WO2017031392A1 - Lipid compounds for treatment of obesity, hypertension and metabolic syndrome - Google Patents

Abstract

The present invention provides compounds, or pharmaceutically acceptable salts or analogs thereof, for the treatment of obesity, hypertension and/or metabolic disorder.

Description

LIPID COMPOUNDS FOR TREATMENT OF OBESITY, HYPERTENSION AND

METABOLIC SYNDROME

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Application Serial No.

62/207,503, filed August 20, 2015, the disclosure of which is hereby incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences.

This invention was made with government support under 1R43 AI094877 awarded by National Institutes of Health, National Institute of Allergy and Infectious Diseases. The government has certain rights in the invention.

BRIEF SUMMARY

Metabolic syndrome is a constellation of disorders manifested as a combination of dyslipidemia, hyperglycemia and obesity commonly accompanied by fatty liver disease, atherosclerosis and hypertension. The metabolic syndrome and obesity is often complicated by life-threatening diseases such as cardiovascular/cerebrovascular insults, type 2 diabetes, hepatobiliary and GI cancers (Chaiteerakij et al., 2013), the most frequent causes of death worldwide (O'Neill, 2015) (Chillaron, 2014; Medina-Santillan, 2013; Fan, 2013; Luo, 2015; Karagozian, 2014). Nearly 47 million individuals in the US have metabolic syndrome, while there is 175 million obese and 198 million overweight subjects in the US, Japan, and five major EU markets (France, Germany, Italy, Spain, and the UK) in 2013 (see Worldwide Website: win.niddk.nih.gov/publications/PDFs/stat904z.pdf). Obesity-related annual healthcare costs in the US equals about 240 billion, and is responsible for 2-8% of healthcare costs and 10-13% of deaths in EU.

At present, the majority of obese subjects are treated with lifestyle correction such as diet and physical activity but the predicted increases in subject populations in the US and worldwide will place an escalating burden on weight management and primary care systems. The treatment of co-morbidities and severe complications such as malignancies, which are spread in the population, dictate a desperate need for additional pharmacological and/or nutraceutical strategies. Pharmaceutical treatment may provide a desperately needed tools to challenge obesity, and the increasing choice between available drugs gives physicians and subjects an opportunity to tackle this condition effectively. However, despite the tremendous subject population, clinical and social needs, the drug market for obesity is not adequate to meet its considerable commercial potential.

Currently available and FDA-approved drugs include two general types of correctors: (i) a combination of stimulators and appetite suppressors (Osymia, Astra Zeneca; Contrave, Orexigen/Takeda) with unfavorable psychoactive effects, and (ii) various agonists of glucagon-like receptor 1 (GLP-1), including liraglutide (Saxenda, Novo Nordisk) and dulaglutide (Trulicity, Eli Lilly). The FDA-approved synthetic GLP-1 agonists have serious gastrointestinal side effects, including vomiting, need to be injected subcutaneously and are very expensive. For example, the least expensive Saxenda is targeted at $9,000/year per subject. A different approach using GPR40 receptor agonist Fasiglifam was proposed and had been in development by Takeda Pharmaceuticals. However, at the end of 2013, it was dropped due to serious safety concerns, primarily hepatotoxicity.

Heterogeneity of metabolic syndrome and obesity regarding molecular mechanisms of its initiation and progression, highlights the need to modulate multiple "targets" for its correction and especially prevention of co-morbidities, including diabetes type 2, fatty liver disease and hepatobiliary and GI tumors. Thus, multifaceted agents, nontoxic for normal cells, including liver and GI cells are needed to overcome molecular diversity of metabolic syndrome and its complications.

It has been demonstrated that AIPs affect multiple targets in vitro and in vivo. For example, the instant application discloses that oral administration of AIPs significantly reduced weight in high fat fed obese mice. Thus, AIPs represent a novel class of non-toxic, orally administered endogenous- like fatty acid analogs, which can be used for correction of metabolic syndrome, hypertension and obesity.

Thus, the present invention provides compounds, or pharmaceutically acceptable salts or analogs thereof, useful for the treatment of obesity, hypertension and metabolic syndrome. By administering the compound of the present invention to a subject (e.g., a human or non-human animal), the invention provides a method for reducing obesity in a subject reducing hypertension in a subject or treating metabolic syndrome in a subject such that improvement in the overall health of the subject is improved.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Chemical structure of AIP prototype and hydrochloride salts of three analogs. AIPs are natural fatty acids linked via ether bond to isopropylamino propanol polar head. The structures were verified by nuclear magnetic resonance ( MR) and mass- spectroscopy.

Figure 2. Weight reduction by oral AIP-1 in obese mice maintained on high fat diet. Obese animals were treated with AIP-1 at a dose of 20 mg/kg every other day for 2 weeks, and then placed on normal diet. Mean of n=6 in each group is shown.

