH ₂ Cl ₂ (20 ml) and under N2 atmosphere, DBU (0.15 G). The reaction mixture was left to warm to room temperature. The solvent was removed under reduced pressure and the resulting residue was purified by flash column chromatography (0 to 10% MeOH in CH ₂ Cl ₂ ), yielding H-Phe-GlyNH(CH ₂ ) ₆ NH-CO-fumagillol as a pale yellow resin (0.45 g, 76%).

Synthesis of poly[HPMA-co-MA-GGFG-N-(6-aminohexyl)carbamoylfumagillol] and general procedure B

H-Phe-Gly-NH (CH ₂ ) ₆ NHCO- fumagillol (0.45 g) in anhydrous DMF (5 ml) followed by the addition of diisopropylethylamine (DIPEA) (0.25 ml). The reaction mixture was allowed to warm to room temperature and after stirring overnight under N ₂ , 3-amino-1-propanol (0.032 g) was added. The mixture was stirred for an additional hour. The solvent was removed under reduced pressure and the resulting residue was dissolved in 300 ml of distilled water and extracted with EtOAc (4 times). A saturated aqueous NaCl solution (50 mL) was used to facilitate phase separation. Traces of EtOAc were removed from the polymer solution by stirring under a stream of nitrogen gas. The polymer solution was filtered through a vacu cap filter (pH=5.56), concentrated to 30 ml with TFF with 10K capsule and washed with water (700 ml) with TFF. The polymer was then lyophilized to obtain the desired polymer-poly[HPMA-co-MA-GGFG-N-(6aminohexyl)carbamoylfumagillol] conjugate as a light pink foam (0.685 g). The spiroepoxide content was measured by reaction with 2-mercaptopyrimidine and determined to be 0.4 mmol/g.

Synthesis of poly[HPMA-co-MA-GGLG-N-(6-aminohexyl)carbamoylfumagillol]

Using standard methods, the dipeptide H-Leu-Gly-NH(CH ₂ ) ₆ NHCO-Fum was prepared and combined with poly(HPMA-co-MA-GG-ONp) using the general procedure B.

Synthesis of poly[HPMA-co-MA-GGVG-N-(6-aminohexyl)carbamoylfumagillolα]

Using standard methods, the dipeptide H-Val-Gly-NH(CH ₂ ) ₆ NHCO-Fum was prepared and combined with poly(HPMA-co-MA-GG-ONp) using the general procedure B.

Synthesis of poly[HPMA-co-MA-GGGG-N-(6-aminohexyl)carbamoylfumagillolα]

- 18 045828

Using standard methods, the dipeptide H-Gly-Gly-NH(CH ₂ )6NHCO-Fum was prepared and combined with poly(HPMA-co-MA-GG-ONp) using the general procedure B.

Synthesis of poly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexyl)carbamoylfumagillol] through the preparation of poly[HPMA-co-MA-GFLG-N-(cis-4-aminohexylamine HCl)] o o V ^x sn ₃ polymer -GFL-NH. /\| CH ₃

NH—/ NH^O OME _-/ '-- T CH ₃

General Procedure C using cis-1,4-diaminocyclohexane (0.914 g) and poly(HPMA-co-MAGFLG-ONp) (1.5 g) produced poly[HPMA-co-MA-GFLG-N-(cis-4 -aminocyclohexylamine-HCl)] as an off-white solid (1.08 g).

Synthesis was carried out by general procedure F using poly[HPMA-co-MA-GFLG-N-(cis-4aminocyclohexylamine-HCl)] (0.98 g), p-nitrophenyl fumagyl-6-yl carbonate (0.465 g) and DIEA (0.268 g ) in 16 ml DMF. The solvent was evaporated and the solution was diluted with water. The aqueous phase (total 500 ml) was extracted with ethyl acetate (total 80 ml) and purified with a TFF filter using an additional 350 ml of water. The remainder was diluted with water, extracted with ethyl acetate and lyophilized to give poly[HPMA-co-MA-GFLG-N-(cis-4-aminocyclohexyl)carbamoylfumagillol] as a light pink solid (0.79 g).

^1H NMR (DMSO- _d6 ): δ 7.90-8.35 (m, 4H, amide-NH), 7.0-7.70 (m, 25H, phenylalanine and amide-NH), 5.26 (m, H-5-Fum), 5.18 (bt, alkene-Fum), 4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H, alpha- phenylalanine proton), 4.10-4.30 (m, 1H, alpha leucine proton), 3.40-3.80 (m, 21H), 3.26 (s, 3H, OMe-Fum), 2, 80-3.10 (m, 31H), 2.17 (m, 2H, allyl-Fum), 0.37-2.0 [m, 166H {1.69 (s, 3H, Fum-Me), 1 .59 (s, 3H, Fum-Me), 1.07 (s, 3H, Fum-Me)}].

Synthesis of poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ NH ₂ HCl) and general procedure for the reaction of diamines with poly(HPMA-co-MA-GFLG-ONp) / ^NH polymer -GFL-ΝΙτ NH ₂

The pH of a solution of ethylenediamine (0.33 g, 5.49 mmol) in water (20 ml) was adjusted to pH 9.1 by adding 37% aqueous HCl (17-18 drops). The solution was cooled in an ice bath and poly(HPMA-co-MA-GFLG-ONp) (1.03 g) in DMF (6 ml) was added dropwise over 20 min while maintaining the temperature below 4°C. The solution was stirred for 20 minutes at 4°C, 50 minutes at room temperature, obtaining a lemon-yellow solution, pH 8.1. The solution was evaporated at 40°C. H ₂ O (3x10 ml) was added and evaporated. The product was diluted with water (60 ml), the pH of the solution was adjusted with NaOH to pH 8.0. The solution was filtered through a VacuCap filter and purified with TFF as follows. The polymer solution was first washed with 25 mM NaCl solution (800 mL) to remove p-nitrophenol. The solution was washed with water (400 ml), then the pH of the solution was adjusted to pH 4 using 0.1 M HCl. The remaining TFF was collected and the filter was washed with 2x10 ml water. The remainder and the washes were combined to obtain a polymer solution, which was lyophilized to isolate the poly compound (HPMA-CO-MA-GFLG-NHCH2CH2NH2 HCl) as a pale yellow solid (0.71 g, 72%).

Synthesis of N-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol and general procedure D o

U ⁴ f CH ₃

I ^dc

ΉΗ .About OMe A

BUT^

Ξ II CH ₃ ⁰ H ₃ C CH ₃

A solution of p-nitrophenylfumagill-6-ylcarbonate (400 mg, 0.89 mmol) and (R)-2-amino-3-methyl-1butanol (280 mg, 2.71 mmol) was stirred in ethanol (10 ml) at room temperature for 12 hours. The yellow solution was concentrated and the residue was purified by flash chromatography (methanol/methylene chloride) to give N-[(2R)-1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol (340 mg, 0.83 mmol) as a colorless oil.

Synthesis of N-(6-hydroxyhexyl)carbamoylfumagillol ^NH .0 OMe x g o

The reaction was carried out according to general procedure D using p-nitrophenyl fumagyl-6-icarbonate

- 19 045828 (150 mg) in ethanol (10 ml) and 6-aminohexanol (48 mg). The product was isolated as a colorless oil (110 mg, 78%).

Synthesis of N-[1-(hydroxymethyl)cyclopentyl]carbamoylfumagillol

The reaction was carried out according to General Procedure D using p-nitrophenyl fumagyl-6-yl carbonate (100 mg) in ethanol (3 ml) and THF (1 ml) and cycloleucinol (52 mg), thereby obtaining N-[1(hydroxymethyl)cyclopentyl] carbamoylfumagillol in the form of oil (50 mg).

Synthesis of N-(1-hydroxy-2-methylpropan-2-yl)carbamoylfumagillol

The reaction was carried out according to general procedure D using p-nitrophenyl fumagyl-6-yl carbonate (100 mg) in ethanol (3 ml) and THF (2 ml) and 2-amino-2-methylpropanol (40 mg), thereby obtaining N-( 1Hydroxy-2-methylpropan-2-yl)carbamoylfumagillol as an oil (37 mg).

Synthesis of fumagill-6-yl-(2S)-2-(hydroxymethyl)pyrrolidine-1-carboxylate

The synthesis was carried out according to General Procedure D. S-Prolinol (68 mg, 0.67 mmol) was reacted with pnitrophenyl fumagill-6-yl carbonate (150 mg, 0.335 mmol) in ethanol (4 ml). The product was purified by flash chromatography (methanol/methylene chloride) to give fumagyl-6-yl-(2S)-2(hydroxymethyl)pyrrolidine-1-carboxylate as a white foam (81 mg, 63%).

