HK1118449B - Methods and compositions for treating diabetes, metabolic syndrome and other conditions - Google Patents

Description

Compositions and methods for treating diabetes, metabolic syndrome and other conditions

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application 60/643,055 filed on 10/1/2005, the entire contents of which are incorporated herein by reference.

Technical Field

The present invention relates to pharmaceutical compositions and methods for treating diabetes and other conditions, including type II diabetes, metabolic syndrome, insulin resistance, obesity, dyslipidemia (lipid disorders), metabolic disease, and other conditions that can be treated by reducing cortisol (cortisol) synthesis, including but not limited to: cushing's Syndrome, osteoporosis, glaucoma and depression. The present invention thus relates to the fields of chemistry, biology, pharmacology and medicine.

Background

Ketoconazole (ketoconazole), 1-acetyl-4- [4- [ [2- (2, 4-dichlorobenzene) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine (1-acetyl-4- [4- [ [2- (2, 4-dichlorphenyl) -2- [ (1H-imidozol-l-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine) is a racemic mixture of the cis enantiomers (-) - (2S, 4R) and (+) - (2R, 4S) sold as an antifungal agent. Ketoconazole is resistant to fungal growth by inhibiting the synthesis of ergosterol (ergosterol). Ergosterol is an important component in fungal cell walls.

More recently, ketoconazole has been found to lower plasma cortisol and can be used alone or in combination with other agents to treat a variety of diseases and conditions, including type II diabetes, metabolic syndrome (otherwise known as insulin resistance syndrome, metabolic disorder syndrome and syndrome X), and other medical conditions associated with elevated levels of cortisol. See U.S. patents 5584790, 6166017, and 6642236, each of which is incorporated herein by reference. Cortisol is a stress-related hormone, secreted from the adrenal cortex. Adrenocorticotropic hormone (ACTH), an adrenocorticotropic hormone, promotes the secretion of cortisol. ACTH is secreted by the pituitary gland, a process triggered by Corticotropin Releasing Hormone (CRH) secreted by the hypothalamus.

Cortisol circulates in the bloodstream and activates specific intracellular receptors, such as the Glucocorticoid Receptor (GR). Disorders in cortisol levels, synthesis rate or activity have been shown to be associated with a number of metabolic complications, including insulin resistance, obesity, diabetes and metabolic syndrome. In addition, these metabolic abnormalities are associated with a substantially increased risk of cardiovascular disease, which is a major cause of death in industrial countries. See Marin P et al, "diagnostic diagnosis in translation to body fat distribution in elementary mouse, 1992," Metabolism; 41: 882-886, Bjorntorp, "neuroendeaperturans as a house of insulin resistance," Diabetes Metab Res Rev 1999; 15(6): 427-41, and Rosmond, "Role of stress in the pathogenesis of the metabolic syndrome," Psychoneuroendocrinology 2005; 30(1): 1-10, each of which is incorporated herein by reference.

While ketoconazole is known to inhibit certain enzymatic steps In the synthesis of cortisol, for example, 17 alpha hydroxylase (Wacharl et al, "Imidazole mutated biphenyls: a new class of high reactivity In visual activity inhibitors of P45017 as a pore thermal regulators, Bioorg Med Chem 1999; 7(9) 1913-24, which is incorporated herein by reference), and 11 b-hydroxylase (Rotstein et al," Stereorgans of ketonic: prediction and biological activity J.J.Med.1992; 35: 2818-25) and 11 beta-hydroxyl steroid dehydrogenase (Diedich-HSD. beta. -histidine) of 11 beta-hydroxyl family (In beta. -cholesterol, beta. -cholesterol synthase, beta. cholesterol synthase, HSD. D.E.D. 2000-11, beta. -hydroxy derivatives of 11-5-11 beta. -hydroxy derivatives of 11-2-5, which is incorporated herein by reference) but there has been no report on the mechanism by which ketoconazole reduces cortisol levels in plasma. For example, the effect of ketoconazole on 11 β -hydroxysteroid dehydrogenase (11 β -HSD) is uncertain. There are two 11 β -HSD enzymes. One of them, 11 β -HSD-I, is primarily a reductase expressed in large amounts in the liver, which converts inactive 11-ketocorticoid (11-keto glucocorticoids) into active glucocorticoids (cortisol in humans and corticosterone in mice). In contrast, 11 β -HSD-II, expressed predominantly in the kidney and acting predominantly as an oxidase, converts active glucocorticoid (cortisol in humans and corticosterone in mice) to inactive 11-ketocorticoid. Thus, the concentration of active glucocorticoid in plasma is influenced by the rate of synthesis and is controlled in part by the activity and rate of interconversion by the adrenal 11 β -hydroxylase and in part by the relative activities of the two 11 β -HSD enzymes. Ketoconazole is known to inhibit these three enzymes (Diederich et al, supra), with the 2S, 4R enantiomer having greater adrenal 11 β -hydroxylase inhibitory activity than the 2R, 4S enantiomer (Rotstein et al, supra). However, there is no report on the effect of these two ketoconazole enantiomers on 11 β -HSD-I or 11 β -HSD-II, and it has therefore not been predicted what, if any, of these two different ketoconazole enantiomers will respectively have an effect on the active glucocorticoid levels in the plasma of mammals.

Ketoconazole is also reported to reduce cholesterol levels in humans (Sonino et al (1991), "Ketoconazole treatment in curing's syndrome: experiment in 34 properties," Clin Endocrinol (Oxf)35 (4): 347-52; Gylling et al (1993) "Effects of Ketoconazole on cholesterol precursors and low intensity lipid metabolism in hypercholesterolemia in JLloyd Res.34 (1): 59-67, each of which is incorporated herein by reference). The 2S, 4R enantiomer has a higher inhibitory activity against the cholesterol synthase 14. alpha. lanosterol demethylase (Rotstein et al infra) than the other enantiomer (2R, 4S). However, since cholesterol levels in patients are controlled by metabolism and excretion rates, as well as synthesis rates, it cannot be predicted from this whether the ketoconazole 2S, 4R enantiomer is more effective in lowering cholesterol levels.

The effect of ketoconazole on the P450 enzymes responsible for drug metabolism complicates the use of ketoconazole as a therapeutic agent. Several of these P450 enzymes are inhibited by ketoconazole (Rotstein et al, supra). This inhibition affects the clearance of ketoconazole itself (Brass et al, "Disposition of ketoconazole, an organic inhibitor, in humans," antimicrobial Agents Chemother 1982; 21 (1): 151-8) and the clearance of several other important drugs, such as Gleevec (Gleeveix) (Dutreix et al, "pharmacological interaction between them and Imatinib mesylate (Glevec) in clinical subjects," cancer Chemother Pharmacol 2004; 54 (4): 290-4) and methylprednisolone (Methylprednisolone) (Glennet et al, "Effects of ketoconazole on methyl cellulose kinase and chemotherapy 654; Effect of ketoconazole and Ketoconazole 654; Glycontrol 654," hydrolysis between them and 9, 1986). As a result, the exposure (exposure) of the patient to ketoconazole increases with repeated dosing, although the amount of drug administered to the patient does not increase. This exposure, and the increase in exposure, can be measured and demonstrated by the "Area under the Curve" (AUC) or the product of the drug concentration in the plasma and the time period over which the measurement is taken. The ketoconazole AUC after the first exposure was much lower than the ketoconazole AUC after repeated exposures. This increase in drug exposure means that it is difficult to provide patients with accurate, consistent doses of the drug. Moreover, increased drug exposure increases the likelihood of adverse side reactions associated with the use of ketoconazole.

Rotstein et al (Rotstein et al, supra) have examined the effects of these two ketoconazole cis enantiomers on the major P450 enzymes responsible for drug metabolism, and reported that "… … showed little selectivity for ketoconazole isomers", and, with respect to drug metabolism P450 enzymes: "the IC50 value for the cis enantiomer is similar to the value reported previously for racemic ketoconazole". This report indicates that both cis enantiomers may play a significant role in the AUC problem observed with ketoconazole racemates.

One of the adverse side effects of ketoconazole administration that is exacerbated by this AUC problem is hepatic reactions. Asymptomatic liver responses can be measured by elevated levels of liver-specific enzymes in serum, which have been found to be elevated in patients treated with ketoconazole (Sohn, "Evaluation of ketoconazole." Clin Pharm 1982; 1 (3): 217-24, and Janssen and Symoens, "cosmetic reactions reducing ketoconazole treatment," Am J Med 1983; 74 (1B): 80-5, each of which is incorporated herein by reference). In addition, patients of 1: 12000 may experience more severe liver failure (Smith and Henry, "Ketoconazole: an organic active anti-hepatic agent. mechanism of action, pharmacology, clinical efficacy and liver effects," Pharmacotherapy 1984; 4 (4): 199. 204, incorporated herein by reference). As mentioned above, the amount of ketoconazole exposed to the patient increases with repeated dosing, although the daily dose does not increase ("AUC problem"). In rabbits where AUC is associated with liver damage (Ma et al, "hepatoxicokinetics and ketoconazole in rabbits," Acta Pharmacol Sin 2003; 24 (8): 778-782, which is incorporated herein by reference), increased drug exposure is believed to increase the frequency of liver damage in patients treated with ketoconazole.

Furthermore, U.S. patent 6040307, which is incorporated herein by reference, reports that the 2S, 4R enantiomer is effective in treating fungal infections. The same patent application also reports studies on isolated guinea pig hearts, indicating that administration of racemic ketoconazole may be accompanied by an increased risk of arrhythmia, but no data is provided to support this proposition. However, as disclosed in this patent, it has not been previously reported that arrhythmia is a side effect of systemic racemic ketoconazole, although it has been reported that a particular subtype of arrhythmia, torsades de pointes, occurs when racemic ketoconazole is administered simultaneously with terfenadine (terfenadine). In addition, several published reports (e.g., Morganroth et al (1997), "rock of effect and ketoconazol coadministration on electrocardiac parameters in health volranters," J Clin Pharmacol.37 (11): 1065-72) have demonstrated that ketoconazole does not increase QTc intervals. This interval is used as an alternative marker for determining whether the drug has a likelihood of causing an arrhythmia. U.S. patent 6040307 also mentions reduced hepatotoxicity associated with the 2S, 4R enantiomer, but does not provide data to support this proposition. The method provided in us patent 6040307 cannot be used to assess hepatotoxicity because microsomes isolated from frozen tissue are used in this method.

Thus, there is a need for new therapeutic agents and methods of treatment for diseases and conditions that are accompanied by elevated cortisol levels or activity, or that can be treated by lowering cortisol levels or activity, that have the same effect as ketoconazole but do not exhibit, or exhibit to a lesser extent, the problems of drug interactions and the adverse side effects of ketoconazole. The present invention meets these and other needs.

Summary of The Invention

The present invention results, in part, from the discovery that the 2S, 4R enantiomer is more effective at reducing plasma concentrations of active glucocorticoid than either racemic ketoconazole or the 2R, 4S enantiomer (the other enantiomer in the racemate) per unit of body weight, and that the 2S, 4R enantiomer does not cause drug accumulation (or accumulates to a much lesser extent) as racemic ketoconazole does.

In a first aspect, the present invention provides methods for treating diseases and conditions associated with elevated cortisol levels, production rates or activity, and other diseases and conditions that can be treated by reducing cortisol, or that can be treated by reducing cholesterol levels, production rates or activity, by administering a pharmaceutical composition comprising a therapeutically effective amount of an enantiomer of 2S, 4R ketoconazole that is substantially free or completely free of the 2R, 4S ketoconazole enantiomer.

In a second aspect, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the 2S, 4R ketoconazole enantiomer, wherein the 2R, 4S ketoconazole enantiomer is substantially or completely absent, said pharmaceutical composition being formulated for use in the methods of the present invention.

Brief Description of Drawings

Figure 1 shows the effect of the four ketoconazole enantiomers 2S, 4S, 2R, 4R, 2R, 4S and 2S, 4R on plasma corticosterone. The figure shows that the 2S, 4R enantiomer is more effective in reducing corticosterone than any of the other three enantiomers. The corticosterone concentration in plasma of Sprague-Dawley rats was determined four hours after administration of 200mg/kg of the indicated enantiomer by oral gavage.

Figure 2 shows the effect of racemic ketoconazole and the two cis enantiomers 2R, 4S and 2S, 4R on plasma corticosterone. The 2S, 4R enantiomer is more effective in reducing corticosterone than either racemic ketoconazole or the other enantiomer present in racemic ketoconazole (2R, 4S). The corticosterone concentration in plasma of Sprague-Dawley rats was determined four hours after administration of the indicated amount of racemic ketoconazole or the two enantiomers present in racemic ketoconazole (2S, 4R and 2R, 4S) by oral gavage.

Figure 3 shows the inhibition of plasma corticosterone by racemic ketoconazole or the two enantiomers 2R, 4S and 2S, 4R over time. The 2S, 4R enantiomer is more effective in reducing corticosterone than either racemic ketoconazole or the other cis enantiomer (2R, 4S) present in racemic ketoconazole. The concentration of corticosterone in plasma of Sprague-Dawley rats was determined at the indicated times after oral feeding of 200mg/kg of racemic ketoconazole or both enantiomers present in racemic ketoconazole (2S, 4R and 2R, 4S).

Figure 4 shows the effect of prior exposure to ketoconazole on the pharmacokinetic profile of racemic ketoconazole in dogs. The pharmacokinetic profile of racemic ketoconazole is clearly altered by prior exposure of racemic ketoconazole. The concentration of racemic ketoconazole in the plasma of dogs administered racemic ketoconazole daily for 28 days (in two different forms: a suspension in olive oil, and a solid tablet) was much higher than the concentration of racemic ketoconazole in the plasma of dogs dosed only once.

Figure 5 shows the effect of prior exposure to racemic ketoconazole on the pharmacokinetic profile of racemic ketoconazole in dogs. Prior exposure to racemic ketoconazole resulted in an increase in the area under the curve (AUC) of racemic ketoconazole. The AUC of the pharmacokinetic profile shown in fig. 4 was calculated according to the trapezoidal rule. The AUC of racemic ketoconazole in dogs treated each day for 28 days was greater than in dogs treated only once. The increase in AUC is not constrained by the administration form of racemic ketoconazole.

Figure 6 shows the effect of prior exposure to 2S, 4R ketoconazole enantiomer on the pharmacokinetic profile of 2S, 4R ketoconazole enantiomer in dogs. The pharmacokinetic profile of the 2S, 4R ketoconazole enantiomer was not altered by prior exposure of the 2S, 4R ketoconazole enantiomer. The dogs were dosed with the 2S, 4R enantiomer once or daily for 28 days, and the concentration of the 2S, 4R ketoconazole enantiomer in the plasma of the dogs was not elevated in the 28 day treated dogs compared to the dogs treated only once.

Figure 7 shows the effect of prior exposure to 2S, 4R ketoconazole enantiomer on AUC of 2S, 4R ketoconazole enantiomer in dogs. The AUC of the 2S, 4R ketoconazole enantiomer was not increased by prior exposure of the 2S, 4R ketoconazole enantiomer. The AUC for the 2S, 4R ketoconazole enantiomer was the same in dogs treated daily for 28 days and in dogs treated only once.

Detailed Description

The present invention provides pharmaceutical compositions comprising the 2S, 4R ketoconazole enantiomer substantially or completely free of the 2R, 4S enantiomer, and methods of using the same. Substantially free of the 2R, 4S enantiomer, meaning in one embodiment that the pharmaceutical composition has a ketoconazole content of less than 2% of the 2R, 4S enantiomer and greater than 98% of the 2S, 4R enantiomer. In another embodiment, substantially free of the 2R, 4S enantiomer means that the pharmaceutical composition has a ketoconazole content of less than 10% of the 2R, 4S enantiomer and greater than 90% of the 2S, 4R enantiomer. In another embodiment, substantially free of the 2R, 4S enantiomer means that the pharmaceutical composition has a ketoconazole content of less than 20% of the 2R, 4S enantiomer and more than 80% of the 2S, 4R enantiomer. The invention also provides methods for treating diseases and conditions associated with elevated cortisol levels or activity, as well as diseases and conditions that can be medically treated by reducing cortisol levels and cortisol activity using these pharmaceutical compositions. To assist in understanding the invention, the structure of the detailed description is as follows. Section I describes the 2S, 4R enantiomer, solvates and salts thereof, and processes for the preparation of pharmaceutical compositions containing the same. Section II, describes the unit dosage form, and the mode of administration of the pharmaceutical compositions of the present invention. In section III, methods of treating diseases and conditions by administering the 2S, 4R ketoconazole enantiomer and pharmaceutical compositions containing the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer are described. Preparation of 2S, 4R ketoconazole enantiomer and pharmaceutical composition containing 2S, 4R ketoconazole enantiomer substantially or completely free of 2R, 4S ketoconazole enantiomer

As used herein, compositions containing the "2S, 4R ketoconazole enantiomer substantially or completely free of the 2R, 4S ketoconazole enantiomer" include compositions free of the 2R, 4S ketoconazole enantiomer, as well as compositions containing substantially less of the 2R, 4S ketoconazole enantiomer than the 2S, 4R enantiomer as compared to the racemic ketoconazole compositions currently approved for therapeutic use. Compositions useful in the methods of the invention include, for example, but are not limited to, compositions having at least 80%, or at least 90%, or at least 99%, or at least 99.5%, or at least 99.9% or more of the total amount of ketoconazole that is the 2S, 4R enantiomer.

The ketoconazole 2S, 4R enantiomer can be obtained by optical resolution of racemic ketoconazole. Such resolution can be achieved by any of a variety of resolution methods well known to those skilled in the art, including, but not limited to, the methods described in Jacques et al, "eneriomers, racemes and solutions," Wiley, New York (1981), which is incorporated herein by reference. For example, resolution can be performed on a chiral column using preparative chromatography. Another example of a suitable resolution method is the formation of diastereomeric salts with chiral acids, e.g. tartaric acid, malic acid, mandelic acid, or N-acetyl derivatives of amino acids, such as N-acetylleucine, followed by separation of the diastereomeric salts of the desired enantiomers by recrystallization. Another method for obtaining compositions containing the 2S, 4R enantiomer substantially free of the 2R, 4S enantiomer is by fractional crystallization of the diastereomeric salts of ketoconazole and (+) -camphor-10-sulfonic acid.

The ketoconazole 2S, 4R enantiomer can also be prepared directly by various methods known to those skilled in the art. For example, the 2S, 4R enantiomer can be prepared directly by transketol reaction (transmotolysis) between 2-bromo-2 ', 4' -dichloroacetophenone and optically pure solketal as described by Rotstein et al (Rotstein et al, supra, which is incorporated herein by reference).