Figure 3. Effect of oral AIP-1 on blood glucose in obese mice. Animals were maintained on high fat diet and treated with AIP-1 at a dose of 20 mg/kg for 2 weeks. Mean of n=6 in each group is shown.

Figures 4A-4B. Lack of AIP toxic effect on normal human hepatocytes. Human donor hepatocytes were obtained from LONZA, Inc. and cultured according to manufacturer's instructions. AIP-1 and AIP-2 were added at different concentrations and incubated overnight, following addition of MTS for additional 2 hours. OD450 nm was counted and data shown as percent of control values (Figure 4B). Phase contrast microphotographs are shown in Figure 4A.

Figure 5. AIPs reduce metabolic fatty acid synthase in human liver cells. Human

HUH-7 cells were cultured under standard conditions as described previously. AIPs were added at final concentration of 10 mM and incubated overnight at 37 °C. Representative immunoblot with FASN antibody is shown out of 3 with similar results.

Figure 6. AIP-1 up-regulates GPR40 expression in human liver cells. The incubations, conditions and AIP concentrations are the same as in autophagy experiments. Note: AIP2 has no effect on GPR40 induction in HUH-7 cells.

Figure 7. AIPs induce autophagy in human liver cells. Cultured HUH-7 cells were incubated with 10 mM of AIP overnight. The cell extract was immunoblotted with LCI and LC3 antibody. A representative western blot out of 3 performed with similar results is shown.

Figure 8. AIPs inhibit beta-2 and beta-3 adrenoreceptors in CHO cells overexpressing adrenoreceptors. AIP1 and AIP-2 stimulate GPR40 receptor signaling in CHO overexpressing the receptor.

DETAILED DISCLOSURE OF THE INVENTION

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms "including", "includes", "having", "has", "with", or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising". The transitional terms/phrases (and any grammatical variations thereof) "comprising", "comprises", "comprise", "consisting essentially of, "consists essentially of, "consisting" and "consists" can be used interchangeably.

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, "about" can mean a range of up to 0-20%, 0 to 10%, 0 to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term "about" meaning within an acceptable error range for the particular value should be assumed. In the context of compositions containing amounts of ingredients where the terms "about" or "approximately" are used, these compositions contain the stated amount of the ingredient with a variation (error range) of 0- 10%) around the value (X±10%>). When ranges are used herein, such as for dose ranges, combinations and subcombinations of ranges (e.g., subranges within the disclosed range), specific embodiments therein are intended to be explicitly included.

"Treatment", "treating", "palliating" and "ameliorating" (and grammatical variants of these terms), as used herein, are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. A therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.

The term "effective amount" or "therapeutically effective amount" refers to that amount of an inhibitor described herein that is sufficient to effect the intended application including but not limited to disease treatment. The therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term "pharmaceutically acceptable salt" refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

"Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions of the invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

"Subject" refers to an animal, such as a mammal, for example a human. The methods described herein can be useful in both pre-clinical human therapeutics and veterinary applications. In some embodiments, the subject is a mammal (such as an animal model of disease), and in some embodiments, the subject is human. The terms "subject" and "patient" can be used interchangeably.

The subject invention pertains to compounds capable of treating obesity, hypertension and metabolic syndrome. In one aspect, the subject invention concerns a pharmaceutical composition comprising compounds of Formula I or a pharmaceutically acceptable salt thereof.

("Formula I")

Preferably R¹ and R² are aliphatic (including, in some embodiments, alicyclic). Preferably R¹ is Ci-C₃₀, and R¹ can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 carbon atoms.

In certain embodiments, R¹ contains no cyclic moieties. In other embodiments, R¹ is straight-chain. In yet other embodiments, R¹ is branched-chain or a non-aromatic cyclic.

R² can contain in some embodiments, no cyclic moieties. In certain other embodiments, R² is straight-chain or branched-chain. Yet other embodiments provide R² as a non-aromatic cyclic.

In certain embodiments, m and n are independently 0-3, and each substituent R³ that is present can independently contain 1-10 non-hydrogen atoms along with 0 or more hydrogen atoms. In determining whether a particular atom is deemed to belong to R¹ or R², on the one hand, or R³ on the other hand, if the particular atom falls within the scope of R¹ or R², then it is deemed to belong to R¹ or R² and not to R³. For example, in an embodiment

where

group is deemed to be isopropyl with n=0, rather than R² = substituted ethyl, n=l, and R³ on R² is methyl. The entire isopropyl group is aliphatic and so is deemed to belong entirely to R². On

the other hand, in the molecule

aldehyde (formyl) group is not aliphatic, and so R² is substituted ethyl, n=l, and R³ on R² is CH=0. In all cases, the atom belonging to R³ that is directly bonded to R² must be (a) a carbon that is itself directly bonded to a heteroatom in R³, for example a carbonyl carbon; (b) a heteroatom, for example halogen, O, S, P, or N; or (c) a carbon that is a ring atom in an aryl or heteroaryl ring.

