AU6301699A - Methods for preparing and use of 1alpha,24(S)-dihydroxy - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Applicant(s): BONE CARE INTERNATIONAL, INC Actual Inventor(s): See attached Address for Service: PATENT ATTORNEY SERVICES 26 Ellingworth Parade Box Hill Victoria 3128 Australia Title: METHODS FOR PREPARING AND USE OF a, 24(S)-DIHYDROXY The following statement is a full description of this invention, including the best method of performing it known to me/us:- -lala.24(S)-DIHYDROXY VITAMIN ITS FORMATION AND USE TECHNICAL FIELD This invention relates to biologically active vitamin D 2 compounds. More specifically, this invention relates to the hormonally active, natural metabolite la,24(S)-dihydroxy vitamin D 2 and to methods of preparing this metabolite and the nonbiological epimer la,24(R)-dihydroxy vitamin D 2 This invention also relates to a pharmaceutical composition which includes a pharmaceutically effective amount of la,24(S)-dihydroxy 25 vitamin D 2 and to a method of controlling abnormal calcium metabolism by administering a pharmaceutically effective amount of the compound.

BACKGROUND OF THE INVENTION Vitamin D and its active metabolites are known to be important in regulating calcium metabolism in animals and humans. The naturally occurring form of vitamin D in animals and humans is vitamin D 3 It has been shown that in animals, including humans, vitamin D 3 is activated by being hydroxylated in the Cz position in the liver, followed by la-hydroxylation in the kidney to I t -2produce the hormone la,25-dihydroxy vitamin D 3 3 See, U.S. Patent No. 3,880,894. The major physiological pathway for catabolism of the vitamin D 3 metabolites, 25-hydroxy vitamin D 3 and la,25-(OH)D, is initiated by C 2 -oxidation.

Holick, Kleiner-Bossallier, Schnoes, H.K., Kasten, Boyle, and DeLuca, J. Biol.

Chem., 248, 6691-6696 (1973).

Vitamin D, is the major, naturally occurring form of vitamin D found in plants. Vitamin D 2 differs structurally from vitamin D 3 in that vitamin D, has a methyl group at C, and has a double bond between C 22 and

C

2 3 Shortly after their discovery, it seemed apparent S: 15 that vitamin D3 and vitamin D, had similar, if not equivalent, biological activity. It has also been commonly believed that the metabolism the activation and catabolism) of vitamin D2 was the same as Sfor vitamin D3. See, Harrison's Principles of Internal 20 Medicine: Part Seven, "Disorders of Bone and Mineral Metabolism: Chap. 35," in E. Braunwald, K.J. Isselbacher, R.G. Petersdorf, J.D. Wilson, J.B.

Martin and H.S. Fauci Calcium, Phosphorus and Bone Metabolism: Calcium Regulating Hormones, McGraw- 25 Hill, New York, pp. 1860-1865. In this regard, the active form of vitamin D 2 is believed to be vitamin D 2 ["la,25-(OH)2D2"]. Further, 24-hydroxy derivatives of 25-hydroxy vitamin D, and 2 D, that is, 24,25-dihydroxy vitamin D2 and la,24,25-trihydroxy vitamin D2, are known, suggesting that catabolism of vitamin D2, like vitamin D3, proceeds through the same C 24 oxidation step. Jones, G., Rosenthal, Segev, Mazur, Frolow, F., Halfon, Robinavich, D. and Shakked, Z., Biochemistry, 18:1094-1101 (1979).

It has recently been found, however, that an active analogue of vitamin D2, la-hydroxy vitamin D2 -3- ["la-(OH)D 2 has pharmacological properties distinctly different than those exhibited by its vitamin D 3 counterpart, la-hydroxy vitamin D 3 ["la-(OH)D 3 U.S. Patent 5,104,864 discloses that la-(OH)D 2 will :reverse the loss of bone mass in human osteoporotic patients when administered at dosages of 2.0 pg/day or higher. Because of toxicity, dosage levels of Ag/day or greater are not safely obtained with la-(OH)D 3 Such distinct pharmacological properties may be explained fully, or in part, by the present inventors' discovery that pharmacological dosages of la-(OH)D 2 administered to humans are metabolized in part to biologically active la,24(S)-dihydroxy vitamin D 2 15 ["la,24(S)-(OH) 2 As explained in more detail below, the hydroxylation at the carbon-24 position of the 1hydroxylated vitamin D, molecule, represents an activation pathway peculiar to the vitamin D, molecule.

While la,24(S)-dihydroxy vitamin D 3 and 20 la,24(R)-dihydroxy vitamin D 3 ["la,24(R/S)-(OH) 2

D

3 have been chemically synthesized Patent No. 4,022,891) it has not been demonstrated that either is a natural .compound found in biological systems. Furthermore, the present inventors have discovered that la,24(S)-(OH) 2

D

2 has distinctly different biological activity from that exhibited by la,24(R/S)-(OH) 2

D

3 For example, Ishizuka et al. have found that la,24(R)-(OH) 2

D

3 binds the 1,25-(OH) 2

D

3 receptor site more tightly than does 1,25-(OH) 2

D

3 itself. Ishizuka, Bannai, K., Naruchi, T. and Hashimoto, Steroids, 37:1,33-42 (1981); Ishizuka, Bannai, Naruchi, T. and Hashimoto, Steroids, 39:1,53-62 (1982). Using a similar assay, the present inventors have discovered that the la,24(S)-(OH),D 2 is two-fold less competitive in binding the 1,25-(OH) 2

D

3 receptor site than is 1,25-(OH) 2

D

3 The present inventors have also found that la,24(S)-(OH),D, shows a relatively poor binding affinity -4for the vitamin D serum binding protein which is evidence of a rather short half life indicative of low toxicity.

The present inventors have demonstrated the presence of circulating la,24(S)-(OH) 2 D, in humans administered la-(OH)D 2 This indicates that in animals and man, vitamin D 2 is naturally metabolized to both 2

D

2 and la,24(S)-(OH) 2

D

2 The relative ratios of the two vitamin D 2 hormones appear to vary according to the precursor and the amount of precursor presented to the C, pathway. Thus it appears that as dosages of la-(OH)D 2 are increased, the ratio of la,24(S)-(OH) 2 D to 2

D

2 increases.

These results which are presented in more detail 15: below, indicate that la,24(S)-(OH) 2 D, has the desirable characteristic of high biological activity with low toxicity. The fact that la,24(S)-(OH) 2 D is a significant metabolite when pharmacological levels of la-(OH)D 2 are administered indicates that la,24(S)-(OH),D, 20 may be mediating the desirable pharmacological effects ,of la-(OH)D 2 and is a useful therapeutic drug for I treating various types of disorders involving calcium metabolism.

SUMMARY OF THE INVENTION The invention provides synthetic la,24(S)-(OH) 2

D

2 which is a biologically produced active form of vitamin D 2 The.biological form may also be referred to as la,24(S)-dihydroxy ergocalciferol and is represented by the structure given hereinafter. The biological form of the compound has potent biological activity and rapid systemic clearance, indicating low toxicity.

