Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/12—Ketones
  • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
  • A61K31/568—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone
  • A61K31/5685—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol substituted in positions 10 and 13 by a chain having at least one carbon atom, e.g. androstanes, e.g. testosterone having an oxo group in position 17, e.g. androsterone
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system

Definitions

• the present invention provides pharmaceutical formulations of DHEA enriched for polymorph form I or form II, which are useful for various therapeutic applications.
• the invention is directed to formulations of DHEA having more consistent bioavailability than previously used formulations.
• DHEA Dehydroepiandrosterone
• DHEA-S inactive sulfate ester
• DHEA + DHEA-S total serum DHEA
• DHEA + DHEA-S total serum DHEA
• DHEA + DHEA-S total serum DHEA
• DHEA + DHEA-S total serum DHEA
• DHEA + DHEA-S declines steadily to about 5 to 10% of peak values by age 60 to 70
• DHEA has been proposed for use in treating many medical conditions, such as systemic lupus erythematosus (U.S. Patent No. 5,817,650), primary adrenal insufficiency (U.S. Patent No. 5,861,391), Addison's disease (ibid.), reduced libido (U.S. Patent No. 5,855,548), obesity (U.S. Patent No. 5,846,962), osteoporosis (U.S. Patent Nos.
• DHEA can be administered by various routes and is orally active.
• the pharmacokinetics of exogenously administered DHEA are complicated by endogenous production of DHEA and by the reversible interconversion between DHEA and DHEA-S, the major metabolite of DHEA, which acts as a reservoir for DHEA.
• DHEA exhibits wide diurnal variations in endogenous production, while DHEA-S levels show little variation during the day.
• DHEA and DHEA-S are bound by serum albumin, globulins, and steroidal sex hormone binding globulin (Meikle et al, 1992; Longcope, 1995). Only a small fraction of orally administered DHEA appears in the blood at any given time as DHEA; most undergoes conversion to DHEA-S by sulfotransferases in the liver and extrahepatic tissues (Barker, 1994; Comer, 1992; Falany, 1995;
• DHEA-S is converted back to DHEA by peripheral tissues containing DHEA sulfatases, including lymphocytes and macrophages. DHEA is subsequently metabolized to androstenedione as well as the potent androgens, testosterone and dihydrotestosterone, and the estrogens, estrone and estradiol. Adipose tissue may serve as a substantial reservoir for adrenal androgens. The aromatization of DHEA in peripheral tissue is thought to account for the majority of estrogen biosynthesis in postmenopausal women (Grodin et al, 1973).
• DHEA occurs in at least three and as many as five anhydrous polymorphic forms and at least three hydrated forms, depending on environmental conditions and the manner of preparation (Chang et al, 1995).
• the known forms have been reported to be distinguishable on the basis of infrared spectroscopy and powder diffraction analysis, except that forms S3 and S4 are indistinguishable using the latter method (ibid.).
• Work conducted in support of the present invention indicates the existence of a sixth anhydrate form designated herein as form VI, which is detectable by solid state NMR but not by infrared spectroscopy or x-ray powder diffraction analysis.
• DHEA formulations enriched in the form I polymorph or the form II polymorph to achieve more consistent bioavailability and reliable efficacy.
• Formulations enriched in the form VI polymorph are also contemplated.
• the present invention includes, in one aspect, a pharmaceutical formulation comprising dehydroepiandrosterone (DHEA), at least 85% of which is present as the form I polymorph, and at least one pharmaceutical excipient.
• DHEA dehydroepiandrosterone
• the invention also includes a composition of matter consisting essentially of the form I polymorph of DHEA.
• the invention also includes, in a second general embodiment, a pharmaceutical formulation comprising DHEA, at least 85 % of which is present as the form II polymorph, and at least one pharmaceutical excipient.
• a pharmaceutical formulation comprising DHEA, at least 85 % of which is present as the form II polymorph, and at least one pharmaceutical excipient.
• at least 90% of the DHEA is present as the form II polymorph, more preferably 95%, and most preferably greater than 99%.
• compositions comprising DHEA, at least 85 % of which is present as the form VI polymorph, and at least one pharmaceutical excipient.
• at least 90% of the DHEA is present as the form VI polymorph, more preferably 95%, and most preferably greater than 99 % .
• the invention also includes a composition of matter consisting essentially of the form VI polymorph of DHEA.
