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[0001] This application claims the benefit of U.S. Ser. No. 09/724,788, filed Nov. 28, 2000, the contents of which are incorporated herein by reference.
[0002] The present invention relates to the use of (−) (3-halomethylphenoxy) (4-halophenyl) acetic acid derivatives and compositions in the treatment of insulin resistance, Type 2 diabetes, hyperlipidemia and hyperuricemia. It further relates to (−) (3-halomethylphenoxy) (4-halophenyl) acetic acid derivatives that are useful for the treatment of insulin resistance, Type 2 diabetes, hyperlipidemia and hyperuricemia.
[0003] Diabetes mellitus, commonly called diabetes, refers to a disease process derived from multiple causative factors and characterized by elevated levels of plasma glucose, referred to as hyperglycemia. See, e.g., LeRoith, D. et al., (eds.), DIABETES MELLITUS (Lippincott-Raven Publishers, Philadelphia, Pa. U.S.A. 1996), and all references cited therein. According to the American Diabetes Association, diabetes mellitus is estimated to affect approximately 6% of the world population. Uncontrolled hyperglycemia is associated with increased and premature mortality due to an increased risk for microvascular and macrovascular diseases, including nephropathy, neuropathy, retinopathy, hypertension, cerebrovascular disease and coronary heart disease. Therefore, control of glucose homeostasis is a critically important approach for the treatment of diabetes.
[0004] There are two major forms of diabetes: Type 1 diabetes (formerly referred to as insulin-dependent diabetes or IDDM); and Type 2 diabetes (formerly referred to as non-insulin dependent diabetes or NIDDM).
[0005] Type 1 diabetes is the result of an absolute deficiency of insulin, the hormone which regulates glucose utilization This insulin deficiency is usually characterized by β-cell destruction within the Islets of Langerhans in the pancreas, which usually leads to absolute insulin deficiency. Type 1 diabetes has two forms: Immune-Mediated Diabetes Mellitus, which results from a cellular mediated autoimmune destruction of the P cells of the pancreas; and Idiopathic Diabetes Mellitus, which refers to forms of the disease that have no known etiologies.
[0006] Type 2 diabetes is a disease characterized by insulin resistance accompanied by relative, rather than absolute, insulin deficiency. Type 2 diabetes can range from predominant insulin resistance with relative insulin deficiency to predominant insulin deficiency with some insulin resistance. Insulin resistance is the diminished ability of insulin to exert its biological action across a broad range of concentrations. In insulin resistant individuals, the body secretes abnormally high amounts of insulin to compensate for this defect. When inadequate amounts of insulin are present to compensate for insulin resistance and adequately control glucose, a state of impaired glucose tolerance develops. In a significant number of individuals, insulin secretion declines further and the plasma glucose level rises, resulting in the clinical state of diabetes. Type 2 diabetes can be due to a profound resistance to insulin stimulating regulatory effects on glucose and lipid metabolism in the main insulin-sensitive tissues: muscle, liver and adipose tissue. This resistance to insulin responsiveness results in insufficient insulin activation of glucose uptake, oxidation and storage in muscle and inadequate insulin repression of lipolysis in adipose tissue and of glucose production and secretion in liver. I: Type 2 diabetes, free fatty acid levels are often elevated in obese and some non-obese patients and lipid oxidation is increased.
[0007] Premature development of atherosclerosis and increased rate of cardiovascular and peripheral vascular diseases are characteristic features of patients with diabetes. Hyperlipidemia is an important precipitating factor for these diseases. Hyperlipidemia is a condition generally characterized by an abnormal increase in serum lipids in the bloodstream and is an important risk factor in developing atherosclerosis and heart disease. For a review of disorders of lipid metabolism, see, e.g., Wilson, J. et al., (ed.),
[0008] Dyslipidemia, or abnormal levels of lipoproteins in blood plasma, is a frequent occurrence among diabetics, and has been shown to be one of the main contributors to the increased incidence of coronary events and deaths among diabetic subjects (see, e.g., Joslin, E.
[0009] Previous studies from the 1970's have demonstrated the effectiveness of racemic 2-acetamidoethyl (4-chlorophenyl) (3-trifluoromethylphenoxy) acetate (also known as “halofenate”) as a potential therapeutic agent to treat Type 2 diabetes, hyperlipidemia and hyperuricemia (see, e.g., Bolhofer, W., U.S. Pat. No. 3,517,050; Jain, A. et al.,
[0010] In addition, there were some indications of drug-drug interactions of racemic halofenate with agents such as warfarin sulfate (also referred to as 3-(alpha-acetonylbenzyl)-4-hydroxycoumarin or Coumadin™ (Dupont Pharmaceuticals, E. I. Dupont de Nemours and Co., Inc., Wilmington, Del. U.S.A.) (see, e.g., Vesell, E. S. and Passantanti, G. T.,
[0011] Drugs that inhibit the metabolism of Coumadin™ result in a further decrease in vitamin K dependent clotting factors that prevents coagulation more than desired in patients receiving such therapy (i.e., patients at risk for pulmonary or cerebral embolism from blood clots in their lower extremities, heart or other sites). Simple reduction of the dose of anticoagulant is often difficult as one needs to maintain adequate anticoagulation to prevent blood clots from forming. The increased anticoagulation from drug-drug interaction results in a significant risk to such patients with the possibility of severe bleeding from soft tissue injuries, gastrointestinal sites (i.e., gastric or duodenal ulcers) or other lesions (i.e., aortic aneurysm). Bleeding in the face of too much anticoagulation constitutes a medical emergency and can result in death if it is not treated immediately with appropriate therapy.
[0012] Cytochrome P450 2C9 is also known to be involved in the metabolism of several other commonly used drugs, including dilantin, sulfonylureas, such as tolbutamide and several nonsteroidal anti-inflammatory agents, such as ibuprofen. Inhibition of this enzyme has the potential to cause other adverse effects related to drug-drug interactions, in addition to those described above for Coumadin™ (see, e.g., Pelkonen, O. et al.,
[0013] Solutions to the above difficulties and deficiencies are needed before halofenate becomes effective for routine treatment of insulin resistance, Type 2 diabetes, hyperlipidemia and hyperuricemia The present invention fulfills this and other needs by providing compounds, compositions and methods for alleviating insulin resistance, Type 2 diabetes, hyperlipidemia and hyperuricemia, while presenting a better adverse effect profile.
[0014] This present invention provides a method of modulating Type 2 diabetes in a mammal. The method comprises administering-to the mammal a therapeutically effective amount of the (−) stereoisomer of a compound of Formula I,
[0015] wherein R is a member selected from the group consisting of a alkoxy (e.g., OCH
[0016] Some such methods further comprise a compound of Formula II:
[0017] wherein R
[0018] Examples of such R
[0019] Subscripts m, o, q, s, t, u, v and w are integers as follows: m is 0 to 2; o and q are O to 5; p is 1 to 5; s is 1 to 3; t is 1 to 5; u is 0 to 1; v is 1 to 3; and w is 1 to (2v+1). R
[0020] R
[0021] In other aspects, the general methods will use a compound of Formula III:
[0022] The preferred compound of Formula III is known as “(−) 2-acetamidoethyl 4-chlorophenyl-(3-trifluoromethylphenoxy)-acetate” or “(−) halofenate.”
[0023] The present invention further provides a method for modulating insulin resistance in a mammal. This method comprises administering to the mammal a therapeutically effective amount of the (−) stereoisomer of a compound of Formula I. In certain preferred embodiments, the methods use a compound of Formula II.
[0024] The present invention further provides a method of alleviating hyperlipidemia in a mammal. This method comprises administering to the mammal a therapeutically effective amount of a compound of Formula I. In certain preferred embodiments, the methods use a compound of Formula II.
[0025] The present invention further provides a method of modulating hyperuricemia in a mammal. This method comprises administering to the mammal a therapeutically effective amount of a compound of Formula I. In certain preferred embodiments, the methods use a compound of Formula II.
[0026] The present invention also provides compounds and pharmaceutical compositions. The compounds are of Formula I, Formula II or Formula III. The pharmaceutical compositions comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of a compound of Formula I, Formula II or Formula III.
[0027]
[0028]
[0029]
[0030]
[0031]
[0032]
[0033]
[0034]
[0035]
[0036]
[0037]
[0038]
[0039]
[0040]
[0041]
[0042]
[0043]
[0044]
[0045]
[0046] FIGS.
[0047] The term “mammal” includes, without limitation, humans, domestic animals (e.g., dogs or cats), farm animals (cows, horses, or pigs), monkeys, rabbits, mice, and laboratory animals.
[0048] The term “insulin resistance” can be defined generally as a disorder of glucose metabolism. More specifically, insulin resistance can be defined as the diminished ability of insulin to exert its biological action across a broad range of concentrations producing less than the expected biologic effect. (see, e.g., Reaven, G. M.,
[0049] The term “diabetes mellitus” or “diabetes” means a disease or condition that is generally characterized by metabolic defects in production and utilization of glucose which result in the failure to maintain appropriate blood sugar levels in the body. The result of these defects is elevated blood glucose, referred to as “hyperglycemia.” Two major forms of diabetes are Type 1 diabetes and Type 2 diabetes. As described above, Type 1 diabetes is generally the result of an absolute deficiency of insulin, the hormone which regulates glucose utilization. Type 2 diabetes often occurs in the face of normal, or even elevated levels of insulin and can result from the inability of tissues to respond appropriately to insulin. Most Type 2 diabetic patients are insulin resistant and have a relative deficiency of insulin, in that insulin secretion can not compensate for the resistance of peripheral tissues to respond to insulin. In addition, many Type 2 diabetics are obese. Other types of disorders of glucose homeostasis include Impaired Glucose Tolerance, which is a metabolic stage intermediate between normal glucose homeostasis and diabetes, and Gestational Diabetes Mellitus, which is glucose intolerance in pregnancy in women with no previous history of Type 1 or Type 2 diabetes.
[0050] The term “secondary diabetes” is diabetes resulting from other identifiable etiologies which include: genetic defects of β cell function (e.g. maturity onset-type diabetes of youth, referred to as “MODY,” which is an early-onset form of Type 2 diabetes with autosomal inheritance; see, e.g., Fajans S. et al.,
[0051] The guidelines for diagnosis for Type 2 diabetes, impaired glucose tolerance, and gestational diabetes have been outlined by the American Diabetes Association (see, e.g., The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus,
[0052] The term “halofenic acid” refers to the acid form of 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetic acid.
[0053] The term “hyperinsulinemia” refers to the presence of an abnormally elevated level of insulin in the blood.
[0054] The term “hyperuricemia” refers to the presence of an abnormally elevated level of uric acid in the blood.
[0055] The term “secretagogue” means a substance or compound that stimulates secretion. For example, an insulin secretagogue is a substance or compound that stimulates secretion of insulin.
[0056] The term “hemoglobin” or “Hb” refers to a respiratory pigment present in erythrocytes, which is largely responsible for oxygen transport. A hemoglobin molecule comprises four polypeptide subunits (two a chain systems and two β chain systems, respectively). Each subunit is formed by association of one globin protein and one heme molecule which is an iron-protoporphyrin complex. The major class of hemoglobin found in normal adult hemolysate is adult hemoglobin (referred to as “HbA”; also referred to HbA
[0057] Among classes of adult hemoglobin HbAs, there is a glycated hemoglobin (referred to as “HbA
[0058] The term “glycosylated hemoglobin” (also referred to as “HbA
[0059] The term “symptom” of diabetes, includes, but is not limited to, polyuria, polydipsia, and polyphagia, as used herein, incorporating their common usage. For example, “polyuria” means the passage of a large volume of urine during a given period; “polydipsia” means chronic, excessive thirst; and “polyphagia” means excessive eating. Other symptoms of diabetes include, e.g., increased susceptibility to certain infections (especially fungal and staphylococcal infections), nausea, and ketoacidosis (enhanced production of ketone bodies in the blood).