The invention also encompasses a novel method of producing la,24(S)-dihydroxy vitamin D, which entails using ergosterol as a starting material, forming 24-hydroxy vitamin D 2 and then, la-hydroxlyating the 24-hydroxy compounds and separating the la,24(S)-dihydroxy vitamin D, epimer from the la,24(R)-dihydroxy vitamin D, epimer. In the course of this synthesis, novel intermediates are also produced.

The compound of the invention is useful in the treatment of various diseases characterized by vitamin D deficiency and various bone depletive disorders, in particular, treatment without the concomitant incidence of hypercalcemia or hypercalciuria. The compound of the invention is advantageously used as an active ingredient of pharmaceutical compositions for vitamin D deficiency diseases, for reversing or preventing the loss of bone mass or bone mineral content in persons predisposed to developing such loss, and for stabilizing bone density in persons suffering from renal osteodystrophy.

The compound of the invention is also useful as a 15 topical agent for treatment of certain skin disorders.

The compound of the invention is advantageously used as an active ingredient for topical compositions which may also include other agents capable of ameloriating skin disorders.

20 Other advantages and a better appreciation of the .specific adaptations, compositional variations, and physical and chemical attributes of the present invention will be gained upon an examination of the following detailed description of the invention, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will hereinafter be described in conjunction with the appended drawings, wherein like designations refer to like elements throughout and in which: Figure 1 illustrates preparative steps for the synthesis of 24-hydroxy vitamin D 2 Figure 2 illustrates preparative steps for the synthesis of la,24(S)-dihydroxy vitamin D 2 starting with 24-hydroxy vitamin D,; Figure 3 is a reverse phase high pressure liquid chromatography profile of biological la,24-dihydroxy vitamin D, and the R and S epimers of synthetic la,24-dihydroxy vitamin D 2 and Figure 4 is a graph illustrating the relative binding affinities of la,24(S)-(OH) 2 D, and la,24(R)-(OH) 2

D

2 DETAILED DESCRIPTION The present invention provides synthetic la,24(S)-dihydroxy vitamin D, [la,24(S)-(OH),-D 2 As used herein, the terms "biological activity", 10 "biologically active", "bioactive", or "biopotent" are meant to refer to biochemical properties of compounds such as affecting metabolism, affecting serum calcium concentration, or binding to an appropriate receptor protein, binding to vitamin D receptor protein. The term "substantially pure" in reference to compounds or substances means a purity of at least In one of its aspects, the invention encompasses the biologically active compound of the formula HO OH

(I)

la,24(S)-dihydroxy vitamin D,.

In another aspect, the invention involves the preparation of la,24(S)-dihydroxy vitamin Synthesis of la,24(S)-dihydroxy vitamin D 2 is accomplished according to the schema presented in Figures 1 and 2.

Hereinafter when reference is made to a 24-hydroxy compound, unless specified, it will be presumed that the compound is an epimeric mixture of the R and S forms.

As seen in Figure 1, the synthesis uses ergosterol as -7the starting material. Ergosterol is converted to 24-hydroxyergosterol (5,7,22 ergostatriene-33,24-diol by a five-step process. The 24-hydroxy ergosterol is then irradiated and thermally converted by methods well known in the art to yield 24-hydroxy vitamin D 2 As seen in Figure 2, 24-hydroxy vitamin D 2 is then hydroxylated in a five-step process to yield la,24-dihydroxy vitamin using a procedure similar to that described by Paaren, et al., J. Orq. Chem., vol.

45, p. 3253 (1980), from which the epimers are separated.

Specifically, ergosterol is acetylated to form the 3(-acetate An adduct is then formed with the B-ring of the ergosterol structure by reaction of the 15 33-acetate with a triazoline dione. The adduct is then ozonated to truncate the side chain to form a C-21 aldehyde The side chain is reestablished by reaction of the resulting aldehyde with the appropriate keto-compound to yield the 24-enone The enone is 20 then converted to the 24-methyl, 33,24-dihydroxy adduct This adduct is then reacted with a lithium aluminum hydride to deprotect the adduct and yield 24-hydroxy ergosterol The 24-hydroxy ergosterol is then irradiated and thermally treated to form 24-hydroxy vitamin D 2 The 24-hydroxy vitamin D 2 is then tosylated to yield 33-tosylate of the 24-hydroxy vitamin D 2 The tosylate is displaced by solvolysis to yield the 6-methoxy-24-hydroxy-3,5-cyclo vitamin The cyclovitamin D 2 is subjected to allylic oxidation to form the la, 24-dihydroxy cyclovitamin derivative. The la,24-dihydroxy cyclovitamin derivative is sequentially solvolyzed and subjected to a Diels-Alder type reaction which removes the 6-methoxy group and separates the la,24-dihydroxy vitamin D 2 (5,6 cis) from the 5,6 trans la,24-dihydroxy vitamin D 2 The la,24-(OH) 2 D, is subjected to reverse phase high pressure liquid chromatography to separate the two -8epimers and recover the epimeric form of the invention, la,24(S)-(OH) 2

D.

The compound of the invention is applicable to various clinical and veterinary fields, and is particularly useful for the treatment of abnormal metabolism of calcium and phosphorus. Specifically, la,24(S)-dihydroxy vitamin D 2 is intended to be used, for example, to stimulate osteoblastic activity, as measured by serum levels of osteocalcin. Osteocalcin is one of the major proteins in the bone matrix. The la,24(S)-dihydroxy vitamin D 2 binds to the vitamin D serum binding protein more weakly than does 1,25-(OH) 2

D

3 indicative of rapid clearance and low toxicity, which enhances its pharmaceutical properties.

In a further aspect, the invention entails a method of controlling calcium metabolism, such as for treating abnormal calcium metabolism caused, by liver failure, renal failure, gastrointestinal failure, etc.

The la,24(S)-dihydroxy vitamin D, can be used to treat prophylactically or therapeutically vitamin D deficiency diseases and related diseases, for example, renal osteodystrophy, steatorrhea, anticonvulsant osteomalacia, hypophosphatemic vitamin D-resistant rickets, osteoporosis, including postmenopausal osteoporosis, senile osteoporosis, steroid-induced osteoporosis, and other disease states characteristic of loss of bone mass, pseudodeficiency (vitamin Ddependent) rickets, nutritional and malabsorptive rickets, osteomalacia and osteopenias secondary to hypoparathyroidism, post-surgical hypoparathyroidism, idiopathic hypothyroidism, pseudoparathyroidism, and alcoholism.

lo,24(S)-Dihydroxy vitamin D 2 is also of value for the treatment of hyperproliferative skin disorders such as psoriasis, eczema, lack of adequate skin firmness, dermal hydration, and sebum secretion, and is valuable for the treatment of breast and colon cancer.

i I

I

-9l,24(S)-Dihydroxy vitamin D, is useful as an active compound in pharmaceutical compositions having reduced side effects and low toxicity as compared with the known analogs of active forms of vitamin D 3 when applied, for example, to diseases induced by abnormal metabolism of calcium. These pharmaceutical compositions constitute another aspect of the invention.