• the invention also includes a method for preparing a capsular or tablet formulation of DHEA.
• a method for preparing a capsular or tablet formulation of DHEA In the method, at least one solid pharmaceutical excipient is mixed with DHEA, at least 85 % of which is present as a single polymorph selected from form I and form II, and the solid formulation is either placed in a capsular container suitable for oral delivery or compressed to form a tablet.
• the invention includes a method of administering DHEA to a subject to obtain an ameliorative result, wherein a pharmaceutically acceptable amount of DHEA is administered such that at least 85% of the DHEA is present as a single polymorph selected from form I, form II, and form VI, and preferably selected from forms I and II.
• a pharmaceutically acceptable amount of DHEA is administered such that at least 85% of the DHEA is present as a single polymorph selected from form I, form II, and form VI, and preferably selected from forms I and II.
• the invention also includes a method for controlling the bioavailability of a DHEA formulation.
• a therapeutically effective amount of a DHEA formulation is administered to a subject, where the DHEA in the formulation consists of a preselected, known ratio of DHEA polymorphs.
• the compositions and methods are useful for achieving more uniform bioavailabilities in DHEA 4 formulations than have previously been achieved, in light of the applicants' discovery that bioavailability of DHEA in vivo is dependent upon the polymorphic composition of the DHEA.
• Figures 1 and 2 show mean baseline adjusted serum concentrations of DHEA and DHEA-S, respectively, in human subjects up to 72 hours after receiving a single dose of DHEA (formulation 1,
• Figures 3 and 4 show mean baseline adjusted serum concentrations of DHEA and DHEA-S, respectively, in human subjects on days 7 to 10 of a multidose study, starting at 0.5 hours prior to dosing on day 7, using DHEA formulations, as described below, containing a mixture of forms I, II and VI (formulation 3) or pure form I (formulation 4).
• the present invention is directed to pharmaceutical preparations of DHEA having more consistent bioavailabilities and pharmacokinetic properties than preparations available prior to the invention.
• the invention is directed to pharmaceutical compositions comprising DHEA such that at least 85 % , preferably at least 90% , and more preferably at least 95 % to 99% of the DHEA is present as the form I polymorph.
• Such compositions exhibit good uptake by the GI tract upon oral administration, show good therapeutic activity, and are highly stable under ambient conditions.
• the invention is directed to pharmaceutical compositions comprising DHEA such that at least 85 % , preferably at least 90% , and more preferably at least 95 % to 99% of the
• DHEA is present as the form II polymorph.
• Such compositions exhibit good uptake by the GI tract upon oral administration, a rapid rate of absorption (greater than the form I polymorph) and good therapeutic activity, and are also stable under ambient conditions.
• compositions thus enriched in form I or form II, as described herein, provide more predictable pharmacokinetic profiles than are provided by commercially available compositions having random polymorphic compositions.
• Compositions similarly enriched in form VI are also contemplated.
• DHEA is known, via analytical techniques such as x-ray diffraction, infrared (IR) spectroscopy, and differential scanning calorimetry (DSC), to occur in several different hydrate and anhydrate crystal forms.
• the anhydrate forms include forms I, II, III, IV and V, although the latter two forms have been observed only transiently by DSC.
• the hydrates (solvates) include forms SI (1/4 hydrate), S2 (monohydrate), S3 (monohydrate), and S4 (1/2 methanolate).
• DHEA and precursors such as DHEA acetate, are commercially available from various sources (e.g., Sigma Chemical Co., St. Louis, MO; Aldrich Chemical Company, Inc.; Diosynth, Inc.; Pfaltz & Bauer, Inc.; Schering AG).
• DHEA compositions emiched for selected polymorphs can be prepared by crystallization of commercial DHEA in selected solvents under appropriate cooling or evaporation conditions.
• conditions have been reported by others for preparing the above-mentioned stable forms (Chang et al, 1995), it has been found by the present applicants that previously reported methods for preparing form I, including those described by Chang, yield products containing an additional polymorph that is designated herein as form VI. Accordingly, improved methods described herein have been developed for preparing pure form I to the exclusion of other polymorphs.