Synthesis of N-(6-aminohexyl)carbamoylfumagillol

A solution of 1,6-diaminohexane (0.13 g) in methanol (8 ml) was cooled to 0° C. and p-nitrophenyl fumagyl-6-yl carbonate (0.13 g) in methanol (2 ml) was added dropwise. The solvent was removed to approximately 2 ml by rotary evaporation. Ethyl acetate was added and the organic phase was washed with water, 0.1 N NaOH, water, brine and dried over sodium sulfate. The solvent was evaporated and the residue was dissolved in ethanol (15 ml). DL-tartaric acid (16 mg) was added, the solution was kept overnight and then evaporated to approximately 0.5 ml. Ether was added to give a white solid. The solid was collected by filtration, washed with ether and dried to give N-(6-aminohexyl)carbamoylfumagillol tartrate salt (74 mg).

Synthesis of fumagill-6-yl-[trans-(4-aminocyclohexyl)]carbamate

To a solution of trans-1,4-diaminocyclohexane (1.3 g) in methanol (80 ml) at 0-5°C, a solution of fumagillol-6-yl-4-nitrophenylcarbonate (1.0 g) in methanol was added over 30 minutes (20 ml) and CH ₂ Cl ₂ (20 ml) and then stirred for 30 minutes. After concentrating to 20 ml on a rotary evaporator and diluting with ethyl acetate (75 ml), the organic layer was washed with water (30 ml), 0.1 N NaOH (30 ml), water and brine (30 ml), dried (MgSO ₄ ) and concentrated under reduced pressure to obtain 0.78 g of solid. It was dissolved in ethanol (80 ml) and DLtartaric acid (127 mg) was added. After 1 hour, a solution formed and was kept overnight before concentration under reduced pressure to remove virtually all the ethanol. MTBE (100 ml) was added and concentrated followed by MTBE (30 ml). The solids were collected by filtration and washed with MTBE (2x10 ml) and dried in vacuo to give fumagill-6-yl-[trans-(4-aminocyclohexyl)]carbamate hemitartrate (0.73 g); m.p. 180-185°C. Synthesis of poly[HPMA-co-MA-GFLG-NH^¾^¾^^]

The synthesis was carried out according to General Procedure C using 1,6-diaminohexane (621 mg, 5.36 mmol) and poly(HPMA-co-MA-GFLG-ONp) (1.0 g). The crude product was purified with TFF (5K) using aqueous NaCl (25 mM) and then acidified to pH 4.0 with 0.1 M HCl and further purified with TFF with water to give

- 20 045828 this poly[HPMA-co-MA-GFLG-NH(CH2)6NH2 HCl] as an off-white solid (860 mg).

Synthesis of p-nitrophenyl-N-[(2R)-1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol-6-ylcarbonate and general procedure E

To a solution of N-[(2R)1-hydroxy-2-methylbutan-2-yl]carbamoylfumagillol alcohol (1.11 g) in methylene chloride at 0°C under N2 atmosphere was added DMAP (660 mg, 5.40 mmol) followed by adding p-nitrophenyl chloroformate (810 mg) in portions.

The reaction mixture was stirred at 0°C for 1 hour. The solvent was evaporated and the resulting residue was dissolved in EtOAc and washed with water, brine and dried (Na ₂ SO ₄ ). Evaporation of ethyl acetate gave the crude product, which was purified by flash chromatography (silica, eluting with 100% hexane and then 2-30% EtOAc). Fractions containing pure product were combined and evaporated, releasing N-(2R)-1-(p-nitrophenylcarbonylhydroxy-2-methylbutan-2yl]carbamoylfumagillol (1.25 g, 80%) as a white solid.

Synthesis of N-[1-(p-nitrophenoxycarbonylhydroxymethyl)-2-methylpropan-2-yl]carbamoylfumagillol

Using General Procedure E, dimethyl alcohol (60 mg), p-nitrophenyl fumagyl-6yl carbonate (46 mg) and DMAP (37 mg) were reacted in methylene chloride (8 ml). The reaction mixture was diluted with ethyl acetate and washed with water (3 times) and then with brine. The organic phase was dried (Na ₂ SO ₄ ) and evaporated to a yellow foam (87 mg), which was used without further purification.

Synthesis of N-[1-(p-nitrophenoxycarbonylhydroxymethyl)cyclopentyl]carbamoylfumagillol

In General Procedure E, N-[1-(hydroxymethyl)cyclopentyl]carbamoyl fumagillol (product from Example 14, 74 mg), p-nitrophenyl chloroformate (53 mg) and DMAP (43 mg) were reacted in methylene chloride (5 ml). After extraction treatment, N-[1-(p-nitrophenoxyarbonylhydroxymethyl)cyclopentyl]carbamoylfumagillol (100 mg) was used without further purification.

Synthesis of poly[HPMA-co-MA-GFLG-NH( _CH2 ) _6NH -carbamoyl-[1-hydroxy-3-methylbutan-2yl]carbamoylfumagillol] and general procedure F

To a solution of polymer (400 mg) and p-nitrophenyl-N-[(2R)-1-hydroxy-3-methylbutan-2yl]carbamoyl fumagill-6-yl carbonate (240 mg) in DMF (8 ml) at 0°C dropwise DIEA (0.11 g) was added.

The solution was stirred at 0°C for one hour and left to warm to room temperature. After 3 days, the solvent was evaporated and water (80 ml) was added. The aqueous phase was extracted with ethyl acetate (total 500 ml) until the absence of starting carbonate was detected by MS. The aqueous phase was purified with TFF (10K) and the retained portion was lyophilized to give the conjugate as a white solid (380 mg, 77%).

1H NMR (DMCO-d ₆ ): δ 8.25 (b.s., 2H, amide-NH), 8.0 (b.s., 1H, amide-NH), 7.70 (b.s., 2H, amide-NH), 7.10-7.30 (m, 15H, phenylalanine and amide-NH), 7.10 (brt, 1H, NH-Fum), 6.92 (brd, 1H, NH -Fum), 5.26 (m, H-5-Fum), 5.18 (bt, alkene-Fum), 4.50-4.80 (m, 1H, phenylalanine alpha proton), 4, 0-4.21 (m, 1H, alpha proton leucine), 3.50-3.84 (m, 19H), 3.29 (s, 3H, OMe-Fum), 2.80-3.10 (m , 28H), 2.51 (d, 1H, J=4.4 Hz, H-2-Fum), 2.19 (m, 2H, allyl-Fum), 0.82-1.92 (m, 131H ), 1.84 (m, 2H, Fum, 1.72 (s, 3H, Fum-Me), 1.60 (s, 3H, Fum-Me), 1.09 (s, 3H, Fum-Me) , 0.84 (dd, 6H, Fum-isopropyl).

Synthesis of poly[HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoylfumagillol]

- 21 045828

General procedure F was used to react poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ NH ₂ -HCl) (200 mg), p-nitrophenyl fumagyl-6-yl carbonate (100 mg) and DIEA (57 mg) in DMF ( 10 ml). The product was purified with TFF (10K) with water and lyophilized to give the conjugate as a pale yellow solid (160 mg).

Synthesis of poly[HPMA-co-MA-GFLG-N(Me)-(2-methylaminoethyl)carbamoylfumagillol]

General procedure F was used to react poly(HPMA-co-MA-GFLG-N(Me) CH ₂ CH ₂ NHMe-HCl) (200 mg), p-nitrophenyl fumagyl-6-yl carbonate (100 mg) and DIEA (57 mg) in DMF (5 ml). The product was purified with TFF (10K) with water and lyophilized to give the conjugate as an off-white solid (180 mg).

Synthesis of poly(HPMA-co-MA-GFLG-N-(2-aminoethyl)carbamoyldihydrofumagillol

Using General Procedure F, poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ NH ₂ HCl) (200 mg), p-nitrophenyldihydrofumagyl-6-ylcarbonate (200 mg) and DIEA (57 mg) were reacted in DMF ( 10 ml). The product was purified by TFF (10K) with water (150 ml) and lyophilized to give poly(HPMA-co-MA-GFLG-N(2-aminoethyl)carbamoyldihydrofumagillol as a pale yellow solid (160 mg).