In certain embodiments, the compound of the invention is

"l-"hexadecenyloxy-3-isopropylamino-propan-2-ol" (trans or cis);

"l-hexadecyloxy-3-isopropylamino-propan-2-ol";

"l-octyloxy-3-isopropaylamino-propan-2-ol"; or

"l-octadecyenyloxy-3-isopo ylamino-propan-2-oΓ' (cis or trans).

In certain compositions and methods of the invention, a combination of any two of these compounds may be used, or a combination of three of all four of the compounds may be used. Likewise, a combination of the salts could be used. Moreover, it is appreciated that each depicted, above, may exist in more than one stereoisomeric form, including E and Z configurations at double bonds and isomers in which any given chiral center may be (R) or (S), and it is contemplated that any single isomer may be used or any mixture or combination thereof can be used for the treatment of obesity, hypertension and metabolic syndrome.

More specifically, it is to be understood that the compounds disclosed herein may contain chiral centers. Such chiral centers may be of either the (R) or (S) configuration, or may be a mixture thereof. Thus, the compounds provided herein may be enantiomerically pure, or be stereoisomeric or diastereomeric mixtures. It is understood that the disclosure of a compound herein encompasses any racemic, optically active, polymorphic, or stereoisomeric form, or mixtures thereof, which preferably possesses the useful properties described herein, it being well known in the art how to prepare optically active forms and how to determine activity using the standard tests described herein, or using other similar tests which are well known in the art.

In another aspect, the subject invention concerns a method of treating obesity, hypertension and metabolic syndrome in a subject by the administration of an effective amount of a compound of the invention or a pharmaceutical composition comprising a compound of the invention. Preferably, an effective amount of a pure or isolated compound is administered. The method of the subject invention is useful in treating obesity, hypertension and metabolic syndrome. Treatment of obesity, hypertension and metabolic syndrome involves a beneficial change in one or more symptoms associated with the obesity, hypertension and metabolic syndrome. For example, a beneficial change for a subject treated for obesity is a reduction in body mass index (BMI). Likewise, subject treated for hypertension observe a reduction in systolic, diastolic or both systolic and diastolic blood pressure. With respect to metabolic syndrome, beneficial changes include a decrease in blood pressure following treatment with the disclosed compounds, reduction in blood sugar levels, reduction in cholesterol levels, reduction in excess body fat around the waist or any combination of such beneficial effects in a subject to whom the disclosed compounds are administered.

Where the treatment of obesity is contemplated, the subject has a BMI of at least 20. Thus, one embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 25. Another embodiment for the treatment of obesity provides for the treatment of individuals with BMI values of at least 30. Yet another embodiment provides for the treatment of individuals with BMI values of at least 40.

For the purposes of this invention, metabolic syndrome is a cluster of conditions selected from increased blood pressure, a high blood sugar level, excess body fat around the waist and abnormal cholesterol levels that occur together. The development of these conditions increase the risk of heart disease, stroke and diabetes in subjects having metabolic syndrome.

According to the method of the subject invention, a substituted amino-propanol compound, or a pharmaceutically acceptable salt or analog thereof, is administered to a subject in an effective amount to treat the obesity, hypertension or metabolic syndrome. The precise dosage will depend on a number of clinical factors, for example, the type of subject (such as human, non-human mammal, or other animal) and age of the subject. A person having ordinary skill in the art would readily be able to determine, without undue experimentation, the appropriate dosages required to achieve the appropriate clinical effect.

A "subject" refers to a human, non-human mammal, or other animal in which the administration of a compound, as disclosed herein, would have a beneficial effect.

As used herein, the term "treatment" includes amelioration or alleviation of a pathological condition associated with obesity, hypertension or metabolic syndrome. These include, for obesity, a reduction in body mass index (BMI) for the subject when treated with the disclosed compounds; for hypertension, an observed reduction in the measured systolic, diastolic or both systolic and diastolic blood pressure of the subject after treatment with the disclosed compounds; and for metabolic syndrome, a decrease in blood pressure following treatment with the disclosed compounds, a reduction in blood sugar levels following treatment with the disclosed compounds, a reduction in cholesterol levels following treatment with the disclosed compounds, a reduction in excess body fat around the waist following treatment with the disclosed compounds or any combination of these effects on a subject to whom the disclosed compounds are administered. With respect to the treatment of hypertension and/or obesity, the reduction in BMI or systolic, diastolic and/or both systolic and diastolic blood pressure can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 39, 30, 31, 32 or 33% from the originally measured value for a subject.