• pure form I is prepared by (a) crystallizing DHEA from anhydrous 2- propanol (or, alternatively, acetone or acetonitrile) under a nitrogen stream at room temperature over about 2 days, producing a crystalline precipitate that contains predominantly form I and some amount of form VI, followed by (b) suspending the precipitate in ethyl acetate (about 100 mL/30 g of DHEA) and stirring the resulting slurry at room temperature for about one week, followed by filtration. The filter cake is allowed to dry at room temperature overnight. 13 C-SSNMR analysis (discussed below) showed that product prepared by this method consisted of pure or nearly pure (>99%) form I; no other forms were detected by 13 C-SSNMR.
• DHEA highly emiched for form II can be obtained by rapid crystallization from tetrahydrofuran (THF), dioxane, chloroform or mixtures of chloroform and THF.
• THF tetrahydrofuran
• Example 1 provides a specific procedure for crystallization from THF, which produced a product shown by X-ray powder diffraction to be pure form II.
• Other polymorphs can be prepared as follows.
• Form III can be obtained by desolvation of form
• Form VI can be obtained in pure form, or as a mixture with form I, by crystallization of crude DHEA (prepared by saponification of DHEA acetate, as described in Example 1, without the methanol recrystallization step) from isopropanol, without a subsequent slurrying step.
• Form SI can be obtained by crystallization in methylene chloride under conditions of 50-60% relative humidity, or by grinding with methanol for 30 minutes and air drying.
• Form S2 can be prepared by crystallization in 40% ethanol or distilled water, or by slow evaporation in acetonitrile, acetone, ethyl acetate, or THF.
• Form S3 can be prepared by replacing methanol molecules of a methanol half-solvate (form S4) with water under conditions of 60% relative humidity.
• Form S4 (1/2 methanolate) can be obtained by cooling a methanol solution of DHEA. Additional procedures for preparing various polymorphs can be found in Chang et al. (1995).
• DHEA compositions that are enriched for selected DHEA polymorphs in accordance with the invention, it is important to be able to determine quantitatively the polymorph content of DHEA materials to establish the degree of enrichment for the desired polymorph relative to other polymorphs that may be present.
• Any appropriate method can be used for this purpose, provided that the method has a level of sensitivity and accuracy sufficient to permit measurement of the content of the major preferred polymorph within ⁇ 5%, and preferably within ⁇ 2.5% or less.
• a selected technique for measuring the polymorphs may not be capable of measuring all of the possible polymorphs, or it may be able to measure certain individual polymorphs only as a sum of two or more polymorphs.
• these limitations either are not a concern or can be overcome by combining the results of complementary methods.
• X-ray powder diffraction the industry standard for determining polymorphic forms of crystalline substances, can be used to measure -the relative amounts of forms I + VI, II, III, SI, S2, S3, and S4.
• this technique is not able to distinguish forms I and VI, as was shown by solid state nuclear magnetic resonance (see below).
• Table 1 summarizes the distinct reflections found for each polymorph.
• the relative amounts of each polymorph in a DHEA sample can be determined by integrating peak heights or peak areas of reflections that are unique to each DHEA polymorph in the sample, according to standard quantitative x-ray methods, with integration of peak areas generally being preferred.
• the XRPD analysis can be simplified by visually inspecting the diffractogram to determine which polymorphs are present at detectable levels, and then limiting integration to the unique reflections corresponding to those polymorphs.
• the diffractogram usually, for anhydrous preparations, only forms I, II and/or VI are present, to the exclusion of hydrates SI through S4. Minor polymorph components present at levels of about 5 % can be readily quantified, with a lower limit of detection of about 2%.
• Solid state 13 C-NMR can also be used to detect and quantify polymorphs or crystalline substances and is a more sensitive technique than XRPD. However, as it is an expensive and time-consuming procedure, it is not routinely used. In the studies described herein, 13 C-SSNMR was required to distinguish forms I and VI of DHEA, as noted above.
• Unique resonance peaks that can be used to quantify polymorphs I, II, VI, and S1-S4 are provided in Table 2.
• the various polymorphs exhibit unique resonance shifts in the regions of 10-18 ppm (low field) and 115-124 ppm (high field) (ppm relative to adamantane). However, because some of the peaks overlap in the low field region, the high field region generally provides better quantitative results.
• the selected resonance peaks can be quantified by any known quantitation technique, such as the curve-fitting technique described by MacDonald (1980). Studies on mixtures of forms I and VI over a range of different concentrations showed that the responses to unique peaks for form VI are linear, indicating that the integration method is reliable for measuring the proportion of form VI in a sample relative to other polymorphs such as form I. Additional studies conducted in support of the invention established that there is good agreement for polymorph determinations by SSNMR and x-ray diffraction.