The invention also provides various pharmaceutically acceptable salts of the 2S, 4R enantiomer of ketoconazole which may be used in the pharmaceutical compositions of the invention. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable bases or acids, including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, manganese, manganous, potassium, sodium, zinc salts and the like. In particular, ammonium, calcium, magnesium, potassium and sodium salts may be preferred in some pharmaceutical formulations. The salts in solid form may exist in more than one crystal structure, and may also exist in the form of hydrates and polyhydrates. Solvates, especially hydrates, of the 2S, 4R ketoconazole enantiomer are useful in the preparation of the pharmaceutical compositions of the invention.

Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amine salts including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

If the compound to be formulated is basic, salts may be prepared from pharmaceutically acceptable acids, including inorganic and organic acids. These acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic (pamoic acid), pantothenic, phosphoric, succinic, sulfuric, tartaric, and p-toluenesulfonic acid, and the like. Exemplary pharmaceutically acceptable acids include citric acid, hydrobromic acid, hydrochloric acid, maleic acid, phosphoric acid, sulfuric acid, and tartaric acid. Ketoconazole compounds are generally basic because the triazole ring is basic. The 2S, 4R ketoconazole compound may be processed during synthesis as a non-pharmaceutically acceptable salt (e.g., a trifluoroacetate salt) and then converted to a pharmaceutically acceptable salt, as described herein.

Suitable pharmaceutically acceptable salts of the 2S, 4R ketoconazole enantiomer include, but are not limited to, the mesylate, maleate, fumarate, tartrate, hydrochloride, hydrobromide, esylate, p-toluenesulfonate, benzoate, acetate, phosphate and sulfate salts. To prepare a pharmaceutically acceptable acid addition salt of the 2S, 4R ketoconazole compound, the free base may be reacted with the desired acid in a suitable solvent using conventional methods. Similarly, acid addition salts can be converted to the free base form using methods known to those skilled in the art.

The pharmaceutical compositions of the present invention may comprise therapeutically active 2S, 4R ketoconazole enantiomer metabolites or enantiomeric prodrugs (produgs). Prodrugs are compounds that are converted to therapeutically active compounds when administered to a patient or after administration.

Accordingly, the pharmaceutical compositions of the present invention comprise the 2S, 4R ketoconazole enantiomer, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a prodrug or active metabolite thereof, in combination with a pharmaceutically acceptable carrier, wherein the 2R, 4S enantiomer is substantially or completely absent. In one embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the 2S, 4R ketoconazole enantiomer or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. As noted above, pharmaceutically acceptable salts of the 2S, 4R enantiomer useful in such compositions include, but are not limited to, hydrochloride, phosphate, maleate, fumarate, tartrate, mesylate, esylate and sulfate salts.

The "therapeutically effective amount" of the 2S, 4R ketoconazole enantiomer or a pharmaceutically acceptable salt thereof will depend on the condition to be treated, the route and duration of administration, the physical characteristics of the patient, including body weight, and other concomitant medications, and can be determined by methods known to those skilled in the art in light of the teachings of this disclosure (see section II, below). The pharmaceutical compositions of the present invention may be conveniently prepared as unit dosage forms of the drug using methods well known in the art of pharmacy, for oral, parenteral (including subcutaneous, intramuscular, and intravenous), ophthalmic (ophthalmic administration), rectal, pulmonary (nasal or buccal inhalation), topical, transdermal or by oral mucosal delivery.

The pharmaceutical compositions of the present invention may be prepared by mixing the 2S, 4R ketoconazole enantiomer with a selected pharmaceutical carrier according to conventional pharmaceutical techniques. The carrier can take many forms. For example, carriers for oral liquid compositions include, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and other ingredients used in the manufacture of oral liquid suspensions, tinctures, and solutions. Carriers, such as starches, sugars and microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like, are used in the preparation of oral solid dosage forms, such as powders, hard and soft capsules, and tablets. Solid oral formulations are generally preferred over liquid oral formulations.

Thus, in one embodiment, the pharmaceutically acceptable carrier is a solid and the pharmaceutical composition is a tablet for oral administration. Other suitable forms of the pharmaceutical compositions of the invention which may be administered orally include compressed or coated pills, dragees, powders, hard or soft gelatin capsules, sublingual tablets, syrups and suspensions. Oral solid dosage forms may also contain binders, such as tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrating agents, such as corn starch, potato starch or alginic acid; lubricants, such as magnesium stearate; and/or a sweetening agent such as sucrose, lactose or saccharin. The capsule may also contain a liquid carrier such as fatty oil. Various other materials may also be used, either as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac, sugar or both. Tablets may be coated by standard aqueous or non-aqueous techniques. Of course, typical percentages of active compound in these compositions may vary and may be, for example, but not limited to, from about 2% to about 60% by weight.

In another embodiment, the pharmaceutically acceptable carrier is a liquid and the pharmaceutical composition is intended for oral administration. Oral liquids suitable for use in such compositions include syrups and elixirs, and may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and/or a flavoring such as cherry or orange flavor.

In another embodiment, the present invention provides a pharmaceutical composition of the 2S, 4R ketoconazole enantiomer, suitable for parenteral administration. For parenteral administration, the pharmaceutical compositions are usually placed in a syringe or vial, consisting essentially of an aqueous or non-aqueous solution or emulsion. These compositions are usually in the form of solutions or suspensions, usually prepared from water, optionally containing a surfactant such as hydroxypropylcellulose. Dispersions (dispersions) can be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Typically, the preparation in diluted form also contains a preservative.

In another embodiment, the pharmaceutically acceptable carrier is a liquid and the pharmaceutical composition is an injectable solution. The pharmaceutically injectable dosage forms, including aqueous solutions and dispersions and powders for the extemporaneous preparation of injectable solutions or dispersions, are also sterile and have sufficient flowability for injection to permit easy injection by syringe. These compositions remain stable during the manufacturing process and storage conditions and are typically preserved. Thus, the carrier includes a solvent or dispersion medium, including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils.

In another embodiment, the pharmaceutically acceptable carrier is a gel and the pharmaceutical composition is provided in the form of a suppository. For rectal administration, the pharmaceutical compositions are provided as suppositories, and the pharmaceutically acceptable carriers are hydrophilic or hydrophobic carriers. In another embodiment, a pharmaceutical composition useful in the methods of the invention is prepared for topical administration and the 2S, 4R ketoconazole enantiomer is formulated as an ointment. The 2S, 4R enantiomer may also be administered transdermally; suitable transdermal delivery systems are known in the art.

The pharmaceutical compositions of the present invention also include sustained release compositions. Suitable sustained release compositions include those described in U.S. patent application publication nos. 20050013834, 20030190357, and 2002055512 and PCT patent application publication nos. WO 03011258 and 0152833, each of which is incorporated herein by reference. A unit dosage form; frequency and duration of administration

As noted above, any suitable route of administration may be used to provide a therapeutically effective dose of the 2S, 4R enantiomer to a mammal, typically a human, although veterinary important mammals, such as cows, horses, pigs, sheep, dogs and cats, may also benefit from the methods described herein. For example, oral, rectal, topical, parenteral, ocular, pulmonary, or nasal administration may be used. The dosage forms include tablets, buccal tablets, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. In many embodiments of the treatment methods of the present invention, the pharmaceutical composition is administered orally. The therapeutically effective dose of the active ingredient will depend on the particular compound (salt, solvate, prodrug or metabolite) employed, the mode of administration, the condition being treated and the severity of the condition. Such dosages can be readily determined by one of skill in the art in light of the disclosure herein. [0042] Satisfactory results are obtained when the 2S, 4R ketoconazole enantiomer is administered at a daily dose of about 0.1 to about 25 milligrams (mg) per kilogram of body weight (mpk), preferably as a once-daily dose or divided into about 2-6 doses per day, when treating or preventing the diseases and conditions described herein. For oral administration to an adult human patient, a therapeutically effective amount will be administered in the range of substantially 50mg to 800mg per dose, including, but not limited to, 100mg per dose, 200mg per dose, and 400mg per dose, as well as multiple doses, which will typically be administered continuously daily for a course of treatment. The 2S, 4R ketoconazole enantiomer pharmaceutical compositions may be administered at different times of the day. In one embodiment, the optimal therapeutic dose may be administered during the evening hours. In another embodiment, the optimal therapeutic dose can be administered in the morning. Thus, the total daily dose of the 2S, 4R ketoconazole enantiomer may be in one embodiment from about 10mg to about 2g, typically from about 10mg to about 1g, and most typically from about 100mg to about 500 mg. For a typical 70kg adult human, the total daily dose of the 2S, 4R ketoconazole enantiomer may be from about 10mg to about 1000mg, and, as noted above, is typically from about 50mg to about 800 mg. This dosage can be adjusted to provide the best therapeutic response.

In one embodiment, the unit dosage form is suitable for oral administration and contains one or more pharmaceutical excipients. Examples of pharmacologically inactive excipients that may be included in the orally available formulations of 2S, 4R ketoconazole enantiomers for the purposes of the present invention, and their effects are shown in the following table.

No active ingredient	Trade name	Grade	Function of
				Silicified microcrystalline cellulose	Prosolv HD 90	NF	Dilution of
Lactose monohydrate	Modified，316 Fast Flo	NF	Dilution of
				Corn starch	STA-Rx	NF	Disintegrating agent
Magnesium stearate	N/A	NF	Lubricant agent
				Colloidal silicon dioxide	Cab-O-Sil M5P	NF	Glidants

The excipients listed in the above table may be mixed with the 2S, 4R enantiomer in various ratios to give specific tablet and manufacturing characteristics. The size of the tablets may vary from 1mg total weight to 1000mg total weight; such as, but not limited to, 100mg total weight to 800mg total weight. The proportion of the 2S, 4R enantiomer in the tablets may vary from 1% to 100%; such as, but not limited to, 10% to 90%. Examples of 400mg tablets containing 50% of the 2S, 4R enantiomer by weight of the tablet are provided in the following table. In this example, a dry mixture of (-) cis 2S, 4R ketoconazole and the listed inactive excipients was compressed into tablets.

Composition (I)	％W/W	Tablet weight (mg)
			(-) cis 2S, 4R ketoconazole	50.0	200
Lactose monohydrate, NF	22.4	89.6
			Silicified microcrystalline cellulose, NF	16.5	66.0
Corn starch, NF	10.0	40.0
			Colloidal silica, NF	0.5	2.0
Stearic acidMagnesium, NF	0.6	2.4
			Total of	100.0	400.0

In U.S. patent application 6040307, a tablet formulation for 2S, 4R ketoconazole is described. The formulation comprises the active drug substance, (-) ketoconazole, lactose, corn starch, water and magnesium stearate. The wet granulation is made from ketoconazole, lactose, water and corn starch, the granules are oven dried and compressed into tablets by adding magnesium stearate and more corn starch. Tablets were compressed and dried. This process is less desirable than the process of the present invention using a dry blending process as described above because the process of the present invention does not use excess water and elevated temperatures. Ketoconazole may undergo degradation (oxidation) (Farhadi and Maleki (2001). "A newsbepoptometric method for the determination of ketoconazole based on the oxidation reaction", "Analytical Sciences 17 Supplement, i867-i869.the Japan society for Analytical Chemistry), the oxidation reaction being accelerated in the presence of water and at elevated temperatures.

The solid unit dosage form of the pharmaceutical composition of the present invention contains the 2S, 4R ketoconazole enantiomer or its salt or hydrate in an amount of from about 1mg to about 2g, typically from about 1.0mg to about 1.0g, and most typically from about 10mg to about 500 mg. In a liquid pharmaceutical composition of the invention suitable for oral administration, the 2S, 4R ketoconazole enantiomer may be present in an amount of about 1mg/ml to about 200 mg/ml. The therapeutically effective amount may also range from about 10mg/ml to about 100 mg/ml. In one embodiment, the liquid pharmaceutical composition is administered in an amount of 0.5ml to 5.0 ml. In another embodiment, the dose is between about 1ml and 3 ml. In a liquid pharmaceutical composition of the present invention designed for intravenous or subcutaneous injection, the 2S, 4R ketoconazole enantiomer may be administered in an amount of about 0.01 to 1mg/ml, and at a rate of 0.01 to 1ml/min, subcutaneously or intravenously. Alternatively, the 2S, 4R enantiomer may be administered in an amount of about 0.1mg/ml to 10mg/ml, and at a rate of 0.001ml/min to 0.1ml/min, subcutaneously or intravenously.

As noted above, the pharmaceutical compositions of the present invention are typically administered for a continuous period of time ranging from one or more weeks to one month, several months, or longer (e.g., at least 7, 14, 28, 60, or 120 days). In one embodiment, the pharmaceutical composition of the invention is administered for the treatment of a chronic disease, disorder, or condition for a treatment period of one month to twelve months. In another embodiment, the 2S, 4R enantiomer is administered for one year to 5 years. In another embodiment, the 2S, 4R enantiomer is administered for 5 years to 20 years. In another embodiment, the 2S, 4R enantiomer is administered until the patient heals or is administered for life.

The duration of administration according to the methods of the invention depends on the disease or condition being treated, on the extent to which the symptoms of the disease and condition are alleviated by the administration of the pharmaceutical composition, and on the response of the individual patient to the treatment. Methods of treating diseases and conditions using the pharmaceutical compositions of the invention

The 2S, 4R ketoconazole enantiomer is significantly more effective at reducing the concentration of physiologically active glucocorticoids in plasma than racemic ketoconazole or the other enantiomer counterpart 2R, 4S enantiomer in racemic ketoconazole. In addition, as demonstrated in the figures and examples below, unlike racemic ketoconazole, the 2S, 4R enantiomer did not increase exposure to the 2S, 4R enantiomer with time. Thus, the methods of the invention provide significantly better therapeutic benefit in treating diseases and conditions associated with elevated cortisol levels or abnormal cortisol activity, or diseases where benefit can be obtained by lowering normal cortisol levels or activity, as compared to methods involving administration of racemic ketoconazole.

Cortisol promotes the accumulation of adipose tissue and the release of free fatty acids from adipose tissue. When free fatty acids are oxidized, they act in an antagonistic manner on insulin in the liver, reducing insulin sensitivity in the liver (i.e., increasing hepatic insulin resistance). Cortisol also acts directly as an antagonist of insulin action in the liver, and thus insulin sensitivity is further reduced. Cortisol also directly increases the amount of rate limiting enzymes (enzymes) that control glucose production by the liver. These effects lead to an increase in gluconeogenesis (gluconeogenesis) and an increase in the amount of glucose produced by the liver. Hepatic insulin resistance also results in a weakening of the liver to synthesize lipoproteins, and thus, it is a major factor in causing dyslipidemia in type II diabetics and metabolic syndrome patients. Patients who have a reduced glucose tolerance are more likely to develop type II diabetes in the presence of abnormally high levels of cortisol. High cortisol levels can also lead to hypertension, which occurs in part through the activation of mineralocorticoid receptors. Inhibition of the 11 β -HSD-I enzyme alters the ratio of cortisol to cortisone in a particular tissue, promoting an increase in cortisone. The 2S, 4R ketoconazole enantiomer is a cortisol synthesis inhibitor and acts on 11 β hydroxylase and may also exert its therapeutic effect at least in part by inhibiting 11 β -HSD-I enzyme.

The present invention provides methods of using 2S, 4R ketoconazole enantiomer, an inhibitor of cortisol synthesis, to treat, control, ameliorate, prevent, delay the onset of, or reduce the risk of developing diseases and conditions that are caused at least in part by cortisol and/or other corticosteroids in mammalian patients, particularly humans. In one embodiment, the method comprises administering to a patient having a disease or disorder a therapeutically effective amount of the 2S, 4R ketoconazole enantiomer or a pharmaceutically acceptable salt or solvate thereof, substantially or completely free of other ketoconazole enantiomers.

Cortisol activity can lead to a variety of diseases and conditions including, but not limited to, type II diabetes, metabolic syndrome, obesity, dyslipidemia, insulin resistance and hypertension. These and other diseases and conditions that may be treated using the compositions of the present invention according to the methods of the present invention are described below. Diabetes, metabolic syndrome and related diseases and conditions

Diabetes is caused by a number of factors, the simplest of which is represented by elevated glucose levels in the plasma in the fasting state (hyperglycemia). There are two recognized forms of diabetes: type I diabetes, in which case the patient produces little or no insulin, a hormone that regulates the production and utilization of glucose; and type II diabetes, in which case the patient produces insulin and even exhibits hyperinsulinemia (plasma insulin levels may be similar or even higher than in non-diabetic individuals), but at the same time exhibits hyperglycemia. Type II diabetics are often somewhat resistant to the glucose lowering action of insulin. Type I diabetes is typically treated by administration of exogenous insulin by injection.

However, type II diabetics often develop "insulin resistance" such that the effect of insulin in stimulating glucose and lipid metabolism in the major insulin sensitive tissues, i.e., muscle, liver and adipose tissue, is reduced. Patients with insulin resistance but no diabetes have elevated insulin levels, which compensate for their insulin resistance, and thus, serum glucose levels are not elevated. In type II diabetics, even elevated plasma insulin levels are still insufficient to overcome significant insulin resistance, resulting in hyperglycemia. Type II diabetics may also have Elevated levels of circulating Cortisol (circulating Cortisol levels) and/or rates of production (see Lee et al, "plasmid instruments, growing phosphor, colletisol, and central organism analysis of Diabetes Type 2Diabetes instruments," Diabetes cards 1999; 22 (9): 1450-7; Homma et al, "Assessing system 11. beta. -hydraulic Diabetes instruments with collagen/collecting reagent in Diabetes patients and tissues with microorganisms and culturing microorganism, treating nitrogen dioxide, reacting phosphor, and biological tissue, tissue analysis "Clin Endocrinol (Oxf)2005," salivatol levels in elderly large type 2 diabeticiveters; 63(6): 642-9; "OccultCushing's syndrome in type-2diabetes," J Clin Endocrinol Metab 2003; 88(12): 5808-13, each of which is incorporated herein by reference). It is now known that excess cortisol (see us patent 5,849,740, incorporated herein by reference) can induce two basic characteristics of insulin resistance and type II diabetes: decreased peripheral glucose uptake, and increased hepatic glucose production. See also Rizza et al, "ceramic-induced insulin resistance in man: "JClin Endocrinol Metab 1982" copied version of glucose production and simulation of glucose administration to a promoter defect of insulin action; 54(1): 131 to 8; holming and Bjorntorp, "The effects of cortisol on insulin sensitivity in muscle," Acta Physiol Scan and 1992; 144(4): 425-31; lecavalier et al, "Glucagon-cortico interactions on glucose turn over and lactateglucose interactions in normal humans," Am J Physiol 1990; 258(4Pt 1): e569-75; andKhani and-Tayek, "ceramic additives gluconeogenesis in humans: its roll in the metabolic syndrome, "Clin Sci (Lond) 2001; 101(6): 739-47; each of which is incorporated herein by reference.