The substituted amino-propanol compounds of the subject invention, including the compounds disclosed above, with their associated stereoisomers, and analogs or derivatives of the foregoing, can be obtained through a variety of methods known in the art. Derivatives of the subject invention can be synthesized using methods of organic synthesis known to those of ordinary skill in the art.

While the substituted amino-propanol compound can be administered as an isolated compound, it may be preferred to administer these compounds as a pharmaceutical composition. The subject invention thus further provides pharmaceutical compositions comprising a substituted amino-propanol compound, as an active agent, or physiologically acceptable salt(s) thereof, in association with at least one pharmaceutically acceptable carrier or diluent. The pharmaceutical composition can be adapted for various routes of administration, such as enteral, parenteral, intravenous, intramuscular, topical, subcutaneous, and so forth. Administration can be continuous or at distinct intervals, as can be determined by a person of ordinary skill in the art. For example, but without limitation, administration may be at a frequency of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times every 1, 2, 3, 4, 5, or 6 months; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times every 1, 2, 3, 4, 5, or 6 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times every 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 times every 1, 2, 3, 4, 5, or 6 hours; or at similar frequencies.

The compounds of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in a number of sources which are well known and readily available to those skilled in the art. For example, Remington 's Pharmaceutical Science (Martin E.W., Easton Pennsylvania, Mack Publishing Company, 19^th ed., 1995) describes formulations which can be used in connection with the subject invention. Formulations suitable for administration include, for example, aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the condition of the sterile liquid carrier, for example, water for injections, prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powder, granules, tablets, etc. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the subject invention can include other agents conventional in the art having regard to the type of formulation in question.

The substituted amino-propanol compounds of the present invention include all hydrates and salts that can be prepared by those of skill in the art. Under conditions where the compounds of the present invention are sufficiently basic or acidic to form stable nontoxic acid or base salts, administration of the compounds as salts may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiologically acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, alpha- ketoglutarate, and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including chloride, sulfate, nitrate, bicarbonate, and carbonate salts. Pharmaceutically acceptable salts may be obtained by using standard procedures well known in the art, for example by reacting a basic group such as an amine with an acid affording a physiologically acceptable anion or by reacting an acidic group such as a carboxylic acid with a base affording a physiologically acceptable cation. For example, alkali metal (e.g., lithium, sodium, potassium) and alkaline earth metal (e.g., magnesium, calcium) salts of carboxylic acids are commonly used. Physiologically acceptable cations and anions are well known in the art.

The compounds of the present invention can be formulated as pharmaceutical compositions and administered to a subject, such as a human or veterinary subject, in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the subject's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations may contain less than 0.01%, 0.01-0.05%, 0.05-0.1%, 0.1-0.5%, 0.5-1.0%, 1.0-5.0%, 5-10%, 10- 20%, 20-30%, 30-40%, 40-50%, or greater than 50% active ingredient by weight, or any combination of such ranges. The amount of the active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

The active agent (i.e., substituted amino-propanol compound or pharmaceutically acceptable salts thereof) may also be administered intravenously or intraperitoneally by infusion or injection. Dispersions and/or solutions of the active agent or its salts can be prepared in water, preferably mixed with a nontoxic surfactant, in oils, or in glycerol, liquid polyethylene glycols, triacetin, and in mixtures thereof. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating compounds of the invention in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. Accordingly, the present invention includes a pharmaceutical composition comprising substituted amino-propanol compound or pharmaceutically acceptable salts thereof, as described herein, or pharmaceutically acceptable salts thereof, in combination with a pharmaceutically acceptable carrier. Pharmaceutical compositions adapted for oral, topical or parenteral administration, comprising an amount of substituted amino-propanol compound, or a pharmaceutically acceptable salt thereof, constitute a preferred embodiment of the invention. The dose administered to a subject, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the subject over a reasonable time frame.

Mammalian species which benefit from the disclosed methods, include, but are not limited to, primates, such as apes, chimpanzees, orangutans, humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats, guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sea lions, elephant seals, otters, porpoises, dolphins, and whales. The term "subject" is intended to include such human and non-human mammalian species.

According to the method of the subject invention, a substituted amino-propanol compound or a pharmaceutically acceptable salt or derivative thereof can be administered to a subject by itself, or co-administered with one or more other compounds, including one or more other substituted amino-propanol compounds, or a pharmaceutically acceptable salt or analog thereof. The terms "co-administration," "administered in combination with," and their grammatical equivalents encompass administration of two or more agents to a subject so that both agents and/or their metabolites are present in the subject at the same time. Coadministration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present. Co-administered agents may be in the same formulation. Co-administered agents may also be in different formulations.