• Infrared spectroscopy provides yet another method for determining polymorph content of DHEA compositions. Details of this technique can be found in Chang et al. (1995). However, like x-ray diffraction, it appears that this technique does not distinguish form I from form VI.
• forms I and II are both stable under ambient conditions and under heat at non- saturating humidity; form I exhibits greater overall stability.
• (b) temperature was 40°C.
• Formulations containing the DHEA compositions of the invention may be provided in various dosage forms, such as, for example, tablets, capsules, powders, controlled-release formulations, suspensions, emulsions, suppositories, creams, ointments, lotions, or aerosols, and are preferably provided in solid dosage forms suitable for simple administration of precise dosages.
• compositions typically include a conventional pharmaceutical carrier, or excipient, and may additionally include other medicinal agents, carriers, adjuvants, and the like.
• the composition will contain about 0.5 % to 75% by weight, and more preferably about 5 % to 25% by weight, DHEA, with the remainder consisting of suitable pharmaceutical excipients.
• excipients may include pharmaceutical grades of lactose, mannitol, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.
• the formulations of the invention can be administered orally, e.g., by capsule or tablet, for rapid uptake in the blood stream and distribution to various compartments of the body.
• the amount and frequency of administration will vary, depending on the patient and on the therapeutic application as detailed further below.
• solid formulations can be 10 administered as a suppository, e.g., when oral administration is contraindicated.
• the preparations may be tablets, granules, powders, capsules or the like.
• DHEA having the desired polymorph composition is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsular container suitable for delivery to the gastrointestinal tract.
• at least 90%, more preferably at least 95%, and most preferably greater than 99% of the DHEA is present as a single polymorph selected from form I, II or VI, preferably form I or II, and most preferably form I.
• DHEA having the desired polymorph composition is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract.
• the tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations.
• the composition of the invention may also be administered to a subject transdermally or by inhalation. Methods for preparing various conventional dosage forms are known or will be apparent to those skilled in the art; for example, see Remington's Pharmaceutical Sciences (19th Ed., Williams & Wilkins, 1995).
• DHEA formulations enriched for polymorph form I or form II are both absorbed well in vivo and are therapeutically active.
• the applicants have further discovered that commercially available supplies of DHEA can vary significantly in their polymorphic compositions, potentially resulting in significant variations in therapeutic bioavailability and efficacy.
• Example 2 describes a study in which 34 healthy postmenopausal women were administered orally one of three formulations of DHEA, each prepared from commercially available DHEA, in capsular form. The formulations were administered in single dosages of four opaque, size 2 capsules, each capsules containing 50 mg DHEA, 152 mg lactose (169 mg for formulation 3), 108 mg corn starch, and 3 mg magnesium stearate.
• compositions of the formulations differed as summarized in Table 4 below.
• Formulation 1 contained 82% form I, 0% form II, and 18% form VI
• formulation 2 contained 22% form I, 44% form II, and 33% form VI (by SSNMR).
• Formulation 3 fairly similar to formulation 2, contained 18% form I, 43% form II, and 39% form VI.
• the average particle sizes in 1 1 these formulations varied according to the technique used for measurement but generally fell within a range of 50 - 150 ⁇ m, with the particles in formulation 1 being somewhat larger (Table 5).
• Figs. 1 and 2 Time plots of the levels of DHEA and DHEA-S are shown in Figs. 1 and 2, respectively.
• the profile for formulation 1 shows that a maximum concentration was reached about three hours after administration, followed by a somewhat rapid decline to half the maximum concentration after about 12 hours and then a gradual decline to about a third of the maximum concentration after 72 hours.
• Formulations 2 and 3 achieved a maximum concentration more rapidly, within about an hour of administration, followed by a 2-stage decline in concentration similar to that shown by formulation 1.
• formulations 2 and 3 having a high proportion of form II, are absorbed more rapidly and appear to have a higher bioavailability (by about 6-14% based on AUC measurements) relative to formulation 1.
• the profiles for DHEA-S showed similar patterns. All formulations reached a maximum concentration after about three hours, showing that DHEA was converted rapidly to its major metabolite, the sulfate form.