Synthesis of poly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagillol]

General procedure F was used to react poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ CH ₂ NH ₂ HCl) (220 mg), p-nitrophenyl fumagyl-6-yl carbonate (110 mg) and DIEA (63 mg) in DMF (6 ml). The solvent was evaporated and the resulting solution was diluted with water. The aqueous phase was extracted with ethyl acetate and purified with TFF using 350 ml of water. The retained portion was lyophilized to obtain poly[HPMA-co-MA-GFLG-N-(3-aminopropyl)carbamoylfumagillol] as a light pink powder (200 mg).

Synthesis of poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)carbamoylfumagillol]

Using General Procedure F, poly[HPMA-co-MA-GFLG-N-(trans-4 aminocyclohexylamine-HCl)] (1.0 g), p-nitrophenyl fumagyl-6-yl carbonate (0.48 g) and DIEA ( 0.27 g) in DMF (25 ml). The solvent was evaporated and the solution was diluted with water. The aqueous phase (300 ml) was extracted with ethyl acetate (total 700 ml) and purified with TFF using an additional 350 ml of water. The remainder was lyophilized to yield poly[HPMA-co-MA-GFLG-N-(4aminocyclohexyl)carbamoylfumagillol] as a light pink solid (0.9 g).

^1H NMR (DMSO-d6): δ 8.10-8.35 (m, 3H, amide-NH), 7.90-8.10 (m, amide-NH), 7.05-7.32 ( m, 22H, amideNH) 5.27 (m, H-5-Fum), 5.18 (bt, alkene-Fum), 4.60-4.90 (m, 14H), 4.50-4 .60 (m, 1H, phenylalanine alpha proton), 4.10-4.30 (m, 1H, leucine alpha proton), 3.40-3.80 (m, 21H), 3.27 (s, 3H, OMe-Fum), 2.803.20 (m, 33H), 2.56 (d, 1H, H = 3.90 Hz, H-2-Fum), 2.18 (m, 2H, allyl-Fum) , 0.37-2.0 [m, 147H), {1.70 (s, 3H, Fum-Me), 1.60 (s, 3H, Fum-Me), 1.07 (s, 3H, Fum -Me).

Synthesis of poly[HPMA-co-MA-GFLG-N-(trans-4-aminocyclohexyl)carbamoylfumagillol]

General Procedure F was used to react poly[HPMA-co-MA-GFLG-N-(trans-4aminocyclohexylamine-HCl)] (1.0 g), p-nitrophenyl fumagyl-6-yl carbonate (0.48 g) and DIEA (0 .27 g) in 25 ml DMF. The solvent was evaporated and the solution was diluted with water. The aqueous phase (300 ml) was extracted with ethyl acetate (total 700 ml) and purified with TFF using an additional 350 ml of water. The precipitate was lyophilized to yield poly[HPMA-co-MA-GFLG-N-(3-aminohexyl)carbamoylfumagillol] as a light pink solid (0.9 g).

- 22 045828

1H NMR (DMSO-di): δ 7.90-8.35 (m, 4H, amide-NH), 7.0-7.70 (m, 25H, phenylalanine and amide-NH), 5.26 (m , H-5-Fum), 5.18 (bt, alkene-Fum), 4.60-4.90 (m, 14H), 4.50-4.60 (m, 1H, phenylalanine alpha proton ), 4.10-4.30 (m, 1H, leucine alpha proton), 3.40-3.80 (m, 21H), 3.26 (s, 3H, OMe-Fum), 2.80- 3.10 (m, 31H), 2.17 (m, 2H, allyl-Fum), 0.37-2.0 (m, 166H), 1.69 (s, 3H, Fum-Me), 1, 59 (s, 3H, Fum-Me), 1.07 (s, 3H, Fum-Me).

Synthesis of poly[HPMA-co-MA-GFLG-N-[2-(4-aminophenyl)ethyl]carbamoylfumagillol] polymer -GFL-NH.

NH.O

To a suspension of poly[HPMA-co-MA-GFLG-OH] (200 mg), N-[2-(4-aminophenyl)ethyl]carbamoylfumagillol] (100 mg) and DIEA (75 mg) in DMF (6 ml) at 0°C EDCI was added in portions (total 44 mg). The solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated, the residue was suspended in water and the suspension was extracted with EtOAc (7 times, total 250 ml). The aqueous phase was purified with TFF (10K) using water (350 ml). The retained portion was lyophilized to give the polymer as a white fluffy solid (170 mg).

Synthesis of poly[HPMA-CO-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoylfumαgillol] polymer-GFL-NH .NH O OM οι

Conc. HCl solution until the pH value was 9.01 (measured with a pH meter). Poly(HPMA-co-MA-GFLG-ONp) (1.0 g, 0.534 mmol) in DMF (6 ml) and H ₂ O (10 ml) was added dropwise to the amine containing solution over a period of 15 min and the reaction mixture was stirred at 0°C for 15 minutes. The reaction mixture was then left to warm to room temperature and stirred for 2 hours. The pH of the solution was measured and adjusted to 8.15. The reaction mixture was diluted with distilled water (300 ml) and filtered through a VacuCap filter, the reaction flask was washed with water (100 ml). The polymer solution was concentrated to 40 ml TFF (10K) and washed with 25 mM NaCl (800 ml) to remove p-nitrophenol, the pH was then adjusted to 4 with 0.1 M HCl and finally washed with water (400 ml). The pure polymer solution was lyophilized to isolate poly[HPMA-CO-MA-GFLG-NH-2-[2-(2-aminoethoxy)ethoxy]ethylamine·HCl] as a pink solid (800 mg, 78%).

To a mixture of p-nitrophenylfumagill-6-ylcarbonate (93 mg, 0.208 mmol) and poly[HPMA-co-MA-GFLGN-2-[(2-(2-aminoethoxy)]ethoxy)ethylamine HCl] (200 mg, 0.104 mmol) in anhydrous DMF (5 ml) at 0°C under N2 atmosphere was added DIEA (57 mg, 0.416 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours. The solvent was removed under reduced pressure and the resulting residue was suspended in water (30 ml) and extracted with EtOAc (the aqueous and organic phases of the resulting emulsion were separated using a centrifuge) to remove excess pnitrophenyl fumagyl- 6-ylcarbonate and p-nitrophenol. Nitrogen was passed through the aqueous solution to remove traces of EtOAc and the solution was purified using TFF (5K) and washed with water (150 ml) to remove DIEA hydrochloride. The polymer solution was lyophilized to obtain the desired polymer conjugate poly[HPMA-CO-MA-GFLG-N-2-[2-(2-aminoethoxy)ethoxyethyl]carbamoylfumagillol] (220 mg, 95%) as an off-white solid .

Synthesis of poly[HPMA-co-MA-GFLG-NH-(6-aminodecyl)carbamoylfumagillol] polymer -GFL-NH.

,ΝΗ .О

To a mixture of p-nitrophenylfumagill-6-ylcarbonate (300 mg, 0.67 mmol) and poly[HPMA-co-MA-GFLGN-10-[decylamine HCl] (300 mg, 0.15 mmol, prepared similarly to the method of example 33 , except that 1,10-diaminodecane was used as the amine) DIEA (83 mg, 0.64 mmol) was added to anhydrous DMF (6 mL) at 0°C under N2. The reaction mixture was allowed to warm to room temperature and stirred for 12 hours. The solvent was removed under reduced pressure and the resulting residue was suspended in water (30 ml) and extracted with EtOAc (the aqueous and organic phases of the resulting emulsion were separated by centrifuge) to remove excess pnitrophenyl fumagyl- 6-ylcarbonate and p-nitrophenol. Nitrogen was passed through the aqueous solution to remove traces of EtOAc. The crude aqueous solution was purified using TFF (10K) with washing with water (150 ml) to remove DIEA hydrochloride. The polymer solution was lyophilized to obtain the desired polymer conjugate poly[HPMA-co-MA-GFLG-NH-(10-aminodecyl)carbamoylfumagyl- 23 045828 lol] (300 mg, 87%) as an off-white solid. Synthesis of N-(2-acetamidoethyl)carbamoylfumagillol

N(2-aminoethyl)acetamide (0.132 ml) was added to a solution of p-nitrophenylfumagyl-6-ylcarbonate (200 mg) in ethanol (5 ml) at 0°C. The solution was stirred at 0°C for 1 hour and overnight at room temperature. The reaction mixture was diluted with ethyl acetate and washed with water. The aqueous phase was again extracted with ethyl acetate and the combined organic phases were dried (MgSO ₄ ). The crude product is purified by flash chromatography. The product was a yellow solid (120 mg).