The pharmacologically active compound of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, mammals including humans. For example, the la,24(S)-dihydroxy vitamin D, can be employed in admixtures with conventional excipients, pharmaceutically 15 acceptable carrier substances suitable for enteral oral), parenteral, or topical application which do not deleteriously react with the active compound.

Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, 20 alcohols, gum arabic, vegetable oils almond oil, corn oil, cottonseed oil, peanut oil, olive oil, coconut oil), mineral oil, fish liver oils, oily esters such as Polysorbate 80, polyethylene glycols, gelatine, carbohydrates lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.

The pharmaceutical preparations can be sterilized and, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or one or more other active compounds, for example, vitamin D 3 and its la-hydroxylated metabolites, conjugated estrogens or their equivalents, anti-estrogens, calcitonin, biphosphonates, calcium supplements, cobalamin, pertussis toxin and boron.

*1 For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solution, as well as suspensions, emulsions, or implants, including suppositories. Parenteral administration suitably includes subcutaneous, intramuscular, or intravenous injection, nasopharyngeal or mucosal absorption, or transdermal absorption.

Ampoules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, lozenges, powders, or capsules. A syrup, elixir, or the like can be used if a sweetened vehicle is desired.

For topical application, suitable nonsprayable viscous, semi-solid or solid forms can be employed which 15 include a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, for example, mineral oil, almond oil, selfemulsifying beeswax, vegetable oil, white soft paraffin, ;and propylene glycol. Suitable formulations include, 20 but are not limited to, creams, ointments, lotions, solutions, suspensions, emulsions, powders, liniments, salves, aerosols, transdermal patches, etc., which are, if desired, sterilized or mixed with auxiliary agents, preservatives, stabilizers, demulsifiers, wetting agents, etc. A cream preparation in accordance with the present invention suitably includes, for example, mixture of water, almond oil, mineral oil and self- Semulsifying beeswax; an ointment preparation suitably includes, for example, almond oil and white soft paraffin; and a lotion preparation suitably includes, for example, dry propylene glycol.

S Topical preparations of the compound in accordance with the present invention useful for the treatment of skin disorders may also include epithelializationinducing agents such as retinoids vitamin A), chromanols such as vitamin E, 3-agonists such as isoproterenol or cyclic adenosine monophosphate (cAMP), anti-inflammatory agents such as corticosteroids -11hydrocortisone or its acetate, or dexamethasone) and keratoplastic agents such as coal tar or anthralin.

Effective amounts of such agents are, for example, vitamin A about 0.003 to about 0.3% by weight of the composition; vitamin E about 0.1 to about isoproterenol about 0.1 to about cAMP about 0.1 to about hydrocortisone about 0.25 to about coal tar about 0.1 to about 20%; and anthralin about 0.05 to about 2%.

For rectal administration, the compound is formed into a pharmaceutical composition containing a suppository base such as cacao oil or other triglycerides. To prolong storage life, the composition advantageously includes an antioxidant such as ascorbic 6 15 acid, butylated hydroxyanisole or hydroquinone.

For treatment of calcium metabolic disorders, oral administration of the pharmaceutical compositions of the present invention is preferred. Generally, the compound of this invention is dispensed by unit dosage form S 20 comprising about 0.5 pg to about 25 pg in a pharmaceutically acceptable carrier per unit dosage.

The dosage of the compound according to this invention generally is about 0.01 to about 1.0 pg/kg/day, ,preferably about 0.04 to about 0.3 pg/kg/day.

For topical treatment of skin disorders, the dosage of the compound of the present invention in a topical composition generally is about 0.01 pg to about 50 pg per gram of composition.

For treatment of cancers, the dosage of la,24(S)-(OH) 2 D, in a locally applied composition generally is about 0.01 pg to 100 pg per gram composition.

It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the efficacy of the specific compound employed, the particular compositions formulated, the mode of application, and the particular site and organism being treated. For example, the specific dose -12for a particular patient depends on the age, body weight, general state of health and sex, on the diet, on the timing and mode of administration, on the rate of excretion, and on medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given host can be determined using conventional considerations, by customary comparison of the differential activities of the subject compounds and of a known agent, such as by means of an appropriate conventional pharmacological protocol.

In a still further aspect, the compound of the present invention can also be advantageously used in veterinary compositions, for example, feed compositions 15 for domestic animals to treat or prevent hypocalcemia.

Generally, the compound of the present invention is dispensed in animal feed such that normal consumption of such feed provides the animal about 0.01 to about pg/kg/day.

20 The following examples are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. In the following examples proton nuclear magnetic resonance ('H NMR) spectra were recorded with a Bruker AM--400(400 MHz) with aspect 3000 Computer in CDC1 3 solutions with CHC1 3 as an internal standard. Chemical shifts are reported in ppm. Ultraviolet spectra were recorded with a Hitachi U-2000 Spectrophotometer and are reported for ethanol solutions.

Example 1: Generation, purification and identification of la,24(?)-(OH) 2 D, in human liver cells incubated with la-(OH)D 2 Substantially pure la-(OH)D 2 was obtained from Bone Care International, Inc. of Madison, Wisconsin. The la-(OH)D 2 was cultured for 48 hours with cells derived from a human hepatoma, Hep 3B, in medium devoid of fetal calf serum using known methods in the art.

-13- Lipid extracts of the combined medium and cells were generated by known methods in the art and were subjected to high pressure liquid chromatography (HPLC) on Zorbax-SlL developed with hexane/isopropanol/methanol The putative metabolite eluted between the parent la-(OH)D 2 and standard 2

D

2 (also obtained from Bone Care International, Inc. of Madison, Wisconsin). (As used herein, the term 2 is meant to indicate that the epimeric form has not been identified.) The lc,24(?)-(OH) 2

D

2 was further purified by this HPLC system *before the metabolite's identification was undertaken using mass spectrometry analysis.

The purified metabolite was more polar than the starting material, la-(OH)D 2 and thus was tentatively concluded to be a dihydroxy vitamin D, metabolite. This metabolite also possessed the vitamin D chromophore, indicating retention of the cis-triene system of vitamin D. Since the metabolite was derived from 20 la-(OH)D 2 its structure was thus la,X-(OH) 2

D

2 where "X" indicates the position of the second hydroxyl group.

The trimethylsilyl-derivative of the la,X-(OH),D, was prepared according to known methods in the art and mass spectrometry was performed on the TMS-derivative and the native compound. The TMS-derivative was analyzed by GC-MS, and the identification was mainly derived from interpretation of the fragmentation pattern of the pyro-metabolite. The molecular ion possessed a m/z of 644 indicating a dihydroxy vitamin D 2 with addition of three TMS groups accounting for 216 units of additional mass. Since la-(OH)D 2 has 30- and la- groups and the putative metabolite had one additional hydroxyl, all three hydroxyls were thus derivatized. Distinctive fragments were found at m/z 601, 511, 421, 331 representing loss of a 43 mass unit of fragment alone or in addition to one, two or three TMS groups of 90 units each. This pattern was most likely explained by cleavage of the C-24 to C-25 bond loss of C 3

H

7 accounting -14for 43 mass units. This represents loss of the C 26 fragment. Furthermore, the mass spectrum lacked the m/z 131 fragment characteristic of all vitamin D compounds.