Persistent, stable or uncontrolled hyperglycemia that occurs in diabetes is associated with increased morbidity and early mortality. Abnormal glucose homeostasis is also directly and indirectly accompanied by obesity, hypertension, and alterations in lipid, lipoprotein, and apolipoprotein metabolism. Patients with type II diabetes have an increased risk of developing cardiovascular complications, such as atherosclerosis, coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy and retinopathy. Therefore, the therapeutic control of glucose homeostasis, lipid metabolism, obesity, and hypertension is extremely important in the clinical control and treatment of diabetes. The present invention provides such a method of therapeutic control by administering a therapeutically effective amount of the 2S, 4R ketoconazole enantiomer substantially or completely free of the 2R, 4S enantiomer.

Many patients who suffer from insulin resistance but have not (yet) developed type II diabetes are also at risk of developing precursors or symptoms previously referred to as "insulin resistance syndrome, metabolic abnormality syndrome or syndrome X", now more commonly referred to as "metabolic syndrome". Metabolic syndrome is characterized by insulin resistance, as well as abdominal obesity, high blood insulin, high blood pressure, low HDL levels, high VLDL triglycerides and small density LDL particles (small dense LDL particles) and elevated glucose levels. These patients, regardless of whether they develop overt diabetes, are at an increased risk of developing the cardiovascular complications described above. Patients with metabolic syndrome are reported to have abnormal Cortisol levels, production or catabolism (see Bersearch-Gabriesecu et al, "hypercorticillim- -a risk factor in acute hypertension and atheroclysis." Endocrinologies 1981; 19 (2): 123-7; Phillips et al, "Elevated plasma coagulation ratios: a link between low biological height and the insulin resistance syndrome," jin Endocrinol Metab 1998; 83 (3): 757-60; and Ward et al, "Cortisol and metabolic syndrome in South Asia Clin Oxf) (2003; 58-5, each of which is incorporated herein by reference).

Treatment of type II diabetes typically involves dietary therapy and increased physical exercise, either alone or in combination with drug therapy. Sufficiently high concentrations of insulin to stimulate insulin resistant tissues can be obtained by administering sulfonylurea(s) drugs (e.g., tolbutamide and glipizide) or meglitinide(s) drugs that stimulate pancreatic beta cells to secrete more insulin, and/or by injecting insulin to increase the level of insulin in the plasma when sulfonylurea and meglitinide drugs become ineffective. However, it may lead to too low a level of glucose in the plasma and a higher degree of insulin resistance may eventually occur.

Biguanide (biguanidines) drugs reduce overproduction of glucose by the liver and increase insulin sensitivity, thereby altering hyperglycemia to some extent. However, many biguanide drugs, for example, phenformin and metformin, can cause lactic acidosis, nausea and diarrhea.

Thiazolidinediones (thiazolidinediones) or glitazones (i.e., 5-benzyl thiazolidine-2, 4-diones) are a newer class of compounds that have been identified as having the potential to ameliorate hyperglycemia and other symptoms of type II diabetes. These agents increase insulin sensitivity in muscle, liver and adipose tissue, thereby partially or completely neutralizing elevated glucose levels in plasma, and substantially not causing hypoglycemia. Glitazones are currently marketed as agonists of the peroxisome proliferator-activated receptor (PPAR) gamma subtype. PPAR γ agonism is generally thought to contribute to the increased insulin sensitivity observed following administration of glitazones. More recently PPAR agonists developed for the treatment of type II diabetes and/or dyslipidemia are agonists of one or more of the PPAR α, γ and δ subtypes. One disadvantage of all known glitazones is their weight-increasing effect, which is caused by an increase in adipose tissue mass. Another disadvantage is that glitazones have been associated with an increased risk of heart failure, which is caused by fluid retention.

There remains a need for new methods of treating diabetes and related conditions, such as various conditions that individually and collectively lead to metabolic syndrome. The present invention satisfies this need. The present invention provides a method of treating diabetes and conditions associated with hyperglycemia and insulin resistance in a mammalian patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. In one embodiment, the method is used to treat type II diabetes. Administering a therapeutically effective amount of an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, effective to treat, control and ameliorate symptoms of diabetes, particularly type II diabetes, wherein administering a therapeutically effective amount of an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, on a regular daily basis delays or prevents the onset of type II diabetes.

The pharmaceutical compositions of the present invention are also useful for the treatment and prevention of conditions associated with type II diabetes and insulin resistance, including obesity (typically abdominal obesity), metabolic syndrome ("syndrome X"), including the various symptoms and conditions that this syndrome, diabetic retinopathy, neuropathy, nephropathy and early cardiovascular disease have, by lowering insulin resistance and maintaining normal serum glucose levels.

Excessive cortisol levels have been associated with obesity, which may be related to the ability of cortisol to stimulate adipogenesis in obesity in general and visceral (also known as abdominal) obesity in particular. Visceral/abdominal obesity is closely linked to glucose intolerance, hyperinsulinemia, hypertriglyceridemia and other factors (symptoms and conditions) of the metabolic syndrome, such as hypertension, elevated VLDL and reduced HDL, and diabetes. Thus, administration of an effective amount of an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, is useful for treating or controlling obesity (e.g., abdominal obesity) and metabolic syndrome. The use of 11 β -hydroxylase inhibitors, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, for long-term treatment in accordance with the methods of the present invention may also be useful in delaying or preventing the development of obesity, particularly when the patient is administered 11 β -hydroxylase inhibitors, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, in combination with dietary management and exercise.

Thus, in another embodiment, the invention provides a method of treating obesity (e.g., abdominal obesity) in a mammalian patient in need of such treatment, the method comprising administering to the patient a therapeutically effective amount of the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Also, in another embodiment, the present invention provides a method of treating metabolic syndrome in a mammalian patient in need of such treatment, the method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Arteriosclerosis, Lipid Disorders (Lipid Disorders), hypertension

Inhibition of 14 α lanosterol demethylase (14 α lanosterol demethylase) and reduction of cholesterol, as well as inhibition of 11 β -hydroxylase activity and reduction of cortisol content, are beneficial in the treatment or control of hypertension and dyslipidemia (dyslipemia). Since hypertension and dyslipidemia contribute to the development of arteriosclerosis, administration of therapeutically effective amounts of a 14 α lanosterol demethylase inhibitor and an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, may be beneficial in treating, controlling, delaying the onset of or preventing hypertension, dyslipidemia, and atherosclerosis. In one embodiment, the present invention provides a method of treating atherosclerosis in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer.

In another embodiment, the present invention provides a method of treating a lipid abnormality selected from the group consisting of dyslipidemia (dyslipemia), hyperlipidemia (hyperlipidemia), hypertriglyceridemia (hypertriglyceridemia), hypercholesterolemia (hypercholesteremia), low HDL and high LDL in a mammalian patient in need of such treatment, comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Apoplexy (apoplexy)

Inhibition of 14 α lanosterol demethylase and reduction of cholesterol, as well as inhibition of 11 β -hydroxylase activity and reduction of cortisol content, are beneficial in the treatment of ischemic stroke (ischemic stroke). Since cortisol, hypertension and dyslipidemia (dyslipemia) contribute to the exacerbation and mortality of ischemic stroke, administration of therapeutically effective amounts of a 14 α lanosterol demethylase inhibitor and an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, may be beneficial in treating or lessening the severity of ischemic stroke. In one embodiment, the present invention provides a method of treating an ischemic stroke (ischemic stroke) event in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Alzheimer's disease (Alzheimer's disease)

Inhibition of 14 alpha lanosterol demethylase and reduction of cholesterol, as well as inhibition of 11 beta-hydroxylase activity and reduction of cortisol content, are beneficial in the treatment of alzheimer's disease. Since elevated cortisol is associated with the development of alzheimer ' S disease and the severity of alzheimer ' S disease can be reduced by lowering cholesterol using statins, administration of therapeutically effective amounts of a 14 α lanosterol demethylase inhibitor and an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, may be beneficial in treating or reducing the severity of alzheimer ' S disease. In one embodiment, the present invention provides a method of treating alzheimer' S disease in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Cognitive Impairment (Cognitive Impairment), Dementia (Dementia) and Depression (suppression)

Excessive levels of cortisol in the brain can also lead to neuronal loss or dysfunction by enhancing the ability of the neurotoxin. Cognitive impairment is associated with aging and excessive cortisol levels in the brain (see Seckl Walker, "Minireview: 11. beta. -hydraulic dehydrogenation type 1-a tissue-specific activity of hypercortiology 2001; 142 (4): 1371-6, which is incorporated herein by reference). Administration of an effective amount of an11 β -hydroxylase inhibitor, such as the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S enantiomer, reduces, ameliorates, controls or prevents age-related cognitive impairment and neuronal dysfunction (neuronal dysfunction). In one embodiment, the present invention provides a method of treating cognitive impairment, neuronal dysfunction and/or dementia in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer.

Another condition is depression, in which high cortisol levels are reported to be important in a causal relationship. Muck-Seler et al (Muck-Seler et al, "Platelet serotonin and plasma prolactin and cortisol in health, decompressed and schizophrenic who," Psychiatry Res 2004; 127 (3): 217-26, which is incorporated herein by reference) reported a substantial increase in plasma cortisol levels in schizophrenic and hypochondric patients as compared to the values of healthy controls. In one embodiment, the present invention provides a method of treating depression in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer.

Cushing's Syndrome (Cushing's Syndrome)

Cushing's syndrome is a metabolic disease or disorder in which patients have high cortisol levels in the bloodstream. These high cortisol levels may be caused by adrenal dysfunction due to pituitary or secondary tumors, both of which produce excess cortisol secretagogue ACTH, or by tumors or disorders of the adrenal gland itself, which directly produce excess cortisol. Patients with cushing's syndrome often develop type II diabetes. Treatment of cushing's syndrome may include removal of aggressive tumors and/or treatment with cortisol synthesis inhibitors such as metyrapone, ketoconazole, or aminoglutethimide (see Murphy, "Steroids and deprension." J Steroid Biochem Mol Biol 1991; 38 (5): 537-59, which is incorporated herein by reference). In one embodiment, the present invention provides a method of treating cushing' S syndrome in a patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer, alone or in combination with other cortisol synthesis inhibitors, such as metyrapone or aminoglutethimide. Reduced insulin secretion

Glucocorticoids (glucocorticoids) have been shown to reduce insulin secretion in vivo (see Billuudel and Sun, "Direct effect of corticosterone upper insulin secretion student biological tissue techniques," Hot Metab Res 1979; 11 (10): 555-60, which is incorporated herein by reference). Therefore, inhibition of cortisol synthesis by the pharmaceutical composition used in the method of the present invention may be beneficial in the treatment of decreased insulin secretion. In addition, decreased 11 β -HSD-I activity was observed to improve glucose-stimulated insulin secretion in isolated murine pancreatic β cells (see Davani et al, "Type 111 beta-hydroscopic enzymes glucose activity and insulin in pancreatic islets," J Biol Chem 2000; 275 (45): 34841-4, incorporated herein by reference). In one embodiment, the present invention provides a method of treating decreased insulin secretion in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Glaucoma and intraocular pressure

The levels of glucocorticoid target receptors (glucocorticoids target receptors) and 11 β -HSD-I enzyme are associated with sensitivity to glaucoma. (see Stokes et al, "Altered university sensitivity touchpoints in primary open-angle glaucoma," Invest Ophthalmol Vis Sci 2003; 44 (12): 5163-7, which is incorporated herein by reference). High cortisol levels are reported to be important in the causal relationship for glaucoma. Median total plasma values (mean total plasma), plasma free values (plasma free), and percent free cortisol levels (percent free cortisol levels) are higher in patients with ocular hypertension and glaucoma. The most significant differences are manifested in the percent free cortisol values (percent free cortisol values) in normal individuals and glaucoma patients (see Schwartz et al, "induced plasma free cortisol in oculorhentens and open angle glaucomatous," Arch Ophthalmol 1987; l 05 (8): 1060-5, which is incorporated herein by reference).

The use of the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer to inhibit 11 β hydroxylase activity in accordance with the methods of the invention is useful for lowering intraocular pressure and treating glaucoma. In one embodiment, the present invention provides a method of treating glaucoma and lowering intraocular pressure in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the enantiomer of 2S, 4R ketoconazole substantially free of the 2R, 4S enantiomer. Immunomodulation

In certain disease states, such as tuberculosis, psoriasis, and even under extreme stress, high glucocorticoid activity shifts the immune response to humoral, where in fact cell-based responses may be more beneficial to the patient. Inhibition of 11 β -HSD-I activity and the concomitant reduction in glucocorticoid levels diverts the immune response towards a cell-based response (see Mason, "Genetic variation in the stress response: sub-regulatory exogenous Genetic inflammation and immunization for human infllamatory disease," immunological day 1991; 12 (2): 57-60; and Rook, "glucoricoides and immunology," Baillieres test practice in endocrine Metab 1999; 13 (4): 567-81; all of which are incorporated herein by reference). In one embodiment, the present invention provides a method of modulating an immune response to a cell-based response in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Impaired renal function

Increased intrarenal blood pressure can lead to renal injury. Cortisol competes with the authentic mineralocorticoid for contact with aldosterone receptors and raises blood pressure. Ketoconazole has been tested in patients with renal failure and has been shown to increase glomerular filtration rate. Ketoconazole has also been shown to reduce albumin leakage in the kidney of patients with type II diabetes without renal failure. Accordingly, in one embodiment, the present invention provides a method of treating impaired renal function or reducing albumin leakage in a mammalian patient in need of such treatment, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Therapeutic use of 2S, 4R ketoconazole enantiomers

In view of the foregoing, it will be appreciated by those skilled in the art that the present invention provides a method of treating a condition selected from the group consisting of: (1) hyperglycemia, (2) impaired glucose tolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders, (6) dyslipidemia (dyslipemia), (7) hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12) signs of arteriosclerosis and its sequelae, (13) vascular restenosis (vascular restenosis), (14) pancreatitis, (15) abdominal obesity, (16) neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19) neuropathy, (20) metabolic syndrome, and (21) other conditions and abnormalities having an insulin resistance component, which comprises administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer.

In another aspect, the invention provides a method of delaying the onset of a condition selected from the group consisting of: (1) hyperglycemia, (2) impaired glucose tolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders, (6) dyslipidemia (dyslipemia), (7) hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12) signs of arteriosclerosis and its sequelae, (13) vascular restenosis (vascular restenosis), (14) pancreatitis, (15) abdominal obesity, (16) neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19) neuropathy, (20) metabolic syndrome, and (21) other conditions and abnormalities having an insulin resistance component, which comprises administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer.

In another aspect, the invention provides a method of reducing the risk of developing a condition selected from the group consisting of: (1) hyperglycemia, (2) impaired glucose tolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders, (6) dyslipidemia (dyslipemia), (7) hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12) signs of arteriosclerosis and its sequelae, (13) vascular restenosis (vascellaresenosis), (14) pancreatitis, (15) abdominal obesity, (16) neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19) neuropathy, (20) metabolic syndrome, and (21) other conditions and abnormalities having an insulin resistance component, which comprises administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. Other disorders

The present invention provides a method of reducing plasma cortisol levels in a subject not diagnosed with or treated for a fungal infection by administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S ketoconazole enantiomer. For example, the methods of the invention may also be used to treat diseases and conditions in which cortisol levels are not elevated (e.g., normal or below normal levels), but therapeutic benefit may be achieved by lowering cortisol levels. Additional optional object features

In certain aspects of the invention, a patient being treated with a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer is not diagnosed with and/or is not treated for a fungal infection. In certain aspects of the invention, a patient being treated with a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer is not diagnosed with and/or is not treated for hypercholesterolemia. In certain aspects of the invention, a patient being treated with a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer is not diagnosed with and/or treated for one or more of the following diseases, disorders or conditions independently selected from the group consisting of: (1) hyperglycemia, (2) impaired glucose tolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders, (6) dyslipidemia (dyslipemia), (7) hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12) signs of arteriosclerosis and its sequelae, (13) vascular restenosis, (14) pancreatitis, (15) abdominal obesity, (16) neurodegenerative disease, (17) retinopathy, (18) nephropathy, (19) neuropathy, (20) metabolic syndrome, (21) prostate cancer, (22) benign prostatic hyperplasia, and (23) other conditions and disorders having an insulin resistant component. Reduction of cortisol levels in a subject by providing sustained exposure (constant exposure) to 1-acetyl-4- [4- [ [2- (2, -dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine (1-acetyl-4- [4- [ [2- (2, 4-dithophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine)

In one aspect, the present invention provides methods for reducing cortisol levels in a subject by sustained exposure to 1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine by administering to the patient an agent that is the 2S, 4R enantiomer and is substantially free of the 2R, 4S enantiomer. In this case, a sustained exposure to 1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine is provided, meaning that the drug does not accumulate in the subject to which it is administered.

In one embodiment, 2S, 4R is administered for at least 14 days (e.g., 14 days), preferably at least 28 days (e.g., 28 days). In one embodiment, the 2S, 4R enantiomer is administered daily (one or more doses per day). In one embodiment, 2S, 4R enantiomeric doses are administered every other day. In one embodiment, the 2S, 4R enantiomeric dose is administered according to another schedule as part of a course of treatment, wherein the course of treatment lasts at least 28 days, and wherein administration of an equal weight (or double weight) of racemic ketoconazole results in stacking of the drug in the subject.

Drug accumulation, or lack thereof, can be determined by measuring plasma drug levels on the first day and one or more subsequent days. For example, if plasma levels are determined on day 1, indicated as day 1, then measurements may be taken on day 7 and/or day 14 and/or day 28, or daily measurements for 1, 2 or 4 weeks. In one embodiment, determining plasma levels involves the measurement of 12 hour or 24 hour AUC. In one embodiment, the difference between the cortisol plasma level on day 1 and at least one subsequent day selected from day 7, 14 and 28 is less than about 50%, preferably less than about 25%, and sometimes less than about 15%. It will be appreciated by guidance of the present disclosure that constant exposure of a particular subject to 1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine (constant exposure) can also be deduced from the dosing shown in pharmacokinetic studies, thereby achieving constant exposure in a statistically large number of similar subjects.

In a preferred embodiment, constant exposure is achieved by administering a constant total periodic dose of the 2S, 4R enantiomer, for example a constant total daily dose (administered in one or more divided doses per day). In one embodiment, the subject has not been previously treated with racemic ketoconazole or enantiomeric ketoconazole. In one embodiment, the subject has not taken medication at least 14 days, at least 28 days, or at least 6 months prior to day 1. In one embodiment, the subject is a human patient. In another embodiment, the subject is a dog or Sprague-Dawley mouse. In one embodiment, the subject is diagnosed with a disorder characterized by elevated cortisol levels. Combination therapy (Combination therapeutics)

Thus, a number of diseases, disorders and conditions may be treated, controlled, prevented or delayed using the pharmaceutical compositions and methods of the present invention, including but not limited to: (1) hyperglycemia, (2) impaired glucose tolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders, (6) dyslipidemia (dyslipemia), (7) hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12) signs of arteriosclerosis and its sequelae, (13) vascular restenosis (vascularesenosis), (14) pancreatitis, (15) abdominal obesity, (16) neurodegenerative diseases, (17) retinopathy, (18) nephropathy, (19) neuropathy, (20) metabolic syndrome, and (21) other disorders having an insulin resistant component. In one embodiment, the methods of the invention are applied to patients who are concurrently receiving additional treatment for one or more of the above-described conditions.