• the shapes of the profiles were also very similar, except that formulation 1 showed consistently lower levels of DHEA-S than formulations 2 and 3, consistent with the results for DHEA shown in Fig. 1. The results show again that formulation 1 appears to have a lower bioavailability than formulations 2 and 3.
• the estimated mean pharmacokinetic parameters (see Example 1) from this study are presented in Tables 6 and 7 below.
• AUC area under curve
• C max and T max are indicators of rate of absorption.
• Serum DHEA concentrations for all samples were above the assay 12 limit of quantification. Baseline-adjusted values were obtained by subtracting the day 1 pre-dosing serum concentration from each serum concentration value.
• Example 3 describes an open-label, randomized, three period crossover pharmacokinetic study in which 39 healthy postmenopausal women were administered orally one of two formulations of DHEA in capsular form.
• the formulations were administered in single dosages of four opaque, size 2 capsules, each capsule containing 50 mg DHEA, 152 mg (Formulation 4) or 169 mg (Formulation 3) lactose, 108 mg corn starch, and 3 mg magnesium stearate.
• the DHEA polymorph composition of formulation 3 was as shown in Table 4 above (18% form I, 43 % form II, and 39% form VI by SSNMR).
• Formulation 4 contained substantially pure ( " 100%) form I DHEA, prepared by methods described herein.
• Tables 8 and 9 show baseline-adjusted pharmacokinetic parameters AUC ]44 -i6 8 (extent of absorption), T max and C max (rate of absorption) for the two formulations.
• the AUC ⁇ 44 - ]68 value represents a single dosing interval (day 7), as is standard for multidose studies.
• Table 8. Baseline Adjusted Mean Pharmacokinetic Parameters for Serum DHEA:
• formulation 4 (100% form I) showed somewhat reduced bioavailability, in terms of rate and degree of absorption, compared to formulation 3 (mixture of polymorphs).
• the invention is directed to methods of controlling bioavailability of DHEA formulations and achieving bioequivalence between different formulations.
• different DHEA formulations containing DHEA and excipients in the same proportions may be prepared from different batches of DHEA and thus may exhibit different bioavailabilities in vivo.
• pharmaceutical formulations prepared from commercially available supplies of DHEA can vary significantly in their in vivo bioavailabilities.
• the invention also includes a method for controlling the bioavailability of a DHEA formulation.
• a therapeutically effective amount of a DHEA formulation is administered such that the formulation comprises a preselected, known ratio of DHEA polymorphs. That is, a DHEA formulation is prepared having known proportions (and amounts) of one or more DHEA polymorphs, e.g., by using appropriate crystallization conditions as discussed above, or by mixing appropriate amounts of the desired polymorphs.
• the polymorphic ratio can be selected to provide a desired bioavailability of the DHEA, e.g., based on assessment of the bioavailability by methods such as described above and in Example 2.
• the method is useful for achieving more uniform and predictable bioavailabilities of DHEA formulations than have previously been achieved, in light of the applicants' discovery that bioavailability of DHEA in vivo is dependent upon the polymorphic composition of the DHEA.
• DHEA is reported to be useful in treating many medical conditions.
• Exemplary conditions which can be treated include, for example, systemic lupus erythematosus (McGuire et al, U.S. Patent No. 5,817,650), where a dose of about 25 to 500 mg/day is administered, optionally concomitantly with a glucocorticoid such as prednisone.
• Corticosteroids may also be used to supplement DHEA, administered at 0.25 to 2.0 mg/kg, in treatment of primary adrenal insufficiency or Addison's disease (S.S.C. Yen et al, U.S. Patent No. 5,861,391).
• DHEA can also be used to enhance the immune response to viral infection (U.S.
• Patent No. 5,077,284 e.g. in treatment of immunocompromised subjects, such as AIDS patients, e.g. with dosages of 400 mg/day. Diabetes can be treated by administration of 120-480 mg/kg daily, for example, or in an amount equaling approximately 0.1% to 0.4% by weight of food intake, according to Coleman et al. (U.S. Patent No. 4,518,595).
• Loss of bone density, as in osteoporosis or osteopenia, can also be treated with DHEA (see, for example, Labrie, U.S. Patent No. 5,776,923), with a typical dose being about 32 mg/kg daily.
• DHEA is also effective for treatment of chronic fatigue syndrome or fibromyalgia; see, for example, White, U.S. Patent No. 5,935,949.