[0060] The term “complication” of diabetes includes, but is not limited to, microvascular complications and macrovascular complications. Microvascular complications are those complications which generally result in small blood vessel damage. These complications include, e.g., retinopathy (the impairment or loss of vision due to blood vessel damage in the eyes); neuropathy (nerve damage and foot problems due to blood vessel damage to the nervous system); and nephropathy (kidney disease due to blood vessel damage in the kidneys). Macrovascular complications are those complications which generally result from large blood vessel damage. These complications include, e.g., cardiovascular disease and peripheral vascular disease. Cardiovascular disease refers to diseases of blood vessels of the heart. See. e.g. Kaplan, R. M., et al., “Cardiovascular diseases” in HEALTH AND HUMAN BEHAVIOR, pp. 206-242 (McGraw-Hill, New York 1993). Cardiovascular disease is generally one of several forms, including, e.g., hypertension (also referred to as high blood pressure), coronary heart disease, stroke, and rheumatic heart disease. Peripheral vascular disease refers to diseases of any of the blood vessels outside of the heart. It is often a narrowing of the blood vessels that carry blood to leg and arm muscles.
[0061] The term “atherosclerosis” encompasses vascular diseases and conditions that are recognized and understood by physicians practicing in the relevant fields of medicine. Atherosclerotic cardiovascular disease, coronary heart disease (also known as coronary artery disease or ischemic heart disease); cerebrovascular disease and peripheral vessel disease are all clinical manifestations of atherosclerosis and are therefore encompassed by the terms “atherosclerosis” and “atherosclerotic disease”.
[0062] The term “antihyperlipidemic” refers to the lowering of excessive lipid concentrations in blood to desired levels.
[0063] The term “antiuricemic” refers to the lowering of excessive uric acid concentrations in blood to desired levels.
[0064] The term “hyperlipidemia” refers to the presence of an abnormally elevated level of lipids in the blood. Hyperlipidemia can appear in at least three forms: (1) hypercholesterolemia, i.e., an elevated cholesterol level; (2) hypertriglyceridemia, i.e., an elevated triglyceride level; and (3) combined hyperlipidemia, i.e., a combination of hypercholesterolemia and hypertriglyceridemia.
[0065] The term “modulate” refers to the treating, prevention, suppression, enhancement or induction of a function or condition. For example, the compounds of the present invention can modulate hyperlipidemia by lowering cholesterol in a human, thereby suppressing hyperlipidemia.
[0066] The term “treating” means the management and care of a human subject for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of the present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
[0067] The term “preventing” means the management and care of a human subject such that the onset of symptoms of a disease, condition or disorder does not occur.
[0068] The term “cholesterol” refers to a steroid alcohol that is an essential component of cell membranes and myelin sheaths and, as used herein, incorporates its common usage. Cholesterol also serves as a precursor for steroid hormones and bile acids.
[0069] The term “triglyceride(s)” (“TGs”), as used herein, incorporates its common usage. TGs consist of three fatty acid molecules esterified to a glycerol molecule and serve to store fatty acids which are used by muscle cells for energy production or are taken up and stored in adipose tissue.
[0070] Because cholesterol and TGs are water insoluble, they must be packaged in special molecular complexes known as “lipoproteins” in order to be transported in the plasma. Lipoproteins can accumulate in the plasma due to overproduction and/or deficient removal. There are at least five distinct lipoproteins differing in size, composition, density, and function. In the cells of the small of the intestine, dietary lipids are packaged into large lipoprotein complexes called “chylomicrons”, which have a high TG and low-cholesterol content. In the liver, TG and cholesterol esters are packaged and released into plasma as TG-rich lipoprotein called very low density lipoprotein (“VLDL”), whose primary function is the endogenous transport of TGs made in the liver or released by adipose tissue. Through enzymatic action, VLDL can be either reduced and taken up by the liver, or transformed into intermediate density lipoprotein (“IDL”). IDL, is in turn, either taken up by the liver, or is further modified to form the low density lipoprotein (“LDL”). LDL is either taken up and broken down by the liver, or is taken up by extrahepatic tissue. High density lipoprotein (“HDL”) helps remove cholesterol from peripheral tissues in a process called reverse cholesterol transport.
[0071] The term “dyslipidemia” refers to abnormal levels of lipoproteins in blood plasma including both depressed and/or elevated levels of lipoproteins (e.g., elevated levels of LDL, VLDL and depressed levels of HDL).
[0072] Exemplary Primary Hyperlipidemia include, but are not limited to, the following:
[0073] (1) Familial Hyperchylomicronemia, a rare genetic disorder which causes a deficiency in an enzyme, LP lipase, that breaks down fat molecules. The LP lipase deficiency can cause the accumulation of large quantities of fat or lipoproteins in the blood;
[0074] (2) Familial Hypercholesterolemia, a relatively common genetic disorder caused where the underlying defect is a series of mutations in the LDL receptor gene that result in malfunctioning LDL receptors and/or absence of the LDL receptors. This brings about ineffective clearance of LDL by the LDL receptors resulting in elevated LDL and total cholesterol levels in the plasma;
[0075] (3) Familial Combined Hyperlipidemia, also known as multiple lipoprotein-type hyperlipidemia; an inherited disorder where patients and their affected first-degree relatives can at various times manifest high cholesterol and high triglycerides. Levels of HDL cholesterol are often moderately decreased;
[0076] (4) Familial Defective Apolipoprotein B-100 is a relatively common autosomal dominant genetic abnormality. The defect is caused by a single nucleotide mutation that produces a substitution of glutamine for arginine which can cause reduced affinity of LDL particles for the LDL receptor. Consequently, this can cause high plasma LDL and total cholesterol levels;
[0077] (5) Familial Dysbetaliproteinemia, also referred to as Type III Hyperlipoproteinemia, is an uncommon inherited disorder resulting in moderate to severe elevations of serum TG and cholesterol levels with abnormal apolipoprotein E function. HDL levels are usually normal; and
[0078] (6) Familial Hypertriglyceridemia, is a common inherited disorder in which the concentration of plasma VLDL is elevated. This can cause mild to moderately elevated triglyceride levels (and usually not cholesterol levels) and can often be associated with low plasma HDL levels.
[0079] Risk factors in exemplary Secondary Hyperlipidemia include, but are not limited to, the following: (1) disease risk factors, such as a history of Type 1 diabetes, Type 2 diabetes, Cushing's syndrome, hypothroidism and certain types of renal failure; (2) drug risk factors, which include, birth control pills; hormones, such as estrogen, and corticosteroids; certain diuretics; and various β blockers; (3) dietary risk factors include dietary fat intake per total calories greater than 40%; saturated fat intake per total calories greater than 10%; cholesterol intake greater than 300 mg per day; habitual and excessive alcohol use; and obesity.
[0080] The terms “obese” and “obesity” refers to, according to the World Health Organization, a Body Mass Index (BMI) greater than 27.8 kg/m
[0081] “Pharmaceutically acceptable salts” refer to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art. The term also includes non-toxic acid addition salts, which are generally prepared by reacting the compounds of the present invention with a suitable organic or inorganic acid. Representative salts include, but are not limited to, the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napsylate, and the like.
[0082] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, menthanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. For a description of pharmaceutically acceptable acid addition salts as prodrugs. See, e.g., Bundgaard, H., ed.,
[0083] “Pharmaceutically acceptable ester” refers to those esters which retain, upon hydrolysis of the ester bond, the biological effectiveness and properties of the carboxylic acid or alcohol and are not biologically or otherwise undesirable. For a description of pharmaceutically acceptable esters as prodrugs, see Bundgaard, H., supra. These esters are typically formed from the corresponding carboxylic acid and an alcohol. Generally, ester formation can be accomplished via conventional synthetic techniques. (See, e.g., March
[0084] “Pharmaceutically acceptable amide” refers to those amides which retain, upon hydrolysis of the amide bond, the biological effectiveness and properties of the carboxylic acid or amine and are not biologically or otherwise undesirable. For a description of pharmaceutically acceptable amides as prodrugs, see, Bundgaard, H., ed., supra. These amides are typically formed from the corresponding carboxylic acid and an amine. Generally, amide formation can be accomplished via conventional synthetic techniques. See, e.g., March et al.,
[0085] (1) General
[0086] The present invention is directed to use of a preferred (−) (3-halomethylphenoxy) (4-halophenyl) acetic acid derivatives having the following general formula:
[0087] In Formula I, R is a functional group including, but not limited to, the following: alkoxy, heteroalkoxy, aryloxy, heteroaryloxy, lower aralkoxy, e.g., phenyl-lower alkoxy such as benzyloxy, phenethyloxy; di-lower alkylamino-lower alkoxy and the nontoxic, pharmacologically acceptable acid addition salts thereof, e.g., dimethylaminoethoxy, diethylaminoethoxy hydrochloride, diethylaminoethoxy citrate, diethylaminopropoxy; benzamido-lower alkoxy, e.g., benzamidoethoxy or benzamidopropoxy; ureido-lower alkoxy, e.g., ureidoethoxy or 1-methyl-2-ureidoethoxy; N′-lower alkyl-ureido-lower alkoxy, i.e., R
[0088] In a preferred embodiment, the present invention relates to use of the (−) (3-halomethylphenoxy)(4-halophenyl) acetic acid derivatives having the following general formula:
[0089] In Formula II, R
[0090] Returning to a discussion of R
[0091] Subscripts m, o, q, s, t, u, v and w are integers as follows: m is 0 to 2; o and q are 0 to 5; p is 1 to 5; s is 1 to 3; t is 1 to 5; u is 0 to 1; v is 1 to 3; and w is 1 to (2v+1). R
[0092] R
[0093] In particularly preferred embodiments, R
[0094] in which the alkyl portions of any R groups have from one to four carbon atoms, and in further preferred embodiments are unsubstituted alkyl portions of from 1 to 4 carbon atoms.
[0095] In the most preferred embodiments, the compound is one of compounds 19.1 through 19.29 in the examples below.
[0096] In a further preferred embodiment, the present invention relates to the use of a compound having the formula:
[0097] The compound of Formula III is referred to as “(−) 2-acetamidoethyl 4-chlorophenyl-(3-trifluoromethylphenoxy) acetate” (also referred to as “(−) halofenate”).
[0098] With reference to each of the formulae above, the substituent groups have art recognized meanings. More particularly, terms such as alkyl are meant to be groups having from 1 to 8 carbon atoms, unless otherwise noted. Similarly, “alkylene” and the like (e.g., alkoxy, heteroalkyl) refer to groups having 8 or fewer carbon atoms, unless otherwise stated. When the term “lower” is used to refer to an alkyl group (or variant thereof, such as alkoxy, alkanamido, etc.), the group is intended to have from 1 to 4 carbon atoms. The terms “aryl” and “heteroaryl” refer to monocyclic and fused bicyclic groups such as phenyl, naphthyl, pyridyl, benzimidazolyl, quinolinyl, and the like. Preferred groups for aryl are phenyl and naphthyl, while a preferred heteroaryl group is pyridyl.