As is clear from the figures and examples herein, the 2S, 4R ketoconazole enantiomer does not alter the pharmacokinetics of the 2S, 4R enantiomer, and, by extension, the 2S, 4R ketoconazole enantiomer will not alter other drugs that use the same metabolic and excretory pathways as the 2S, 4R enantiomer. Thus, the present invention provides a method of co-administering a drug commonly co-administered with racemic ketoconazole that does not suffer from the abnormal pharmacokinetics of co-administration of the drug or racemic ketoconazole that occurs when racemic ketoconazole is administered.

The pharmaceutical compositions of the invention may be co-administered or otherwise used in combination with one or more other drugs for the treatment, prevention, suppression, or amelioration of the diseases, disorders, and conditions described herein that are susceptible to therapeutic intervention according to the methods of the invention. In general, the methods of the invention provide a combination of drugs that is safer or more efficacious than either the drug alone or the combination of the non-2S, 4R ketoconazole enantiomer drug with racemic ketoconazole, or the combination is safer or more efficacious than would be expected based on the additive properties of each drug. These other drugs may be administered in commonly used amounts, by conventional routes, simultaneously with or subsequently to a pharmaceutical composition containing the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. When a pharmaceutical composition of the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer is used contemporaneously with one or more other drugs, if the two active drugs can be formulated together, a combined product containing such other drugs and the 2S, 4R ketoconazole enantiomer may be used. Combination therapy according to the methods of the present invention also includes treatments wherein the pharmaceutical composition useful in the methods of the present invention and one or more other drugs are administered according to different overlapping schedules. It is contemplated that the pharmaceutical composition used in the methods of the present invention, or the other active ingredient or both, may be used effectively at lower dosages than when each is used alone, when used in combination with other active ingredients. Accordingly, pharmaceutical compositions useful in the methods of the invention include those that contain one or more additional active ingredients in addition to the 2S, 4R ketoconazole enantiomer.

Examples of other drugs that may be administered in combination with the pharmaceutical compositions of the present invention, either independently or, in some cases, in the same pharmaceutical composition, include, but are not limited to:

(a) dipeptidyl peptidase IV (DPP-IV) inhibitors; (b) an insulin sensitiser comprising: (i) PPAR γ agonists, such as glitazones (glitazones) (e.g., pioglitazone (pioglitazone), rosiglitazone (rosiglitazone) and the like) and other PPAR ligands, including PPAR α/γ dual agonists, such as KRP-297, and PPAR α agonists, such as gemfibrozil (gemfibrozil), clofibrate (clofibrate), fenofibrate (fenofibrate) and bezafibrate (bezafibrate), and (ii) biguanides (biguanidines), such as metformin (metformin) and phenformin (phenformin); (c) insulin, insulin analogs (insulin mimetics) or insulin mimetics (insulin mimetics);

(d) sulfonylureas and other insulin secretagogues, such as tolbutamide (tolbutamide), glipizide (glipizide), glyburide (glyburide), meglitinide (meglitinide), and related substances; (e) α -glucosidase inhibitors (e.g., acarbose); (f) glucagon receptor antagonists, such as those disclosed in the following PCT patent application publications: WO98/04528, WO99/01423, WO00/39088 and WO00/69810, all of which are incorporated herein by reference; (g) GLP-1, GLP-1 analogs and mimetics, and GLP-1 receptor agonists, such as those disclosed in the following PCT patent application publications: WO00/42026 and WO00/59887, both incorporated herein by reference; (h) GIP, GIP analogs and mimetics, including but not limited to those disclosed in PCT patent application publication No. WO 00/58360, which is incorporated herein by reference, and GIP receptor agonists; (i) PACAP, PACAP analogs and mimetics, and PACAP receptor 3 agonists, such as those disclosed in PCT patent application publication No. WO01/23420, which is incorporated herein by reference; (j) cholesterol lowering agents, such as (i) HMG-CoA reductase inhibitors (lovastatin, simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), atorvastatin (atorvastatin), rosuvastatin (atorvastatin), and other statins (statins)), (ii) chelating agents (cholestyramine, colestipol, and dialkylaminoalkyl derivatives of cross-linked dextran (diamylaminoalkivalalderivatives of a cross-linked dextran), (iii) nicotinol (nicotinyl alcohol), nicotinic acid or salts thereof, (iv) cholesterol absorption coenzyme inhibitors, e.g., ezetimibe (ezetimibe) and β -ketosterol), (a): cholesterol acyltransferase inhibitors, such as avasimibe, (vi) antioxidants, such as probucol (probucol); (k) PPAR delta agonists such as those disclosed in PCT patent application publication No. WO97/28149, which is incorporated herein by reference; (l) Anti-obesity compounds, such as fenfluramine (fenfluramine), dexfenfluramine (dexfenfluramine), phentermine (phentermine), sibutramine (sibutramine), orlistat (orlistat), neuropeptide Y5 inhibitors, CB1 receptor inverse agonists and antagonists, and β 3 adrenergic receptor agonists; (m) ileal bile acid transporter inhibitors; (n) agents intended for inflammatory conditions and not glucocorticoids, such as aspirin, nonsteroidal anti-inflammatory drugs, sulfasalazine (azulfidine), and cyclooxygenase-2 selective inhibitors, and (o) protein tyrosine phosphatase-1B (PTP-1B) inhibitors.

Accordingly, in one embodiment, the present invention provides a pharmaceutical composition comprising: (1) a therapeutically effective amount of the 2S, 4R ketoconazole enantiomer, substantially free of the 2R, 4S ketoconazole enantiomer; (2) a therapeutically effective amount of a compound selected from: (a) a DPP-IV inhibitor; (b) an insulin sensitiser selected from: (i) a PPAR agonist and (ii) a biguanide; (c) insulin, insulin analogs and insulin mimetics; (d) sulfonylureas and other insulin secretagogues; (e) an alpha-glucosidase inhibitor; (f) a glucagon receptor antagonist; (g) GLP-1, GLP-1 analogs and mimetics, and GLP-1 receptor agonists; (h) GIP, GIP analogs and mimetics, and GIP receptor agonists; (i) PACAP, PACAP analogs and mimetics, and PACAP receptor 3 agonists; (j) a cholesterol lowering agent selected from: (i) HMG-CoA reductase inhibitors, (ii) chelating agents, (iii) nicotinyl alcohol, nicotinic acid or salts thereof, (iv) PPAR α agonists, (v) PPAR α/γ dual agonists, (vi) cholesterol absorption inhibitors, (vii) acyl-CoA: (viii) a cholesterol acyltransferase inhibitor, and (viii) an antioxidant; (k) PPAR δ agonists; (l) Anti-obesity compounds; (m) ileal bile acid transporter inhibitors; (n) anti-inflammatory agents that are not glucocorticoids; and (o) inhibitors of protein tyrosine phosphatase-1B (PTP-1B); and (3) a pharmaceutically acceptable carrier.

The above pharmaceutical compositions and combination therapies comprise the 2S, 4R ketoconazole enantiomer substantially free or completely free of the 2R, 4S enantiomer, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, co-formulated or co-administered with one or more other active compounds. Non-limiting examples include 2S, 4R ketoconazole enantiomers in combination with two or more active compounds selected from biguanides, sulfonylureas, HMG-CoA reductase inhibitors, PPAR agonists, PTP-1B inhibitors, DPP-IV inhibitors and anti-obesity compounds. [0093] Accordingly, in one embodiment, the present invention provides a method of treating a disorder selected from the group consisting of: (1) hyperglycemia, (2) impaired glucose tolerance, (3) insulin resistance, (4) obesity, (5) lipid disorders (lipid disorders), (6) dyslipidemia (dyslipemia), (7) hyperlipidemia, (8) hypertriglyceridemia, (9) hypercholesterolemia, (10) low HDL levels, (11) high LDL levels, (12) signs of arteriosclerosis and its sequelae, (13) vascular restenosis, (14) pancreatitis, (15) abdominal obesity, (16) neurodegenerative diseases, (17) retinopathy, (18) nephropathy, (19) neuropathy, (20) metabolic syndrome, and (21) other conditions and disorders having an insulin resistant component, said method comprising administering to said patient a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer and a therapeutically effective amount of a compound or a pharmaceutical composition comprising a compound, the compound is selected from: (a) a DPP-IV inhibitor; (b) an insulin sensitiser selected from: (i) a PPAR agonist and (ii) a biguanide; (c) insulin, insulin analogs and mimetics; (d) sulfonylureas and other insulin secretagogues; (e) an alpha-glucosidase inhibitor; (f) a glucagon receptor antagonist; (g) GLP-1, GLP-1 analogs and mimetics, and GLP-1 receptor agonists; (h) GIP, GIP analogs and mimetics, and GIP receptor agonists; (i) PACAP, PACAP analogs and mimetics, and PACAP receptor 3 agonists; (j) a cholesterol lowering agent selected from: (i) HMG-CoA reductase inhibitors, (ii) chelating agents, (iii) nicotinyl alcohol, nicotinic acid or salts thereof, (iv) PPAR α agonists, (v) PPAR α/γ dual agonists, (vi) cholesterol absorption inhibitors, (vii) acyl-CoA: (viii) a cholesterol acyltransferase inhibitor, and (viii) an antioxidant; (k) PPAR δ agonists; (l) Anti-obesity compounds; (m) ileal bile acid transporter inhibitors; (n) anti-inflammatory agents that are not glucocorticoids; and (o) inhibitors of protein tyrosine phosphatase-1B (PTP-1B).

In another embodiment, the present invention provides a method of treating a condition selected from the group consisting of: hypercholesterolemia, atherosclerosis, low HDL levels, high LDL levels, hyperlipidemia, hypertriglyceridemia and dyslipidemia (dyslipemia), comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition of the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer and an HMG-CoA reductase inhibitor. In one embodiment, the HMG-CoA reductase inhibitor is a statin. In one embodiment, the statin is selected from: lovastatin (lovastatin), simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), atorvastatin (atorvastatin), itavastatin (itavastatin), ZD-4522, rosuvastatin (rosuvastatin) and rivastatin (rivastatin).

In another embodiment, the present invention provides a method of reducing the risk of developing a condition selected from the group consisting of: hypercholesterolemia, atherosclerosis, low HDL levels, high LDL levels, hyperlipidemia, hypertriglyceridemia and dyslipidemia (dyslipemia), and the sequelae of these symptoms, comprising administering to a mammalian patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer and an HMG-CoA reductase inhibitor. In another embodiment, the method of delaying the onset or reducing the risk of developing atherosclerosis in a human patient in need of such treatment further comprises administering a cholesterol absorption inhibitor in combination with a statin HMG-CoA reductase inhibitor, and a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. In one embodiment, the cholesterol absorption inhibitor is a cholesterol transesterification protein (CTEP) inhibitor. In another embodiment, the CTEP inhibitor is ezetimibe (ezetimibe).

In another embodiment, the invention provides a method of delaying the onset or reducing the risk of developing atherosclerosis in a human patient in need of such treatment, said method comprising administering to said patient an effective amount of a pharmaceutical composition comprising the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer and an HMG-CoA reductase inhibitor. In one embodiment, the HMG-CoA reductase inhibitor is a statin. In one embodiment, the statin is selected from: lovastatin (lovastatin), simvastatin (simvastatin), pravastatin (pravastatin), fluvastatin (fluvastatin), atorvastatin (atorvastatin), itavastatin (itavastatin), ZD-4522, rosuvastatin (rosuvastatin) and rivastatin (rivastatin). In one embodiment, the statin is simvastatin (simvastatin).

The various embodiments of the present invention have been described above, and the present invention may be further appreciated and understood by reference to the following examples, which demonstrate that the 2S, 4R enantiomer is more effective at reducing plasma concentrations of active glucocorticoids than racemic ketoconazole or the 2R, 4S enantiomer in the racemate, and does not impair (or impair to a much lesser extent) drug metabolism as racemic ketoconazole does. Examples example 1 measurement of Cortisone and Cholesterol after administration of racemic Ketoconazole and Ketoconazole enantiomers

The effect of ketoconazole and the ketoconazole enantiomers on the level of corticosterone in plasma of Sprague Dawley rats was determined. In the experiment depicted in fig. 1, the four enantiomers and racemic ketoconazole were suspended in olive oil. To obtain the results shown in fig. 1, five groups of mice (eight per group) were used. The mice were housed in an 14/10 hour light/dark cycle and were free to feed and water. Each mouse was administered via a gastric tube (200mg drug/kg body weight). The mice in group 1 received the vehicle (olive oil) while the mice in the other four groups received one of the four ketoconazole enantiomers, respectively, as indicated. All mice were dosed at 2 to 3 pm and sacrificed four hours later (between 6 and 7 pm). Plasma was prepared and corticosterone concentrations were determined using an enzyme-linked immunoassay (ELISA). In the mouse, the most active glucocorticoid is corticosterone; in humans, the most predominant active glucocorticoid is the molecular cortisol closely related to it. The results presented in figure 1 demonstrate that both trans enantiomers (2S4S and 2R4R), when administered to mice at 200mg/kg, had little effect on corticosterone levels in the blood compared to vehicle controls. In contrast, both cis enantiomers reduce corticosterone content, 2S, 4R is much more effective than 2R, 4S.

In the experiment summarized in fig. 2, 9 mice consisted of one vehicle (olive oil) group, and 15 groups (10/group) were treated with a specific dose of ketoconazole or one of the two cis ketoconazole enantiomers (2S, 4R and 2R, 4S). These mice were fed and dosed using the methods described above. Plasma was prepared and the concentration of corticosterone in the plasma was determined by ELISA. The results presented in figure 2 demonstrate that the effect of ketoconazole and its enantiomers on corticosterone levels is dose-dependent and that the 2S, 4R enantiomer is much more potent than both ketoconazole racemate and the other cis enantiomer (2R, 4S).

In the experiments summarized in fig. 3 and 8, six groups of mice, 10/group, were treated with vehicle (olive oil); 18 groups of mice, 10/group, were treated with ketoconazole or one of the two cis ketoconazole enantiomers (2S, 4R and 2R, 4S). Mice were fed by the above method; the drug was suspended in olive oil and administered once per mouse via a gastric tube to a dose of 200 mg/kg. All mice were dosed at the specified time, so that all endpoints were between 6 and 7 pm. For example, mice treated for 24 hours were administered from 6 pm to 7 pm the day before being sacrificed, and mice treated for 12 hours were administered from 6 am to 7 am the day after being sacrificed. After sacrifice, plasma was prepared and the concentration of corticosterone in the plasma was determined using ELISA. In the same plasma samples, total cholesterol levels were also determined. The results presented in figure 3 demonstrate that the 2S, 4R enantiomer is much more effective in reducing the effect of corticosterone than the 2R, 4S enantiomer, and that this enhanced efficacy lasts at least 24 hours. The racemate has an efficacy intermediate between these two enantiomers. Similarly, the results shown in the table below demonstrate that 2S, 4R is much more effective in reducing cholesterol than the 2R, 4S enantiomer. The results show that the racemate potency is intermediate between these two enantiomers. Effect of racemic Ketoconazole, 2S, 4R and 2R, 4S enantiomers on the Cholesterol level in mice at the indicated times after oral administration of 200mg of the indicated drug (or vehicle) to miceExample 2 racemic ketoconazole and cis ketoconazole enantiomeric formsMeasurement of drug Exposure (drug Exposure) after drug administration

In this example, dogs were treated with ketoconazole alone or the 2S, 4R enantiomer alone and the amount of the corresponding drug in plasma was determined.Pharmacokinetics of racemic ketoconazole

Two groups of dogs, three male dogs and three female dogs each, were studied. Each dog was dosed with racemic ketoconazole, the concentration of racemic ketoconazole in the dog plasma, measured on the first day of dosing and again after four weeks for daily dosing. The difference between the two groups is that in one group, racemic ketoconazole is provided as a gelatin capsule containing a dry white powder, and in the other group, racemic ketoconazole is suspended in olive oil.

These dogs are purposely bred beagle dogs available from Covance Research Products, inc. These dogs were 4.5 to 5 months old at the beginning of dosing. These dogs were fed in suspended stainless steel cages. The air conditioner provides a minimum of 10 air changes per hour. The ranges of temperature and relative humidity are 18 to 29 degrees celsius and 30 to 70 percent, respectively. A few exceptions are the cycles of automatic control of fluorescent lamps to 12 hours light (0700-1900) and 12 hours dark when manual (manual-edge) is used for the study-related activities. Certified dog food (#8727C, Harlan Teklad) was used ad libitum. Water is provided ad libitum through an automatic water supply system. Upon arrival at the testing laboratory, dogs were acclimated to the new environment for 19 days, then randomized, and divided into treatment groups as needed using a computerized modular program designed to achieve weight balance. After distribution, the average body weight was calculated and checked to ensure that there were no unacceptable differences between groups. These animals are identified individually by the electronic implant.

In the first group, dogs were dosed daily by oral gelatin capsules (size 13, Torpac, New Jersey, USA). The capsules contained enough racemic ketoconazole to provide a dose of 40mg drug/kg body weight/day. Capsules were prepared weekly for each animal according to the individual body weight. The capsules and tablets were stored at room temperature in sealed containers. For the second group, the gelatin capsules contained sufficient racemic ketoconazole suspended in olive oil to provide a dose of 40mg drug/kg body weight/day. Animals were observed daily about 1 to 2 hours after dosing and throughout the experiment. Blood samples (1ml in lithium heparin) were taken from the jugular vein of each animal and blood was taken 0 (pre-dose), 1, 2, 4, 8, 12 and 24 hours after dosing on the first day and week 4 (28 days after daily dosing). On week 4, the pre-dose sample was timed 24 hours after the previous day of dosing. Plasma samples were stored frozen at-70 degrees celsius until analyzed for use. Plasma samples were analyzed for racemic ketoconazole by the following method using racemic ketoconazole as a standard.