• the invention provides pharmaceutical formulations of DHEA that are enriched for polymorph form I, form II, or form VI, preferably form I or form II, and most preferably form I. These formulations provide consistent bioavailabilities, in contrast to previously utilized DHEA formulations containing varying amounts of multiple DHEA polymorphs.
• Formulations enriched in DHEA form I are highly stable and provide pharmaceutical compositions that retain a constant level of DHEA activity over long periods of time (e.g., more than 1 or 2 years). Accordingly, formulations enriched in form I have long shelf-lives, which is highly desirable in a pharmaceutical product.
• Formulations enriched in form II are stable, though somewhat less stable than form I formulations, and can have a faster rate of absorption (and higher efficacy) in vivo than form I formulations. Accordingly, formulations enriched in form II are also therapeutically beneficial.
• XRPD X-Ray Powder Diffraction
• the Siemens instrument was equipped with an IBM-compatible interface and utilized DIFFRAC AT software (SOCABIM, 1994). Slits I and II were set at 1° and the radiation was electronically filtered by a Kevex Psi Peltier cooled silicon detector with slits III at 1° and IV at 0.15°. A continuous theta-two theta scan at 6°/min (0.4 sec/0.04° step) from 4 to 40 °2 ⁇ was used. A silicon standard was analyzed each day to check the instrument alignment. Each sample was prepared by pressing a small amount of the powder onto a zero- background quartz-in-aluminum sample holder.
• the Shimadzu instrument was equipped with a fine- focus X-ray tube.
• the tube power was set at 40 kV, 40 mA.
• the divergence and scattering slits were set at 1° and the receiving slit was set at 0.15 mm.
• Diffracted radiation was detected by an Nal scintillation detector.
• a theta-two theta continuous scan at 3°/min (0.4 sec/0.02° step) from 4 to 40 °2 ⁇ was used.
• a silicon standard was analyzed each day to check the instrument alignment, and an alumina standard was analyzed each day to check the X- ray tube output.
• Each sample was prepared for analysis by pressing it with a spatula onto a glass or quartz sample holder.
• Quantitative work was generally done on the Shimadzu instrument. Measurements of XRPD peak heights and areas were carried out using GRAMS/32 version 5.05 running on Windows NT.
• GRAMS/32 version 5.05 running on Windows NT.
• theta-two theta continuous scans were performed over a selected range (e.g., from 17 to 23 °2 ⁇ ) at a selected scan range of from 0.5 to 3.0 min, typically 1 min (0.4 sec/0.02° step), three times for each sample.
• the XRPD files were converted to ASCII format and read into GRAMS/32 version 5.05 running on Windows NT.
• a unique form I peak typically either 18.5 or 20.2 °2 ⁇
• a unique form II peak typically 20.8 °2 ⁇ as measured over 20.5-21.2 °2 ⁇
• a standard curve was generated using standardized mixtures of FI and FII ranging from 0 to 100% FII in FI at 10% intervals, except that a sample containing 5% FII was also included.
• the three heights (or areas) measured for each sample were averaged, and the averages were input into the equation defined by the calibration curve.
• SSNMR Solid State NMR Method
• DHEA was prepared from DHEA acetate (obtained from Diosynth, Chicago, IL or Berlichem, Montville, NJ) by saponification using potassium carbonate in methanol. The product was dissolved in 6 parts methanol at reflux, and charcoal was added and removed by filtration. The methanol was evaporated until a volume of 3 parts remained, and the solution was cooled to 15°C, maintained at this temperature for 1 hour, and filtered. The wet product was refluxed with 8.5 parts of water to remove the methanol, filtered, and dried under vacuum at 90°C. The loss on drying specification for the final product was ⁇ 0.5%, and the specification for residual methanol was ⁇ 0.01%. Preparation of Form I.
• the filter cake was allowed to dry under ambient conditions overnight and then passed through a single sieve (75 ⁇ m), yielding 9.0 g of particles with sizes > 75 ⁇ m and 3.2 g of particles with sizes ⁇ 75 ⁇ m. 13 C-SSNMR analysis of the fraction with particle size > 75 ⁇ m showed that only form I was present.