The mass spectrum showed the m/z 513 fragment indicating loss of 131 mass units due to A-ring cleavage with loss of C 2

-C

3

-C

4 also characteristic of vitamin D compounds. The mass spectrum also contained m/z 143 which was probably derived from C-24 to C-23 cleavage and a loss of a methyl group. The unusual loss of 43 units indicating C 2 4

-C

2 fragility coupled with the loss of a fragment due to CB-C 2 4 cleavage indicated that the extra hydroxyl in la,X-(OH) 2

D

2 was at carbon-24.

Thus, the structure was identified as la,24(?)-(OH) 2

D

2 15 The native metabolite was analyzed by direct probe mass spectrometry. This analysis was consistent with a hydroxyl in the 24 position, and was also consistent with the GC-MS analysis of the TMS-derivative described above. The native metabolite showed the expected 20 molecular ion at m/z 428 and a distinctive fragment at m/z 367, indicating the loss of one water and the C 25

-C

26 C fragment of 43 mass units.

Example 2: Synthesis of la,24(S)-dihydroxy vitamin D, (22E)-5,7,22-ergostatriene-33-yl acetate (2) To a solution of 50 gm (0.13 mol) of ergosterol (1) in 300 ml of anhydrous pyridine was added 33.3 ml (0.35 mol) of acetic anhydride. The mixture was stirred at room temperature overnight and then 600 ml of water was added. The precipitate was filtered and washed three times with 200 ml portions of acetonitrile and then air dried to yield 42.0 g of 22-oxo-5a,8a-(4-phenyl-3.5-dioxo-1,2,4-triazolidine-1.2diyl)23,24-dinor-6-cholene-33-yl acetate (4) To a solution of 33.0 g (0.075 mol) of ergosterol acetate in 1000 ml of chloroform was added 13.2 g (0.075 mol) of 4-phenyl-1,2,4-triazoline-3,5-dione. The ;solution of the thus formed was stirred at room temperature for 30 min. and then 5 ml of pyridine was added. The solution was cooled to -78°C and treated at -78 0 C with an ozone-oxygen mixture for 2 hours and then thoroughly purged with nitrogen. Then 50 ml of dimethylsulfoxide was added and the mixture was washed with 300 ml of water, then twice with 200 ml of 2F HC1 and finally 300 ml of water. The organic layer was separated, dried over anhydrous MgSO 4 and concentrated to dryness in vacuo. The residue was purified on a silica gel column using 30% ethyl acetate in hexane to yield 16.0 g of the title compound as a foamy solid.

I'H NMR: (400 MHz; CDC1 3 6ppm 0.85 (3H,-s, 15 18-CH 3 1.10 (3H, s, 19-CH 3 1.15 (3H, d, 21-CH 3 1.99 (3H, s, 3f-CH 3 CO), 5.45 (1H, m, 3a-H), 6.26 (1H, d. 7-H), 6.40 (1H, d, 7.42 (5H, m, Ph), 9.58 (1H, d, HCO).

S (22E)5a,8a-(4-phenyl-3,5-dioxo-l,2,4-triazolidine-1,2diyl) cholesta-6,22-diene-24-one-33-yl acetate 20 Butyllithium (1.6M solution in hexane 8.94 ml, 0.014 mol) was added to a stirred, cooled (0 0 C) solution of diisopropylamine (1.45 g, 0.014 mol) in dry tetrahydrofuran (20 ml) under nitrogen.

3-Methylbutan-2-one (1.23 g, 0.014 mol) in dry tetrahydrofuran (6 ml) was added dropwise at OOC over min. The solution was stirred at 0OC for 1 hr. more, then cooled to -70 0 C and a solution of the aldehyde (4) g, 0.011 mol) in dry tetrahydrofuran (60 ml) was added. The temperature was raised to -20 0 C and kept at this temperature for 3 hrs. Then glacial acetic acid ml) was added at -20 0 C and the solution was brought to room temperature. Ether (800 ml) and water (400 ml) were added and the organic layer was separated and washed with 10% hydrochloric acid (2 x 300 ml), saturated sodium bicarbonate solution (2 x 300 ml), and water (2 x 300 ml). Concentration gave the crude product (7.5 g) which was dissolved in tetrahydrofuran I -16- (100 ml) containing 1.5 N-hydrochloric acid (12 ml).

After refluxing for 1.5 hrs., the mixture was diluted with ether (600 ml), washed with a 5% sodium carbonate solution (2 x 200 ml) and water (2 x 200 ml), and dried (anhydrous MgSO 4 Concentration under reduced pressure gave the crude product (7.0 Chromatography over silica gel (50% ethyl acetate in hexane) gave the enone 4.0 g 'H NMR: (400 MHz): 6ppm 0.83 (3H, s. 18-CH 3 0.99 (3H, s, 19-CH 3 1.09 (6H, dd, 26 and 27-CH 3 1.12 (3H, d, 21-CH 3 2.0.(3H, s, 30-CH 3 CO), 2.84 (1H, m, 5.45 (1H, m, 3a-H), 6.06 (1H, d, 23-H), 6.24 (1H, d, 6.39 (1H, d, 6.71 (1H, dd, 22-H), 7.42 m, Ph).

*ee (22E)-5,8a-(4-phenyl-3,5-dioxo-1,2,4-triazolidine- 1,2-diyl)-6,22-ergostadiene-3 ,24-diol (6) C. The enone (3.5 g, 5.7 mmol) in dry ether (100 ml) was cooled to 0°C and methylmagnesium bromide (3.0 M solution in ether 6.8 ml, 0.02 mol) was added 20 dropwise. After 1 hr. at 0°C, saturated ammonium chloride (100 ml) was added. The organic layer was separated. The aqueous layer was extracted with ether (2x200 ml). The combined ether phases were dried over anhydrous MgSO 4 and concentrated to dryness in vacuo to yield the crude product 3.0 g of (22E)-5,7,22-ergostatriene-33,24-diol (7) To a solution of 3.0 g (5.1 mmol) of in dry tetrahydrofuran (250 ml) was added 3.6 g (0.09 mol) of lithium aluminum hydride. The mixture was heated under reflux for 3 hrs., cooled with ice water bath and reaction mixture decomposed by the cautious dropwise addition of ice water (5 ml). The mixture was filtered and the filtrate was concentrated in vacuo to remove most of the tetrahydrofuran. The residue was dissolved in 200 ml of ethyl acetate and washed twice with -17saturated NaCl solution (2x200 ml), dried over anhydrous MgSO 4 and concentrated in vacuo. The residue was purified on a silica gel column using 30% ethyl acetate in hexane to yield 1.5 g of H NMR: (400 MHz, CDC1 3 6ppm 0.64 (3H, s, 18-H), 0.88 (6H, dd, 26 and 27-CH 3 0.93 (3H, g, 19-CH3), 1.06 (3H, d, 21-CH 3 1.19 (3H, g, 28-CH 3 3.55 (1H, m, 3a-H), 5.36 (1H, d, 5.42 (2H, m, 22 and 23-H), 5.52 (1H, d, UV (ethanol) 282 nm.