[0099] Changes in drug metabolism mediated by inhibition of cytochrome P450 enzymes has a very high potential to precipitate significant adverse effects in patients. Such effects were previously noted in patients treated with racemic halofenate. In the present studies, racemic halofenic acid was found to inhibit cytochrome P450 2C9, an enzyme known to play a significant role in the metabolism of specific drugs. This can lead to significant problems with drug interactions with anticoagulants, anti-inflammatory agents and other drugs metabolized by this enzyme. However, quite surprisingly, a substantial difference was observed between the enantiomers of halofenic acid in their inability to inhibit cytochrome P450 2C9, the (−) enantiomer being about twenty-fold less active whereas the (+) enantiomer was quite potent (see Example 7). Thus, use of the (−) enantiomer of compounds in Formula I, Formula II or Formula III will avoid the inhibition of this enzyme and the adverse effects on drug metabolism previously observed with racemic halofenate.
[0100] The present invention encompasses a method of modulating insulin resistance in a mammal, the method comprising: administering to the mammal a therapeutically effective amount of a compound having the general structure of Formula I or a pharmaceutically acceptable salt thereof. In a presently preferred embodiment, the compound has the general structure of Formula II. In a further preferred embodiment, the compound has the structure of Formula III. Quite surprisingly, the method avoids the adverse effects associated with the administration of a racemic mixture of halofenate by providing an amount of the (−) stereoisomer of the compounds in Formula I, Formula II or Formula III which is insufficient to cause the adverse effects associated with the inhibition of cytochrome P450 2C9.
[0101] The present invention also encompasses a method of modulating Type 2 diabetes in a mammal, the method comprising: administering to the mammal a therapeutically effective amount of a compound having the general structure of Formula I or a pharmaceutically acceptable salt thereof. In a presently preferred embodiment, the compound has the general structure of Formula II; In a further preferred embodiment, the compound has the structure of Formula III. Quite surprisingly, the method avoids the adverse effects associated with the administration of a racemic mixture of halofenate by providing an amount of the (−) stereoisomer of the compounds in Formula I, Formula II or Formula III which is insufficient to cause the adverse effects associated with the inhibition of cytochrome P450 2C9.
[0102] The present invention further encompasses a method of modulating hyperlipidemia in a mammal, the method comprising: administering to the mammal a therapeutically effective amount of a compound having the general structure of Formula I or a pharmaceutically acceptable salt thereof. In a presently preferred embodiment, the compound has the general structure of Formula II. In a further preferred embodiment, the compound has the structure of Formula III. Quite surprisingly, the method avoids the adverse effects associated with the administration of a racemic mixture of halofenate by providing an amount of the (−) stereoisomer of the compounds in Formula I, Formula II or Formula III which is insufficient to cause the adverse effects associated with the inhibition of cytochrome P450 2C9.
[0103] The racemic mixture of the halofenate (i.e., a 1:1 racemic mixture of the two enantiomers) possesses antihyperlipidemic activity and provides therapy and a reduction of hyperglycemia related to diabetes when combined with certain other drugs commonly used to treat this disease. However, this racemic mixture, while offering the expectation of efficacy, causes adverse effects. The term “adverse effects” includes, but is not limited to, nausea, gastrointestinal ulcers, and gastrointestinal bleeding. Other side effects that have been reported with racemic halofenate include potential problems with drug-drug interactions, especially including difficulties controlling anticoagulation with Coumadin™. Utilizing the substantially pure compounds of the present invention results in clearer dose related definitions of efficacy, diminished adverse effects, and accordingly, an improved therapeutic index. As such, it has now been discovered that it is more desirable and advantageous to administer the (−) enantiomer of halofenate instead of racemic halofenate.
[0104] The present invention further encompasses a method of modulating hyperuricemia in a mammal, the method comprising: administering to the mammal a therapeutically effective amount of a compound having the general structure of Formula I or a pharmaceutically acceptable salt thereof. In a presently preferred embodiment, the compound has the general structure of Formula II. In a further preferred embodiment, the compound has the structure of Formula III. Quite surprisingly, the method avoids the adverse effects associated with the administration of a racemic mixture of halofenate by providing an amount of the (−) stereoisomer of the compounds in Formula I, Formula II or Formula III which is insufficient to cause the adverse effects associated with the inhibition of cytochrome P450 2C9.
[0105] (2) (−) Enantiomers of Formula I, Formula II and Formula III
[0106] Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes “d” and “l” or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or l meaning that the compound is “levorotatory” and with (+) or d is meaning that the compound is “dextrorotatory”. There is no correlation between nomenclature for the absolute stereochemistry and for the rotation of an enantiomer. For a given chemical structure, these compounds, called “stereoisomers,” are identical except that they are mirror images of one another. A specific stereoisomer can also be referred to as an “enantiomer,” and a mixture of such isomers is often called an “enantiomeric” or “racemic” mixture. See, e.g., Streitwiesser, A. & Heathcock, C. H.,
[0107] The chemical synthesis of the racemic mixture of halofenates (3-trihalomethylphenoxy) (4-halophenyl) acetic acid derivatives can be performed by the methods described in U.S. Pat. No. 3,517,050, the teaching of which are incorporated herein by reference. The synthesis of the compounds of the present invention is further described in the Examples, supra. The individual enantiomers can be obtained by resolution of the racemic mixture of enantiomers using conventional means known to and used by those of skill in the art. See, e.g., Jaques, J., et al., in
[0108] The term “substantially free of its (+) stereoisomer,” as used herein, means that the compositions contain a substantially greater proportion of the (−) isomer of halofenate in relation to the (+) isomer. In a preferred embodiment, the term “substantially free of its (+) stereoisomer,” as used herein, means that the composition is at least 90% by weight of the (−) isomer and 10% by weight or less of the (+) isomer. In a more preferred embodiment, the term “substantially free of its (+) stereoisomer,” as used herein, means that the composition contains at least 99% by weight of the (−) isomer and 1% by weight or less of the (+) isomer. In the most preferred embodiment, the term “substantially free of its (+) stereoisomer,” means that the composition contains greater than 99% by weight of the (−) isomer. These percentages are based upon the total amount of halofenate in the composition. The terms “substantially optically pure (l) isomer of halofenate,” “substantially optically pure (1) halofenate,” “optically pure (l) isomer of halofenate” and “optically pure (1) halofenate” all refer to the (−) isomer and are encompassed by the above-described amounts. In addition, the terms “substantially optically pure (d) isomer of halofenate,” “substantially optically pure (d) halofenate,” “optically pure (d) isomer of halofenate” and “optically pure (d) halofenate” all refer to the (+) isomer and are encompassed by the above-described amounts.
[0109] The term “enantiomeric excess” or “ee” is related to the term “optical purity” in that both are measures of the same phenomenon. The value of ee will be a number from 0 to 100, 0 being racemic and 100 being pure, single enantiomer. A compound that is referred to as 98% optically pure can be described as 96% ee.
[0110] (3) Combination Therapy With Additional Active Agents
[0111] The compositions can be formulated and administered in the same manner as detailed below. “Formulation” is defined as a pharmaceutical preparation that contains a mixture of various excipients and key ingredients that provide a relatively stable, desirable and useful form of a compound or drug. For the present invention, “formulation” is included within the meaning of the term “composition.” The compounds of the present invention can be used effectively alone or in combination with one or more additional active agents depending on the desired target therapy (see, e.g., Turner, N. et al.
[0112] An example of combination therapy that modulates (prevents the onset of the symptoms or complications associated) atherosclerosis, wherein a compound of Formula I is administered in combination with one or more of the following active agents: an antihyperlipidemic agent; a plasma HDL-raising agent; an antihypercholesterolemic agent, such as a cholesterol biosynthesis inhibitor, e.g., an hydroxymethylglutaryl (HMG) CoA reductase inhibitor (also referred to as statins, such as lovastatin, simvastatin, pravastatin, fluvastatin, and atorvastatin), an HMG-CoA synthase inhibitor, a squalene epoxidase inhibitor, or a squalene synthetase inhibitor (also known as squalene synthase inhibitor); an acyl-coenzyme A cholesterol acyltransferase (ACAT) inhibitor, such as melinamide; probucol; nicotinic acid and the salts thereof and niacinamide; a cholesterol absorption inhibitor, such as β-sitosterol; a bile acid sequestrant anion exchange resin, such as cholestyramine, colestipol or dialkylaminoalkyl derivatives of a cross-linked dextran; an LDL (low density lipoprotein) receptor inducer; fibrates, such as clofibrate, bezafibrate, fenofibrate, and gemfibrizol; vitamin B
[0113] Another example of combination therapy can be seen in treating obesity or obesity-related disorders, wherein the compounds of Formula I can be effectively used in combination with, for example, phenylpropanolamine, phentermine, diethylpropion, mazindol; fenfluramine, dexfenfluramine, phentiramine, β
[0114] Still another example of combination therapy can be seen in modulating diabetes (or treating diabetes and its related symptoms, complications, and disorders), wherein the compounds of Formula I can be effectively used in combination with, for example, sulfonylureas (such as chlorpropamide, tolbutamide, acetohexamide, tolazamide, glyburide, gliclazide, glynase, glimepiride, and glipizide), biguanides (such as metformin), thiazolidinediones (such as ciglitazone, pioglitazone, troglitazone, and rosiglitazone); dehydroepiandrosterone (also referred to as DHEA or its conjugated sulphate ester, DHEA-SO
[0115] A further example of combination therapy can be seen in modulating hyperlipidemia (treating hyperlipidemia and its related complications), wherein the compounds of Formula I can be effectively used in combination with, for example, statins (such as fluvastatin, lovastatin, pravastatin or simvastatin), bile acid-binding resins (such as colestipol or cholestyramine), nicotinic acid, probucol, betacarotene, vitamin E, or vitamin C.
[0116] In accordance with the present invention, a therapeutically effective amount of a compound of Formula I (or Formula II or Formula III) can be used for the preparation of a pharmaceutical composition useful for treating diabetes, treating hyperlipidemia, treating hyperuricemia, treating obesity, lowering triglyceride levels, lowering cholesterol levels, raising the plasma level of high density lipoprotein, and for treating, preventing or reducing the risk of developing atherosclerosis.
[0117] Additionally, an effective amount of a compound of Formula I (or Formula II or Formula III) and a therapeutically effective amount of one or more active agents selected from the group consisting of: an antihyperlipidemic agent; a plasma HDL-raising agent; an antihypercholesterolemic agent, such as a cholesterol biosynthesis inhibitor, for example, an HMG-CoA reductase inhibitor, an HMG-CoA synthase inhibitor, a squalene epoxidase inhibitor, or a squalene synthetase inhibitor (also known as squalene synthase inhibitor); an acyl-coenzyme A cholesterol acyltransferase inhibitor; probucol; nicotinic acid and the salts thereof; niacinamide; a cholesterol absorption inhibitor; a bile acid sequestrant anion exchange resin; a low density lipoprotein receptor inducer; clofibrate, fenofibrate, and gemfibrozil; vitamin B
[0118] (4) Pharmaceutical Formulations and Methods of Administration
[0119] In the methods of the present invention, the compounds of Formula I, Formula II, and Formula III can be delivered or administered to a mammal, e.g., a human patient or subject, alone, in the form of a pharmaceutically acceptable salt or hydrolyzable precursor thereof, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount. By a “therapeutically effective dose”, “therapeutically effective amount”, or, interchangeably, “pharmacologically acceptable dose” or “pharmacologically acceptable amount”, it is meant that a sufficient amount of the compound of the present invention, alternatively, a combination, for example, a compound of the present invention, which is substantially free of its (+) stereoisomer, and a pharmaceutically acceptable carrier, will be present in order to achieve a desired result, e.g., alleviating a symptom or complication of Type 2 diabetes.