Furthermore, according to the method of the subject invention, the substituted amino- propanol compound, or a pharmaceutically acceptable salt or analog thereof, can be administered to a subject as adjunctive therapy. For example, a substituted amino-propanol compound, or a pharmaceutically acceptable salt or analog thereof, can be administered to a subject in conjunction with various approved treatments for obesity, hypertension or metabolic syndrome.

For example, the disclosed compounds can be co-administered with thiazide diuretics, such as hydrochlorothiazide (Microzide), chlorthalidone and others; beta blockers, such as propranolol (Inderal), acebutolol (Sectral ), atenolol (Tenormin) and others; angiotensin-converting enzyme (ACE) inhibitors, such as lisinopril (Zestril), benazepril (Lotensin), captopril (Capoten) and others; angiotensin 11 receptor blockers (ARBs), such as candesartan (Atacand), losartan (Cozaar) and others; calcium channel blockers, such as amlodipine (Norvasc), diltiazem (Cardizem or Tiazac); and iRenin inhibitors (e.g., Aliskiren (Tekturna)); alpha blockers, such as doxazosin (Cardura), prazosin (Minipress) and others; alpha-beta blockers, such as carvedilol (Coreg) and labetalol (Trandate); central nervous system -acting agents, such as clonidine (Catapres, Kapvay), guanfacine ( Intuniv, Ten ex) and methyldopa; vasodilators, such as hydralazine and minoxidil; and aldosterone antagonists, such as spironolactone (Aldactone) and eplerenone ( Inspra) for the treatment of hypertension. For the treatment of metabolic syndrome, the disclosed compounds can be co-administered with statins, fibrates, or nicotinic acid, diuretics or ACE inhibitors, such as metformin, insulin injections, any combination of such treatments.

All patents, patent applications, provisional applications, and publications referred to or cited herein, supra or infra, are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Example 1

AIPs analogs with C16:0 (AIP-1), C18: l (AIP-2)and C8:0 (AIP-3) fatty acids, were prepared, purified and tested as previously described by us in detail (Cao et al, 2013). The chemical structures of AIPs were verified by mass-spectrometry (MS) and Nuclear Magnetic Resonance (NMR), and hydrochloride salts obtained as described previously (Cao et al, 2013). For in vivo gavage, AIPs will be solubilized ex tempora in sterile saline/1% low molecular weight polyvinylpyrrolidone at 1.0 and 5.0 mg/ml, heated and kept at 40°C during the procedure.

Pathogen-free male C57BL/6 J mice were obtained from Harlan (Indianapolis, IN) at 4 weeks of age. After 1 week of adaptation, the animals were divided into control, normal diet (n=40) and experimental groups, high fat diet (n=70). The experimental group was placed on a high-fat diet (D 12492; Research Diets, New Brunswick, NJ), while the control group was placed on standard laboratory rodent chow (ProLab RMH 3000; LabDiet, Richmond, VA). In the high-fat diet, 20% of energy is from carbohydrates, 20% from protein and 60% from fat. The standard low-fat diet supplies 60% of energy from carbohydrates, 26%) from protein and 14% from fat. The body weight and food intake was measured using a digital weighing scale every other day at which time urine was collected from lightly anesthetized mice by massaging the lower abdominal part of the body, while a drop of blood was collected twice a week to measure glucose and lipids by glucose monitor and lipid assay micro-method.

Treatment with AIPs or vehicle began 8 weeks after initiation of high fat diet, when body weight increase in obese mice attains about 45% over normal diet animals. Before initiation of the treatments, 70 high fat diet mice were randomly divided into 7 equal groups (n=10 mice each). For gavage, AIP hydrochloride salts were be solubilized in sterile saline/1.0%) low molecular weight polyvinylpyrrolidone at 1.0 and 5 mg/ml, heated and maintained at 40° C throughout the procedure. In 6 groups of obese mice, AIPs were administered at 5 and 25 mg/kg of each ADM, AIP -2 and AIP-3 every other day for 3 weeks. Group 7 received a vehicle (saline/1%) PVP).

AIP-1 administration in obese mice for 2 weeks decreased body mass by nearly 9% compared to untreated obese mice (Fig. 2). Blood glucose levels were significantly higher during the first 10-14 days in mice on high fat diet compared to normal diet animals: 187.3 ± 3.26 vs. 159.4 ± 4.54 mg/dL (p=0.0021) at day 7. Blood glucose levels varied substantially during the whole observation period in both normal diet mice and high fat diet group. Ten weeks after initiation of high fat diet and beginning the treatment with AIP-1, blood glucose levels were higher in obese mice vs. normal dietl68.6+6.5 mg/dL vs. 143. 7+3.4 mg/dL. Two weeks treatment with AIP-1 reduced weight (Fig. 2) and showed a trend to decrease blood glucose, but not in a consistently significant fashion: 148.1 ± 7.2 mg/dL 7 days after beginning of AIP-1 treatment and 153.2+4.9 mg/dL at 14 day of treatment. (Fig. 3).