As shown in figure 4, the pharmacokinetic profile (concentration as a function of time) of racemic ketoconazole in canine plasma dosed only once (plasma was analyzed within the first 24 hours after dosing) was significantly reduced compared to the pharmacokinetic profile of racemic ketoconazole in canine plasma dosed daily for 28 days (plasma was analyzed within 24 hours after the last of 28 doses). This effect was seen in both groups (racemic ketoconazole was administered as a dry powder and racemic ketoconazole was administered as a suspension in olive oil). The area under the curve (AUC) is calculated using the linear trapezoidal rule (linear trapezoidal rule). The AUC measured after one dose was much smaller than the AUC measured after 28 days of daily dosing (see fig. 5). Furthermore, this effect was found in both groups (racemic ketoconazole was administered as a dry powder and racemic ketoconazole was administered as a suspension in olive oil).Pharmacokinetics of the 2S, 4R enantiomer

Another group of three female dogs and three male dogs was administered 2S, 4R ketoconazole enantiomer and the concentration of the enantiomer in the plasma of dogs was determined daily on the first and four weeks of administration.

These dogs were purposely bred beagle dogs obtained from Harlan, bicenter, Kent, England. These dogs were 4.5 to 5 months old at the time of arrival at the test laboratory and weighed between 6.7 and 8.85 kg. At the beginning of the administration, their age is approximately 6 to 6.5 months. Dogs were fed to a single and unique room and the air conditioner provided a minimum of 10 air changes per hour. The temperature and relative humidity range from 16 to 24 degrees celsius and 30% to 80%, respectively. A few exceptions are the cycles of automatic control of fluorescent lamps to 12 hours light (0700-1900) and 12 hours dark when manual (manual over-ride) is used for the study-related activities. Animals were housed individually in 2.25m2 pens during the day and animals of the same experimental group and sex were housed in at least 4.5m2 pens at night.

After administration of ketoconazole or the 2S, 4R enantiomer, 400g Harlan Teklad Dog Maintenance Diet (Harlan, Teklad, Bicester, England) and Winalt Shapes biscuits (Friskies Pet Care, Suffllk, England) were provided to each animal daily in the morning. Water is optionally supplied through an automatic water supply system. The sleeping mats were changed daily for each animal using clean wood flakes/chips (Datesand ltd. manchester, England). After arrival at the test laboratory, dogs were acclimated for 7 weeks and then randomized, and assigned to treatment groups according to a stratified randomization program, using littermate data and latest body weight data, as needed. After distribution, the average body weight was calculated and checked to ensure that there were no unacceptable differences between groups. These animals are identified individually by the electronic implant.

Three male dogs and three female dogs were dosed daily by oral gelatin capsules (size 13, Torpac, New Jersey, USA). The capsules contained enough of the 2S, 4R enantiomer to provide a dose of 20mg drug/kg body weight/day. Capsules were prepared weekly for each animal according to the individual body weight. The capsules and tablets were stored at room temperature in sealed containers. Animals were observed daily about 1 to 2 hours after dosing and throughout the experiment. Blood samples (1ml in lithium heparin) were taken from the jugular vein of each animal and at 0 (pre-dose), 1, 2, 4, 8 and 24 hours post-dose on the first day and week 4 (28 days post daily dose). On week 4, the pre-dose sample was timed 24 hours after the previous day of dosing. Plasma samples were stored frozen at-70 degrees celsius until used for analysis. Plasma samples were analyzed for the 2S, 4R enantiomer using racemic ketoconazole as a standard as follows.

As shown in figure 6, the pharmacokinetic profile (concentration as a function of time) of the 2S, 4R enantiomer in canine plasma dosed only once (plasma analyzed within the first 24 hours after dosing) was indistinguishable from the pharmacokinetic profile of the 2S, 4R enantiomer in canine plasma dosed daily for 28 days (plasma analyzed within 24 hours after the last of 28 doses). The area under the curve (AUC) is calculated using the linear trapezoidal rule. The AUC measured after one dose was not different from the AUC measured after 28 days of daily dosing (see fig. 7).Ketoconazole assay procedure

The analytical method was established and validated using racemic ketoconazole. Plasma from dogs treated with racemic ketoconazole, 2S, 4R enantiomer or vehicle control was prepared by standard methods and frozen at-70 degrees celsius until analyzed. To analyze the concentration of racemic ketoconazole (or the 2S, 4R enantiomer), plasma samples were thawed and vortexed briefly and 100 microliters of sample was taken. An internal standard (clotrimazole 25 μ l, 100 μ g/ml, Sigma Aldrich) was added to the sample and mixed briefly. The samples were subjected to solid phase extraction using OASIS HLB (Waters Ltd.730-740 Centenial Cort, Centenial Park, Elstree, Hertsfordshire WD63SZ England). The eluent was evaporated to dryness at nominal 40 degrees celsius under a stream of nitrogen and the residue redissolved in a mobile phase and then analyzed by liquid chromatography with uv detection.

The concentrations of racemic ketoconazole and the 2S, 4R ketoconazole enantiomer in the calibration standard and the study sample were determined using least squares regression with the reciprocal of the concentration (1/x) as the weighting (weighing) to improve the accuracy at low concentrations. The lower limit of quantitation (LLOQ) of ketoconazole in canine plasma was 0.25 micrograms/ml, which could be linearly characterized to 25 micrograms/ml. Coefficient of decidable (r)²) Greater than or equal to 0.99226. Example 3 formulation of the 2S, 4R enantiomer substantially free of the 2R, 4S ketoconazole enantiomer and clinical trial a in type II diabetes.Abbreviations

The following abbreviations are used in the present examples.B. Overview

An exemplary formulation of the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer (hereinafter DIO-902) is described in this example, with preclinical data also included to support a trial new drug test in human clinical trials for the treatment of hyperglycemia associated with type II diabetes. All documents cited herein are incorporated herein by reference. Indirect effects of this candidate would be expected to include lowering total and LDL cholesterol, lowering blood pressure and reducing visceral adiposity. Racemic ketoconazole (a mixture of two enantiomers, 2S, 4R and 2R, 4S) is an approved drug for the treatment of various fungal infections ((R))). Since racemic ketoconazole also inhibits cortisol synthesis, this drug is used as an unauthorized treatment for patients with cushing's syndrome. In these patients, racemic ketoconazole reduces glucose, cholesterol and blood pressure. Since cortisol may be a contributing cause in the development of type II diabetes, clinical trials of racemic ketoconazole have been conducted in these patients. These clinical trial results demonstrate that type II diabetes can be treated by lowering plasma cortisol. However, racemic ketoconazole is associated with hepatotoxicity. Preclinical results demonstrate that DIO-902 can be safer and more effective than racemic mixtures.

DIO-902 is the 2S, 4R ketoconazole enantiomer (2S, 4R cis-1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-ylmethyl) -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine ]. ketoconazole, an approved drug, is a racemic mixture of the 2S, 4R enantiomer and the 2R, 4S enantiomer DIO-902 has been purified from the racemic mixture and is largely (greater than 99%) free of the 2R, 4S enantiomer After 28 days at the mg/kg/day dose, the only significant effects were the tendency for reduced feeding and weight loss and reduced cholesterol. Other serum chemistries or measured hematological parameters did not change significantly. Higher single doses were used in rats. DIO-902 inhibited testosterone to 10% of baseline at 200mg/kg body weight. Inhibition occurred within 4 hours after administration and testosterone levels returned to normal within 8 hours. DIO-902 can be administered orally and reaches maximum plasma concentrations in dogs between 2 and 8 hours. DIO-902, 200mg drug/kg body weight, reduced the active glucocorticoid (corticosterone) levels in rodent serum to 25% of baseline within 4 hours after oral administration. This drug dose also inhibits cholesterol in plasma. Thus, DIO-902(2S, 4R) was much more effective in reducing corticosterone in rats than the other enantiomer (2R, 4S) and was also more effective in reducing cholesterol in rats than the other enantiomer.

DIO-902 has not previously been administered to humans as a separate chemical entity. However, such molecules have been widely administered as part of an approved racemic mixture. When the racemic mixture was administered to normal volunteers, both enantiomers were orally available, and, after 200mg administration, the maximum plasma concentration of DIO-902 (about 3.6 μ g/mL) was reached after two hours. An approved use for the racemic mixture is in the treatment of fungal infections, and the approved dose is 200mg BID. In addition, higher doses of the racemic mixture (up to 2000 mg/day) have been used. Racemic mixtures have also been used for unapproved symptoms including cushing's syndrome and prostate cancer. Racemic mixtures can cause hepatotoxicity and reduce testosterone, and 1, 25 dihydroxy vitamin D.

The diagnostic criteria for type II diabetes is hyperglycemia. More specifically, the American diabetes Association confirms that a patient exhibits one of three characteristics: a) occasionally (at any time of day or night) plasma glucose values in excess of 200mg/dL (11.1mmol/L) with or without the signs of diabetes (polyuria, polydipsia, or famous weight loss) occurred on two separate occasions, or b) fasting (8 hours) plasma glucose values in excess of 126mg/dL (7mmol/L), or c) plasma glucose values in excess of 200mg/dL (11.1mmol/L) 2 hours after 75 grams of glucose had been orally administered. Prospective studies have strongly demonstrated that hyperglycemia is causally linked to long-term microvascular complications, including nephropathy and retinopathy. In addition to diagnosed hyperglycemia, type II diabetic patients also have increased incidence of hypertension, hypertriglyceridemia, and hypercholesterolemia. These increase the risk of macroangiopathy and microvascular disease to a large extent.

The most important acquired risk factor for developing type II diabetes is obesity, more specifically visceral obesity. Genetic susceptibilities also exist. Most genes responsible for the development of type II Diabetes have not been identified except for a few well-defined syndromes such as adult-Onset Diabetes of Young (moderate on sets Diabetes of the Young, MODY, mainly caused by mutations in the gene encoding glucokinase). Physiologically, hyperglycemia in type II diabetics is primarily caused by insulin resistance, the relative failure of insulin to stimulate glucose absorption and inhibit glucose production. This insulin resistance is initially partially compensated by increased insulin synthesis. In many patients, there is a late phase in which insulin production is reduced and with the concomitant drastic worsening of hyperglycemia. There is some uncertainty about the cause of insulin resistance, and there is evidence supporting a significant role for intracellular lipids and a direct change in the activity of insulin signaling molecules. Increased glucocorticoid biological activity may also be a direct or indirect cause of insulin resistance and beta cell failure.

Important treatment options for type II diabetics are diet modification, increased exercise and weight loss. Unfortunately, this option, while effective, has proven difficult to implement. Drug therapies include metformin, sulphonylureas (and Meglitinide) and Nateglinide (Nateglinide), which increase insulin secretion like sulphonylureas), glitazones (pioglitazone and rosiglitazone), and insulin. Although effective, glucose control is still not optimal. In 2005, the American Association of Clinical Endocrinologists (AACE) announced a new glycosylated hemoglobin standard of 6.5% or less in type II diabetic patients at their annual meeting. This new standard is part of the efforts made to prevent diabetic complications. Paul Jellinger, the incumbent American society for internal medicine (ACE) school, said that AACE is beginning to address this effort after a study has shown that two-thirds of Americans with type II diabetes are not adequately treating the disease. In addition, there does not appear to be a root cause of insulin resistance in drugs approved for glucose control, and some (e.g., insulin and glitazones) may cause weight gain, which may exacerbate insulin resistance. One advantage of DIO-902 over currently available therapeutics is that this drug is believed to target one of the etiologies of type II diabetes.

Another potential advantage of DIO-902 is that this drug is believed to significantly ameliorate other cardiovascular risk factors, including hypercholesterolemia and hypertension. Most type II diabetic patients have coexisting cardiovascular risk factors including hypertension, dyslipidemia, and microalbuminuria (Alexander et al (2003), "NCEP-defined metabolic syndrome, Diabetes, and the Presence of cardiovascular disease analysis NHANES III therapeutics 50years and older," Diabetes 52 (5): 1210-4). Independent of glycemic control (glycemic control), controlling hypertension and microalbuminuria has been shown to prevent microvascular and macrovascular diabetic complications. In addition, control of dyslipidemia promotes a reduction in cardiovascular risk and also reduces the risk of development of diabetic nephropathy (Bell (2002). "pharmaceutical compositions of diabetes." South Med J95 (1): 30-4). Racemic Ketoconazole reduces blood pressure and Cholesterol in patients with Cushing's syndrome (Sonino et al (1991)., "Ketoconazole treatment in curing's syndrome: experiment in 34 patents," Clin Endocrinol (Oxf)35 (4): 347-52), and reduces Cholesterol in patients with hypercholesterolemia (Gyrling et al (1993) "Effects of Ketoconazole on Cholesterol precorrents and low coherence lipolytics in hypercholesterolema" J Lipid Res 34 (1): 59-67) and prostate cancer (Miettin (1988) "Cholesterol treatment along with Cholesterol experiment in J Lipid resin 1 (43): 51). Data obtained in phase 2 clinical trial described by IND60874 also supports that racemic ketoconazole reduces total and LDL cholesterol and blood pressure in type II diabetics. The preclinical results described here and in example 1 indicate that DIO-902 will have enhanced activity in terms of blood pressure and cholesterol.

As mentioned above, the behavioral and therapeutic options available for type II diabetic patients are insufficient. Lifestyle changes have proven difficult to achieve. Treatment options do not target the underlying cause of the disease, and some therapies, such as insulin and glitazones, may exacerbate certain factors that contribute to underlying insulin resistance, such as body weight. In addition, most treatment options reduce one (hyperglycemia) or at most two (hyperglycemia and hypertension or dyslipidemia) factors that cause microvascular and macrovascular complications. DIO-902 is believed to target an important causative component of type II diabetes (increased cortisol bioactivity) and can treat hyperglycemia, hypertension, and dyslipidemia in these patients.

Glucocorticoids reduce insulin sensitivity and increase plasma glucose levels by acting on the liver, fat, muscle and pancreatic beta cells of humans (and experimental animals), as well established (McMahon et al (1988), "Effects of glucose on carbohydrate metabolism," Diabetes methods Rev 4 (1): 17-30). In rodent models, glucocorticoids are essential for the development of obesity, glucose intolerance and diabetes, and in some cases, elevated glucocorticoid activity is sufficient to trigger diabetes. In humans, a pathological increase in glucocorticoid levels (as seen in Cushing's syndrome patients) can also trigger diabetes. Recently, it has become increasingly recognized that patients with adrenal sporadic tumors (incidenttalomas) and with more subtle increases in cortisol activity have a significantly increased risk of developing diabetes, glucose intolerance, hypertension, obesity (difuse obesity) and dyslipidemia (Terzololoet al (1998) 'Subclinic curing's syndrome in acquired abnormality, 'ClinEndocrinol (Oxf)48 (1): 89-97; Rossi et al (2000)' Subclinic curing's syndrome in surgery with acquired abnormalities: clinical and biochemical experiments' J ClinEndocrinol ab 85 (4): 1440-8).

Several reports suggest that type II diabetic patients have elevated plasma cortisol levels, especially during the period from the lowest point in the daily rhythm occurring around midnight to the morning rise in cortisol. Cameron (Cameron et al (1987). "Hypercortisolism in Diabetes mellitus systems" Diabetes Care 10 (5): 662-4) reported that at 24 hour cortisol levels, at all time points, diabetic patients had higher levels than non-diabetic patients, with the greatest difference at 8 AM. This study also examined the cortisol levels of diabetic patients following the dexamethasone inhibition test. Cortisol levels in diabetic patients were still significantly elevated in the morning after ingestion of 1mg dexamethasone, but not in the controls. Similarly, The Cortisol level in type II diabetics is higher in The evening (Lentle And Thomas (1964), "additional Function And The compatibility Of Diabetes mellitis," Lancet 14: 544-9; Vakov (1984), "English translation Of clinical trial in Diabetes patents"), And in The morning (Lee et al (1999) "Plasma in growth hormone, Cortisol, And central object about Chinese type 2Diabetes patents," Diabetes Care 22 (9): 1450-7) than in The control.

The relationship of these parameters to cortisol has been studied in type II diabetics, since cortisol increases blood pressure and plasma glucose. One study reported that among type II diabetics, those with hypertension had greater fluctuations in the early-late rhythm of cortisol than those with normal blood pressure (Kostican Secen (1997) "circumcadian rhythm of blood pressure and day medical variations" Med Pregl 50 (1-2): 37-40). One study reported that adult, slightly overweight, non-insulin-requiring diabetic patients had higher cortisol levels than non-diabetic patients, and that diabetic patients had clear glucose early-late rhythmicity with their glucose peaks consistent with cortisol peaks (Faiman and moorohouse (1967). "neural variability in the levels of glucose and related disorders in the health and diabetes subjects along with their glucose peaks regulation" Clin Sci 32 (1): 111-26). Similarly, another study reported a close correlation of cortisol to glucose concentration at 8 am in type II diabetics (r 0.82; p < 0.01) (Atiea et al (1992). "The dawn phenomenon and Diabetes control in treated NIDDM and IDDM activities" Diabetes Res clean practice 16 (3): 183-90). One study found elevated cortisol levels at 6 Am in relatively lean Type II diabetics, as well as a correlation of plasma cortisol with glucose production rate (r 0.55; p < 0.05), as determined using tracer dilutions (Richardson and teak (2002). "Type 2diabetes patients mass had a long time for an in jet response: acetic research center study," Am J physical Endocrinol ab 282 (6): E1286-90).

Adrenocorticotropic hormone (ACTH, a pituitary hormone that regulates the production of adrenocorticosteriods) has also been measured in a few studies. One study assayed cortisol and ACTH in normal volunteers and in diabetic patients with and without Autonomic Neuropathy (AN). Diabetic patients with AN have higher HbA1c levels than Diabetic patients without AN, and also have higher ACTH and cortisol levels than both patients without AN and controls (Tsigos et al (1993). "diabetes neuropathic associated activity of the hypothalamic-pituitary-acquired activity", J Clin Endocrinolob 76 (3): 554-8). ACTH was elevated in patients with diabetes and AN, compared to controls, and did not reach statistical significance. One study reported that ACTH was elevated in patients with type II diabetes (but not type I diabetes) (Vermes et al (1985). "induced plasma levels of immunoreactive beta-endorphin and corticotropin in non-insulin-dependent diabetes." Lancet 2 (8457): 725-6).

In contrast to these major positive correlations, another study (Serio et al (1968). "Plasmacortisol response to insulin and cyclic human in metabolic subjects", "Diabetes 17 (3): 124-6) reported normal plasma cortisol levels in diabetic patients. These patients suffer from very mild diabetes, as their glucose is controlled by diet alone. Similarly, another study (for a lesser number of individuals) did not find elevated levels of circulating cortisol in type II diabetic patients (Kerstens et al (2000). "rock of relationship between 11 beta-hydro-specific gene therapy and insulin sensitivity in the basal state and after 24h of insulin infusion in health injections and type 2diabetes patents" Clin endocrine (Oxf)52 (4): 403-11).