Synthesis of a compound of the following formula

DIEA was added to a solution of poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ NH ₂ HCl) (200 mg) and N-(5-carboxypentyl)carbamoylfumagillol (96 mg) in DMF (6 ml) at 0°C (104 mg) followed by the addition of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (42 mg). The solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated and the residue was dissolved in water (50 ml) and extracted with ethyl acetate (200 ml). The aqueous phase was purified with TFF with water (450 ml). The retained portion was lyophilized to give the polymer (200 mg) as a pale yellow solid.

Synthesis of a compound of the following formula

DIEA (118 mg) was added to a solution of poly[HPMA-co-MA-GFLG-N(CH ₂ ) ₆ NH ₂ HCl] (216 mg), 2-carboxyethylcarbamoylfumagillol (91 mg) in DMF (8 ml) at 0°C ) followed by the addition of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (88 mg). The solution was allowed to warm to room temperature and stirred overnight. The solvent was evaporated and the residue was dissolved in water (50 ml) and extracted with ethyl acetate (200 ml). The aqueous phase was purified with TFF (10K) with water (1 L). The retained portion was lyophilized to give the polymer (170 mg) as a pale yellow solid.

Synthesis of a compound of the following formula

Using General Procedure F, poly(HPMA-co-MA-GFLG-NHCH ₂ CH ₂ CH ₂ NH ₂ HCl) (220 mg) and carbonate (Example 24, 100 mg) were reacted in DMF (6 ml) with DIEA (63 mg). The reaction mixture was extracted with ethyl acetate. After purification of TFF (10K) using water and lyophilization, the product was isolated as a light pink powder (140 mg).

BocNHCH ₂ CH ₂ N(Me)CH ₂ C(O)NHC(O) ₂ -fumagill-6-yl (alkylation of N-BOC-N'-methylethylenediamine with chloroacetylcarbamoylfumagillol)

A solution of TNP-470 (0.2 g) and DIEA (0.105 g) in DMF (3 ml) was cooled to 0°C. A solution of tert-butyl-N-[2-(methylamino)ethyl]carbamate (0.105 g) in DMF (3 ml) was added and the mixture was stirred for 3 hours at 0° C. and then overnight. The reaction mixture was diluted with ethyl acetate and extracted with water. The aqueous phase was again extracted with ethyl acetate and the combined organic phases were extracted with brine, dried (MgSO ₄ ) and evaporated to give an oil. Purification by chromatography on silica gel (methanol/methylene chloride) and evaporation of product fractions gave BocNHCH ₂ CH ₂ N(Me) CH ₂ C (O)NHC (O) 2-fumagill-6-yl as a white foam (0.16 g, 60 %).

- 24 045828

Reaction of tert-butyl-N-[2-aminoethyl]carbamate with chloroacetylcarbamoylfumagillol

An aliquot of 1 M solution of Boc-ethylenediamine in DMF in an amount of 30 μl was added to DMF (270 μl). The solution was cooled to 0°C and a solution of TNP-470 (48 mg) in DMF (600 μL) was added dropwise over 2 min. The reaction was monitored using LC/MS. The highest amount of alkylation product required was 34%. Carbamoyl fumagillol was also obtained. The ratio of the target product to carbamoylfumagillol was from 1.0 to 0.4. An attempt to isolate the target product led to the isolation of hydantoin and fumagillol. Thus, the target product cannot be isolated due to the rate of its decomposition. Thus, TNP-470 cannot be alkylated according to the described method.

In vivo testing of DIO C57B16 mice - changes in weight, food intake, body composition

Male C57B16 mice (N=6), thirteen weeks old, with an average weight of 34 g, were fed ad libitum with a high fat diet, TD.06414, containing 60% of calories from fat (Harlan food). On day 1 of the study, animals were randomized into groups such that the average weight of the mouse in each group was 33.9 g. Mice were administered either phosphate buffered saline (vehicle), TNP470, or compound 16 (spinal, subcutaneous injection). Administration was continued for 31 days at the doses and schedule shown in the table below. Animals were weighed every day. Feed intake was measured weekly. On day 33, macroscopic pathology was performed to determine body habitus.

In fig. 1 compares body weight loss in an obese DIO mouse following administration of a fumagillol conjugate of a compound of the present invention (compound 16) or TNP-470 (a synthetic fumagillin analogue) at various dosages/schedules, as shown in Table 1. 2.

table 2

Body mass Day 33 Dose mg/kg/day Dose* Administration scheme Medicine 39.5 0 0 Qod Filler 34.1 0.5 1, 12 Qod TNP-470 30.4 0.5 0.18 Qod Compound 16 27.4 1.5 0.56 Q4d Compound 16

* Daily dose of fumagillol in µmol/kg

The results in Fig. 1 show a 16% increase in weight in the vehicle control group and a 19% decrease in weight when compound 16 was administered according to the q4d administration regimen. Administration of compound 16 causes both therapeutic and prophylactic effects. In particular, compound 16 induces or increases body weight loss and also prevents weight gain. Compound 16 is superior to TNP-470 in terms of weight loss. Compound 16 is superior to TNP-470 in that fumagillol dosages are reduced.

Table 3 compares body fat composition in an obese DIO mouse following treatment with Compound 16 or TNP-470 at various doses/schedules described in the context. The analysis was performed on day 33 of macroscopic pathology. Total fat content as a percentage of body weight in the vehicle group was 13.2%, whereas total fat content in the Compound 16-treated groups was 8.2% (1 mg/kg, qod) or 5.6% (6 mg/kg q4d).

- 25 045828

Table 3. Weight in grams, group averages

Weight body Total fat Epidermal fat Inguinal fat Retroperitoneal fat Liver Filler 39.6 5.24 2.21 1.93 1.10 1.46 TNP, qod 34.1 3.55 1.54 1.21 0.80 1.15 Conn. 16 1 mg/kg qod 30.4 2.52 1.18 0.92 0.42 1.0 Conn. 16 6 mg/kg q4d 27.4 1.55 0.78 0.53 0.23 1.17

In fig. Figure 2 compares the average daily food intake of a DIO mouse following treatment with Compound 16 or TNP-470 at various dosages/schedules. The results in Figure 2 show a reduction in feed intake after treatment with Compound 16, and Compound 16 caused a greater reduction in feed intake than TNP-470.

In fig. 3 shows a comparison of body composition (fat content versus body weight) of an obese DIO mouse following treatment with Compound 16 or TNP-470 at various dosages/schedules. The results in Fig. 3 show that quantitative weight loss directly correlates with fat loss.

In vivo testing of DIO C57B16 mice -- changes in body weight, food intake, glucose tolerance and body composition response to dose

Fifteen-week-old male C57B16 mice (N=6) with an average weight of 42 g were fed ad libitum with the high-fat diet TD.06414, which contained 60% calories from fat (Harlan diet). On day 1 of the study, animals were administered either phosphate buffered solution (vehicle) or compound 16 at various doses (spinal, subcutaneous injection). Treatment was continued for 29 days at the doses and schedules shown in the table below. Animals were weighed every day. Feed intake was measured weekly. On day 24 (mice were most recently treated with compound 16, on day 21), overnight fasted mice were subjected to intraperitoneal IP glucose tolerance testing (GTT) in the vehicle group and the four compound 16 groups each. the animal was weighed and glucose measurements were collected as a baseline in the fasted state. Each animal was administered a dose of dextrose 1 g per kilogram of body weight in the form of a 25% solution by intraperitoneal injection. Blood glucose levels were measured at 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after intraperitoneal glucose administration (blood samples were collected from the tail vein using an AlphaTRAK blood glucose monitoring system (including a meter and test strips) from Abbott Laboratories, (North Chicago, Illinois, USA) The AlphaTRAK system showed results between 20 and 750 mg/dL (1.1-41.7 mmol/L) on day 32 (mice were most recently dosed on day 29) animals that fasted for three hours were weighed, bled by cardiac puncture, and gross pathology was performed to determine body composition.Blood analysis was performed by Idexx Laboratories. Blood glucose levels were 278, 290, 265, 259, and 227 mg/dL for doses 0, 0.2, 0.6, 2.0 and 6.0 respectively.BUN was 21.8, 22.0, 19.7, 15.3 and 16.5 for doses 0, 0.2, 0. 6, 2.0 and 6.0 respectively.

In table Table 4 shows the blood glucose level as a function of time and dose of compound 16. Table. 4 shows that higher doses of compound 16 lead to lower blood glucose levels even at the lowest dose of 0.2 mg/kg; these results are shown in Fig. 4.