[0120] The compounds of Formula I, Formula II, and Formula III that are used in the methods of the present invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of Formula I (or Formula II or Formula III) can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and can be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intratracheal administration. Moreover, the compound can be administered in a local rather than systemic manner, in a depot or sustained release formulation. In addition, the compounds can be administered in a liposome.
[0121] In addition, the compounds of Formula I, Formula II or Formula III can be formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration, or administered by the intramuscular or intravenous routes. The compounds can be administered transdermally, and can be formulated as sustained release dosage forms and the like.
[0122] Compounds of Formula I, Formula II, or Formula III can be administered alone, in combination with each other, or they can be used in combination with other known compounds (discussed supra). In pharmaceutical dosage forms, the compounds can be administered in the form of their pharmaceutically acceptable salts thereof. They can contain hydrolyzable moieties. They can also be used alone or in appropriate association, as well as in combination with, other pharmaceutically active compounds.
[0123] Suitable formulations for use in the present invention are found in
[0124] For injection, the compounds can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Preferably, the compounds of the present invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[0125] For oral administration, the compounds of Formula I, Formula II, or Formula III can be formulated readily by combining with pharmaceutically acceptable carriers that are well known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[0126] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions can be used, which can optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestiffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
[0127] Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. All formulations for oral administration should be in dosages suitable for such administration.
[0128] For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.
[0129] For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or from propellant-free, dry-powder inhalers. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0130] The compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles; and can contain formulator agents such as suspending, stabilizing and/or dispersing agents.
[0131] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
[0132] The compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols or other glycerides, all of which melt at body temperature, yet are solidified at room temperature.
[0133] In addition to the formulations described previously, the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
[0134] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds can be employed. Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs. In a presently preferred embodiment, long-circulating, i.e., stealth, liposomes can be employed. Such liposomes are generally described in Woodle, et al., U.S. Pat. No. 5,013,556, the teaching of which is hereby incorporated by reference. The compounds of the present invention can also be administered by controlled release means and/or delivery devices such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719; the disclosures of which are hereby incorporated by reference.
[0135] Certain organic solvents such as dimethylsulfoxide (DMSO) also can be employed, although usually at the cost of greater toxicity. Additionally, the compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various types of sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules can, depending on their chemical nature, release the compounds for a few hours up to over 100 days.
[0136] The pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
[0137] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a therapeutically effective amount. The amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
[0138] For any compound used in the method of the present invention, a therapeutically effective dose can be estimated initially from cell culture assays or animal models.
[0139] Moreover, toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD
[0140] The amount of active compound that can be combined with a carrier material to produce a single dosage form will vary depending upon the disease treated, the mammalian species, and the particular mode of administration. However, as a general guide, suitable unit doses for the compounds of the present invention can, for example, preferably contain between 100 mg to about 3000 mg of the active compound. A preferred unit dose is between 500 mg to about 1500 mg. A more preferred unit dose is between 500 to about 1000 mg. Such unit doses can be administered more than once a day, for example 2, 3, 4, 5 or 6 times a day, but preferably 1 or 2 times per day, so that the total daily dosage for a 70 kg adult is in the range of 0.1 to about 250 mg per kg weight of subject per administration. A preferred dosage is 5 to about 250 mg per kg weight of subject per administration, and such therapy can extend for a number of weeks or months, and in some cases, years. It will be understood, however, that the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound employed; the age, body weight, general health, sex and diet of the individual being treated; the time and route of administration; the rate of excretion; other drugs which have previously been administered; and the severity of the particular disease undergoing therapy, as is well understood by those of skill in the area.
[0141] A typical dosage can be one 10 to about 1500 mg tablet taken once a day, or, multiple times per day, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect can be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
[0142] It can be necessary to use dosages outside these ranges in some cases as will be apparent to those skilled in the art. Further, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with individual patient response.
[0143] (5) Protecting Groups
[0144] Certain compounds having the general structure of Formula I and II may require the use of protecting groups to enable their successful elaboration into the desired structure. Protecting groups can be chosen with reference to Greene, T. W., et al.,
[0145] Examples of suitable hydroxylprotecting groups are: trimethylsilyl, triethylsilyl, o-nitrobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, t-butyldiphenylsilyl, t-butyldimethylsilyl, benzyloxycarbonyl, t-butyloxycarbonyl, 2,2,2-trichloroethyloxycarbonyl, and allyloxycarbonyl. Examples of suitable carboxyl protecting groups are benzhydryl, o-nitrobenzyl, p-nitrobenzyl, 2-naphthylmethyl, allyl, 2-chloroallyl, benzyl, 2,2,2-trichloroethyl, trimethylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, 2-(trimethylsilyl)ethyl, phenacyl, p-methoxybenzyl, acetonyl, p-methoxyphenyl, 4-pyridylmethyl and t-butyl.
[0146] (6) Process
[0147] Processes for making the compounds of the present invention are generally depicted in Schemes 1 and 2 (and further described in the Examples):
[0148] According to Scheme 1, a substituted phenyl acetonitrile is converted to a substituted phenyl acetic acid. The substituted phenyl acetic acid is converted to an activated acid derivative (e.g., acid chloride), followed by halogenation at the alpha-carbon and esterification with an alcohol. The halogenated ester is treated with a substituted phenol (e.g., 3-trifluoromethylphenol), yielding an aryl ether, which is hydrolyzed to form a carboxylic acid derivative. The acid derivated is converted to an activated acid derivative and subsequently treated with a nucleophile (e.g., N-acetylethanolamine) to afford the desired product.
[0149] According to Scheme 2, a substituted phenyl acetic acid is converted to an activated acid derivative (e.g., acid chloride) followed by halogenation at the alpha-carbon. The activated acid portion of the molecule is reacted with a nucleophile (e.g., N-acetylethanolamine) to provide a protected acid. The halogenated, protected acid is treated with a substituted phenol (e.g., 3-trifluoromethylphenol), yielding the desired product.
[0150] The stereoisomers of the compounds of the present invention can be prepared by using reactants or reagents or catalysts in their single enantiomeric form in the process wherever possible or by resolving the mixture of stereoisomers by conventional methods, discussed supra and in the Examples. Some of the preferred methods include use of microbial resolution, resolving the diastereomeric salts formed with chiral acids or chiral bases and chromatography using chiral supports.
[0151] (7) Kits
[0152] In addition, the present invention provides for kits with unit doses of the compounds of Formula I, Formula II, or Formula III either in oral or injectable doses. In addition to the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the drugs in alleviating symptoms and/or complications associated with Type 2 diabetes as well as in alleviating hyperlipidemia and hyperuricemia. Preferred compounds and unit doses are those described herein above.
[0153] The compounds of Formula I, Formula II, or Formula III of the present invention can be readily prepared using the process set forth in Scheme 1, supra, and from the following examples.
[0154] This example relates to the preparation of Methyl Bromo-(4-chlorophenyl)-acetate.
[0155] The initial compound listed in Scheme 1, i.e., 4-chlorophenylacetic acid, is readily available from several commercial sources (e.g., Aldrich and Fluka).
[0156] A 5-L Morton reactor equipped with a magnetic stirrer, a pot temperature control, and addition funnel was vented through a gas scrubber and charged with p-chlorophenylacetic acid (720 gm, 4.2 moles) and SOCl
[0157] This example relates to the preparation of Methyl 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetate.
[0158] This step was similar to the same step in U.S. Pat. No. 3,517,050 with one exception, potassium t-butoxide was used in place of sodium methoxide to prevent generation of the corresponding methyl ether. A 5-L Morton reactor equipped with an overhead stirrer, a pot temperature detector, and addition funnel and under a nitrogen atmosphere was charged with methyl bromo-(4-chlorophenyl)-acetate (830 gm, 3.0 moles) and THF (600 ml). The reactor was cooled to 14±3° C. in an ice-water bath and then a similarly cooled solution of trifluoromethyl-m-cresol (530 gm, 3.3 moles) in 1.0 M potassium t-butoxide in THF (3.1 L, 3.1 moles) was added. The reaction proceeded exothermically with a typical temperature rise exceeding 250 C and the addition was controlled to maintain a temperature of 15°±2° C. and stirred at ambient temperature for 2 hours. HPLC was run on a Zorbax SB-C8 column at 300 C measuring 250×4.6 mm and 5μ particle size. The mobile phase was 60:40 (v:v) acetonitrile: 0.1% H
[0159] This example relates to the preparation of 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetic Acid
[0160] A 12-L Morton reactor with magnetic stirrer, pot temperature controller, a reflux condenser and under a nitrogen atmosphere was charged with methyl 4-chlorophenyl-(3-trifluoromethylphenoxy)-acetate (810 gm, 2.3 moles) and absolute ethanol (5.8 L) and heated with stirring to 57° C. to dissolve the solid. A solution of KOH (520 gm, 9.3 moles) in 0.98 L water was added. The solution was refluxed for 30 min. and solvent was stripped by a rotary evaporator to obtain 2.03 kg of a mixture of two nearly colorless liquids. These were dissolved in water (16 L) and treated with 16 gm neutral Norit, then filtered through a pad of infusorial earth retained on Whatman #1 filter paper. The pH of the filtrate was lowered from an initial range of 13 to a range of 1 to 2 by adding a total of 2.75 L of 3 M HCl (8.25 moles). A very sticky solid formed after the addition of the first 2.30 L of acid and ether (7 L) was added at this point. The two layers were separated and the organic layer was dried over MgSO
[0161] This example relates to the preparation of resolved enantiomers of 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetic Acid.
[0162] A 12-L open-top Morton reactor with an overhead stirrer was charged with 4-chlorophenyl-(3-trifluoromethyl-phenoxy)-acetic acid (350 gm, 1.06 moles) and isopropanol (4.0 L) and heated to 65°±3° C. A slurry of (−) cinchonidine (300 gm, 1.02 moles) in isopropanol (2.0 L) was added, rinsing all solid into the reactor with an additional 0.8 L of isopropanol. The temperature dropped from 65° to 56° C. and a transparent, orange solution ultimately formed and the mixture was held at 55°±5° C. for 2 hours. Fine crystals were collected by filtration through Whatman #1 filter paper, washing once with 0.7 L hot (55° C.) isopropanol. The crystals were dried for 16 hours at ambient temperature in a 12.6-L vacuum oven under a 5 LPM nitrogen flow. The dry solid weighed 0.37 kg and had an 80% enantiomeric excess (ee) of the (+) enantiomer. The enantiomeric excess was determined by HPLC using a 250×4.6 mm R,R-WhelkO-1 column at ambient temperature. Injected samples were 20 μl of 2 mg/ml solutions of the samples in ethanol. The column was eluted with 95:5:0.4, hexane:isopropanol:acetic acid at a flow of 1 ml/min. Detection was at 210 nm. The (+) enantiomer eluted at 7 to 8 min. and the (−) enantiomer at 11 to 13 min. The mother liquor dropped a second crop almost immediately that was filtered, washed, and dried to afford 0.06 kg salt that has a 90% ee of the (−)-enantiomer. Similarly third, fourth and fifth crops weighing 0.03 kg, 0.03 kg and 0.7 kg, respectively, were obtained; with (−) enantiomer excesses of 88%, 89% and 92%, respectively.