Lowering plasma cortisol by drug intervention has been shown to be effective in treating diabetes, hypertension and dyslipidemia in patients with cushing's syndrome. Sonino et al 1991supra reported that 34patients with Cushing's syndrome had reduced hypercortisolemia (hypercortisoliemia) by using ketoconazole at doses between 400 and 800 mg/day. Three patients with hyperglycemia who did not receive any diabetic medication became normoglycemic; of the other three hyperglycemic patients undergoing diabetic medication, one may discontinue medication and the other two may reduce their use of hypoglycemic medications. Similar results have been reported by Winqist (Winqist et al 1995, "Ketoconazole in the management of regional assessment's synthetic indirect diagnosis," J Clin Oncol 13 (1): 157-64). Ketoconazole also reduces blood pressure in most patients with Cushing's syndrome (Sonino et al 1991supra; Fallo et al (1993)' Response of hypertension in hypertension's absolute treatment.' J Intern Med 234 (6): 595-8).

Pharmacological reduction of cortisol synthesis has also been assessed in type II diabetic patients. Metyrapone also inhibits the 11 β hydroxylase, the last step in cortisol synthesis, and is used in short-term studies to determine whether acute inhibition of cortisol would have a beneficial effect on glucose homeostasis. One study (Atiea et al, (1990). "Early moving hyperglyceremia" dawn phenomenon "in non-insulin dependent diabetes mellitis (NIDDM): effects of cholesterol depression by metacoping" diabetes Res 14 (4): 181-5) use of metpirone to inhibit the normal rise of cortisol in the morning and reports that this interference prevents the normal rise of glucose in this time period. One study used metyrapone to inhibit endogenous cortisol synthesis in type I diabetic subjects, followed by infusion of cortisol to mimic normal nocturnal cortisol elevation, or to produce lower basal levels of cortisol. In patients with the "inhibited" cortisol profile, the rate of glucose production is significantly reduced (Dinneen et al (1995) "Effects of the normal organ in cardiovascular carbohydrate and fast metabolism in IDDM." Am J Physiol 268(4Pt 1): E595-603). Carbenoxolone (carbenoxolone) inhibits the activity of HSD1 and HSD2 and thus reduces exposure of liver and fat to cortisol. Another study used carbenoxolone for 7 days of treatment in normal volunteers and type II diabetic patients (Andrews et al (2003), "Effects of the 11 beta-hydrologic deficient diabetes mellitus in the responsive in the men with type 2diabetes. Type II diabetic patients (not normal volunteers) showed a decrease in the rate of glucose production during euglycemia hyperinsulinemic hyperglycemic blood group clamps. Racemic ketoconazole has also been tested in type II diabetics. These tests are consistent with the following conclusions: the therapeutic use of the drug to inhibit cortisol synthesis may have beneficial effects on glucose, blood pressure and cholesterol in type II diabetics. Although an increase in cortisol levels or activity may occur in type II diabetics, therapeutic benefit may be obtained by further reducing cortisol levels or activity, even in patients with normal cortisol levels or activity.

Although therapeutic use of racemic ketoconazole in type II diabetics has produced encouraging results, DIO-902 will be more effective and safer. DIO-902 has a much lower IC than the 2R, 4S enantiomer for the enzyme important in cortisol synthesis (11. beta. -hydroxylase)₅₀And has a lower IC for an important enzyme in cholesterol synthesis (14 alpha-lanosterol demethylase)₅₀(Rotstein et al (1992) "Stereomers of ketoconazoles: preparation and biological activity," J Med Chem 35 (15): 2818-25), it is therefore possible to allow lower doses of drug to be used to achieve the same effect. As demonstrated in example 1, DIO-902 was more effective than the 2R, 4S enantiomer in reducing corticosterone and cholesterol in rats.

In addition, DIO-902 is present in combination with the 2R, 4S enantiomer (IC)₅₀0.195 μ M) has a 12-fold higher IC for CYP7A₅₀(IC50 ═ 2.4 μ M) (Rotstein et al 1992, supra). Without intending to be limited by a particular mechanism, CYP7A inhibition can lead to functional cholestasis, and as a result, accumulation of potentially toxic metabolites, such as oxygenated sterols and bilirubin and xenobiotics (ketoconazole itself) in the liver and plasma can occur. The reduced CYP7A inhibition associated with DIO-902 (compared to racemic ketoconazole) may, at least in part, result in unaltered DIO-902 toxicokinetics observed after repeated dosing.

Preclinical studies have linked glucocorticoid activity to insulin resistance, hyperglycemia, and increased obesity, supporting the rationale for the use of cortisol synthesis inhibitors such as ketoconazole as a therapeutic option for type II diabetics. Preclinical studies have shown that DIO-902 is safer and more potent than racemic ketoconazole. C.Physical, chemical and pharmaceutical Properties of an exemplary pharmaceutical formulation of the invention-DIO 902

DIO-902 isThe single 2S, 4R ketoconazole enantiomer was obtained from racemic ketoconazole. It is prepared from cellulose, lactose, corn starch, colloidal silicon dioxide and magnesium stearate into a quick-release hard tablet of 200 mg. The chemical name is 2S, 4R cis-1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- (1H-imidazol-1-ylmethyl) -1, 3-dioxolan-4-yl]Methoxy radical]Phenyl radical]Piperazine of formula C₂₆H₂₈Cl₂N₄O₄And the molecular weight is 531.44. CAS number 65277-42-1, the structure is shown below. The chiral centers are at the carbon atoms numbered 2 and 4 in the figure.

Ketoconazole is an antifungal compound containing imidazole. DIO-902 is an orally available immediate release tablet and is formulated as shown in the table below.

Composition (I)	Percentage of
		2S, 4R ketoconazole; DIO-902	50％
Silicified microcrystalline cellulose, NF (Prosolv HD90)	16.5
		Lactose monohydrate, NF (316Fast-Flo)	22.4
Corn starch, NF (STA-Rx)	10
		Colloidal silica, NF (Cab-O-Sil M5P)	0.5
Magnesium stearate, NF	0.6

The drug product can be stored at room temperature and is expected to remain stable for at least 2 years at 25 degrees celsius and 50% RH. The medication is packaged as blister tablets. D.Non-clinical studies1. Overview of non-clinical studies

This section includes the pharmacological and toxicological information of DIO-902 and racemic ketoconazole. Pharmacological studies include studies conducted to demonstrate the inhibitory effect of DIO-902 on corticosterone synthesis, serum cholesterol and testosterone levels in rats. The antifungal activity of DIO-902 was also tested in vitro studies. The toxicology studies of DIO-902 in canines included MTD studies, 7-day studies, and 28-day studies (with toxicokinetics). Genotoxicity (genotoxicity) studies were also performed with DIO-902. Since DIO-902 is purified from racemic ketoconazole, the safety of the mixture is related to the safety of DIO-902. Thus, this section includes a Summary of pharmacological and toxicological data obtained primarily from the Summary Basis of approach for NDA18-533 with respect to oral ketoconazole, as well as a Summary of data from the scientific literature and from a 28-day toxicity study in dogs. 2. Non-clinical pharmacology

The main pharmacological effects of DIO-902 will be achieved by inhibition of cortisol synthesis. The reduction of plasma cortisol by means of drug intervention has proven to be effective in the treatment of diabetes, hypertension and dyslipidaemia in patients with Cushing' S syndrome (S oa et al 1991, supra; Winquist et al 1995, supra). Preclinical studies have linked glucocorticoid activity to insulin resistance, hyperglycemia, and increased obesity (reviewed in (McMahon et al 1988, supra.) secondary effects of DIO-902 administration would include reduced cholesterol levels, reduced visceral adiposity, and reduced blood pressure.

One important enzymatic activity associated with the therapeutic effect of DIO-902 is 11 β hydroxylase, an enzyme that catalyzes the final step in cortisol synthesis. DIO-902 has been shown to inhibit this enzyme with an IC50 of 0.15. mu.M (see Table below). Since in rats the major glucocorticoid is corticosterone (in humans the major glucocorticoid is cortisol), the inhibitory effect of DIO-902 on corticosterone synthesis was studied in rats. In one study, male Sprague Dawley mice (10/group) received 2S, 4R-ketoconazole (DIO-902), 2R, 4S-ketoconazole or racemic ketoconazole at single oral (via gastric tube) doses of 0, 50, 100, 200, 400 and 600mg/kg and were sacrificed 4 hours after dosing. In another study, male Sprague Dawley mice (10/group) received a single oral (via gastric tube) dose of 0 or 200mg/kg of 2S, 4R-ketoconazole (DIO-902), 2R, 4S-ketoconazole or racemate and were sacrificed at 4, 8, 12, 16, 20 and 24 hours post-dose. The results show that DIO-902(2S, 4R enantiomer) reduces plasma corticosterone and is more potent than the 2R, 4S enantiomer as shown in the table below. See example 1 for more details. Inhibition of enzymes catalyzing glucocorticoid Synthesis (gluconolactol Synthesis) by DIO-902

Enzyme	Ketoconazole IC50	2S，4R(DIO-902)IC50	2R，4SIC50	Reference to the literature
					17 alpha hydroxylase	0.91	NAV	NAV	(Ideyama et al.1999*)
17, 20 lyase	0.17	0.05	2.38	(Rotstein et al.1992，supra；Ideyama et al.1999)
					11beta hydroxylase	NAV	0.15	0.61	(Rotstein et al.1992)
Aromatase enzymes	NAV	110	39.6	(Rotstein et al.1992)

All ICs in the above table₅₀Values are given in μ M. Although there may be a single enzyme or complex responsible for 17. beta. hydroxylase and 17, 20 lyase activity, the different ICs of several inhibitors₅₀Values have been reported.NAV means that data is not available.^*Ideyama et al.(1999).“YM116，2-(1H-imidazol-4-ylmethyl)-9H-carbazole，decreases adrenal androgen synthesis by inhibiting C17-20lyase activity in NCI-H295human adrenocortical carcinoma cells.”Jpn J Pharmacol 79(2)：213-20

In the table below, the effect of ketoconazole enantiomers on corticosteroid hormone levels in rats is reported. In the table, corticosterone levels (mean ± SEM; ng/mL) were determined four hours after oral gavage of the indicated drugs (N10/group). There was a separate control group (vehicle). Effect of ketoconazole enantiomers on corticosteroid hormone levels in mice

The following table presents data from a study of the inhibition of corticosterone in rats over time following a single oral dose of 200mg/kg of the ketoconazole enantiomer. Corticosterone levels (mean. + -. SEM; ng/mL) were determined at the indicated times following oral administration of 200mg/kg of the indicated drug. To minimize the confounding effect of corticosterone daily rhythms, all mice were sacrificed at the same time of day (18:00) and the time of administration was determined based on this point (N10/group). The mean value of vehicle-treated groups was used as the zero time point control point. Cortisone inhibition in mice over time following a single oral dose of 200mg/kg ketoconazole enantiomer

Subsequent elicited effects of administration of DIO-902 include reduced LDL and total cholesterol, reduced visceral adiposity, reduced blood pressure, and antifungal activity. The mechanism of action of DIO-902 induced cholesterol inhibition and pharmacological studies demonstrating the effect of DIO-902 on serum cholesterol and testosterone levels in rats are discussed below.

Racemic ketoconazole can be used for inhibiting lanosterol 14 alpha demethylationEnzymatic activity, directly cholesterol-lowering, and the 2S, 4R enantiomer has twice the IC for this enzyme compared to the other enantiomers₅₀(Rotstein et al 1992, supra). The cholesterol lowering activity of the 2S, 4R enantiomer is expected to be further enhanced by reduced inhibition of CYP7A, CYP7A being the major enzyme controlling cholesterol catabolism. Reduced CYP7A activity ("Human cholestol 7alpha-hydroxylase (CYP7a1) hyperfocal present type" J Clin Invest 110 (1): 109-17) in humans and "Hypercholesterolemia and changes in Lipid and Lipid metabolism in mice (Erickson et al (2003)" Hypercholesterolemia and Lipid cycle 7a 1-default mice, "J Lipid Res 44 (5): 1001-9)) in humans, thus inhibition of CYP7A by ketoconazole (pulringer et al 2002, supra) in humans is expected to diminish cholesterol lowering effects of this drug. The 2S, 4R ketoconazole enantiomer alone was not expected to reduce CYP7A activity to the same extent as racemic ketoconazole. One study showed that IC of 2S, 4R ketoconazole (DIO-902) for CYP7A₅₀(determined by cholesterol 7. alpha. -hydroxylase Activity) is 2.4. mu.M, IC for 2R, 4S ketoconazole₅₀Is 0.195. mu.M, which supports a 12-fold higher IC for CYP7A for DIO-902 compared to 2R, 4S-ketoconazole₅₀(Rotstein et al.1992，supra)。

A study was conducted to demonstrate the effect of DIO-902 on cholesterol levels in rats. In this study, male Sprague Dawley mice (10/group) were orally administered (via gastric tube) a single dose of 0 or 200mg/kg of 2S, 4R-ketoconazole (DIO-902), 2R, 4S-ketoconazole or racemate and sacrificed at 4, 8, 12, 16, 20 and 24 hours post-administration. The results are shown in the table below, showing small reductions in cholesterol levels at 16, 20 and 24 hours after treatment with the 2S, 4R enantiomer, whereas no such reductions were seen with the racemate or the other enantiomer. Cholesterol levels (mean. + -. SEM; mg/dL) were determined at the indicated times following oral gavage with 200mg/kg of the indicated drug. All mice were sacrificed at the same time of day (18:00) and the dosing time was appropriately determined (N ═ 10/group). Ketoconazole enantiomers in rats on serum bileEffect of sterols

Two studies were conducted to study the effect of DIO-902 on testosterone levels in rats. In one study, male Sprague Dawley mice (10/group) were dosed orally (via gastric tube) with 0, 50, 100, 200, 400 and 600mg/kg of 2S, 4R-ketoconazole (DIO-902), 2R, 4S-ketoconazole or racemate in a single dose and sacrificed 4 hours after dosing. In another study, male Sprague Dawley mice (10/group) were dosed orally (via gastric tube) with 0 or 200mg/kg of 2S, 4R-ketoconazole (DIO-902), 2R, 4S-ketoconazole or racemate in a single dose and sacrificed at 4, 8, 12, 16, 20 and 24 hours post-dose. The results shown in the table below indicate that the 2S, 4R enantiomer (DIO-902) is more effective in inhibiting testosterone than the other enantiomer (2R, 4S). For the results shown in the following table, testosterone levels (mean ± SEM; nmol/L) were determined four hours after oral gavage of the indicated drug (N10/group). There was a separate control group (vehicle). Effect of ketoconazole enantiomers on Testosterone in rats

For the results shown in the table below, testosterone levels (mean. + -. SEM; nmol/L) were determined at the indicated times after oral feeding of 200mg/kg of the indicated drug. All mice were sacrificed at the same time of day (18:00) and the dosing time was determined appropriately (N-10/group). The mean of vehicle treated groups was used as time zero control point. Although 2S, 4R is more effective than 2R, 4S in acute suppression of testosterone, the overall physiological outcome using 2S, 4R may be reduced, as opposed to 2R, 4S. As mentioned in example 2, the concentration of the 2S, 4R enantiomer did not increase with repeated dosing. This is different from the concentration of the racemic mixture, which increases with repeated dosing. Therefore, with the elimination of the external medicineWith repeated administration of the rotamixte, testosterone inhibition will become more pronounced over time. As also mentioned in example 3, the concentration of the racemic mixture 24 hours after dosing increased significantly between the first and the next dose. Thus, testosterone suppression will continue for longer and longer days in the inter-drug interval. Since the 2S, 4R enantiomer does not inhibit its own clearance, the time during which testosterone production is inhibited will not become longer and longer. Testosterone inhibition in rats as a function of time after a single administration (200mg/kg) of the ketoconazole enantiomer3. Antifungal Activity

In vitro studies, DIO-902 and 2R, 4S-ketoconazole both exhibited antifungal activity, as shown in the following table. In this study, yeast isolates were incubated with racemic ketoconazole, DIO-902(2S, 4R-ketoconazole), 2R, 4S-ketoconazole or solvent (DMSO) for 48 hours at 36 ± 1 ℃ and the Minimum Inhibitory Concentration (MIC) was determined. MIC is defined as the lowest concentration that substantially inhibits the growth of an organism (i.e., the concentration that causes a significant reduction in turbidity of greater than or equal to 80% compared to a control). Antifungal Activity of DIO-902

Although the antifungal activity of the 2S, 4R enantiomer has been asserted without evidence, these results demonstrate for the first time that this enantiomer is much more effective as an antifungal agent than the racemate and/or the 2R, 4S enantiomer against a variety of fungi, including Issatchenkia orientalis, Cryptococcus neoformans, Candida tropicalis, Candida parapsilosis, Candida utilis and Candida albicans, or certain strains thereof. In one embodiment, the present invention provides a method of treating a fungal infection by one of these fungi or fungal strains by administering a therapeutically effective amount of a pharmaceutical composition of the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. 4. Safety Pharmacology (Safety Pharmacology)

The inhibitory potential of DIO-902 for CYP3A inhibitory activity was investigated. In this study, the DIO-902 and 2R, 4S ketoconazole enantiomers showed IC' S comparable to each other and to the racemic mixture₅₀Value, despite the IC of the 2S, 4R-enantiomer for CYP3A5₅₀A small (2X) increase in value occurs. DIO-902 (0.005-50. mu.M for CYP3A4, 0.01-100. mu.M for CYP3A 5) was added to microsomes prepared from human liver or to recombinant 3A4 and 3A 5. The activity of the other enantiomer (2R, 4S) and the racemic mixture was also determined as a positive control and as a comparator. The substrates used in these experiments were quinones, an established CYP3A4 and CYP3A5 substrate (Mirghani et al (2002). "Enzyme kinetics for the formation of 3-hydroxyquinoline and three new metabolites of quinoline in vitro; 3-hydroxylation by CYP3A4is and the major metabolic pathway" Drug Metab disorders 30 (12): 1368-71). Activity of DIO-902 on the hydroxylation of quinine

	HLM pool quinine 160. mu.M IC50μM	rCYP3A4 quinine 30. mu.M IC50μM	rCYP3A5 quinine 20. mu.M IC50μM
				Racemic substance	0.27	0.12	0.38
2R，4S	0.37	0.14	0.40
				2S，4R(DIO-902)	0.29	0.10	0.71

HLM: human liver microsomes

Scientific literature reportsInhibitory activity of 2S, 4R enantiomer on cytochrome P450 inhibition. One study (Rotstein et al 1992, supra) evaluated the inhibitory activity of two ketoconazole enantiomers (2S, 4R and 2R, 4S ketoconazole) on the hydroxylation of progesterone, lauric acid and cholesterol, which are markers for various P450 enzymes. IC of 2S, 4R enantiomer₅₀Slightly higher than 2R, 4S. IC of CYP3A4 inhibition (by 6 beta-hydroxylase)₅₀Similar to that of racemic ketoconazole, as reported by Swinney, 1990. In particular, IC for inhibition of progesterone 6 beta-hydroxylase metabolism in rat liver microsomes₅₀Is 1.4. mu.M. CYP4503A4 inhibition by 2S, 4R enantiomer and racemic ketoconazole has similar IC₅₀The potential for drug metabolism interactions of the two compounds is therefore expected to be similar. However, as described below and in example 2, DIO-902 has a significantly reduced potential for causing a PK profile (PK profile) change in the administered drug through inhibition of drug excretion as compared to the other enantiomers.