Table 4. Average blood glucose levels in groups

Dose Average body weight (g) Average blood glucose in mg/dl±SD mg/kg Starvation 15 minutes 30 min 60 min 90 min 120 min Filler 43, 0 226±22.9 502±30.3 621±38.4* 580±48.7* 459±54.0 297±33.7 0.2 43.4 254±5.8 505±33.9 446±20.5 456±25.9 341±15.5 241±16.8 0.6 40, 9 206±14.9 442±50.0 441±35.54 388±56.7 302±43.3 192±12.9 2.0 36, 8 198±11.6 388±20.7 361±24.3 327±26.7 244±24.0 16b±b,4 6, 0 33, 0 185±6.5 473±39.1 355±23.8 324±19.1 240±6.7 177±9.3

*one mouse in each group had a blood glucose level >750 mg/dL; the treated mouse had 750 mg/dL

In table Figure 5 shows that increasing doses of compound 16 leads to a significant increase in weight loss at doses above 0.2 mg/kg, scheme q4d. In table Figure 6 shows that doses of 2 mg/kg regimen q4d and 6 mg/kg regimen q4d were associated with significant reductions in feed intake relative to vehicle controls and that feed intake was dose-dependent. After days 9-29, weekly feed intake in the 2 mg/kg group was 90% of the vehicle group, whereas weekly feed intake in the 6 mg/kg group was 75% of the vehicle group.

Table 5. Body weights per day 32

Dose mg/kg Group body weight (d)± rms Weight change from 1 day Fumagillol* Filler - 0 45.7+1.12 + 8.3% 0.00 0.2 46.5+0.65 +10.5% 0.02 0.6 43.3+0.42 + 2.9% 0.06 2.0 38.8+0.70 -7.4% 0.18 6, 0 33.6+0.72 -20.0% 0.54

*micromoles/kg/day;

q4d dosing scheme

Table 6. Weekly feed intake, group mean

Day 1-8 Day 9-15 Day 15-22 Day 22-29 Group Feed (g) Feed (g) Feed (g) Feed (g)

Filler 22.4 23, 3 22.4 21.5 0.2 mg/kg 25, 8 22, 6 20.3 19, 7 0.6 mg/kg 19, 5 21.5 20.2 18.4 2 mg/kg 14.3 22.2 19, 9 18.5 6 mg/kg 9, 0 17.5 17.8 16, 1

In table Figure 7 shows that adipose tissue is lost preferentially over other tissues, mice in the control group had approximately 13% fat content, while mice in the 2 mg/kg q4d group and 6 mg/kg q4d group had 11 and 10% fat. % respectively.

Table 7. Average tissue masses of groups on day 32

Dose mg/kg Average body weight (g) Total average fat mass Average tissue weights (g) % Body Fat Epid. fat Groin. fat Retroabdominal Fat Liver 0, 0 45.70 5, 88 1.88 2, 65 1.35 2.00 12.9% 0.2 46, 50 6, 02 2.01 2.54 1.48 2.06 13.0% 0.6 43.32 6, 01 2.24 2.51 1.26 1.75 13.9% 2.0 38.82 4.42 1.72 1.81 0.89 1.44 11.4% 6, 0 33, 60 3.25 1.11 1.54 0.60 1.30 9.7%

The results in Fig. 5 show increased body weight loss following treatment with compound 16 at doses greater than or equal to 0.6 mg/kg using the q4d dosage regimen. Weight loss is dose dependent, with higher doses causing greater weight loss.

Results in table. 8 shows decreases in cholesterol, triglycerides, HDL, LDL and HDL/LDL ratio associated with increasing dose of Compound 16. These results are depicted in FIG. 6.

Table 8. Blood lipids on day 32

Dose mg/kg CHOLESTEROL mg/dl TRIGLYCRIDE mg/dL CHOLESTEROL HLD mg/dl LDL mg/dl Attitude HDL/LDL 0 244+12.6 140+7.7 112+4.0 24+2.2 4.8+0.4 0.2 253+8.4 158+10.9 117+2.7 24+1.1 4.9+0.2 0.6 210+3.7 116+3.1 106+1.2 20+0.3 5.4+0.1 2 149+4.7 95+8.7 92+2.6 10+0.5 9.0+0.4 6 109+2.6 81+13.6 72+1.3 8+0.5 9, 0+0.5

Values are indicated with correction ± standard deviation

Results in table. 9 show favorable changes in alkaline phosphatase, SGPT, SGOT and IBS associated with increasing dosage of compound 16.

Table 9. Blood enzyme analysis

Dose mg/kg ALKALINE PHOSPHOTASE SGPT (ALT) SGOT (AST) IBS 0 61+1.2 74+11.2 72+5.4 34+2.5 0.2 51+3.2 79+16.8 77+10.1 74+18.5 0.6 44±1.4 50±2.3 57±2.4 66±11.3 2 43±2.4 35±3.1 53±3.7 68±17.8 6 36±0.9 42±5.9 59±2.2 84±13.3

- 27 045828

Example.

Fifteen-week-old male C57B16 mice (N=6) with an average weight of 42 g were fed ad libitum with the high-fat diet TD.06414, which contained 60% calories from fat (Harlan diet). On day 1 of the study, animals were treated q4d with either phosphate buffered saline (vehicle) or compounds 16, 28, 29 or 30 at 2 mg/kg or compound 31 at 6 mg/kg (spinal, subcutaneous administration). Animals were weighed every second day. Figure 10 shows a comparison of weight loss in an obese DIO mouse after treatment for 23 days with various conjugates of the present invention. The results in Fig. 10 show that changes only in the bond (linker) lead to changes in the rate of mass loss.

Example.

Male Sprague Dawley rats (n=3), nine to ten weeks old, with an average weight of 300 g, were fed ad libitum with a standard rodent chow (PharmaServ lab diet 5001). Fig. 7 - rats were treated with compound 16 at either 100 mg/kg or 200 mg/kg (intravenous, tail vein) on days 1, 8, 15, 22 and 29. The rats were weighed periodically and a blood sample was taken on day 10, day 17, and day 24. To collect blood collections from living rats, they were anesthetized with an inhaled mixture of 4% isoflurane and 1.5% oxygen, and then blood was collected by retroorbital plexus puncture in a volume of at least 1 ml. On day 31, animals were weighed, blood was collected by cardiac puncture, and gross pathology was performed to determine body composition. Within the limits of comparisons to normal ranges and pre-dose data, clinical parameters of albumin, albumin/globulin ratio, alkalinity, phosphatase, ALT (SGPT), AST (SGOT), bicarbonate, direct bilirubin, indirect bilirubin, total bilirubin, BUN, ratio BUN/creatinine, calcium, chloride, cholesterol, SC, creatinine, globulin, glucose, phosphorus, potassium, sodium, sodium/potassium ratio, total protein were not noticeable. Apart from weight loss and other indicators indicated in the context, the animals were found to be quite normal and did not show any signs of neurotoxicity such as ataxia, disorientation, tremors or convulsions. The results in Fig. 7 show that compound 16 is tolerable at high doses and drug regimen q7d.

Example.

Male Sprague Dawley rats (n=3, mean weight 350 g) were intravenously administered a single bolus of either vehicle, Compound 1 (30 mg/kg), or Compound 16 (200 mg/kg). Blood samples were collected by saphenous vein puncture at 0, 0.25, 0.5, 1, 2, 4, 8, 24 and 48 hours. An aliquot of each sample was diluted with methanol containing propranolol as an internal standard and analyzed by LC/MS. /MS with a lower limit of quantification of 2.5 nM. When either compound 1 or compound 16 was administered, the analyte was compound 1. The small molecule half-life of compound 1 is in the range of 10-15 min; Cmax is approximately 15 µM and is present at T ₀ . For the polymer conjugate, compound 16, the released small molecule has a Cmax of approximately 0.3 μM in approximately 3 hours and a terminal half-life of 10 hours. These results are shown in FIG. 9.