[0163] The crude (+) salt (320 gm) was recrystallized from a mixture of ethanol (5.9 L) methanol (1.2 L). The mixture was heated with overhead stirring to dissolve, cooled at ambient temperature for 16 hours, filtered and washed twice with 0.20 L of 5:1 (v:v) ethanol:methanol. The crystals were dried to obtain 0.24 kg of the (+) enantiomer that had an ee of 97%. This corresponded to an 80% recovery of this isomer. The resolved salt was suspended in a mixture of ether (6.5 L) and water (4.0 L) with overhead stirring. The pH was lowered to 0-1 as measured by pH indicating strips with a solution of concentrated H
[0164] The combined, crude (−) salt (200 gm) was recrystallized from isopropanol (3.1 L). The mixture was heated to dissolve almost all of the solid and fast-filtered to remove insoluble solids. The mixture was then cooled with stirring at ambient temperature for 16 hours, filtered, washed, and dried to obtain 0.16 kg of the (−) enantiomer that has an ee of 97%. This corresponds to a 49% recovery of this isomer. The (−) enantiomer of the acid was isolated in the same manner as described above for the (+) acid. The resolved salt was suspended in ether and water, the pH lowered with concentrated H
[0165] A. Preparation of (−) 4-Chlorophenyl-(3-tritluoromethylphenoxy)-acetyl Chloride
[0166] A 2-L evaporation flask with magnetic stirrer, Claissen adapter, pot thermometer and a reflux condenser routed to a gas scrubber was charged with (−) 4-chlorophenyl-(3-trifluoromethylphenoxy)-acetic acid (143 g, 0.42 mole based on 97% purity) and CHCl
[0167] B. Preparation of (+) 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetyl Chloride
[0168] A 3-L evaporation flask with magnetic stirrer, Claissen adapter, pot thermometer and a reflux condenser routed to a gas scrubber was charged with (+) 4-chlorophenyl-(3-trifluoromethylphenoxy)-acetic acid (131 g, 0.37 mole) and CHCl
[0169] A. Preparation of (−) 2-Acetamidoethyl 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetate
[0170] A 3-L round-bottom flask with magnetic stirrer, pot thermometer, under a nitrogen atmosphere and in an ice-water bath was charged with DMF (420 ml), pyridine (37 ml, 36 g, 0.46 mole) and N-acetoethanolamine (39 ml, 43 g, 0.42 mole). The mixture was cooled to 0° to 5° C. and a solution of crude (−) 4-chlorophenyl-(3-trifuoromethylphenoxy)-acetyl chloride (151 gm, 0.42 mole based on 100% yield of previous step) in ether (170 ml) was added over a 40 min. period so as to maintain the pot temperature below 13° C. The mixture was stirred at ambient temperature for 16 hours and dissolved by adding water (960 ml) followed by ethyl acetate (630 ml). The water addition proceeded exothermically raising the temperature from 24° to 34° C. Ethyl acetate addition caused a temperature drop to 30° C. The layers were separated and the aqueous phase extracted once with ethyl acetate (125 ml). The combined organic layers were extracted once with 7% (w:w) aqueous NaHCO
[0171] B. Preparation of (+) 2-Acetamidoethyl 4-Chlorophenyl-(3-trifluoromethylphenoxy)-acetate
[0172] A 3-L round-bottom flask with magnetic stirrer, pot thermometer, under a nitrogen atmosphere and in an ice-water bath was charged with DMF (365 ml), pyridine (33 ml, 32.3 g, 0.41 mole) and N-acetoethanolamine (34 ml, 38.1 g, 0.37 mole). The mixture was cooled to 0° to 5° C. and a solution of crude (+) 4-chlorophenyl-(3-trifluoromethylphenoxy)-acetyl chloride (139 gm, 0.37 mole based on 100% yield of previous step) in ether (155 ml) was added over a 25 min. period so as to maintain the pot temperature below 13° C. The mixture was stirred at ambient temperature 40 hours and dissolved by adding water (850 ml) followed by ethyl acetate (550 ml). The water addition proceeded exothermically raising the temperature from 24° to 34° C. Ethyl acetate addition caused a temperature drop to 30° C. The layers were separated and the aqueous phase extracted once with ethyl acetate (110 ml). The combined organic layers were washed twice with 55 ml portions of water and then five times with 55 ml portions of 25% (w:w) aqueous NaCl and dried over 30 g MgSO
[0173] This example relates to the inhibition of cytochrome P450 2C9 (CYP2C9) by the compounds of the present invention.
[0174] Tolbutamide hydroxylation activity (100 μM C-tolbutamide; 1 mM NADPH) was assayed in pooled human liver microsomes (0.6 mg protein/ml)) for 60 minutes at 37° C. both with and without test compounds. Racemic halofenic acid, (−) halofenic acid and (+) halofenic acid were tested (0.25 μM to 40 μM. As shown in
[0175] This example relates to the time course of glucose-lowering for the compounds of the present invention.
[0176] A. Material and Methods
[0177] Male, 9-10 weeks old, C57BL/6J ob/ob mice were purchased from The Jackson Laboratory (Bar Harbor, Me., USA). Animals were housed (4-5 mice/cage) under standard laboratory conditions at 22° C. and 50% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal. Mice that had non-fasting plasma glucose levels between 300 and 500 mg/dl were used. Each treatment group consisted of 10 mice that were distributed so that the mean glucose levels were equivalent in each group at the start of the study. Mice were dosed orally once by gavage with either vehicle, racemic halofenate (250 mg/kg), (−) halofenate (250 mg/kg) or (+) halofenate (250 mg/kg). All compounds were delivered in a liquid formulation contained 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 2.7% (w/v) methylcellulose. The gavage volume was 10 ml/kg. Blood samples were taken at 1.5, 3, 4.5, 6, 7.5, 9 and 24 hour after the dose and analyzed for plasma glucose. Plasma glucose concentrations were determined calorimetrically using glucose oxidase method (Sigma Chemical Co, St. Louis, Mo., USA). Significance difference between groups (comparing drug-treated to vehicle-treated or between drug-treated groups) was evaluated using Student unpaired t-test.
[0178] B. Results
[0179] As illustrated in
[0180] This example relates to the Glucose lowering activity of the compounds of the present invention.
[0181] A. Materials and Methods
[0182] Male, 8-9 weeks old, C573L/6J ob/ob mice were purchased from The Jackson Laboratory (Bar Harbor, Me., USA). Animals were housed (4-5 mice/cage) under standard laboratory conditions at 22° C. and 50% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal. Mice that had non-fasting plasma glucose levels between 300 and 520 mg/dL were used. Each treatment group consisted of 10 mice that were distributed so that the mean glucose levels were equivalent in each group as the start of the study. Mice were dosed orally by gavage once a day for 5 days with either vehicle, racemic halofenate (250 mg/kg), (−) halofenate (125 and 250 mg/kg) or (+) halofenate (125 and 250 mg/kg). Racemic halofenate was delivered in 2.7% (w/v) methylcellulose and both the (−) enantiomer and (+) enantiomer were delivered in a liquid formulation contained 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 2.7% (w/v) methylcellulose. The gavage volume was 10 ml/kg. Blood samples were taken at 3, 6, 27, 30 and 120 hour after the first dose and analyzed for plasma glucose and insulin. The animals were fasted overnight (14 hours) before the 120 hours sampling. Plasma glucose concentrations were determined calorimetrically using glucose oxidase method (Sigma Chemical Co, St. Louis, Mo., USA). Plasma insulin concentrations were determined by using the Rat Insulin RIA Kit from Linco Research Inc. (St. Charles, Mo., USA). Significance difference between groups (comparing drug-treated to vehicle-treated) was evaluated using Student unpaired t-test.
[0183] B. Results
[0184] As illustrated in
[0185] This example relates to the improvement in Insulin Resistance and Impaired Glucose Tolerance for the compounds of the present invention.
[0186] A. Materials and Methods
[0187] Male, 8-9 weeks old Zucker fa/fa rats (Charles River,) were housed (2-3 rats/cage) under standard laboratory conditions at 22° C. and 50% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, rats were assigned to 6 groups based on body weight. Each treatment group consisted of 8 rats. Rats were dosed orally once by gavage with either vehicle, racemic halofenate (100 mg/kg), (−) halofenate (50 or 100 mg/kg) or (+) halofenate (50 or 100 mg/kg). All compounds were delivered in a liquid formulation contained 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 2.7% (w/v) methylcellulose. The gavage volume was 10 ml/kg. All rats received an oral glucose challenge (1.9 g/kg) 5.5 hours after the treatment and 4 hours after withdrawal of the food. Blood samples were taken at 0, 15, 30, 60, 90, 120, and 180 minutes following the glucose challenge for plasma glucose measurement. The vehicle, (−) halofenate (50 mg/kg) and (+) halofenate (50 mg/kg) groups were subjected to an insulin challenge following daily gavage of the respective treatments for 5 days. On day 5, rats received the intravenous insulin (0.75 U/kg) 5.5 hours after the last dose and 4 hours after withdrawal of the food. Blood samples were taken at 3, 6, 9, 12, 15 and 18 minutes following the insulin injection for plasma glucose measurement. Plasma glucose concentrations were determined colorimetrically using glucose oxidase method (Sigma Chemical Co, St. Louis, Mo., U.S.A.). Significance difference between groups (comparing drug-treated to vehicle-treated or between drug-treated groups) was evaluated using Student unpaired t-test.
[0188] B. Results
[0189] As illustrated in
[0190] Changes in insulin sensitivity were assessed by monitoring the fall in glucose after an intravenous injection of insulin. The slope of the line is a direct indication of the insulin sensitivity of the test animal. As shown in
[0191] This example relates to the lipid lowering activity of the compounds of the present invention.
[0192] A. Materials and methods
[0193] Male Zucker diabetic fatty (ZDF) rats were obtained from GMI Laboratories (Indianapolis, Ind.) at 9 weeks of age. Vehicle or enantiomers of halofenate administered by oral gavage on a daily basis starting at 74 days of age. Initial blood samples were obtained for analysis one day before treatment and at the indicated times in the treatment protocol. Blood was analyzed for plasma triglyceride and cholesterol by standard techniques.
[0194] B. Results
[0195] In experiment I animals received a dose of 25 mg/kg/day. As shown in
[0196] This example relates to the glucose lowering activity of (±) halofenate analogs and (−) halofenate analogs.
[0197] A. Materials and methods
[0198] Male, 8-9 weeks old, C57BL/6J ob/ob mice were purchased from The Jackson Laboratory (Bar Harbor, Me., USA). Animals were housed (4-5 mice/cage) under standard laboratory conditions at 22±3° C. temperature and 50±20% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal. Mice that had non-fasting plasma glucose levels between 250 and 500 mg/dl were used. Each treatment group consisted of 8-10 mice that were distributed so that the mean glucose levels were equivalent in each group at the start of the study. Mice were dosed orally by gavage once a day for 1-3 days with either vehicle, (−) halofenic acid, (±) analog 14, 29, 33, 34, 35, 36, 37, or 38 at 125 mg/kg or (−) analog 29, 36, 37 or 38 at 150 mg/kg. Compounds were delivered in a liquid formulation containing 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 0.9% (w/v) methylcellulose. The gavage volume was 10 ml/kg. Blood samples were taken at 6 hours after the each dose and analyzed for plasma glucose. Food intake and body weight were measured daily. Plasma glucose concentrations were determined calorimetrically using glucose oxidase method (Sigma Chemical Co, St. Louis, Mo., USA). Significant difference between groups (comparing ding-treated to vehicle-treated) was evaluated using the Student unpaired t-test.