24-hydroxyvitamin D, (8) One gram (2.4 mmol) of was dissolved in 250 ml of ether and benzene and irradiated with stirring under nitrogen in a water-cooled quartz immersion well using a Hanovia medium-pressure UV lamp for 2 hrs. The solution was concentrated in vacuo, redissolved in 100 ml of ethanol and heated under reflux overnight.

S' The solution was concentrated to dryness in vacuo and the residue was purified on a silica gel column using ethyl acetate in hexane to yield 0.55 g of 20 'H NMR: (400 MHz, CDC1 3 3ppm 0.57 (3H, s, 18-CH 3 0.92 (6H, dd, 26 and 27-CH 3 1.06 (3H, d, 21-CH3), 1.20 (3H, s, 28-CH 3 3.93 (1H, m, 4.79 (1H, m (sharp), 19-H), 5.01 (1H, m, (sharp), 19-H), 5.43 (2H, m, 22 and 23-H), 6.02 (1H, d, 6.22 (1H, d, 6-H) UV (ethanol) 265 nm.

24-hydroxyvitamin D 2 tosylate (9) To a solution of 0.55 g (1.3 mmol) of dissolved in 5 ml of anhydrous pyridine was added 0.6 g (3.2 mmol) of tosyl chloride. The mixture was stirred under nitrogen at 5 0 C for 20 hrs. The reaction mixture was poured into 100 ml of cold saturated NaHCO 3 solution and extracted with ether (3 x 100 ml). The combined organic extracts were washed with 5% HC1 solution (2 x 200 ml) saturated sodium bicarbonate solution (2 x 200 ml) and -18- .i saturated NaCl solution (2 x 200 ml), dried over anhydrous MgSO 4 and concentrated in vacuo to yield 0.62 g of 'H NMR: (400 MHz, CDC1 3 Sppm 0.57 (3H, s, 18-CH 3 0.92 (6H, dd, 26 and 27-CH,), 1.08 (3H, d, 21-CH3), 1.24 (3H, s, 28-CH 3 2.43 (3H, s, CH3 (tosylate)), 4.69 (1H, m, 4.77 (1H, m, (sharp), 19-H), 5.0 (1H, m, (sharp), 19-H), 5.42 (2H, M, 22 and 23-H), 6.03 d, 6.25 d, 6-H) 7.31 and 7.83 (4H, d, aromatic).

6-methoxy-24-hydroxy-3,5-cyclovitamin D, To a solution of 0.6 g (1.06 mmol) of dissolved in 50 ml of anhydrous methanol was added sodium bicarbonate 4.0 (0.047 mol). The mixture was heated at 15 reflux for 6 hrs. The reaction mixture was concentrated in vacuo. Water (100 ml) was added followed by extraction with ether (2 x 200 ml). The combined ether extracts were dried over anhydrous MgSO 4 and concentrated to dryness in vacuo to yield 450 mg (100%) of (10) as an oil.

6-methoxy-la,24-dihydroxy-3,5-cyclovitamin

D

2 (11) ter-Butyl hydroperoxide (870 m1 (2.61 mmol); 3M in toluene) was added to a suspension of 73 mg (0.66 mmol) of selenium dioxide in 50 ml of anhydrous dichloromethane under nitrogen. The mixture was stirred at room temperature under nitrogen for 3 hrs. Then 0.1 ml of anhydrous pyridine was added followed by a solution of 450 mg (1.06 mmol) of (10) dissolved in ml of anhydrous dichloromethane. The mixture was stirred under nitrogen at room temperature for 10 min.

then 25 ml of 10% NaOH solution was added and the mixture was extracted with ether (3 x 100 ml). The combined ether extracts were washed with 10% NaOH solution (2 x 100 ml), water (2 x 100 ml), saturated sodium chloride solution (2 x 100 ml), dried over -19anhydrous MgSO 4 and concentrated to dryness in vacuo.

The residue was purified on a silica gel column using a mixture of 30% ethyl acetate in hexane to yield 110 mng of (11).

'H NMR: (400 M4Hz, CDCl 3 6ppm, 0. 55 (3H, s, 18CH 3 0.90 (6H, dd, 26 and 27-C! 3 1.03. (3H, !I, 21-CH 3 1.19 (3H, g, 28-CH 3 3.25 (3H, g, -OCH 3 4.19 (111, 4, 4.19 (1H, M, 4.92 (211, 5.15 (1H1, M, (sharp), 19-H), 5.2 (1H, Mn, (sharp), 19-H), 5.42 (2H, mn, 22 and'23-H).

S,6-is and 5,6-trnn-lc,24-dihydroxy vitamin (12, 13) la,24-dihydroxy-3,5-cyclovitamjn

D

2 (11) 110 mig .(0.25 mmol) was dissolved in 2.0 ml of dimethylsulf oxide .and 1.5 ml of acetic acid and heated at 50 0 C under nitrogen for 1 hr. The solution was poured over ice and ml of saturated NaHCO 3 solution. The mixture was *extracted with ether (3 x 100 ml). The combined ether extracts were washed with saturated NaHCO 3 solution (3 x 100 ml), water (2 x.100 ml), saturated NaCi solution (2 x 200 ml) dried over anhydrous MgSO 4 and concentrated in vacua, to yield the crude product 100 mig of (12) and (13).

1 6-cis-lc,24-dihydroxry vitamin D. (12) To a solution of (12) and (13) in 5 ml of ethyl acetate was added 20 mig (0.2 minol) of maleic anhydride and the mixture was stirred at 35 0 C for 24 hrs. under nitrogen. The solution was concentrated to dryness in vacua. The residue-was purified on a silica gel column using 50% ethyl acetate in hexane to yield 20 mg of 'H NMR: (400 M4Hz, CDCl 3 6ppm 0.57 (311, s, 18-C! 3 0.89 (611, d.4, 26 and 27-C! 3 1.04 (311, d, 21-CH 3 1.21 (311, g, 28-C! 3 4.23 (1H, M, 4.40 (111, m, 5.0 (lH, M, (sharp), 19-H1), 5.33_.(11, m, (sharp), 19-H), 5.44 (2H, m, 22 and 23-H), 6.01 (1H, d, 6.37. (1H, d, UV (ethanol) 265 nm.

la,24(B)-dihydroxy vitamin D, (14) The 24 epimers of la,24-(OH) 2

D

2 were separated by high pressure liquid chromatography, performed on a Waters instrument using a reverse-phase Supelco C-8 prep. column (25 cm x 21.2 mm; particle size 12 pm) with the solvent system, acetonitrile:water, 60:40, mL/min. The epimers were given the designations epimer 1 and epimer 2. Under these conditions the retention time of epimer 1 was 63 min., and the retention time of epimer 2 was 71 min. Using x-ray crystallography, it was determined that the stereochemistry of epimer 2 was The stereochemistry of epimer 1 was therefore known to be la,24(S)-(OH) 2

D

2 Example 3: Identification of the stereochemistry and the biologically derived la,24(?)-(OH) 2

D,

metabolite by comparison to the 20 chemically synthesized epimers, Sla,24(S)-(OH) 2

D

2 and la,24(R)-(OH) 2

D.