Example of in vivo testing in DIO Levin rats - changes in weight, food intake, body composition, response to drug regimen, leptin levels

A study was conducted to evaluate the relative potency of the polymeric conjugate of fumagillol, compound 16, and the small molecule fumagillol derivatives, compound 1 and CKD732 (also known as beloranib and ZGN-433). CKD-732, as indicated in the context, is the hemitartrate salt of the following structure:

O

O

Compound 1 was also tested in the form of the hemitartrate salt. Test compounds were administered subcutaneously every 4 days according to the drug regimen (q4d) of the Levin-DS rat model (DIO) of diet-induced obesity. The efficacy of compound 16 was also assessed using a weekly dosing schedule (q7d). Feed effects (standard food, laboratory food 5001; 3.4 kcal/g) were included for comparison with drug effects. After three weeks of age, male rats were fed an assortment of Harlan TD.06414 pellets, 60% fat calories, 21% carbohydrate calories; 5.1 kcal/g. Before dosing, rats were divided into groups of three animals with an average body weight of 595 gm. Rats were administered phosphate buffered saline (vehicle), compound 16, compound 1, or CKD-732 (spinal, subcutaneous administration). Compound 16 was dissolved in vehicle, Compound 1 hemitartrate and CKD-732 hemitartrate were dissolved in ethanol before dilution with vehicle. All doses were administered in a volume of 5.0 ml/kg. Treatment continued for 68 days at the doses and schedules shown in Table 1 below. 10. Since the molecular weight of CKD-732 is 15% greater than the molecular weight of compound 1,

- 28 045828 the dose of CKD-732 was 1.15 mg/kg, while the dose of compound 1 was 1 mg/kg for the purpose of comparing them on a molar basis. On Day 1 of the first dose, the rats were 14 weeks old. In addition, on day 1, rats in group 2 were switched from high-fat chow to standard chow; the remaining groups maintained the high-fat diet throughout the study.

Table 10

No. groups Test connection Number of rats Route of administration Feed Dosing frequency Dose volume (ml/kg) Conc. (mg/ml) Dose (mg/kg) 1 Filler 3 Subcutaneous 60% fat q4d 5.0 0 0 2 Filler 3 Subcutaneous 0% fat H 4d 5.0 0 0 3 Conn. 16 3 Subcutaneous 60% fat q4d 5.0 0.067 0.3 4 Conn. 16 3 Subcutaneous 60% fat q4d 5.0 0.2 1.0 5 Conn. 16 3 Subcutaneous 60% fat q4d 5.0 0.6 3.0 6 Conn. 16 3 Subcutaneous 60% fat q4d 5.0 1.2 6, 0 7 Conn. 1 3 Subcutaneous 60% fat q4d 5.0 0.2 1.0 8 Conn. 1 3 Subcutaneous 60% fat q4d 5.0 0.6 3.0 9 CKD-732 3 Subcutaneous 60% fat q4d 5.0 0.23 1.15

Animals were weighed every day. Feed intake was measured weekly. Blood samples were collected approximately weekly throughout the study for biochemical assessments, including glucose and insulin determinations. On day 48, all rats that had fasted for 4 hours were subjected to an oral glucose tolerance test (OGTT). The animals were administered a dose (orally, PO) of 8 ml/kg of a 25% glucose solution (2 g/kg). On day 68, macroscopic pathology was performed to determine body habitus.

In fig. Figure 11 shows the change in body weight depending on the day of the study for each group. Significant reductions in body weight were demonstrated in groups that received both the Q4D and Q7D dosing regimens of the polymer conjugate. Treatment with compound 16 at a dose of 3 mg/kg (Q4D) or with a dose of 6 mg/kg (Q7D) showed greater body weight loss than the change with standard chow feeding. At the end of the study, Compound 16 at 3 mg/kg and the Q4D drug regimen showed a 22.1% reduction in body weight compared to vehicle control and a 6.2% lower body weight than rats. when feeding with standard food. After approximately ten weeks, treatment with compound 16 at 6 mg/kg and the Q7D regimen resulted in body weight comparable to treatment at 3 mg/kg with the Q4D regimen. Compound 1 at 1 mg/kg and CDK-732 at 1.15 mg/kg in the Q4D regimen showed a weight loss of 3.9% or 3.2% lower than vehicle body weight. A dose of compound 1 3 mg/kg when administered according to the Q4D regimen showed an 8.9% reduction in body weight compared to vehicle administration. The polymer conjugates contained approximately 1/6 of the active fumagillol derivative by weight.

In fig. Figure 12 shows final body weight at day 68 for all groups as a function of average daily fumagillol exposure. Two groups, both vehicle and standard feed, were not exposed to fumagillol. Compound 16 showed higher weight loss with significantly lower fumagillol exposure compared to Compound 1 or CKD-732 using the same dosing schedule as the polymer conjugate. All groups were dosed using the q4D dosing regimen, with the exception of compound 16, which was dosed at 6 mg/kg using the Q7D dosing schedule.

- 29 045828

Table 11. Average values of final body weight (g) of the group (n=3) of DIO rats depending on the dose of fumagillol

Weight loss by exposure 68 days after administration Group Dose, mg/kg Administration scheme mkM fumagillol kg/day Final body weight (g) HF filler 0 q4d 0 742.3 Standard feed 0 q4d 0 645.0 Conn. 16 0.3 q4d 25 727.3 Conn. 16 1.0 q4d 83 668.6 Conn. 16 3.0 q4d 250 604.7 Conn. 16 6, 0 Q7d 286 596, 7 Conn. 1 1.0 q4d 500 717.3 Conn. 1 3.0 q4d 1500 684.0 CKD-732 1.15 q4d 500 714, 0

In fig. 13 shows the decrease in serum insulin levels in male Levin DIO rats when consuming a 60% fat diet and dosing the compounds of the present invention at q4d (3 mg/kg) and q7d (6 mg/kg) dosing regimens compared to the standard intake groups. feed and filler introduction.

Table 12. Average insulin content in DIO rats (n=3) depending on time Average insulin content (ng/ml) in male Levin DIO rats

Before introduction doses Day 7 Day 15 Day 23 Day 48 Filler 1.74 2, 66 3.37 3, 15 3.00 Standard feed 1.96 1, 68 1.40 1.78 1.51 Conn. 16, 0.3 mg/kg 1.76 2.27 1.65 1, 66 1.70 Conn. 16, 0.6 mg/kg 2.95 2.09 1.38 1.72 1.91 Conn. 16.3 mg/kg 1.49 0.66 0.54 1.26 1.08 Conn. 16.6 mg, q7d 3, 06 1.96 1.00 1.11 1.32 Conn. 1.1 mg/kg 1.92 1.83 1.39 1.20 1.75 Conn. 1.3 mg/kg 2.10 1.35 1, 68 1.27 1.40 CKD-732, 1.15 mg/kg 2.58 2.08 1.91 1, 60 1.56

In table Figure 12 shows the changes in fasting insulin levels of rats for each group. All groups (except the vehicle control group) showed reduced insulin levels, demonstrating that the compounds of the present invention reduce insulin levels in an infrequent dosing regimen.

In fig. 14 shows the results of oral glucose tolerance testing (OGTT) on insulin levels in rats treated with compounds of the present invention on schedules q4d and q7d, compared to the standard chow and vehicle groups. Consumption of standard feed also resulted in decreased insulin levels. Insulin levels maintained at lower levels than when vehicle was administered in the presence of abnormally high glucose levels indicate that lower insulin levels were necessary to reduce blood glucose levels (see FIG. 15), suggesting improved performance. restored insulin sensitivity.

In fig. 15 shows reduced glucose levels over time for various treatments following oral glucose administration.

Table 13. Blood glucose levels following oral glucose tolerance testing (OGTT) in DIO rats fed a high-fat 60% fat diet.

Blood glucose (mg/ml) in male Levin DIO rats during glucose administration Group Time BEFORE OGTT 15 thirty 60 90 120 Mol. mass (g) HF filler 126 158 153 163 160 132 702 Standard feed 110 147 145 134 124 129 617 Conn. 16, 0.3 mg/kg 121 156 145 157 149 147 697 Conn. 16, 0.6 mg/kg 121 150 148 149 143 150 662

- 30 045828

Conn. 16.3 mg/kg 120 157 144 140 127 129 582 Conn. 16.6 mg, q7d 128 161 141 145 145 138 604 Conn. 1.1 mg/kg 124 156 142 152 151 142 686 Conn. 1.3 mg/kg 123 175 171 149 147 129 668 CKD-732, 1.15 mg/kg 123 174 171 158 153 144 686

In fig. 16 shows the product of glucose (mM/l) x insulin (μE/ml)/22.5 in male DIO Levin rats as an acceptable value for insulin sensitivity (Matthews et al., Diabetologia (1985) 28, 412±419 ;Pickavance et al., British Journal of Pharmacology (1999) 128, 1570±1576).