As for the activity of the P450 enzyme, CYP7A (cholesterol 7. alpha. hydroxylase), the results shown in the following table indicate the IC of the 2S, 4R enantiomer₅₀IC to 2R, 4S enantiomer₅₀Is about 12 times higher. CYP7A is associated with drug interactions because this enzyme controls bile formation and, therefore, exposure of drugs that are normally cleared by bile may be altered with reduced bile formation and bile flow. Racemic ketoconazole has been shown to inhibit bile formation by inhibition of CYP 7A. Racemic Ketoconazole has been shown to reduce bile flow and clearance of endogenous metabolites (cholesterol) and xenobiotics (sodium tetrabromophthalide sulfonate) into bile (Princen et al (1986), "Ketoconazole blocks double acid synthesis in bile salts and in vitamin a inhibiting cholesterol 7 alpha-hydrolyase," J Clin Invest 78 (4): 1064-71; Gaeta and Tripodi (1987), "Ketoconazole ligands bilier experiment function in the isolated functional yield." nauneyn Schmiedeeberg Arcol 335 (6): 697). The 2S, 4R enantiomer has a reduced effect on the pharmacokinetics of the drug normally cleared by bile (ketoconazole), probably byIn this observation, the IC of the 2S, 4R enantiomer for CYP7A₅₀IC to 2R, 4S enantiomer₅₀Is about 12 times higher. As a result of this reduced inhibition of drug clearance, the 2S, 4R enantiomer will have a significantly reduced risk of liver damage compared to the other enantiomer or the racemic mixture of the two enantiomers that make up ketoconazole. P450 inhibitory Activity of Ketoconazole enantiomers (Rotstein et al 1992, supra)5. Non-clinical pharmacokinetics

The absorption of DIO-902(2S, 4R enantiomer) was studied in a 28-day canine toxicology study. In this study, dogs were treated with DIO-902 orally at doses of 2, 6.5 and 20 mg/kg. Serum samples were taken after the 1 st and 28 th daily 2S, 4R enantiomer administrations. For comparison, one group of dogs will receive racemic ketoconazole at a dose of 40 mg/kg/day for 28 days. This dose was administered on the first 9 days of the study as scheduled; however, due to toxicity, the dose of 40mg/kg was stopped after day 9, and animals in this group were not treated for the next 5 days (days 10 to 14). Animals were treated with 20mg/kg of ketoconazole from study day 15 until study day 28. The toxicokinetic parameters are summarized in the table below.

Plasma levels in dogs dosed with 2 mg/kg/day DIO-902 were below the lower limit of detection for the majority of the 24 hour curve. Therefore, the exact AUC for this dose cannot be calculated. Where AUC can be calculated, it is based on values above the limit of detection over a time range of 0 to 12 hours post-dose (see table below). Thus, the AUC for the 2mg/kg dose cannot be reliably compared to the AUC for other dose levels. AUC and C at 2, 6.5 and 20mg/kg for each DIO-902 dose level_maxValues were comparable between day 1 and day 28, indicating minimal to no accumulation with repeated dosing. No gender differences were found in the DIO-902 treated animals. In animals treated with 2mg/kg or 6.5mg/kg DIO-902, C_maxAnd AUC levels are substantially proportional to dose. AUC and C at 6.5 and 20mg/kg dose levels_maxThe increase in level is greater than the increase in dose. T is_maxValues ranged from 1 to 8 hours on day 1 and from 1 to 12 hours on day 28 (see second of the two tables below).

For racemic ketoconazole, AUC and plasma drug levels were significantly lower on day 28 than on day 1 due to discontinuation of dosing and reduction in dosing. However, AUC and C from day 1 to day 28_maxThe values all decreased more than the dose. Thus, the racemic ketoconazole data on day 1 and day 28 could not be reliably compared. AUC and C in animals treated with racemic ketoconazole when data from a 40mg/kg dose of ketoconazole on day 1 was compared to data from a 20mg/kg dose of DIO-902_maxThe value was about twice that of the animals treated with 20mg/kg DIO-902. AUC and C in animals treated with 20mg/kg racemic ketoconazole on day 28_maxValues substantially lower than those for animals treated with 20mg/kg DIO-902.

Due to the problems with racemic ketoconazole dosage discussed above, additional racemic ketoconazole data from another 28-day toxicity study in dogs was obtained for comparison purposes. In this study dogs (3/sex/group) were treated with 2.5, 10 or 40mg/kg of racemic ketoconazole in the form of an oral powder dispersion or 2.5, 10 or 40mg/kg of racemic ketoconazole in the form of an oily suspension once daily for 28 days. Toxicological kinetic samples were collected on day 1 and at weeks 2 and 4. For comparison with the current data, day 1 and day 28 data were obtained from ketoconazole powder dispersions (10 and 40mg/kg) taken. The data obtained from the oil suspension was similar to the powder dispersion. DIO-902C at day 28 for dogs dosed at 20 mg/kg/day_maxValues were between 9.94. mu.g/ml and 9.95. mu.g/ml (see second of the two tables below). For comparison, a dose of 10mg/kg of racemic ketoconazole produced C_max7.52 to 9.20. mu.g/ml (on day 28), a 40mg/kg dose resulted in a C of 42.78 to 46.75. mu.g/ml_max(on day 28). In contrast to racemic ketoconazole, it is evident that AUC and C of 2S, 4R ketoconazole (DIO-902)_maxOn day 28 withThere was no significant difference at day 1. A significant increase in racemic ketoconazole was noted between day 1 and day 28 (see second of the two tables below). For the following table: days of treatment. The lower limit of detection was 0.25. mu.g/ml. a: racemic ketoconazole data. b: racemic ketoconazole data. Values represent the average of 3 animals. Plasma drug levels of DIO-902 and racemic ketoconazole after single and repeated oral drug administrations in dogsToxicological kinetics of DIO-902 following single and repeated oral dosing in dogsFor the above tables, the data for the first of the two tables is used to obtain AUC and Cmax values for the first day of administration and 28 days post daily administration. Values represent the average of 3 animals.^*n＝1，^**n is 2. a: data for racemic ketoconazole. b: data for racemic ketoconazole. AUC data were from 0 to 24 hours. Toxicity of repeated doses of DIO-902

Toxicity of DIO-902 has been studied in dogs in the maximum tolerated dose study, the 7 day study, and the 28 day study. The MTD study and the 7 day study were conducted as two separate phases in a single study.

In the GLP maximum tolerated dose study Beagle dogs (2/sex) were treated with oral (capsule) increasing doses (20, 40, 60 and 80mg/kg) of the 2S, 4R enantiomer. As a control, another group comprising 2 male dogs and 2 female dogs was treated with vehicle. Animals were treated with each dose for three days before the next higher dose was used. No mortality occurred during the increment phase. Clinical symptoms (vomiting) were found at 40 mg/kg. At higher doses, head shaking, tremors, salivation, colored urine and liquid feces were found. From an animal welfare perspective, the 80mg/kg dose was abandoned. Food intake and weight gain were reduced at all doses.

After the end of the MTD study, 4 vehicle-treated animals were treated by oral administration (capsule) of 40mg/kg of enantiomer for 7 days. The control group was not included. All animals were alive at the scheduled time to sacrifice. During the fixed dose period (7 days, 40 mg/kg/day), one dog was found to be thin and one dog was found to have tears. No post-dose observation was made. Food consumption was reduced in all four animals, which lost weight during the 7 day study period. Hematological analysis showed a decrease in reticulocytes (absolute and relative) and a 20% decrease in the total white blood cell count in one dog. The treated dogs had an increase in mean ALT levels of less than twice that determined prior to dosing. The measurements of other liver enzymes did not change significantly. Visual findings at necropsy were limited to GI inflamed areas. The weight of the liver and kidneys may increase, but in the absence of concurrent controls, this is not conclusive.

In a 28-day GLP study, Beagle dogs (3/gender/group) were orally dosed with the 2S, 4R enantiomer daily at 0 (placebo), 2, 6.5, or 20 mg/kg. A separate control group (3/sex) was included and treated by oral administration of racemic ketoconazole with a starting dose of 40 mg/kg/day. At 40mg/kg of racemic ketoconazole, a significant weight loss (up to 17.3%) resulted in discontinuation of the administration after 9 days. Dogs (3/sex) in this group were discontinued for 6 days and then restarted at 20 mg/kg/day. The toxicological kinetics at day 28 curve indicates that the C of racemic ketoconazole at day 28_maxIs 5% or less of the value measured on the first day. Thus, for data comparison, this group cannot be used as a comparator with confidence. All further references to drugs and dosages in this study will refer to the individual 2S, 4R enantiomers unless otherwise indicated below.

The toxicological kinetic data indicated that DIO-902 was absorbed systemically. At a dose level of 2mg/kg DIO-902, the plasma drug level was below the lower limit of detection, as was the case at various time points between 1 and 12 hours post-dose. Thus, AUC was calculated using the time point at which the plasma drug level was above the lower limit of detection. No gender differences were observed for each dose, and no accumulation occurred during the 28 day dosing period.

Dogs dosed with 20 mg/kg/day DIO-902 ate approximately 23-35% less food than dogs in the placebo control group. Weight gain for dogs at the dose of 20 mg/kg/day was 0.25kg (male) and 0.14kg (female), compared to 1.1kg (male) and 0.9kg (female) for placebo-treated dogs. This trend indicates that the majority of the effect on body weight was in the first two weeks of the study, at the end of which the rate of increase in body weight of dogs at the dose of 20 mg/kg/day was similar to that of the placebo control group. Food intake was also increased in the group of 20 mg/kg/day, although still lower than the placebo control group. At the intermediate dose, there was no significant effect on food intake or weight gain.

DIO-902 had no measurable effect on any of the measured ophthalmic or electrocardiographic parameters at these doses. Specifically, dogs treated with DIO-902 at a dose of 20 mg/kg/day did not have significant QTc prolongation. No hematological changes were found. There was no change in the urinalysis. In any serum chemistry assay, the only change is a decrease in cholesterol. In female dogs there is a tendency for the kidney weight to decrease and in male and female dogs there is a tendency for the relative (but not absolute) weight of the liver and adrenal glands to increase. At any dose, no unusual microscopic findings were observed. 7. Other toxicity tests

No reproductive toxicology studies were performed on DIO-902; however, the reproductive toxicology of racemic ketoconazole has been extensively studied.

DIO-902 was found to be negative for genotoxicity in the Ames (Ames) assay and in the murine lymphoma assay. In the Ames test, DIO-902 was tested for mutation induction in five different strains of Salmonella typhimurium requiring histidine. Exposure to DIO-902 did not produce dose-related increases in the number of reproducible back mutations. In lymphoma assays, DIO-902 (with and without S-9 activation) was tested against mutation induction of the thymidine kinase locus in murine L5178Y lymphoma cells. In three separate experiments lacking S-9, and two separate experiments with S-9 present, DIO-902 also did not reproducibly or meaningfully induce mutations in the TK locus when tested up to toxic doses.

No carcinogenic studies have been conducted on DIO-902. Racemic ketoconazole has been found to be non-carcinogenic (sbafonda 18-533).

Administration of the 2S, 4R enantiomer substantially free of the 2R, 4S enantiomer is believed to reduce the risk of Hepatic reactions that sometimes occur after administration of racemic Ketoconazole (Streeker et al (1986) "A clinical associated with Hepatic infection. A clinical ocular study of 55cases," JHinjector 3 (3): 399. 406; Lake-Bakaar et al (1987) "Hepatic microorganisms associated with Hepatic infection in the same kingdom. Br. D. J. 294 (6569): 419-22; Vancauteren et al (1990)" therapeutic articles of biological microorganisms "biological antibiotic J. reaction J. 1997" M. reaction J. 1997 and chemical reaction J. 1997: 19. sodium chloride, and sodium chloride, 35. 9. sodium chloride, 1997 and 9. biological conjugates: 19. 3. A. a mixture of biological microorganisms, and 3. A. a mixture of biological microorganisms of 3. A. biological assay of biological microorganisms of the same kind, A. Ketoconazole-induced hepatic responses are often characterized as very heterogeneous responses (Strieker et al 1986, supra), meaning that the underlying mechanism is not yet clear. Racemic ketoconazole has been shown to inhibit bile formation in rats by inhibiting CYP7A (Princen et al 1986, supra). Racemic ketoconazole was shown to inhibit human CYP7A (Rotstein et al 1992, supra), reduce bile acid synthesis by human hepatocytes (Princen et al 1986, supra), and inhibit bile acid production in treated patients (Miettinen 1988, supra). We believe that a major factor in ketoconazole-induced hepatotoxicity is inhibition of CYP 7A. Because DIO-902 has a higher enantiomeric ratio to CYP7A than the other enantiomer, 2R, 4S (IC)₅₀0.195 μ M) 12 times higher IC₅₀(IC₅₀2.4 μ M) and no increase in drug concentration over time like the racemate occurs, DIO-902 will be accompanied by a much lower incidence of hepatic reactions. Both effects should be interactive; that is, the racemate will accumulate more than DIO-902, the racemate being higherDrug accumulation will result in a stronger relative inhibition of CYP7A than was shown in the cell-free experiment. The corresponding drug concentrations, relative levels of the two enantiomers in plasma and relative IC obtained in humans₅₀The values are consistent with this expectation. E.Pharmacokinetics of DIO-902 in humans

No clinical trials have been conducted with DIO-902. However, after the first and fifth 200mg doses of racemic ketoconazole (administered once every 12 hours), pharmacokinetic profiles of the individual enantiomers have been proposed in a short reported form (Gerber (2003), "Stereoselective Pharmaceutical (PK) of organic ketoconazole (K) in clinical subjects," ACAAF spot "). The pharmacokinetic data are summarized in the following table. The exposure to the DIO-902, 2S, 4R enantiomer was about 2.5 times that of the 2R, 4S enantiomer. It is unclear whether this is due to differences in bioavailability or clearance. AUC and Cmax values for both enantiomers increased after five doses. This result is not necessarily inconsistent with the pharmacokinetic data obtained from preclinical results obtained from dogs dosed with the individual enantiomer DIO-902, since exposure to the 2R, 4S enantiomer may alter clearance of the 2S, 4R and 2R, 4S enantiomers. Pharmacokinetic data (Gerber 2003, supra)F. Idiosyncratic liver reactions in humans

A very heterogeneous hepatic response to racemic ketoconazole has been described (Strieker et al 1986, supra). The description of these reactions as very heterogeneous means that the mechanism is not clearly understood. Any relevant mechanistic explanation should include an asymptomatic increase in liver enzymes, which occurs in 1-10% of treated patients within a short time after the first drug exposure, and a more severe response with a relatively infrequent incidence. There is no reliable evidence to link ketoconazole to immune-mediated mechanisms.

Although there is no description of the relationship of dose and hepatotoxicity in humans, there is a clear correlation between AUC and liver injury in rabbitsIn the union (Ma et al (2003), "hepatoxicity and toxicokinetics of ketoconazoles in rabbits." Acta Pharmacol Sin 24 (8): 778-82). These authors reported that 40mg/kg ketoconazole caused morphological changes in hepatocytes and increased serum liver enzymes in rabbits. This dose is comparable to the highest dose tested in a one year canine study. Acute in vitro hepatotoxicity was studied by others (Rodriguez and dAcosta 1997, supra, and Rodriguez and Acosta (1997), "N-deacetylketoconjugate-induced hepatotoxicity in a basic culture system of rats 117 (2-3): 123-31). In these studies, rat hepatocytes were cultured in the presence of increasing doses of ketoconazole (up to 200 μ M) for periods varying from 0.5 hours to 4 hours. These authors found that both dose and time factors influence the release of Lactate Dehydrogenase (LDH). In the longest exposure studied (four hours), there was no detectable effect at ketoconazole concentrations below 75 μ M (39 μ g/mL). It has also been suggested from preclinical animal models that ketoconazole metabolites, in particular Deacetylketoconazole (DAK), are more potent mitochondrial inhibitors than ketoconazole (Rodriquez and Acosta (1996). "Inhibition of mitochondal function in anaerobic microorganisms": JBiochem Toxicol 11 (3): 127-31). In vitro IC for DAK inhibition of succinate dehydrogenase₅₀Is 350. mu.M (corresponding to 12.3. mu.M of C of the unmetabolized ketoconazole after administration of 400mg in humans_maxComparison of values (Huang et al (1986). "pharmacy and dose reporting of ketoconazole in northern laboratories" analytical Agents Chemother 30 (2): 206-10). These and related direct effects of ketoconazole (and metabolites) may lead to a very heterogeneous response if it is a much more sensitive patient than the general population.

The materials presented here and in example 2 show that the main factor of ketoconazole-induced hepatotoxicity is inhibition of CYP 7A. Because of the IC of DIO-902 for CYP7A₅₀(IC₅₀2.4 μ M) to the other enantiomer 2R, 4S (IC)₅₀0.195. mu.M) 12 times higher (Rotstein et al 1992, supra), andunlike the racemate, where the drug concentration increases over time, DIO-902 will be associated with a much lower incidence of hepatic reactions. As described above, both actions will be interactive; the racemate will accumulate more than DIO-902 and the higher drug accumulation of the racemate will result in a stronger relative inhibition of CYP7A than suggested by the cell-free experiments. Inhibition of CYP7A by racemic ketoconazole may cause a hepatic reaction indirectly through reduced bile acid synthesis and consequent reduction in bile flow and increase in potentially toxic metabolites. Ketoconazole may further exacerbate this process by directly increasing the potentially hepatotoxic oxysterols (oxysterols).

Racemic ketoconazole inhibits bile formation in mice by inhibiting CYP7A (Princen et al 1986, supra) (when cholesterol is used as a substrate bile synthesis is blocked, but not when 7 a-cholesterol is used as a substrate). The inhibition of Bile acid synthesis by ketoconazole is a direct effect on hepatocytes (whitening et al (1989). "Bile synthesis and differentiation by platelet hepatocytes in primary monolayerculture" Biochim Biophys Acta 1001 (2): 176-84). Bile flow is also reduced by ketoconazole and clearance of endogenous metabolites (cholesterol) (Princen et al 1986, supra) and xenobiotics (gauta and Tripodi 1987, supra) to bile is reduced. Since ketoconazole is secreted into bile, it is understood that ketoconazole may inhibit its own clearance and lead to an increase in plasma concentration. This increase in drug concentration has been noted in humans and dogs. Inhibition of CYP7A causes functional cholestasis (reduced bile acid synthesis and bile flow) is consistent with the recognition that CYP7A is the rate-limiting step in bile acid synthesis, and that bile acid synthesis appears to be the rate-limiting step in bile flow. In humans, genetic deficiencies of functional CYP7A cause a substantial reduction in fecal bile acids (Pullinger et al 2002, supra) and in rats, genetic deficiencies of CYP7A can cause cholestasis (Arnon et al (1998). "Cholesterol 7-hydroxyase knockout mouse: a model for monohydroxy biologic associated cholestatis" Gastroenterology 115 (5): 1223-8).