The stereochemistry of the biologically generated metabolite obtained as described in example 1, above, was compared by high pressure liquid chromatography and gas chromatography to the chemically synthesized epimers obtained as described in example 2, above. Based on Sthese comparisons, it was determined that the biologically produced metabolite has the structure, la,24(S)-(OH) 2

D

2 Figure 3 shows a profile of the high pressure liquid chromatography experiment making this comparison. In Figure 3, epimer 1 is the chemically synthesized la,24(S)-(OH) 2

D

2 High pressure liquid chromatographic comparisons utilized two different columns and solvent systems. On the reverse-phase column Zorbax-ODS (Dupont Instruments; 3 p; 6.2 mm x 8 cm) utilizing the solvent S system, acetonitrile:water, 60:40, 1 ml/min., the -21biological metabolite emerged at 14.3 min. and la,24(S)-(OH) 2

D

2 ran at 14.2 min.; however, la,24(R)-(OH) 2

D

2 ran at 15.7 min.

On the straight-phase column Zorbax-SIL (Dupont Instruments; 3 6.2 mm x 8 cm) utilizing the solvent system, hexane:isopropanol:methanol, 94:5:1, 1 ml/min., the biological metabolite emerged at 22.4 min. and la,24(S)-(OH) 2

D

2 ran at 22.4 min.; however, la,24(R)-(OH) 2 DZ ran at 22.8.

With gas chromatography, la,24(S)-(OH) 2

D

2 co-migrated with the biologically generated compound whereas the retention time of la,24(R)-(OH),D 2 was quite different (Table 1).

Table 1: Gas Chromatography Retention Times of 15 Pyro-Derivatives Relative to Pyro-la, 25-(OH) 2

D

3 Compound Relative Retention Time* la,24(S)-(OH) 2 D 1.0165 la,24(R)-(OH) 2

D

2 1.0098 Biological Metabolite 1.0163 *Retention time is expressed relative to an internal standard la,25-(OH) 2

D

3 where the pyro-derivatives are compared.

Example 4: Comparison of the biological activity of 1,24(S)-(OH),D 2 and la,24(R)-(OH) 2

D

2 The biological activity in vitro of chemically synthesized la,24(S)-(OH) 2

D

2 and la,24(R)-(OH),D, was measured using a vitamin D-dependent transcriptional activation model system in which a vitamin D receptor (VDR)-expressing plasmid pSG5-hVDR1/3 and a plasmid p(CT4) 4 TKGH containing a Growth Hormone (GH)-gene, under the control of a vitamin D-responsive element (VDRE) were co-transfected into Green monkey kidney, COS-1 cells. DNA's for these two vectors were supplied by Dr. Mark Haussler, Department of Biochemistry, S University of Arizona, Tucson, Arizona.

-22- Tranfected cells were incubated with vitamin D metabolites and growth hormone production was measured.

As shown in Table 2, la-,24(S)-(OH) 2 2 has significantly more activity in this system than la,24(R)-(OH) 2

D

2 Table 2: Vitamin D Inducible Growth Hormone Production in Transfected COS-1 Cells.

Vitamin D-Inducible Growth Hormone Production Net Total GH vitamin 0-inducible Molar Production* GH-productioh Inducer Con centration (nci/ml) 1nci/ml) :1 10 Ethanol 44 0 3 0- 245 201 10-6 1100 1056 .10-1 775 731 2

D

3 10-10 74 10'9 925 881 10'8 1475 1441 la,24(S)-(OH) 2 DA 5x10-1 0 425 381 5kl0' 1350 1306 5x10'4 1182 1138 15 1a,24(R)-(OH) 2

D

2 10-9 80 36 10'8 1100 1056 10.7 1300 1256 *Averages of duplicate determinations Example 5: Affinity of lcz,24(S)-(OH) 2

D

2 for the vitamin D receptor.

The affinity of la,24(S)-(OH) 2 0A for the mammalian vitamin D receptor (VDR) was assessed using a commercially available kit of bovine thymus VDR and standard l,25-(OH) 2

-D

3 solutions from Incstar (Stillwater, Minnesota). Purified 1a,24(S)-(OH) 2

D

2 was quantitated by photodiode array spectrophotometry and assayed in the'radioreceptor assay. The half-maximal binding of la,24(S)-(OH) 2

D

2 was approximately 150 pg/ml whereas that of la,25-(QH) 2

D

2 was 80 pg/ml. Thus, the 1 a,24(S)-(OH) 2

D

2 had a two-fold lower affinity for bovine thymus VDR than does la,25-(1-) 2

D

3 indicating that la,24(S)-(OH) 2

D

2 had potent biological activity.

-23- Example 6: Relative affinities of la,24(S)-(OH) 2

D

2 and la,24(R)-(OH) 2 D, for the vitamin D receptor.

The relative affinities of la,24(R)-(OH),D 2 and la,24(S)-(OH) 2 D, for the vitamin D receptor (VDR) were assessed using commercially available reagents of bovine thymus VDR and standard la,25-(OH) 2

D

3 solutions from Incstar (Stillwater, Minnesota). The purified la,24(R)-(OH) 2 D, and la,24(S)-(OH) 2

D

2 epimers were quantitated by ultraviolet spectroscopy. The concentration of la,24(R)-(OH) 2

D

2 required to produce the same displacement of 3 H-la,25-(OH) 2

D

3 tracer from the receptor was 20 to 30 times that required for ;o la,24(S)-(OH) 2 as shown in Figure 4. These data 15 indicate that the activity of the la,24(S)-(OH) 2

D

2 epimer is significantly greater than that of the la,24(R)-(OH) 2

D

2 epimer.

Example 7: Affinity of la,24(S)-(OH) 2 D, for the vitamin D serum binding protein.

20 The affinity of la,24(S)-(OH),D 2 for the vitamin D serum binding protein (DBP) was assessed using vitamin D deficient rat serum according to known methods in the art. The data indicated that the la,24(S)-(OH) 2

D,

binding of DBP was at least 1000 times weaker than that for 25-OH-D 3 Given the strong binding of la,24(S)-(OH) 2

D

2 for the VDR and weak binding for the DBP, this compound would tend to be taken up by target cells, thus possessing a potent biological activity. In addition, the weak binding by the DBP was indicative of more rapid clearance, allowing for low toxicity.

Thus, the pr(.:.:eding assays demonstrated that the new la,24(S)-(OH) 2

D

2 exhibited a distinct and unique spectrum of activities-namely, high biological potency and low toxicity which clearly distinguished the compound from those of the prior art and from its 24(R) epimer.

-24- Example 8: Generation of la,24(S)-(OH),D, from vitamin

D

2 and 24-OH-D.