Initial experiments were also performed to assess acute AIP-1 toxicity in normal wild type C57/BL/J6 mice. Mice received AIP-1 at maximal dose of lg/kg in gavage (-20 mg/mouse, n=5). All animals remained alive at 72 hours and did not exhibit visible signs of abnormalities. Blood, tissues and urine were collected at 72 h and are being analyzed (data not shown). AIPs also did not have significant cytotoxic effects on cultured primary human hepatocytes up to 100 μΜ (Fig. 4). In contrast, AIPs killed human HUH-7 liver tumor cells with EC50 of 6.85 μΜ, 5.3 μΜ and 11.3 μΜ for AIP-1, AIP-2, and AIP-3, respectively.

Example 2

Human pre-adipocytes and adipocyte differentiation Kits were obtained from ATCC. The cells will be analyzed by FACS, induced to differentiate to mature adipocytes as described according to manufacturer's instructions and adapted from Church et al. (2014). The effect of AIPs (1-25 uM) on adipocyte precursors was assessed using Sca-1 and CD34 markers during time-course of incubation. At defined times, media aliquots were removed and cytotoxicity assessed by LDH activity. At the end of incubations, MTS tests were conducted to assess cell survival/proliferation.

Cells were collected for analysis of FASN, mTOR, CD36, and GPR40 expression by Flow Cytometry, immunocytochemistry and western blot. Autophagy was assessed by LC3 expression and apoptosis by expression of αΙΙ-spectrin breakdown products. AIP-1 and particularly AIP-3, but not AIP-2 substantially down-regulated FASN expression in human liver cell line HUH-7 upon overnight treatment (Fig. 5). AIP-1 significantly up-regulated GPR40 in these cells in a dose-dependent fashion (Fig. 6). In these cultured cells, AIP-1 and AIP-2 stimulated autophagy as indicated by up-regulation of LC3 isoform 1 and 2 (Fig. 7).

Importantly, we found that in CHO cells overexpressing recombinant GPR40 receptor, AIP-1 and AIP-2 (10 uM) induced Ca²⁺ mobilization as assessed by fluorescent FLIPR assay, while in the cells expressing adrenoreceptors, AIPs (10 μΜ), particularly AIP- 3, suppressed agonist-induced calcium fluxes (FLIPR assay, Millipore, Inc., Fig. 8).

REFERENCES

Cao, M. et al. "Composite fatty acid ether amides suppress growth of liver cancer cells in vitro and in an in vivo allograft mouse model" Cell Oncol (Dordr)., 2013, Vol. 36, No. 3, pp. 247-257.

Church, CD. et al. "Isolation and study of adipocyte precursors" Methods Enzymol, 2014, Vol. 537, pp. 31-46.

Claims

We claim:

1. A method of treating obesity, metabolic disorder or diabetes in a subject comprising administering a compound according to Formula I to a subject that is obese, has metabolic disorder or is diabetic, wherein said compound has the formula

ⁿ ("Formula I"), or a pharmaceutically acceptable salt thereof, wherein:

R¹ is (Ci-C₃₀) aliphatic,

R² is (C1-C30) aliphatic,

m and n are independently 0-3, and

each substituent R³ that is present independently consists of 1-10 non-hydrogen atoms along with 0 or more hydrogen atoms.

2. The method of claim 1, wherein R¹:

(i) contains no annulation, or

(ii) contains 1 annulation, or

(iii) contains 2 annulations, or

(iv) contains 3 annulations, or

(v) contains 4 or more annulations.

3. The method of claim 1 or 2, wherein R¹:

(i) contains no branch points, or

(ii) contains 1 branch point, or

(iii) contains 2 branch points, or

(iv) contains 3 branch points, or

(v) contains 4 branch points, or (vi contains 5 branch points, or

(vii contains 6 branch points, or

(viii contains 7 branch points, or

(ix contains 8 branch points, or

(x contains 9 or more branch points.