The relationship between CYP7A inhibition, cholestasis and liver injury is also consistent with other rodent models that do not use ketoconazole as an experimental tool. Thus, cholestasis induced by ethinylestradiol (ethinylestradiol) in rats was associated with bile flow suppression, hepatic bile acid levels, and hepatic cholesterol levels. Epomediol prevents ethinyl estradiol induced cholestasis and produces a clear (though small) reversal in these three measurements. CYP7A activity is inhibited by ethinyl estradiol and returns to normal under the action of epoxol (Cuevas et al (2001). "Effect of epoxy on ethinyl estradiol-induced changes in acid and cholesterol metabolism in rates", "Clin Exp pharmaceutical 28 (8): 637-42). Ketoconazole inhibits human microsomal CYP7A, reduces bile acid synthesis by human hepatocytes in treated patients (Princen et al 1986, supra), and inhibits bile acid production (Miettinen 1988, supra). Functional cholestasis can cause subsequent liver damage through reduced clearance of endogenous metabolites such as oxysterol (below) and bilirubin, and through reduced clearance of exogenous metabolites such as ketoconazole.

In addition to the broader effects of ketoconazole-mediated inhibition of CYP7A described above, there may also be more specific effects through reduced clearance of oxysterols. Oxidized sterols (hydroxylated sterols) are formed as precursors to cholesterol or by subsequent hydroxylation of cholesterol. They are removed from the liver by conversion to bile acids or dissolution in bile. The most abundant enzyme in humans that can initiate the conversion of Oxysterol to bile acids is CYP7A (Norlin et al (2000). "Oxysterol 7 alpha-hydrolases activity by cholestol 7 alpha-hydrolases (CYP 7A." J Biol Chem 275 (44): 34046-53), and, as mentioned above, ketoconazole inhibits this enzyme and increases the levels of some oxysterols (Miettinen 1988, supra). If the conversion fails or bile flow decreases, oxysterols can accumulate and liver damage may occur. Oxysterols are cytotoxic to a variety of Cell types, including hepatoma Cell lines (Hietter et al (1984), "anticancer action of cholesterol and the toxicity of hydroxyl on concentrated hepatoma cells," Biochem Biophys Commun 120(2), "657-64," "Leighton et al (1991)," Activation of the cationic cholesterol-7-alpha-hydroxyhydrolase in hepatoma cells: a new differentiation group survival Cell 25-hydroxyhydrolase, Mol Cell Biol11 (4): 2049-56; pGO' calcium et al 1999) oxygen-dehydrogenase nucleotide Numbers 2. mu.M: 255-62). More specifically, one study has reported that H35 murine hepatoma cells die in the presence of the oxidized sterol 25-hydroxycholesterol, and resistance to 25-hydroxycholesterol can be produced by expression of human CYP 7. Ketoconazole abrogates this resistance mediated by CYP7 (Leighton et al 1991, supra).

The extent of the decrease in bile acid synthesis and increase in oxysterol following ketoconazole-mediated CYP7A inhibition will depend on CYP7B (oxysterol 7alpha hydroxylase) levels. Since CYP7B is under genetic and physiological control (Ren et al (2003), "Regulation of oxysterol 7al rhoa-hydrolases (CYP7B 1)" Metabolism 52 (5): 636-42; Jakobsson et al (2004), "A functional C-G polyraphism inter CYP7B1 promoter region and its differential distribution in organisms and Caucasians," pharmacogenerics J4 (4): 245-50), it is expected that the level of CYP7B will not be sufficient to compensate for the inhibition of CYP7A mediated by ketoconazole in patients treated with a certain proportion of ketoconazole. It is known that insufficient CYP7B may cause liver damage if CYP7A activity is significantly reduced. As an extreme of this deficiency, a complete deficiency of CYP7B can be fatal. Thus, one study reported fatal liver damage in infants lacking a functional copy of CYP7B (functional copy). Liver damage is thought to occur as a direct toxic effect, also resulting from inhibition of bile acid formation, and possibly from induction of oxidant stress (oxidant stress). The accumulated oxysterol cannot be further metabolized by CYP7A because this enzyme is not expressed in infants (Setchell et al (1998). "Identification of a new oligonucleotide in biological acid synthesis: simulation of the oxysterol 7 alpha-hydroxyase gene expression", "J Clin Invest 102 (9): 1690) -703).

The observations made in human patients receiving ketoconazole treatment require an explanation for why only a fraction of patients develop brief mild elevation of serum liver enzymes and why a smaller fraction develop more severe reactions. It is likely that CYP7A was inhibited, bile formation and bile flow were reduced, and oxysterol and other potentially toxic metabolites began to accumulate during the first ketoconazole exposure. In most patients, CYP7B is expressed at sufficient levels, or is induced rapidly enough that liver damage is undetectable. It has been shown that in the complete absence of CYP7A, an alternative pathway for bile acid synthesis is upregulated (Pullinger et al 2002, supra). In this model, CYP7B is expressed at lower levels and/or CYP7B induction is delayed, and as a result, less liver damage occurs in about 1% to 10% of individuals. However, CYP7B will subsequently be upregulated, lesions are restricted, and subside, even with continued ketoconazole exposure. In a smaller number of patients, induction of CYP7B may not be sufficient to compensate for inhibition by CYP7A, and more severe liver damage occurs. In particularly sensitive patients, ketoconazole-mediated inhibition of CYP7A can lead to accumulation of ketoconazole and drug concentrations high enough to induce direct toxicity.

It is important to note that, although ketoconazole is an important, commercially available antifungal drug and the hepatic response elicited by ketoconazole may be life threatening, there is no evidence reported in the literature of a direct link between ketoconazole and the hepatic response produced by inhibition of CYP7A, and there is no evidence in the literature of a lower IC of CYP7A based on the 2S, 4R enantiomer₅₀The 2S, 4R enantiomer is proposed to be a safer drug report. U.S. patent 6040307 describes a method for determining drugs using liver microsomes obtained from frozen tissuesWhether the substance can cause hepatotoxicity. However, hepatotoxicity can only be measured using intact live hepatocytes, preferably in live animals.

The materials provided herein and in example 3 provide an inherently consistent mechanism that explains the hepatic reaction initiated by racemic ketoconazole. Because of the IC of DIO-902 for CYP7A₅₀12 times higher than the 2R, 4S enantiomer, patients treated with DIO-902 will have a much lower incidence of hepatic reactions. The relative drug concentrations, the relative levels of the two enantiomers in plasma, and the relative IC achieved in humans₅₀The values are consistent with this possibility. The pharmacokinetic curves for the two enantiomers after five BID administrations of 200mg of racemate have been obtained. For the 2R, 4S enantiomer, IC for CYP7A₅₀Is 0.195 μ M, and if the intrahepatic concentration of the drug is about 20% of the total plasma drug concentration (Venkatakrishnan et al (2000). "Effects of the anti-inflammatory agent active drug metabolism" clinical release. "Clin pharmaceutical 38 (2): 111-80), the enantiomer will need to reach a total plasma concentration of about 1 μ M (about 0.5 μ g/mL) to effectively inhibit intrahepatic CYP 7A. This is within the concentration range of this enantiomer after administration with 200mg of racemate. In contrast, IC of DIO-902₅₀At 2.4. mu.M. Thus, assuming similar drug availability, the total plasma concentration of DIO-902 required for significant inhibition of CYP7A would be 12 μ M (about 6.3 μ g/mL). This enantiomeric C, even with significantly greater exposure to DIO-902_maxThere is also only 65% of this level, and thus CYP7A is unlikely to be inhibited by DIO-902 at these doses. Clinical study of DIO-902

Phase I tests of type II diabetic patients can be conducted to study the safety and tolerability of DIO-902. A summary of this test is provided below. This trial was the first human clinical study with the 2S, 4R ketoconazole enantiomer substantially free of the 2R, 4S enantiomer. The primary objective was to assess the safety and tolerability of the daily administration of the 2S, 4R enantiomer in type II diabetic subjects for 14 days. The next objective was to determine the Pharmacokinetic (PK) profile of the 2S, 4R enantiomer in plasma after a single dose and after 14 days of daily dosing. In addition, the pharmacodynamic activity of the 2S, 4R enantiomer administered daily for 14 days was determined, as reflected by blood pressure, cholesterol, plasma and salivary cortisol, changes in cortisol-binding globulin, measurement of glycemic control (glycemic control) (fructosamine, continuous glucose monitoring, insulin levels and fasting blood glucose), and plasma free fatty acids.

Seven (7) dose groups were studied. Each dose group included six subjects. The dose groups were: ketoconazole 400mg po QD2S, 4R enantiomer 200mg po QD2S, 4R enantiomer 400mg po QD2S, 4R enantiomer 600mg po QD2S, 4R enantiomer 800mg po QD2S, 4R enantiomer 400mg po BID placebo po QD2S

The dose of ketoconazole is the maximum dose recommended based on the label of the product for fungal infection. The dosage levels of the 2S, 4R enantiomer to be studied were based on the knowledge that 50% of racemic ketoconazole is the 2S, 4R enantiomer, extensive clinical experience with racemic ketoconazole at doses significantly higher than the recommended dose on the drug label, the pharmacokinetic profile of racemic ketoconazole and the 2S, 4R enantiomer in dogs, and a 28 day toxicology study of the 2S, 4R enantiomer in dogs. The 2S, 4R enantiomer and racemic ketoconazole were provided in the form of 200mg tablets for oral administration. Placebo tablets matching both the 2S, 4R enantiomer tablets and the racemic ketoconazole tablets are also provided.

The invention, which has been described and illustrated in detail hereinabove, is capable of many embodiments; thus, while certain embodiments of the invention have been described in detail herein, it is intended that numerous alternative embodiments be included within the scope of the following claims.

All publications and patent documents (patents, published patent applications, and unpublished patent applications) cited herein are incorporated by reference as if each such publication or document were specifically and individually indicated to be incorporated by reference. Citation of publications and patent documents is not intended as an admission that any such documents are pertinent prior art, nor does it constitute any admission as to the contents or date of them.

Claims (33)

1. Use of a 2S, 4R ketoconazole enantiomer in the preparation of a medicament for treating, delaying the onset of, or reducing the risk of developing a disease or condition associated with elevated cortisol levels or activity without significant accumulation of the drug in a subject to which the medicament is administered, wherein the disease or condition is selected from the group consisting of hyperglycemia, diabetes, hyperinsulinemia, hypertension, insulin resistance, metabolic syndrome, obesity, hyperlipidemia, atherosclerosis, vascular restenosis, diabetic retinopathy, diabetic nephropathy and diabetic neuropathy, and wherein the total ketoconazole amount of the medicament comprises at least 80% of the 2S, 4R ketoconazole enantiomer.
2. 2. The use as claimed in claim 1, wherein the obesity is visceral obesity, abdominal obesity or central obesity.
3. 3. The use of claim 1, wherein the diabetes is type II diabetes.
4. 4. The use as described in claim 1, wherein said hyperlipidemia is hypertriglyceridemia or hypercholesterolemia.
5. 5. The use of any preceding claim, wherein the medicament further comprises a compound selected from the group consisting of: (a) an insulin sensitiser selected from: (i) pioglitazone, rosiglitazone, 5- [ (2, 4-dioxo-1, 3-thiazolidin-5-yl) methyl ] -2-methoxy-N- [ [4- (trifluoromethyl) phenyl ] methyl ] benzamide, gemfibrozil, clofibrate, fenofibrate and bezafibrate and (ii) biguanide; (b) insulin; (c) tolbutamide, glipizide, glibenclamide, and meglitinide; (d) acarbose; (e) glucagon-like peptide-1; (f) glucose-dependent insulin-releasing peptide; (g) pituitary adenylyl cyclase activating protein; (h) a cholesterol lowering agent selected from: (i) 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitor, (ii) cholestyramine, colestipol, (iii) nicotinol, nicotinic acid and their salts, (iv) cholesterol absorption inhibitor, (v) avasimibe, and (vi) probucol; (i) an anti-obesity compound selected from fenfluramine, dexfenfluramine, phentermine, sibutramine, orlistat; and (j) an inflammatory agent selected from aspirin and sulfasalazine.
6. 6. The use of claim 5, wherein the biguanide is metformin or phenformin.
7. 7. The use of claim 5, wherein the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor is lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, itavastatin or rosuvastatin.
8. 8. The use of claim 5, wherein the cholesterol absorption inhibitor is ezetimibe or beta-sitosterol.
9. 9.The use of claim 5, wherein the medicament or formulation is for the treatment of a disease or condition selected from: atherosclerosis and hyperlipidemia, and said compounds are 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitors.
10. 10. The use as described in claim 9, wherein said hyperlipidemia is hypertriglyceridemia or hypercholesterolemia.
11. 11. The use of claim 5, wherein the medicament is for delaying the onset of or reducing the risk of developing arteriosclerosis in a human patient, and the compound is a 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor.
12. 12. The use of claim 11, wherein the 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, itavastatin and rosuvastatin.
13. 13. The use according to claim 12, wherein the medicament is for delaying the onset or reducing the risk of the occurrence of arteriosclerosis in a human patient, which medicament or formulation further comprises a cholesterol absorption inhibitor.
14. 14. The use of claim 13, wherein the cholesterol absorption inhibitor is ezetimibe.
15. 15. The use according to claim 5, wherein the medicament or formulation is for reducing the risk of the occurrence of a condition selected from the group consisting of: atherosclerosis and hyperlipidemia, and said compounds are 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitors.
16. 16. The use as described in claim 15, wherein said hyperlipidemia is hypertriglyceridemia or hypercholesterolemia.
17. Use of the enantiomers of S, 4R ketoconazole and a compound selected from the group consisting of: (a) an insulin sensitiser selected from: (i) pioglitazone, rosiglitazone, 5- [ (2, 4-dioxo-1, 3-thiazolidin-5-yl) methyl ] -2-methoxy-N- [ [4- (trifluoromethyl) phenyl ] methyl ] benzamide, gemfibrozil, clofibrate, fenofibrate and bezafibrate and (ii) biguanide; (b) insulin; (c) tolbutamide, glipizide, glibenclamide, and meglitinide; (d) acarbose; (e) glucagon-like peptide-1; (f) glucose-dependent insulin-releasing peptide; (g) pituitary adenylyl cyclase activating protein; (h) a cholesterol lowering agent selected from: (i) 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitor, (ii) cholestyramine, colestipol, (iii) nicotinol, nicotinic acid and their salts, (iv) cholesterol absorption inhibitor, (v) avasimibe, and (vi) probucol; (i) an anti-obesity compound selected from fenfluramine, dexfenfluramine, phentermine, sibutramine, orlistat; and (j) an inflammatory agent selected from aspirin and sulfasalazine, for simultaneous separate or sequential administration to a subject, for use in treating, delaying the onset of, or reducing the risk of developing a disease or condition as defined in any one of claims 1 to 5, and wherein the total amount of ketoconazole of the combined preparation comprises at least 80% of the 2S, 4R ketoconazole enantiomer.
18. 18. The use of claim 17, wherein the biguanide is metformin or phenformin.
19. 19. The use of claim 17, wherein the 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitor is lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, itavastatin or rosuvastatin.
20. 20. The use of claim 17, wherein the cholesterol absorption inhibitor is ezetimibe or β -sitosterol.
21. 21. The use of claim 17, wherein the formulation is for the treatment of a disease or condition selected from the group consisting of: atherosclerosis and hyperlipidemia, and said compounds are 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitors.
22. 22. The use as described in claim 21, wherein said hyperlipidemia is hypertriglyceridemia or hypercholesterolemia.
23. 23. The use of claim 17, wherein the formulation is for delaying the onset of or reducing the risk of developing arteriosclerosis in a human patient, and the compound is a 3-hydroxy-3-methyl-glutaryl-coa reductase inhibitor.
24. 24. The use of claim 23, wherein the 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, rosuvastatin, itavastatin and rosuvastatin.
25. 25. The use of claim 24, wherein the formulation is for delaying the onset or reducing the risk of the occurrence of arteriosclerosis in a human patient, the medicament or formulation further comprising a cholesterol absorption inhibitor.
26. 26. The use of claim 25, wherein the cholesterol absorption inhibitor is ezetimibe.
27. 27. The use of claim 17, wherein the formulation is for reducing the risk of the occurrence of a condition selected from the group consisting of: atherosclerosis and hyperlipidemia, and said compounds are 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase inhibitors.
28. 28. The use as described in claim 27, wherein said hyperlipidemia is hypertriglyceridemia or hypercholesterolemia.
29. Use of a 2S, 4R ketoconazole enantiomer in the preparation of a medicament for the treatment of a disease or condition associated with elevated cortisol levels or activity without significant accumulation of the drug in a subject to which the medicament is administered, wherein the disease or condition is selected from depression, cushing' S syndrome, glaucoma, stroke, renal failure or psoriasis, and wherein the total amount of ketoconazole of the medicament comprises at least 80% of the 2S, 4R ketoconazole enantiomer.
30. Use of the 2S, 4R ketoconazole enantiomer in the manufacture of a medicament for use in a method of reducing cortisol levels in a patient diagnosed with a condition characterised by elevated cortisol levels, without significant accumulation of said medicament in the subject to whom said medicament is administered, wherein said condition is hyperglycemia, diabetes or insulin resistance, and said patient is not receiving treatment for a fungal infection, said method comprising providing said patient with sustained exposure to 1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine for a period of at least 14 days, wherein said sustained daily exposure is achieved by administering a constant daily dose of said 2S, 4R enantiomer, and wherein the total amount of ketoconazole of said drug comprises at least 80% of said 2S, 4R ketoconazole enantiomer.
31. 31. The use of claim 30, wherein the period of time is at least 28 days.
32. 32. The use of claim 30 or 31, wherein the period of at least 14 days or at least 28 days begins on day 1 and 1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine is not administered to the patient at least 28 days prior to day 1.
33. 33. The use of claim 31, wherein said 1-acetyl-4- [4- [ [2- (2, 4-dichlorophenyl) -2- [ (1H-imidazol-1-yl) -methyl ] -1, 3-dioxolan-4-yl ] methoxy ] phenyl ] piperazine was not administered to said patient for at least 6 months prior to day 1.