Vitamin D, or 24-OH-D 2 was administered (either oral or intraperitoneal supplementation) to vitamin Ddeficient rats. Lipid extracts of the plasma were prepared and the metabolites purified by the method of Horst et al. (Horst, R. Koszewski, N. J. and Reinhardt, T. Biochem., 29:578-82 (1990)) described below for synthesyzing standard biological la,24-(OH) 2

D

2 Standard biological la,24-(OH) 2 D was synthesized in vitro from 24-OH-D 2 by incubating 10 pg of 24-OH-D in flask containing 5 ml of 20% kidney homogenates made from vitamin D-deficient chicks. The product of this reaction was isolated by HPLC and identified by mass 15 spectrometry. In the lipid extracts of the plasma from the vitamin D-deficient rats administered vitamin D, or 24-OH-D,, one metabolite isolated co-migrated on HPLC with the standard 1a,24-(OH) 2

D

2 indicating that la,24-(OH),D 2 is a natural metabolite of vitamin Dz. In 20 contrast, comparable rats administered vitamin D 3 had no detectable 24-OH-D 3 Example 9: Preferential production of la,24(S)-

(OH)

2 D, with increased substrate concentrations in vitro.

Hep 3B cells were incubated with la-OH-D 2 as described above, at final concentrations of 1, 10, or 100 nM (Experiment and 1 or 10pM (Experiment 2) and la,24(S)-(OH) 2

D

2 was extracted and purified. The la,24(S)-(OH) 2

D

2 and la,25-(OH) 2

D

2 metabolites were quantitated by recovered radiolabel (Experiment 1) or by photodiode array spectrophotometry (Experiment As shown in Table 3, the amount of la,24(S)-(OH) 2

D,

increased relative to the amount of la,25-(OH) 2

D

2 as the substrate concentration was raised. This indicates that in this system la,24(S)-(OH) 2

D

2 was the predominant natural active metabolite of la-OH-D, at higher substrate concentrations.

TABLE 3

SUBSTRATE

EXPERIMENT CONCENTRATION PRODUCT FORMED 1 TM Ratio of lcr,24(S)-(OH) 2

D

2 -to la,25-(OH) 2

D,_

1 1:4 1:1 1.5:1 2 PM Rate of Production, pmol 106 cells/day la,24(S)-

(OH)

2 DA (OH) 2

DA

1 4.9N.* 10 59 7.4 *Sq.

*5*a D. means not detectable Production of 1a,24(S)(O) 2

D

2 in osteoporotic women administered lar- (OH) A Example 10: An increase in the production of la,24(S)-(OH) 2

D

2 relative to la,25-(OH) 2

D

2 has also been observed by the present inventors in human females who received lcr-OH-D 2 as part of an investigation of that drug for the treatment of osteoporosis. Following either a single dose of 2 pg of lcx-OH-D 2 or daily doses of 8 pg/day f or one week, blood was collected and analyzed for the metabolites lcr,24(S)-(OH) 2

D

2 and lcr,25-(OH) 2

D

2 Lipid was extracted from the blood, and the metabolites were purified by HPLC using standard methods and quantified with the radioreceptor assay produced by Incstar (Stillwater, Minnesota). One day after a single 2 pg dose, the level of lax,24 -(OH) 2 DA was undetectable with the la,25-(OH) 2

D

2 level being approximately 11 pg/ml. In contrast, one day following the last dose of 8 pg, the level of lcx,24(S)-(OH) 2

D

2 averaged 9 pg/ml with the la,25-(OH) 2

D

2 level averaging 30 pg/ml.

-26- Example 11: Dose ranging study in postmenopausal osteoporotic women.

Twenty postmenopausal osteoporotic women are enrolled in an open label study. The selected patients have ages between 55 and 75 years, and exhibit L2-L3 vertebral bone mineral density between 0.7 and 1.05 g/cm 2 as determined by measurements with a LUNAR Bone Densitometer (Lunar Corporation, Madison, Wisconsin).

On admission to the study, all patients receive instruction on selecting a daily diet containing 400 to 600 mg of calcium. Compliance to this diet is verified at weekly intervals by 24-hour food records and by interviews with each patient.

15 All patients complete a one-week baseline period, a five-week treatment period, and a one-week posttreatment observation period. During the treatment period, patients orally self-administer la,24(S)-dihydroxy vitamin D, at an initial dose of 20 0.5 pg/day for the first week, and at successively higher doses of 1.0, 2.0, 4.0, and 8.0 pg/day in each of the following four weeks. All doses are administered before breakfast.

Blood and urine chemistries are monitored on a weekly basis throughout the study. Key blood chemistries include fasting serum levels of calcium, phosphorus, osteocalcin, creatinine, and blood urea nitrogen. Key.urine chemistries include 24-hour excretion of calcium, phosphorus, and creatinine.

Blood and urine data from this clinical study indicate that this compound does not adversely affect I kidney function, as determined by creatinine clearance and blood levels of urea nitrogen; nor does it increase urinary excretion of hydroxyproline, indicating the absence of any stimulatory effect on bone resorption.

The compound has no effect on any routinely monitored serum parameters, indicating the absence of adverse metabolic effects.

-27- A positive effect of la,24(S)-dihydroxy vitamin D 2 on calcium homeostasis is evident from modest increases in 24-hour urinary calcium levels, confirming that the compound increases intestinal calcium absorption, and from increases in serum osteocalcin levels, indicating that the compound stimulates the osteoblasts.

Example 12: Preventive treatment of bone mass loss in postmenopausal osteoporotic women.

A clinical study is conducted with postmenopausal 10 osteoporotic out-patients having ages between 55 and 75 years. The study involves up to 120 patients randomly divided into three treatment groups and continues for 24 to 36 months. Two of the treatment Sgroups receive constant dosages of la,24(S)-dihydroxy vitamin D 2 two different dose levels at or above Ag/day) and the other group receives a matching Splacebo. All patients maintain a normal intake of dietary calcium (500 to 800 mg/day) and refrain from using calcium supplements. Efficacy is evaluated by S 20 pre-and post-treatment comparisons of the patient groups with regard to total body calcium retention, and i(b) radial and spinal bone mineral density as determined by dual-photon absorptiometry (DPA) or dual-energy x-ray absorptiometry (DEXA). Safety is evaluated by comparisons of urinary hydroxyproline excretion, serum and urine calcium levels, creatinine clearance, blood urea nitrogen, and other routine determinations.

The results show that patients treated with la,24(S)-dihydroxy vitamin D, exhibit significantly higher total body calcium, and radial and spinal bone densities relative to patients treated with placebo.

The monitored safety parameters confirm an insignificant Sincidence of hypercalcemia or hypercalciuria, or any other metabolic disturbance with la,24(S)-dihydroxy vitamin D 2 therapy.

-28- Example 13: Prophylaxis of postmenopausal bone loss.

A clinical study is conducted with healthy postmenopausal women having ages between 55 and years. The study involves up to 80 patients randomly divided into two treatment groups, and continues for 24 to 36 months. One treatment group receives a constant dosage of la,24(S)-dihydroxy vitamin D 2 a dose level at or above 1.0 gg/day) and the other receives a matching placebo. The study is conducted as indicated I* in Example 2 above.