4. The method of any of claims 1-3, wherein R¹:

(i) contains no carbon-carbon double bond, or

(ϋ) contains 1 carbon-carbon double bond, or

(iii) contains 2 carbon-carbon double bonds, or

(iv) contains 3 carbon-carbon double bonds, or

(v) contains 4 carbon-carbon double bonds, or

(vi) contains 5 carbon-carbon double bonds, or

(νϋ) contains 6 carbon-carbon double bonds, or

(viii) contains 7 carbon-carbon double bonds, or

(ix) contains 8 carbon-carbon double bonds, or

(x) contains 9 or more carbon-carbon double bonds

The method of any of claims 1-4, wherein R¹:

(i) contains no carbon-carbon triple bond, or

(ϋ) contains 1 carbon-carbon triple bond, or

(iii) contains 2 carbon-carbon triple bonds, or

(iv) contains 3 carbon-carbon triple bonds, or

(v) contains 4 carbon-carbon triple bonds, or

(vi) contains 5 carbon-carbon triple bonds, or

(νϋ) contains 6 carbon-carbon triple bonds, or

(viii) contains 7 carbon-carbon triple bonds, or

(ix) contains 8 carbon-carbon triple bonds, or

(x) contains 9 or more carbon-carbon triple bonds

6. The method of any of claims 1-5, wherein R¹:

(i is Ci aliphatic, or

(ϋ is C₂ aliphatic, or (iii) is C₃ aliphatic, or

(iv) is C₄ aliphatic, or

(v) is C₅ aliphatic, or

(vi) is C₆ aliphatic, or

(vii) is C₇ aliphatic, or

(viii) is C₈ aliphatic, or

(ix) is C9 aliphatic, or

(x) is Cio aliphatic or

(xi) is C11 aliphatic or

(xii) is C12 aliphatic or

(xiii) is Ci3 aliphatic or

(xiv) is Ci4 aliphatic or

(xv) is Ci5 aliphatic or

(xvi) is Ci6 aliphatic or

(xvii) is Ci7 aliphatic or

(xviii) is Ci₈ aliphatic or

(xix) is Ci9 aliphatic or

(xx) is C20 aliphatic or

(xxi) is C21 aliphatic or

(xxii) is C22 aliphatic or

(xxiii) is C23 aliphatic or

(xxiv) is C24 aliphatic or

(xxv) is C25 aliphatic or

(xxvi) is C26 aliphatic or

(xxvii) is C27 aliphatic or

(xxviii) is C2₈ aliphatic or

(xxix) is C29 aliphatic or

(xxx) is C30 aliphatic

7. The method of any of claims 1-6, wherein R²:

(i) contains no annulation, or

(ii) contains 1 annulation, or

(iii) contains 2 annulations, or (iv contains 3 annulations, or

(V contains 4 or more annulations.

The method of any of claims 1-7, wherein R²:

(i) contains no branch points, or

(ϋ) contains 1 branch point, or

(iii) contains 2 branch points, or

(iv) contains 3 branch points, or

(v) contains 4 branch points, or

(vi) contains 5 branch points, or

(νϋ) contains 6 branch points, or

(viii) contains 7 branch points, or

(ix) contains 8 branch points, or

(x) contains 9 or more branch points

9. The method of any of claims 1-8, wherein R²:

(i contains no carbon-carbon double bond, or

(ϋ contains 1 carbon-carbon double bond, or

(iii contains 2 carbon-carbon double bonds, or

(iv contains 3 carbon-carbon double bonds, or

(v contains 4 carbon-carbon double bonds, or

(vi contains 5 carbon-carbon double bonds, or

(vii contains 6 carbon-carbon double bonds, or

(viii contains 7 carbon-carbon double bonds, or

(ix contains 8 carbon-carbon double bonds, or

(x contains 9 or more carbon-carbon double bonds.

10. The method of any of claims 1-9, wherein R²:

(i contains no carbon-carbon triple bond, or

(ϋ contains 1 carbon-carbon triple bond, or

(iii contains 2 carbon-carbon triple bonds, or

(iv contains 3 carbon-carbon triple bonds, or

(V contains 4 carbon-carbon triple bonds, or (vi) contains 5 carbon-carbon triple bonds, or

(vii) contains 6 carbon-carbon triple bonds, or

(viii) contains 7 carbon-carbon triple bonds, or

(ix) contains 8 carbon-carbon triple bonds, or

(x) contains 9 or more carbon-carbon triple bonds.

11. The method of any of claims 1-10 wherein R²:

(i) is Ci aliphatic, or

(ϋ) is C₂ aliphatic, or

(iii) is C₃ aliphatic, or

(iv) is C₄ aliphatic, or

(v) is C₅ aliphatic, or

(vi) is C₆ aliphatic, or

(vii) is C₇ aliphatic, or

(viii) is C₈ aliphatic, or

(ix) is C₉ aliphatic, or

(x) is Cio aliphatic or

(xi) is Cii aliphatic or

(xii) is Ci₂ aliphatic or

(xiii) is Ci₃ aliphatic or

(xiv) is C_i4 aliphatic or

(xv) is Ci₅ aliphatic or

(xvi) is Ci6 aliphatic or

(xvii) is On aliphatic or

(xviii) is Ci₈ aliphatic or

(xix) is Ci9 aliphatic or

(xx) is C₂o aliphatic or

(xxi) is C₂i aliphatic or

(xxii) is C₂₂ aliphatic or

(xxiii) is C₂₃ aliphatic or

(xxiv) is C₂₄ aliphatic or

(xxv) is C₂5 aliphatic or

(xxvi) is C₂₆ aliphatic or (xxvii) is C27 aliphatic, or

(xxviii) is C₂₈ aliphatic, or

(xxix) is C29 aliphatic, or

(xxx) is C₃₀ aliphatic.