[0199] B. Results
[0200] As illustrated in Table 2, compounds were evaluated in 5 different experiments. Single dose (−) halofenic acid significantly reduced plasma glucose concentrations at 6 hours. Analog 14 significantly lowered plasma glucose concentrations at 6, 30 and 54 hours. Analog 33 significantly lowered plasma glucose concentrations at 6 and 54 hours. Analog 29 and 38 significantly lowered plasma glucose concentrations at 6, 30 and 54 hours. Analog 35 and 36 significantly lowered plasma glucose concentrations at 30 and 54 hours. Analog 37 significantly lowered plasma glucose concentrations at 54 hours. Single dose (−) analogs 29, 36, 37 and 38 significantly reduced plasma glucose concentrations at 6 hours. Compound treatments did not affect the animal's food intake and body weight.
TABLE 1 (±) and (−) Halofenate analogs. Compounds described in reference to Formula II. Formula II
Cmpd No. X CX R halofenic Cl CF H acid 14 F CF (CH 29 Br CF (CH 33 Cl CF (CH 35 Cl CF (CH 36 Cl CF (CH 37 Cl CF CH 38 Cl CF CH
[0201]
TABLE 2 Glucose-lowering Activities of (±)Halofenate and (−)Halofenate Analogs 6 hours 30 hours 54 hours Predose P P P Glucose Glucose VALUE Glucose VALUE Glucose VALUE (mg/dl) (mg/dl) vs. veh (mg/dl) vs. veh (mg/dl) vs. veh Vehicle 313 ± 18 303 ± 19.8 NA NA (−)halofenic 312.9 ± 17.7 163.8 ± 11.8 0.0011 NA NA acid Vehicle 360.2 ± 27.8 405.8 ± 25.8 356.0 ± 27.6 386.1 ± 20.6 (±)Analog 14 361.0 ± 17.1 328.9 ± 34.1 0.0444 267.0 ± 21.3 0.0099 293.0 ± 29.4 0.0092 Vehicle 291.6 ± 18.5 363.0 ± 25.1 340.8 ± 30.0 351.5 ± 23.8 (±)Analog 33 292.0 ± 19.1 227.5 ± 13.2 0.0001 298.0 ± 15.3 0.1119 286.6 ± 9.9 0.0125 Vehicle 387.1 ± 14.3 371.5 ± 24.2 326.2 ± 22.5 374.0 ± 37.9 (±)Analog 29 387.1 ± 16.0 299.7 ± 24.5 0.0259 237.4 ± 14.9 0.0020 293.3 ± 9.7 0.0268 (±)Analog 35 387.0 ± 18.0 319.6 ± 26.7 0.0834 276.8 ± 17.6 0.0504 286.2 ± 31.5 0.0458 (±)Analog 37 387.4 ± 18.8 345.4 ± 19.7 NS 312.5 ± 21.7 NS 285.1 ± 14.7 0.0210 Vehicle 329.6 ± 16.1 361.8 ± 23.2 346.5 ± 24.6 379.2 ± 24.4 (±)Analog 36 329.7 ± 17.6 300.5 ± 27.3 0.0522 249.7 ± 8.6 0.0008 272.2 ± 18.4 0.0013 (±)Analog 38 329.4 ± 18.9 303.2 ± 18.2 0.0312 245.6 ± 15.6 0.0014 243.1 ± 10.6 0.0000 Vehicle 373.0 ± 13.6 405.8 ± 33.7 NA NA (−)Analog 36 373.2 ± 15.5 281.1 ± 18.2 0.0019 NA NA (−)Analog 37 373.4 ± 16.1 271.7 ± 22.5 0.0018 NA NA (−)Analog 38 373.4 ± 16.1 251.2 ± 23.6 0.0007 NA NA (−)Analog 29 372.2 ± 17.1 333.5 ± 16.1 0.0353 NA NA
[0202] This example relates to a comparison between the activities of (−) halofenate and (+) halofenate.
[0203] A. Materials and methods
[0204] Male 8-9 week old ZDF rats were purchased from Genetic Models, Inc. (Indianapolis, Ind.). Animals were housed (3 rats/cage) under standard laboratory conditions at 22±3° C. temperature and 50±20% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal. Rats that had 4-hour fasting plasma glucose levels between 200 and 500 mg/dL were used. Each treatment group consisted of 8-10 rats that were distributed so that the mean glucose levels were equivalent in each group at the start of the study. Rats were dosed orally by gavage once a day for 3 days with either vehicle, (−) halofenate or (+) halofenate at 50 mg/kg. Compounds were delivered in a liquid formulation containing 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 0.9% (w/v) methylcellulose. The gavage volume was 5 ml/kg. Blood samples were taken at 5 hours post dose on day 2 and 3. Plasma glucose concentrations were determined colorimetrically using glucose oxidase method (Sigma Chemical Co, St. Louis, Mo., USA). Significant difference between groups (comparing drug-treated to vehicle-treated) was evaluated using the Student unpaired t-test.
[0205] B. Results
[0206] Oral administration of (−) halofenate at 50 mg/kg significantly reduced plasma glucose concentrations, while (+) halofenate at the same dosage levels failed to reduce plasma glucose concentrations as compared to vehicle-treated animals (
[0207] This example relates to a pharmacokinetic study of (i) halofenate and (−) halofenate.
[0208] A. Materials and methods
[0209] Male 225-250 g SD rats were purchased from Charles River. Animals were housed (3 rats/cage) under standard laboratory conditions at 22±3° C. temperature and 50±20% relative humidity, and were maintained on a diet of Purina rodent chow and water ad libitum. A catheter was placed in the left carotid artery under sodium pentobarbital (50 mg/kg i.p.) and animals were allowed to recover for 2 days before treatment. Single dose of (±) halofenate or (−) halofenate at 50 mg/kg were administered by oral gavage. Compounds were delivered in a liquid formulation containing 5% (v/v) dimethyl sulfoxide (DMSO), 1% (v/v) tween 80 and 0.9% (w/v) methylcellulose. The gavage volume was 5 ml/kg. Blood samples were collected at 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 and 120 hours post dose. The plasma samples were analyzed for each enantiomeric acid ((−) halofenic acid and (+) halofenic acid) by a chiral specific HPLC assay, since the esters are prodrugs, which are designed to convert to their respective enantiomeric acids in vivo.
[0210] B. Results
[0211] After oral administration of (±) halofenate, both (−) halofenic acid and (+) halofenic acid were detected in the plasma samples. As shown in table 3, it appeared that the two enantiomeric acids had different dispositional profiles. The elimination of (−) halofenic acid was much slower than (+) halofenic acid. As a result, the AUC of (−) halofenic acid was significantly higher than the AUC for (+) halofenic acid, 4708.0 vs. 758.0 fig-h/mL and the terminal half-life was 46.8 vs. 14.3 hours.
[0212] After oral administration of (−) halofenate, the dispositional profile of (−) halofenic acid was basically identical to the administration of (±) halofenate as the terminal half-life is the same (Table 2). The Cmax and AUC of (−) halofenic acid were proportionally higher simply due to higher amount of (−) halofenate administered (Table 3). (+) Halofenic acid was also detected in the plasma but the concentration was much lower than (−) halofenic acid. It is speculated that (+) halofenic acid was formed in vivo since the terminal half-life (T
[0213] These results suggest the use of (−) halofenate is more desirable since the AUC of (−) halofenic acid was significantly higher than the AUC for (+) halofenic acid.
TABLE 3 Pharmacokinetic Analysis of(−) Halofenate (− Enantiomer) and (+) Halofenate (+ Enantiomer) Drug administered (-) Halofenate (n = 3) (±) Halofenate (n = 1) Enantiomer − + − + Dose administered* 50 mg/kg 0 (metabolite) 25 mg/kg 25 mg/kg C 114.6 ± 29.7 2.4 ± 0.5 65.2 30.5 T 8 − 12 6 − 12 12 6 AUC (μg · h/mL) 7159 ± 1103 164.3 ± 79.3 4708 758 T 46.4 ± 4.7 41.7 ± 11.8 46.8 14.3
[0214]
TABLE 4 Plasma Concentrations of (−) halofenic acid and (+) halofenic acid following a single dose of (−) halofenate. Compound Analyzed (μg/mL) (−) halofenic acid (+) halofenic acid Time (hour) Rat 8 Rat 9 Rat 11 Rat 8 Rat 9 Rat 11 0 BQL BQL BQL BQL BQL BQL 1 81.2 23.7 61.0 1.12 BQL BQL 2 100.1 30.4 87.8 1.27 BQL 1.09 4 122.3 36.9 94.5 1.67 BQL 1.95 6 128.3 56.5 116.3 2.96 BQL 1.73 8 128.2 79.0 127.8 2.58 BQL 2.06 12 135.3 80.6 104.8 2.85 2.23 2.08 24 82.5 73.1 66.5 2.22 1.29 1.86 48 56.2 44.5 47.1 1.64 1.03 1.14 72 39.7 37.4 30.8 1.25 BQL BQL 96 31.1 N/A 24.6 BQL N/A BQL 120 20.3 N/A N/A BQL N/A N/A
[0215] This example relates to the prevention of the development of diabetes and the alleviation of hypertriglyceridemia by (−) halofenate.
[0216] A. Materials and methods
[0217] Male, 4 weeks old, C57BL/6J db/db mice were purchased from The Jackson Laboratory (Bar Harbor, Me., USA). Animals were housed (5 mice/cage) under standard laboratory conditions at 22±3° C. temperature and 50±20% relative humidity, and were maintained on a powder diet of Purina rodent chow (#8640) and water ad libitum. Prior to treatment, blood was collected from the tail vein of each animal for plasma glucose, insulin and triglyceride concentrations. Mice were distributed so that the mean glucose levels and body weight were equivalent in each group at the start of the study. The control group (20 mice) was put on powder chow mixed with 5% sucrose and the treatment group (20 mice) was put on powder chow mixed with 5% sucrose and (−) halofenate. The amount of (−) halofenate in the chow was adjusted continuously according the animal's body weight and food intake to meet the target dosage of 150 mg/kg/day. Blood samples were taken at 8-10 AM once a week for 9 weeks under non-fasting condition. Food intake and body weight were measured every 1-3 days. Plasma glucose and triglyceride concentrations were determined calorimetrically using kits from Sigma Chemical Co (No. 315 and No. 339, St. Louis, Mo., USA). Plasma insulin levels were measured using RIA assay kit purchased from Linco Research (St. Charles, Mo.). Significant differences between groups (comparing drug-treated to vehicle-treated) was evaluated using Student unpaired t-test.
[0218] B. Results
[0219] C57BL/6J db/db mice at 4 weeks of age are in a pre-diabetic state. Their plasma glucose concentrations are normal, but the plasma insulin concentrations are significantly elevated. As illustrated in
[0220]
[0221] This example describes the preparation of (−) 2-Acetamidoethyl 4-Chlorophenyl-(3-trifluoro methylphenoxy)-acetate ((−) halofenate).
[0222] 4-Chlorophenylacetic acid was combined with 1,2-dichloroethane and the resulting solution was heated to 45° C. Thionyl chloride was added to the reaction mixture, which was heated at 60° C. for 18 hours. The reaction was allowed to cool to room temperature and was then added slowly to a solution of N-acetylethanolamine in dichloromethane. After stirring 30 min., the reaction was quenched with aqueous potassium carbonate and sodium thiosulfate. The organic layer was washed with water, dried over magnesium sulfate and filtered. Removal of the solvent by rotary evaporation provided N-acetylaminoethyl 2-bromo-2-(4-chlorophenyl)acetate as an oil.