• DHEA levels were determined by radioimmunoassay (RIA) at Endocrine Sciences Inc., after non- polar solvent extraction. Method validation data demonstrated a recovery range of 92-99%, a limit of detection (LOD) of 18.9 ng/dL, a limit of quantitation (LOQ) of 87.5 ng/dL, intra-assay precision ⁇ 5%, and inter-assay precision ⁇ 10%. Internal assay controls were monitored via control charts. DHEA-S levels were determined by radioimmunoassay (RIA) at Endocrine Sciences Inc., after enzymolysis of DHEA-S.
• RIA radioimmunoassay
• Method validation data demonstrated recovery range 86-106%, limit of detection (LOD) 3.6 ⁇ g/dL, intra-assay precision ⁇ 7%, and inter-assay precision ⁇ 10%.
• Internal assay controls were monitored via control charts.
• Each capsule contained 50 mg DHEA (formulation 1, 2 or 3) and excipients consisting of 152 mg lactose (169 mg for formulation 3), 108 mg corn starch, and 3 mg magnesium stearate.
• the DHEA polymorph contents of formulations 1 -3 are listed in Table 4 in Section III above.
• the formulations were prepared in bulk amounts (e.g., 150 kg) by weighing and sieving all individual components through a size 14 stainless steel mesh screen to de-lump.
• magnesium stearate All components except magnesium stearate were blended together for at least 10 minutes using a Patterson-Kelly Twin Shell 10 cubic ft dry V- blender. Magnesium stearate was added and blended into the mixture for at least 5 minutes. Homogeneity was verified by taking samples from different regions of the blender and quantifying the DHEA content on a weight basis by HPLC (isocratic, 1 mL/min 45:45:10 acetonitrile:water:methyl t- butylether, Synchropak column (C18 RP-P-100, 5 micron, 250 x 4.6 mm i.d. from Thinchrom, Inc., Lafayette, IN). After weighing, the blend was placed in opaque size 2 gelatin capsules (Capsugel or Shionogi) with 313 mg fill-weights.
• DHEA and DHEA-S AUC (area under curve) values were determined by the linear trapezoidal method from hour 0 to hour 72 (AUC(o. 7 ) ) or the last measurable concentration.
• the area under the concentration time curve from zero to infinity (AUCo---) was determined using the following equation:
• AUC 0 - ⁇ ) AUCo-, + Oha/ d
• AUC 0 . t is the area under the concentration-time curve from zero to the last measurable concentration (C ⁇ ast ) as determined by the linear trapezoidal method
• k e i is the terminal elimination rate constant.
• the terminal elimination rate constant was determined by using linear regression of the individual log concentrations versus time during the terminal elimination phase.
• the elimination rate constant was estimated by performing a regression of the natural logarithm of the concentrations on sampling time, for times in the specified range. Regressions were repeated first using the last three points, then the last four points.
• the half-life (t y ,) can be calculated by dividing 0.693 by k e i.
• the main pharmacokinetic parameters used to describe DHEA and DHEA-S pharmacokinetics during treatments are the area under serum concentration curve from hour 0 to hour 72 (AUC (0 . 72) ), the maximum serum concentration (C max ), and the time to maximum serum concentration (T max ). Comparisons of these parameters were made with an analysis of variance (ANOVA) model using the PROC GLM software (PC SAS, version 6.10).
• This study was an open-label, randomized, steady-state, two-treatment cross-over study of DHEA pharmacokinetics/pharmacodynamics in healthy postmenopausal females.
• DHEA was administered on each of 7 days of two study periods, with an intervening 7-day washout period (Days 1 to 7 and Days 15 to 21).
• Subjects received a single 200 mg oral dose (four 50 mg capsules) of DHEA at the same time each morning for the 7 days of each study period. Subjects were instructed to fast for 10 hours prior to each DHEA dose.
• Each capsule contained 50 mg of DHEA and pharmaceutical excipients (169 mg (Formulation 3) or 152 mg (Formulation 4) lactose, 108 mg corn starch, and 3 mg magnesium stearate) to result in a total capsule fill weight of 330 and 313 mg for formulations 3 and 4, respectively.
• Subjects were randomized to receive one of the two formulations during each study period.
• the DHEA polymorph composition of formulation 3 was as shown in Table 4 above (18% form I, 43% form II, and 39 % form VI).
• Formulation 4 contained substantially pure (-100%) form I DHEA, prepared by methods described herein. Serum samples for the measurement of trough levels of DHEA and DHEA-S were obtained five minutes prior to the 200 mg oral dose of DHEA on days 1 to 6 of each study period.

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