12. The method of any of claims 1-11, wherein m:

(i) is zero, or

(ii) is one, or

(iii) is two, or

(iv) is three.

13. The method of any of claims 1-12, wherein any R³ substituted on R¹:

(i) contains at most a single non-hydrogen atom, or

(ii) contains at most 2 non-hydrogen atoms, or

(iii) contains at most 3 non-hydrogen atoms, or

(iv) contains at most 4 non-hydrogen atoms, or

(v) contains at most 5 non-hydrogen atoms, or

(vi) contains at most 6 non-hydrogen atoms, or

(vii) contains at most 7 non-hydrogen atoms, or

(viii) contains at most 8 non-hydrogen atoms, or

(ix) contains at most 9 non-hydrogen atoms, or

(x) contains at most 10 non-hydrogen atoms.

14. The method of any of claims 1-13, wherein n:

(i) is zero, or

(ii) is one, or

(iii) is two, or

(iv) is three.

15. The method of any of claims 1-14, wherein any R³ substituted on R²:

(i) contains at most a single non-hydrogen atom, or

(ii) contains at most 2 non-hydrogen atoms, or

(iii) contains at most 3 non-hydrogen atoms, or (iv) contains at most 4 non-hydrogen atoms, or

(v) contains at most 5 non-hydrogen atoms, or

(vi) contains at most 6 non-hydrogen atoms, or

(vii) contains at most 7 non-hydrogen atoms, or

(viii) contains at most 8 non-hydrogen atoms, or

(ix) contains at most 9 non-hydrogen atoms, or

(x) contains at most 10 non-hydrogen atoms.

16. The method of any of claims 1-15, wherein for any R³ substituted on R¹:

(i) each non-hydrogen atom is halogen, C, N, O, P, or S; or

(ii) each non-hydrogen atom is halogen, C, N, O, or P; or

(iii) each non-hydrogen atom is halogen, C, N, O, or S; or

(iv) each non-hydrogen atom is halogen, C, N, P, or S; or

(v) each non-hydrogen atom is halogen, C, O, P, or S; or

(vi) each non-hydrogen atom is halogen, N, O, P, or S; or

(vii) each non-hydrogen atom is C, N, O, P, or S.

17. The method of any of claims 1-16, wherein for any R³ substituted on R²:

(i) each non-hydrogen atom is halogen, C, N, O, P, or S; or

(ii) each non-hydrogen atom is halogen, C, N, O, or P; or

(iii) each non-hydrogen atom is halogen, C, N, O, or S; or

(iv) each non-hydrogen atom is halogen, C, N, P, or S; or

(v) each non-hydrogen atom is halogen, C, O, P, or S; or

(vi) each non-hydrogen atom is halogen, N, O, P, or S; or

(vii) each non-hydrogen atom is C, N, O, P, or S.

18. The method of any of claims 1-17, wherein no R³ substituted on R¹ contains an aryl group.

19. The method of any of claims 1-18, wherein no R³ substituted on R¹ contains a heteroaryl group. The method of any of claims 1-19, wherein no R³ substituted on R² contains an aryl group.

21. The method of any of claims 1-20, wherein no R³ substituted on R² contains a heteroaryl group.

The method of claim 1, wherein R¹ is hexadecenyl, octadecenyl, hexadecyl, octyl; R² is i sopropyl; and neither R¹ nor R² is substituted with R³.

23. The method of claim 1, wherein said compound is:

24. The method of claim 1, wherein said compound is:

25. The method of claim 1, wherein said compound is:

the cis isomer thereof.

26. The method of claim 1, wherein said compound is

"l-octadecyenyloxy-3-isopo ylamino-propan-2-oΓ' (cis or trans).

27. The method of claims 1-26, wherein said compound is co-administered with a thiazide diuretics; beta blocker; angiotensin-converting enzyme (ACE) inhibitor; angiotensin 11 receptor blocker (ARB ); calcium channel blocker; renin inhibitor; alpha blocker; alpha- beta blocker; central nervous system-acting agent; vasodilator; aldosterone antagonist; statin; fibrate; nicotinic acid; insulin or any combination thereof.