[0223] 3-Hydroxybenzotrifluoride was added to a solution of potassium hydroxide in isopropanol. N-acetylaminoethyl 2-bromo-2-(4-chlorophenyl)acetate in isopropanol was added to the isopropanol/phenoxide solution and stirred at room temperature for 4 hours. The isopropanol was removed by vacuum distillation, and the resulting slush was dissolved in ethyl acetate and washed twice with water and once with brine. After drying over magnesium sulfate and filtration; the solvent was removed to give crude product as an oil. The crude product was dissolved in hot toluene/hexanes (1:1 v/v) and cooled to between 0 and 10° C. to crystallize the product. The filter cake was washed with hexanes/toluene (1:1 v/v) and then dried under vacuum at 50° C. The isolated solid was dissolved in hot 1:6 (v/v) isopropanol in hexanes. After cooling, the pure racemic 2-Acetamidoethyl 4-Chlorophenyl-(3-trifluoro methylphenoxy)-acetate formed as a crystalline solid. The solid was collected by filtration, the filter cake washed with 1:6 (v/v) isopropanol in hexanes and dried under vacuum at 50° C.
[0224] The racemic compound was dissolved in a solution of 20% isopropanol (IPA) and 80% hexane at 2.5% (wt/wt). The resulting solution was passed over a Whelk-O R,R Chiral Stationary Phase (CSP) in continuous fashion until >98% ee extract could be removed. The solvent was evaporated from the extract under reduced pressure to provide (−) 2-Acetamidoethyl 4-Chlorophenyl-(3-trifluoro methylphenoxy)-acetate. (The Simulated Moving Bed resolution was conducted by Universal Pharm Technologies LLC of 70 Flagship Drive, North Andover, Mass. 01845.)
[0225] This example relates to the lowering of plasma uric acid levels through the administration of (−) halofenate.
[0226] A. Materials and methods
[0227] Male SD rats, weight 275-300 g were purchased from Charles River. Animals were housed (3 rats/cage) under standard laboratory conditions at 22±3° C. temperature and 50±20% relative humidity, and were maintained on a powder diet of Purina rodent chow (#8640) and water ad libitum. To establish a hyperuricemic state, animals were put on a diet containing 2.5% (w/w) of oxonic acid (Sigma Chemical Co, St. Louis, Mo., USA) throughout the experiment. Oxonic acid elevates plasma uric acid by inhibiting uricase. Rats were screened for plasma uric acid levels 3 days after they were placed on the diet, and those that had extreme plasma uric acid levels were excluded. Rats were assigned to one of three groups and the mean uric acid levels were equivalent in each group. Rats were dosed orally by gavage once a day for 3 days with either vehicle, (−) halofenate or (+) halofenate at 50 mg/kg. On the 4
[0228] B. Results
[0229] As shown in
[0230] This example relates to the inhibition of cytochrome P450 isoforms by the compounds of the present invention.
[0231] A. Materials and methods
[0232] The following probe substrates were used to investigate the inhibitory potential of the test article on the cytochrome P450 isoforms 1A2, 2A6, 2C9, 2Cl9, 2D6, 2E1 and 3A4: 100 μM phenacetin (CYP1A2), 1 μM coumarin (CY)2A6), 150 μM tolbutamide (CYP2C9), 50 μM S-mephenytoin (CYP2C19), 16 μM dextromethorphan (CYP2D6), 50 μM chlorzoxazone (CYP2E1), and 80 μM testosterone (CYP3A4). The activity of each isoform was determined in human hepatic microsomes in the presence and absence of the test article.
[0233] Unless otherwise noted, all incubations were conducted at 37° C. The sample size was N=3 for all test and positive control conditions and N=6 for all vehicle control conditions. (−) Halofenic acid (MW=330) was prepared at room temperature as 1000× stocks in methanol, then diluted with Tris buffer to achieve final concentrations of 0.33, 1.0, 3.3, 10 and 33.3 μM, each containing 0.1% methanol. A vehicle control (VC) consisting of microsomes and substrate in Tris buffer containing 0.1% methanol without the test article was included for all experimental groups. Positive control (PC) mixtures were prepared using the following known CYP450 inhibitors: 5 μM furafylline (CYP1A2), 250 μM tranylcypromine (CYP2A6), 50 μM sulfaphenazole (CYP2C9), 10 μM omeprazole (CYP2Cl9), 1 μM quinidine (CYP2D6), 100 μM 4-methylpyrazole (CYP2E1), and 5 μM ketoconazole (CYP3A4). A chromatographic interference control (CIC) was included to investigate the possibility of chromatographic interference by the test article and its metabolites. The test article (at 33.3 μg/mL) was incubated with 1× microsomal protein, 1× NRS, and 10 μL of an appropriate organic for an appropriate time period as described below.
[0234] Stable, frozen lots of pooled adult male and female hepatic microsomes prepared by differential centrifugation of liver homogenates were used in this study (see, e.g., Guengerich, F. P. (1989). Analysis and characterization of enzymes. In
[0235] After each incubation, the activities of the P450 isoforms were determined by measuring the rates of metabolism for the respective probe substrates. The metabolites monitored for each probe substrate were as follows: acetaminophen for CYP1A2; 7-hydroxycoumarin for CYP2A6; 4-hydroxytolbutramide for CYP2C9; 4-hydroxymephenytoin for CYP2Cl9; dextrorphan for CYP2D6; 6-hydroxychlorzoxazone for CYP2E1; and 6β-hydroxytestosterone for CYP3A4. Activities were analyzed using HPLC (In Vitro Technologies, Inc., Baltimore, Md.).
[0236] Inhibition was calculated using the following equation:
[0237] Percent inhibition data for the test article was presented in a tabular format. Descriptive statistics (mean and standard deviation) of each test article concentration were calculated, then presented to show inhibitory potency. IC
[0238] Measures of time, temperature, and concentration in this example are approximate.
[0239] B. Results
[0240] The results for each of the 7 isoforms of cytochrome P450, expressed as metabolic activity and percentage of inhibition, are presented in Tables 5-8. (−) Halofenic acid inhibited 4-hydroxytolbutamide production (CYP2C9, IC50=11 μM) and also inhibited 4-hydroxymephenytoin production (CYP2C19) at the 10 and 33 μM dose levels. Inhibition of other CYP450 isoforms was not observed. It should be noted that the IC50 for CYP2C9 in this experiment was approximately three times that reported in Example 7 (11 μM as compared to 3.6 μM). This result is most likely due, at least in part, to the use of a lower purity (−) halofenic acid (lower ee) in Example 7.
TABLE 5 Hepatic microsomal activities of phenacetin (CYP1A2) and coumarin (CYP2A6) in male and female human microsomes incubated with (−) halofenic acid at doses of 0.33, 1.0, 3.3,10, and 33.3 μM Coumarin Phenacetin 7-HC Control/ AC Production % Production Test Conc. (pmol/mg In- (pmol/mg % Article (μM) protein/min) hibition protein/min) Inhibition CIC 33.3 0.00 ± 0.00 NA 0.00 ± 0.00 NA VC 0.1% 118 ± 2 0 32.0 ± 1.4 0 FUR 5 54.5 ± 1.3 54 NA NA TRAN 250 NA NA 0.00 ± 0.00 100 (−) 0.33 116 ± 2 1 33.3 ± 0.7 −4 halofenic 1.0 118 ± 2 0 32.6 ± 0.7 −2 acid 3.3 119 ± 2 −1 32.1 ± 0.7 0 10 119 ± 2 −1 33.1 ± 0.7 −3 33.3 119 ± 2 −1 32.3 ± 0.7 −1 IC NA NA
[0241]
TABLE 6 Hepatic microsomal activities of tolbutamide (CYP2C9) and S- mephenytoin (CYP2C19) in male and female human microsomes incubated with (−) halofenic acid at doses of 0.33, 1.0, 3.3, 10, and 33.3 μM Tolbutamide S-Mephenytoin 4-OH TB 4-OH ME Control/ Production % Production Test Conc. (pmol/mg In- (pmol/mg % Article (μM) protein/min) hibition protein/min) Inhibition CIC 33.3 0.00 ± 0.00 NA 0.00 ± 0.00 NA VC 0.1% 43.0 ± 1.4 0 3.17 ± 0.29 0 OMP 10 NA NA 1.58 ± 0.05 50 SFZ 50 BQL ˜100 NA NA (−) 0.33 41.0 ± 0.9 5 3.03 ± 0.03 4 halofenic 1.0 38.6 ± 0.5 10 3.01 ± 0.07 5 acid 3.3 34.2 ± 0.2 21 2.69 ± 0.12 15 10 22.7 ± 0.6 47 2.43 ± 0.09 23 33.3 12.7 ± 0.2 71 1.80 ± 0.07 43 IC ˜11.335 μM >33.3 μM
[0242]
TABLE 7 Hepatic microsomal activities of dextromethorphan (CYP2D6) and chlorzoxazone (CYP2E1) in male and female human microsomes incubated with (−) halofenic acid at doses of 0.33, 1.0, 3.3, 10, and 33.3 μM Chlorzoxazone Dextromethorphan 6-OH CZX Control/ DEX Produc- % Production Test Conc. tion (pmol/mg In- (pmol/mg % Article (μM) protein/min) hibition protein/min) Inhibition CIC 33.3 0.00 ± 0.00 NA 0.00 ± 0.00 NA VC 0.1% 111 ± 6 0 246 ± 5 0 4-MP 100 NA NA BQL ˜100 QUIN 1 BQL ˜100 NA NA (−) 0.33 107 ± 4 3 238 ± 4 3 halofenic 1.0 110 ± 2 1 244 ± 1 1 acid 3.3 104 ± 3 6 239 ± 4 3 10 107 ± 1 4 244 ± 6 1 33.3 106 ± 4 5 239 ± 4 3 IC NA NA
[0243]
TABLE 8 Hepatic microsomal activities ol testosterone (CYP3A4) in male and female human microsomes incubated with (−) halofenic acid at doses of 0.33, 1.0, 3.3, 10, and 33.3 μM Testosterone Control/ 6β-OHT Production Test Conc (pmol/mg Article (μM) protein/min) % Inhibition CIC 33.3 0.00 ± 0.00 NA VC 0.1% 1843 ± 9 0 KTZ 5 32.4 ± 0.2 98.2 (−) 0.33 1816 ± 12 1.5 halofenic 1.0 1851 ± 14 0 acid 3.3 1810 ± 3 1.8 10 1819 ± 4 1.3 33.3 1816 ± 6 1.5 IC NA
[0244] This example illustrates the preparation of various prodrug esters of the (−) enantiomer of CPTA. Representative coupling methods are provided (Methods 1-5).