Table 14. Calculation of HOMA-ir for DIO rats

HOMA-ir (insulin sensitivity) Before dosing 15 minutes 30 min HF filler 26, 7 73, 6 24.3 Standard diet 11.7 36.5 11.7 Conn. 16, 0.3 mg/kg 14, 6 40.1 10, 8 Conn. 16.1 mg/kg 16, 4 50.1 18.1 Conn. 16.3 mg/kg 9, 2 13.7 4, 6 Conn. 16.6 mg/kg, q7d 12.0 37.2 3, 5 Conn. 1.1 mg/kg 15, 4 42.7 21.2 Conn. 1.3 mg/kg 12.2 29.2 9, 5 CKD-732, 1.15 mg/kg 13, 6 49, 0 35, 9

The adipocyte hormone leptin is a known appetite suppressor. Leptin resistance (abnormally high levels regardless of feed intake) is known to occur in feed-obese patients and animals (Levin et al., Am J. Physiol. Regul Integr Comp Physiol. 2002 Oct; 283(4):R941-8). Low leptin levels are associated with feeding hyperphagia (Sindelar et al., 1999, Enriori et al., 2006). Food intake was measured weekly. Serum leptin levels were measured on day 29 and plotted against food intake during the week containing day 29. Animals on standard chow were hyperphagic and exhibited significantly greater food intake as a function of leptin levels than when treated with compounds of the present invention. Treatment with compound 1 did not result in significant reductions in leptin levels. In fig. Figure 17 shows the weekly feed intake in grams for each group. The standard feed group showed a significant increase in feed intake after switching from high fat feed to standard feed. Hyperphagia is known to occur to maintain caloric intake levels.

Table 15. Feed consumption

Average feed intake group

Day 1-8 Day 9-15 Day 16-23 Day 24-30 Day 31-36 Day 37-43 Day 44-50 Day 51-57 Day 58-63 Day 63-68 Dose group Feed (G) Feed Food (G) Food (G) Food (G) Food (G) Food (G) Food (G) Food (G) Food (G) Filler Std. feed Conn. 16, 0.3 mg/kg Conn. 16, 0.6 mg/kg Conn. 16.3 mg/kg Conn. 16.6 mg, q7d Conn. 1.1 mg/kg Conn. 1.3 mg/kg CKD-732, 1.15 mg/kg 119, 7 136, 8 146, 9 113, 8 124.0 129, 4 120, 9 131.8 117.9 99, 0 108.3 158, 6 201.7 174, 6 194.8 201.8 25, 4 208.3 201.1 120, 6 126, 2 125, 9 128.2 129, 3 122.4 131.9 135, 6 134.5 134.2 57, 6 121.2 101.0 109, 7 122.1 125, 8 116, 6 118.2 123, 1 130.7 45, 9 97.3 63.2 123.4 108.7 102, 6 105.5 129, 9 115, 2 128.0 43.3 105, 2 48.3 43, 9 139, 8 117.4 126, 3 81.2 107.5 107.7 19, 8 128, 6 109, 3 121.1 145, 9 144, 6 141.7 122.4 147.7 140.7 55, 1 123, 6 98.9 110, 1 149, 3 138.8 130, 9 94.7 116, 2 124.9 49, 8 131.8 110.5 108.7 133, 9 153, 9 148.7 112.3 135.0 125.5 61.9

In fig. 18 shows changes in leptin levels from baseline in male DIO Levin rats fed a high fat diet and treated with the conjugates of the present invention or fed a standard diet. A dose-dependent response was observed as leptin levels changed from baseline for compound 16.

- 31 045828

Table 16. Changes in leptin levels from baseline

Leptin levels, day 29 change from baseline Group % Before Day 29 changes introduction doses HF filler 5.3% 4.43 4, 67 Standard feed -63.9% 3.33 1.20 Conn. 16, 0.3 mg/kg 30.3% 2.53 3.30 Conn. 16.1 mg/kg -2.9% 4, 67 4.53 Conn. 16.3 mg/kg -4.0% 2.47 1.36 Conn. 16.6 mg, q7d -49.2% 3, 93 2.00 Conn. 1.1 mg/kg -1.3% 2.50 2.47 Conn. 1.3 mg/kg 11.3% 3.23 3, 60 CKD-732, 1.15 mg/kg -thirty% 4.43 4.30

Example: In Vivo Testing of DIO Mice - Weight Changes, Feed Intake, Dose Response Schedule

Male C57B1/6 mice (N=9/group), 21 weeks old, with an average body weight of 46.8 g, were fed ad libitum on a high-fat diet with 60% of the kcal coming from fat. The animals were dosed according to the schedule indicated in the table below. 17.

Table 17

Group Substance treatment Dose Filler Frequency mg/kg PBS q4d 1 Filler-1 0 DBS q4d 2 Polymer 12 PBS q4d 3 Connection 16 2 PBS q4d 4 Connection 16 6 PBS q4d 5 Connection 16 12 PBS q8d* 6 CKD-732 1 Filler-2 qod 7 Filler-2 0 Filler-2 qod 8 CKD-732 2 Filler-2 q4d 9 Filler-2 0 Filler-2 q4d

*filler-1 with different q4d

Dosing of the compound was performed between 9 and 10 o'clock on the dosing day. Groups 6 and 7 received a total of 17 doses. The q4d regimen groups (1, 2, 3, 4, 8, 9) received a total of 9 doses. The q8d group (5) received a total of 5 doses of the drug. Body weight and food intake were measured every second day. Blood glucose levels were measured in fed mice on day 7, day 0, day 7, day 14, day 21 and day 28 at 9:00 pm (blood glucose was measured pre-dose on dosing days). Blood glucose was measured with a glucometer. An intraperitoneal glucose tolerance test (ipGTT, 6 h fast) was performed.

The study was stopped on day 34. Liver tissues and epididymal white adipose tissues (eWAT) were collected, weighed and stored at -80°C. Sera were collected for determination of: AST, ALT, ALP, CK, BUN, creatinine, calcium, potassium, sodium, chloride, total protein, albumin, total bilirubin, glucose, triglycerides and cholesterol. The insulin content of the samples was measured using a commercial kit.

The polymer referred to in this example is poly[HPMA-co-MA-GFLG-N-(6aminohexyl)acetamide, a polymer that does not contain fumagillol. The synthesis of poly[HPMA-co-MAGFLG-N-(6-aminohexyl)acetamide is described in WO/2011/150022, which is incorporated by reference in its entirety.

Structure of poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide]

Based on FIG. 19 the surprising and unexpected finding is that the 12 mg/kg dose administered with the Q8D regimen resulted in greater initial weight loss than the 6 mg/kg dose administered with the Q4D regimen. At the end of the study, the Q8D group stopped losing weight, while the 6 mg/kg group continued to lose weight. A polymer that does not contain fumagillol shows mass changes similar to filler.

In fig. 20 shows that a dose of the small molecule CKD-732 (Compound B) administered in a Q2D (QOD) regimen showed a better response than the same average daily dose of the same small molecule administered

- 32 045828 according to the Q4D scheme. As expected, small molecules show better responses with more frequent dosing.

In fig. 21 shows the reduction in feed intake for the compounds of the present invention. Of note is the initial, significant reduction in food intake for the 12 mg/kg Q8D group and subsequent rebound in intake, followed by a pattern of decline-recovery cycles.

In fig. 22 shows significantly reduced insulin levels during ipGTT in male C57B16 mice fed a high-fat diet. The compounds of the present invention significantly reduce the amount of insulin excreted by B cells in the presence of elevated glucose levels, indicating reduced resistance and increased sensitivity to insulin. It should be noted that in fasting assays, insulin was also reduced in mice for all compound 16 groups.

In fig. 23 shows changes in total AUC (area under the curve) of insulin in high-fat chow-fed male C57B16 mice during glucose administration as a function of treatment group.

In fig. Figure 24 shows the reduction in blood glucose levels compared to the vehicle and polymer groups throughout the treatment period.

In fig. Figure 25 shows the product of glucose (mg/dL) x insulin (μE/ml)/405 (Akagiri et al., A Mouse Model of Metabolic Syndrome, J. Clin. Biochem. Nutr., 42, 150-157, March 2008) or the HOMA-ir measurement value, which is a recognized measurement value of insulin resistance and a prognostic factor for cardiovascular disease (Bonora et al., Diabetes Care, 2002, 25, 1135-1141).