[0245] Method 1—Preparation of the Cesium Salt of (−)-CPTA by Sub-Stoichiometric Addition of Base and Reaction With Benzyl Bromide. Preparation of (−)-benzyl 4-chloro-alpha-(3-trifluoromethylphenoxy)benzeneacetate
[0246] To 100.2 mg (0.303 mmol) of (−)-CPTA was added 0.82 ml MeOH and 0.2 ml H
[0247] Method 2: HTU-Mediated Coupling. Preparation of (−)-2-ethoxycarbonyl-aminoethyl 4-chloro-alpha-(3trifluoromethylphenoxy)benzeneacetate
[0248] To a cooled (0-5° C.) solution of (−)-4-chloro-alpha-(3-trifluoromethylphenoxy)benzeneacetic acid (0.100 g, 0.302 mmol) and ethyl N-(2-hydroxyethyl)carbamate (44 mg, 0.333 mmol) in methylene chloride was added pyridine (36 mg, 0.454 mmol) from a 25% stock solution in methylene chloride. HATU (115 mg, 0.302 mmol) was added in one portion and after 20 min the suspension was allowed to warm to room temperature. The reaction was monitored by LC/MS and was complete after 3.5 h. The product mix was partitioned between methylene chloride (5 ml) and 0.2 M NaHCO
[0249] Method 3: p-Toluenesulfonic Acid Catalyzed Esterformation. Preparation of (−)-2-methoxyethyl 4-chloro-alpha-(3-trifluoromethylphenoxy)benzeneacetate
[0250] To 100 mg (0.302 mmol) of (−)—CPTA was added 150 mg (1.97 mmol) of methoxyethanol, 4 ml benzene and 16.1 mg of p-toluenesulfonic acid. The solution was refluxed for 3 h with use of a Dean-Stark trap for removal of the benzene-water azeotrope. The reaction was cooled, evaporated under reduced pressure and partitioned between methylene chloride (5 ml) and 0.2M Na
[0251] Method 4: Conversion of (−)-CPTA to Acid Chloride Via Oxalyl Chloride/DMF and Reaction With Alcohols. Preparation of (−)-propargyl 4-chloro-alpha-(3-trifluoromethylphenoxy)benzeneacetate.
[0252] (−)-CPTA (100 mg, 0.302 mmol) was added to a white suspension prepared by the dropwise addition of oxalyl chloride (43 mg, 0.336 mmol) to DMW (33 mg, 0.453 mmol) in 1 ml of a 60/40 acetonitrile/methylene chloride. The reaction was stirred for 30 min at 0° C. to complete the formation of acid chloride. A solution of propargyl alcohol (18.5 mg, 0.330 mmol) and pyridine (52.4 mg, 0.663 mmol) in 200 μl dry methylene chloride was added dropwise to the cooled solution of the acid chloride. The temperature was maintained at 0° C. for the first hour and then allowed to warm to ambient. After 3 h the reaction was complete (monitored by LC/MS). The reaction mixture was partitioned between methyl chloroform and 0.2 M Na
[0253] Method 5: In situ Generation of the Silver (I) Salt of (−)-CPTA by Sub-Stoichiometric Addition of Ag
[0254] To a cooled (0-5° C.) solution of (−)-4-chloro-alpha-(3-trifluoromethylphenoxy)benzeneacetic acid (0.200 g, 0.605 mmol) in 2 ml acetonitrile was added 1-chloroethyl cyclohexyl carbonate (0.105 g, 0.544 mmol). Ag
[0255] Table 9 provides structures of additional prodrug compounds, along with characterization data and reference to the methods employed for preparation the source of starting materials.
TABLE 9 Illustrative compounds, source of reagents and coupling methods Coupling Reagent and Method Compound Source (vida and number Reference: Structural elucidation infra):
[α] Method 1
[α] Method 2
[α] Method 4
[α] Method 2
[α] Method 1
[α] Method 1
[α] Method 3
[α] Method 3
[α] Method 2
[α] Method 2
[α] Method 1
[α] Method 1
[α] Method 1
[α] Method 3
[α] Method 2
[α] Method 1
[α] Method 2
[α] Method 2
[α] Method 1
[α] Method 2
[α] Method 2
[α] Method 2
[α] Method 4
[α] Method 1
[α] Method 2
[α] Method 2
[α] Method 5
[α] Method 2
[α] Method 2
[0256] This example illustrates methodology and conditions for the chiral analysis of prodrug esters of (−)-CPTA.
[0257] Since the chiral center of the prodrug esters is adjacent to the carbonyl of the acid, the esters were analyzed to confirm that the optical center had not racemized during the coupling. Both MBX102 and the MBX102 acid have been resolved on a normal phase chiral column, but confirmation of the relative retentions of the enantiomeric esters on such a column would require standards of both enantiomers for all the compounds involved. Alternatively, the esters could be hydrolyzed to the known acids and the ratio of the enantiomeric acids could be analyzed, but some degree of racemization is likely under these conditions. Reverse phase chiral columns offer the ability to be interfaced with an LC-MS system, so that the enantiomers could be positively identified without separate standards. Under negative ion, selected ion monitoring (SIM) conditions, an LC-MS would be expected to be an order of magnitude more sensitive than UW detection as well as being much more specific. The application of chiral-column-LC-MS has proven to be an excellent method for characterizing the optical purity of this series of compounds. The quantitation limit has been shown to be well below the 2.5% limit set for the alternate optical isomer, and the extremely low level of detection gives more flexibility in the separation of the series, as well as establishing the relative retentions of the enantiomeric pairs in-most cases without the need to produce racemized material.
[0258] The enantiomers of the prodrug esters synthesized were well separated using one of the columns and solvent systems listed below. Most of the enantiomers were separated using the reverse phase ES-OVM column with the mass spectrometer as a detector. Since this gives the added conformation of the molecular species and isotope ratio, this was the preferred technique. For enantiomeric pairs which were not separated on the reverse phase column, a normal phase system was used and some of the sample was partly racemized to establish the retention time of the enantiomers.
[0259] Reversed Phase Separation:
Column: Shinwa Chemicals Ultron ES-OVM 4.6 × 150 mm with matching pre column (part #712111630). Available from Mac Mod Analytical. Solvents: “A” Acetonitrile, Ethanol or Methanol. “B” Water containing 20 milli molar NH4 OCOCH3 (Ammonium Acetate) adjusted to pH 4.6 with acetic acid. Solvent Flow: 1 milliliter per minute Isocratic as indicated. Detector: UV for method development at 220 or 270 nm or as specified. LC- MS using negative ion electrospray, monitoring two ions, M-H chlorine 37 isotope (M + 1) Mass Spectrometer: Waters/Micromass ZMD Bench top LC-MS with Electrospray source. HPLC: Agilent 1050 or Shimadzu LC-10 as indicated. Software: HP 3396 integrator for method development and Micromass MassLynx V3.4 for quantitation.
[0260] Methods Development:
[0261] Most prodrug esters separated using between 10 and 40% acetonitrile in water with an ammonium acetate buffer at pH 4.6. The free acid was a contaminant in the crude or racemized samples. The retention times of the acids were pH dependant, but the chromatography of the esters was in general not sensitive to pH. Retention and peak shape were affected by the concentration and injection solvent, and the samples were injected using a minimum concentration and the column solvent mixture for best results. The desired enantiomer was retained less and with a retention time of approximately 5 minutes, the second enantiomer eluted from 1 to 3 minutes later with complete separation in most cases. There was some peak tailing from the main enantiomer in some cases. The amount of acetonitrile was adjusted to give a retention time of about 5 minutes for the main component or best peak separation with maximum peak sharpness. Peaks retained more than 10 minutes tended to be too broad to accurately quantify the second enantiomer at levels under 2%.
[0262] Detection Limit:
[0263] Sample 1061-18-05 (the benzyl ester, MW 420) was mixed with racemized material to give an expected concentration of 2.47% of the unwanted enantiomer. The sample was analyzed repeatedly by LC-MS (monitoring ions at 419 and 421) giving an average of 2.6% recovery of the second enantiomer with a 10.9% RSD. See
[0264] Normal Phase Separation:
Column: Analytical Column: Regis (S,S) WHELK-0 4.6 ± 250 mm with Peek Scientific Cyano pre column (part # DC5-CN). Preparative Column Regis (S,S) WHELK-0 20 × 250 mm with Cyano pre column. Solvents: “A” 10% of Solvent B in Hexane. “B” Isopropanol (IPA), Ethanol or Ethyl Acetate buffered with ammonium acetate or triethylamine when indicated. Solvent Flow: 1.2, 1.5 or 25 milliliter per minute Isocratic as indicated. Detector: UV for method development at 270 nm or as specified. HPLC: Agilent 1050, Gilson 321 or Shimadzu LC-10 as indicated. Software: Gilson Unipoint, HP 3396 Integrator or Micromass MassLynx V 3.4 as indicated.
[0265] Methods Development:
[0266] Most prodrug esters separated using between 10 and 40% alcohol in Hexane without a modifier. The desired enantiomer was retained less. With a retention time of approximately 5 minutes for the first enantiomer, the second enantiomer eluted from 1 to 3 minutes later with complete separation. The compounds with a basic amine group required ammonium acetate (0.2%) as a modifier to sharpen the peak shape. Triethyl amine was tried but was not effective as a modifier. IPA was normally the first solvent tried, if the separations were not complete, ethanol or ethylacetate were used. The enantiomeric excess for each compound was measured using reverse phase chromatography when possible, but in several cases, separation was only possible using normal phase. In these cases, the data were collected and integrated using the MicroMass MassLynx software to be consistent with the other analyses
[0267] This example illustrates the plasma hydrolysis of various prodrug esters of the (S)-enantiomer of CPTA at physiological conditions. Hydrolysis was monitored and analyzed by HPLC. The hydrolytic product was identified by comparison to authentic (−) CPTA and the hydrolytic rates were calculated.
[0268] Human plasma (heparinized, pooled) was obtained from Golden West Biololgicals Inc., USA; Plasma incubation was carried out in a Water Bath Shaker (New Brunswick Scientific, Inc.); Sample analyses were carried out on Agilent HPLC 1100 system.
[0269] General Hydrolytic Procedure:
[0270] Prepare 31.25 or 62.5 mM prodrug stock solutions in 100% DMSO. Add 4 μL of 31.25 or 62.5 mM prodrug stock solutions to 1.0 mL of plasma in a microcentrifuge tube to make plasma concentration at 125 or 250 μM, mix gently. Aliquots of 50 μL are transferred to 15 microcentrifuge vials. Three samples are immediately removed to a freezer at about −80° C. (0 time point), the remaining samples are incubated in a bath shaker at 37° C. Three samples are removed at 30 min, 2, 7 and 24 hr, and stored at −80° C. until analysis. The relative concentrations of MBX-compounds are determined by HPLC methods. If there are enough data points, the hydrolytic rate can be calculated using WinNonlin software. Table 10 provides a summary of plasma half-lives of some pro-drug esters and FIGS.
[0271] HPLC: Agilent 1100 HPLC System
Column: Phenomenex Luna C18(2) 5u 150 × 2 mm lot #105554-2 Phenomenex Luna C18(2) 5u 250 × 4.6 mm lot #103992-8 Solvent Flow Rate: 0.25 or 1 ml/minute Injection Volume: 20 or 40 μl RunTime: 7 to 15 min Solvent Composition: 45 to 76% AGN/0.1% TFA in Water (V/V) Detection: Diode Array UV-Visible Detector at 220 mn
[0272]
TABLE 10 Plasma Half-Life of (−) CPTA Prodrugs Compound number Plasma T 19.1 5.5 19.2 <1 19.3 <1 19.4 <1 19.5 <0.5 19.6 6.3 19.7 <7 19.8 <2 19.9 <1 19.10 <1 19.11 <0.5 19.12 <0.5 19.13 <0.5 19.14 <2 19.15 <1 19.16 <0.5 19.17 <1 19.18 <1 19.19 <0.5 19.20 <0.5 19.21 <2 19.22 <1 19.23 <1 19.24 <0.5 19.25 <0.5 19.26 <1 19.27 <1 19.28 <0.5
[0273] Although the foregoing invention has been described in detail for purposes of clarity of understanding, it will be obvious that certain modifications can be practiced within the scope of the appended claims. All publications and patent documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each were so individually denoted.