Table 18. Data used to calculate HOMA-ir

ipGTT Insulin (µU/ml) Glucose (mg/dl) Q4D filler 0 min 205, 0 15 minutes 263, 4 0 min 239, 2 15 min 482.0 Excipient 12 mg/kg, Q4D 291.9 299, 4 215, 6 525.9 Conn. 16.2 mg/kg 104, 6 133, 4 182.8 488, 6 Conn. 16.6 mg/kg 53.2 64.3 179, 8 557.3 Conn. 16, 12 mg/kg 75, 9 96, 0 204, 553, 1 Filler, Q2D (ethanol) 264.7 241, 8 218.0 521.9 Filler, Q2D (ethanol) 249, 4 264, 9 231.0 493, 6 CKD-732, 1 mg/kg, Q2D 54.8 89, 3 161.3 469, 2 CKD-732, 2 mg/kg, Q2D 99, 8 118.3 182, 6 498, 6

Example of the effectiveness of various compounds in the DIO mouse model

Male C57B16 mice (N=6) were fed ad libitum a high-fat diet, TD.06414, with 60% of the Kcal from fat (Harlan diet). On day 1 of the study, animals were divided into groups so that the average weight of the mouse in each group was 47 g. Mice were treated with either phosphate buffered saline (vehicle) or the compounds listed in Table. 19, dissolved in the vehicle (spinal, subcutaneous administration). Treatment was continued for 26 days at the doses and according to the schedule indicated below in the table. 19. The polymer referred to in this example is poly[HPMA-co-MA-GFLG-N-(6-aminohexyl)acetamide, a polymer that does not contain fumagillol.

O polymer

ABOUT

The cis-16 compound is compound 16, in which the 1,4-diaminocyclohexane has a cis configuration rather than the trans configuration as shown for compound 16.

Compound aa is the reaction product of a methoxy-terminated PEG-amine with a molecular weight of 2 KDa and p-nitrophenyl fumagyl-6-yl carbonate

Compound bb is a compound of the formula o

K L\ ⁰ g-m polymer OFL-NH. .ABOUT. .NH .О ОМе о·

ABOUT

-

Claims (20)

The synthesis of poly[HPMA-CO-MA-GFLG-NH-2-[(2-(2-aminoethoxy)ethoxy)ethyl]carbamoylfumagillol] is described in WO/2011/150022, which is incorporated by reference in its entirety. Table 19. Body weight of the DIO mouse model as a function of time Group Dose (q4d) Average body weight of mouse group on the day of analysis (g) Change in body weight depending on vehicle mg/kg Scheme 1 7 15 21 26 Fill tel 0 q4d 47, 0 45, 0 45, 8 46, 1 46, 1 0.0% Connection 16 2 q4d 47.0 43, 4 43.2 42.0 41.7 -9.6% Connection 16, q8d 12 q4d 47, 0 41.3 38.3 36, 2 38, 0 -17.5% Connection tion 32 2 q4d 47, 0 42, 0 40, 6 41, 6 40.5 -12.1% Connection 3 8 2 q4d 47, 0 44.3 43, 8 42, 9 43, 6 -5.5% Polymer 12 q4d 47, 0 44, 8 46, 1 46, 3 46.5 0.8% 0% cis16 connection 2 q4d 47, 0 42.7 42, 6 40, 9 40, 6 -12.0 Connection nie aa 2 q4d 47, 0 44.3 45, 2 45.4 45, 8 -0.8% bb connection 2 q4d 47, 0 43, 6 43, 9 42.2 42.5 -7.8% CLAIM 1. The use of a compound for improving or restoring insulin sensitivity in a patient in need thereof, where the patient is overweight or obese and wherein the compound or a pharmaceutically acceptable salt thereof is selected from the group consisting of - 34 045828 2. Application of compound formula [уНх] HN^^O Z'^O I _ _ η or a pharmaceutically acceptable salt thereof for improving or restoring insulin sensitivity in a patient in need thereof, where the patient is overweight or obese; and where in each case, regardless R4 represents H or _C1 -C6alkyl; R ₅ represents H or C1-C6alkyl; R6 is _C2 -C6hydroxyalkyl; Z represents -NH-AA ₁ -AA ₂ -AA ₃ -AA ₄ -AA ₅ -AA ₆ -C(O)-QXYC(O)-W; AA1 is glycine, alanine or H ₂ N(CH ₂ )mCO ₂ H, where m is 2, 3, 4 or 5; AA ₂ represents a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; AA ₃ is a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; AA ₄ is a bond or alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine; AA ₅ is a bond or glycine, valine, tyrosine, tryptophan, phenylalanine, methionine, leucine, isoleucine or asparagine; AA ₆ is a bond or alanine, asparagine, citrulline, glutamine, glycine, leucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, valine or H2N(CH2)mCO2H, where m is 2, 3, 4 or 5; Q-X-Y represents - 35 045828 W represents OMe\7 x is equal to a number in the range from 1 to 450; y is equal to a number in the range from 1 to 30; n is a number ranging from 1 to 50 and the compound has a molecular weight of less than 60 kDa. 3. Use according to claim 2, wherein R ₄ represents methyl. 4. Use according to claim 2, wherein R5 is methyl. 5. Use according to claim 2, wherein R ₆ represents 2-hydroxypropyl. 6. Use according to claim 2, where AA ₁ represents glycine, AA ₂ , AA ₃ , AA4, AA5 and AA ₆ represent bonds. 7. Use according to claim 2, where AA ₁ represents glycine, AA ₂ , AA ₃ , AA4 and AA5 represent bonds and AA ₆ represents glycine. 8. Use according to claim 2, wherein AA1 is glycine, AA ₂ is a bond, AA ₃ is a bond, AA4 is glycine, AA5 is leucine and AA ₆ is glycine. 9. Use according to claim 2, wherein AA1 is glycine, AA ₂ is a bond, AA ₃ is a bond, AA4 is glycine, AA5 is valine and AA ₆ is glycine. 10. Use according to claim 2, wherein AA1 is glycine, AA ₂ is a bond, AA ₃ is a bond, AA4 is glycine, AA5 is phenylalanine and AA ₆ is glycine. 11. Use according to claim 2, wherein AA1 is glycine, AA ₂ is a bond, AA ₃ is a bond, AA4 is phenylalanine, AA5 is leucine and AA ₆ is glycine. 12. Use according to claim 2, wherein each of AA1, AA4, AA5 and AA ₆ is glycine, AA ₂ is a bond and AA ₃ is a bond. 13. Use according to claim 2, wherein the ratio of x to y is in the range from 20:1 to 4:1, preferably 11:1. 14. Use according to claim 1 or 2, where the patient’s body mass index (BMI) is from 25 to 29.9 kg/m ² ; 30 kg/ ^m2 or more; 35 kg/ ^m2 or more; or 40 kg/ ^m2 or more. 15. Use according to claim 1 or 2, where the patient has at least one concomitant disease induced by or associated with obesity, selected from the group consisting of diabetes, impaired glucose tolerance, impaired fasting glucose, elevated plasma insulin concentrations , insulin resistance syndrome, hyperlipidemia, dyslipidemia, hypertension, hyperuricemia, gout, coronary artery disease, myocardial infarction disease, angina pectoris, sleep apnea, obstructive sleep apnea, Pickwick's syndrome, fatty liver, cerebral infarction, stroke, vascular thrombosis brain, respiratory complications, gallbladder disease, kidney disease, gastroesophageal reflux, stress urinary incontinence, atherosclerosis, heart disease, abnormal heart rhythms, cardiac arrhythmia, transient ischemic attack, orthopedic disorders, osteoarthritis, deforming arthritis, lumbago, menstrual disorders cycle, endocrinopathy, hormonal imbalances and infertility. 16. Use according to claim 1 or 2, wherein the compound is further used to treat, reduce or improve one or more cardiometabolic risk factors in said patient, and the cardiometabolic risk factors are selected from plasma triglyceride levels, LDL cholesterol levels, C-reactive protein (CRP) levels ), systolic blood pressure, diastolic blood pressure, abdominal tissue adiposity, fasting glucose levels, fasting insulin levels, fibrinogen levels, plasminogen activator inhibitor-1 levels, and leptin levels. 17. Use according to claim 1 or 2, wherein the compound is administered in a therapeutically effective amount that is from 0.0001 to 5 mg/kg body weight per day or from 0.001 to 1 mg/kg body weight per day. 18. Use according to claim 1 or 2, wherein said compound is administered 1 to 5 times per week, preferably every two weeks, more preferably administered on a q4d dosage schedule, or most preferably on a q7d dosage schedule. 19. Use according to claim 1 or 2, wherein said compound is administered parenterally or subcutaneously. 20. Use according to claim 1 or 2, wherein said compound is provided in the form of a pharmaceutical composition containing said compound and a pharmaceutically acceptable carrier. -