The results show that patients treated with la,24(S)-dihydroxy vitamin D, exhibit reduced losses in total body calcium, radial or spinal bone densities relative to baseline values. In contrast, patients treated with placebo show significant losses in these parameters relative to baseline values. The monitored o. safety parameters confirm the safety of long-term la,24(S)-dihydroxy vitamin D, administration at this dose I level.

Example 14: Management of hypocalcemia and the resultant metabolic bone disease in chronic hemodialysis patients.

A twelve-month, double-blind, placebo-controlled clinical trial is conducted with thirty men and women with renal disease who are undergoing chronic hemodialysis. All patients enter an 8-week control period during which time they receive a maintenance dose of Vitamin D 3 (400 IU/day). After this control period, the patients are randomized into two treatment groups: one group receives a constant dosage of la,24(S)-dihydroxy vitamin D, a dosage greater than 3.0 pg/day) and the other group receives a matching placebo. Both treatment groups receive a maintenance dosage of Vitamin 03, maintain a normal intake of dietary calcium, and refrain from using calcium supplements.

Efficacy is evaluated by pre- and post-treatment comparisons of the two patient groups with regard to (a) -29- S direct measurements of intestinal calcium absorption, total body calcium retention, radial and spinal bone mineral density, or determinations of serum calcium. Safety is evaluated by regular monitoring of serum calcium.

Analysis of the clinical data show that la,24(S)-dihydroxy vitamin D 2 significantly increases intestinal calcium absorption, as determined by direct measurements using a double-isotope technique. Patients 10 treated with this compound show normalized serum calcium levels, stable values for total body calcium, and stable radial and spinal bone densities relative to baseline Ivalues. In contrast, patients treated with placebo show frequent hypocalcemia, significant reductions in total body calcium and radial and spinal bone density. An insignificant incidence of hypercalcemia is observed in the treated group.

o• Example 15: Medicament preparations.

A topical cream is prepared by dissolving 1.0 mg of la,24(S)-dihydroxy vitamin D 2 in 1 g of almond oil. To this solution'is added 40 gm of mineral oil and 20 gm of self-emulsifying beeswax. The mixture is heated to liquify. After the addition of 40 ml hot water, the mixture is mixed well. The resulting cream contains approximately 10 ig of la,24(S)-dihydroxy vitamin D, per gram of cream.

Example 16: An ointment is prepared by dissolving 1.0 mg of la,24(S)-dihydroxy vitamin D 2 in 30 g of almond oil. To this solution is added 70 gm of white soft paraffin which had been warmed just enough to be liquified. The ointment is mixed well and allowed to cool. This ointment contains approximately 10 ig la,24(S)-dihydroxy vitamin D, per gram of ointment.

Example 17: To the ointment of Example 14 is added with thorough mixing 0.5 g of adenosine and 2.0 g of papaverine base, both dissolved in a minimum quantity of dimethyl sulfoxide. The additional ingredients are present to the extent of about 0.5 wt (adenosine) and 2 wt (papaverine base).

Example 18: To the ointment of Example 14 is added with 10 thorough mixing 10,000 U of Vitamin A dissolved in a minimum quantity of vegetable oil. The resultant ointment contains about 100 U Vitamin A per gram of the ointment.

Example 19: 15 A dermatological lotion is prepared by dissolving 1.0 mg of la,24(S)-dihydroxy vitamin D 2 in 100 g of dry propylene glycol. The lotion is stored in a refrigerator in.a brown bottle and contains about 10 pg of la,24(S)-dihydroxy vitamin D 2 per gram of lotion.

Example In 1 g of almond oil is dissolved 0.2 mg of la,24-dihydroxy vitamin D 2 To the solution is added g of mineral oil and 20 g of self-emulsifying beeswax, followed by 40 ml of hot water. The mixture is mixed well to produce a cosmetic cream containing about 2.0 ig of la,24(S)-dihydroxy vitamin D 2 per gram of cream.

Example 21: To a cosmetic cream prepared according to example 18 is added 100 mg adenosine. The cream is mixed well and contains about 0.1 wt adenosine.

-31- Example 22: An ointment is prepared by dissolving 100 pg of la,24(S)-dihydroxy vitamin D, in 30 g of almond oil. To the solution so produced is added 70 g white soft paraffin which had been warmed just enough to be liquified. The ointment is mixed well and allowed to cool. The ointment so produced contains about 1.0 pg of la,24-dihydroxy vitamin D 2 per gram of ointment.

Example 23: 10 To the cosmetic ointment of Example 18 is added with thorough mixing 200 U/g Vitamin A dissolved in a minimum amount of vegetable oil.

Example 24: A cosmetic lotion is prepared by dissolving 300 pg 15 of la,24-dihydroxy vitamin D, in 100 g of dry propylene glycol. The lotion is stored in a refrigerator in a brown bottle and contains about 3.0 Mg la,24(S)-dihydroxy vitamin D 2 per gram of lotion.

Example 25: Dermatological testing.

Compositions containing la,24(S)-dihydroxy vitamin D, are evaluated for therapeutic efficacy of the composition in the topical treatment of dermatitis (contact and ectopic). The composition evaluated is an ointment containing 10 Mg of la,24-dihydroxy vitamin D, per gram of ointment in a petrolatum-almond oil base.

The control composition is identical except that it does not contain the active agent la,24(S)-dihydroxy vitamin D 2 The patients are treated in an out-patient clinic. They are instructed to use the preparation two times a day.

The ointment is as far as possible applied to a single lesion, or to an area of the disease. The ointment and its container are weighed before the -32treatment starts and returned with any unused contents for reweighing at the end of the treatment.

The area of the lesion treated is estimated and recorded, and the lesion is photographed as required, together with suitable "control" lesions. The latter are preferably lesions of similar size and stage of development, either in the vicinity of the treated lesion or symmetrically contralateral. Relevant details of the photographic procedure are recorded so as to be 10 reproduced when the lesions are next photographed (distance, aperture, angle, background, etc.). The ointment is applied twice daily and preferably left uncovered. The "control" lesions are left untreated, but if this is not possible, the treatment used on them is noted.

Evaluations of erythema, scaling, and thickness are conducted at weekly intervals by a physician, with the severity of the lesion rated from 0 to 3. The final evaluation is usually carried out at the end of four to 20 six weeks of treatment. Those lesions treated with la,24(S)-(OH) 2 D, have lower scores than the control lesions. An insignificant incidence of hypercalcemia is also observed.

Example 26: Epidermal cell differentiation and proliferation testing.

Human keratinocytes are cultured according to known modifications of the system originally described by Rheinwald and Green (Cell, vol. 6, p. 331 (1975)). The la,24(S)-dihydroxy vitamin D 2 dissolved in ethanol, is added to cells to yield a variety of concentrations between 0.05 and 5 Ig/ml with the ethanol concentration not to exceed 0.5% v/v. Control cultures are supplemented with ethanol at a final concentration of v/v.

Differentiation and proliferation of epidermal cells in culture is examined by: 1. quantitation of cornified envelopes; -33- 2. quantitation of cell density of cells attached to disks; 3. monitoring transglutaminase activity; or 4. monitoring DNA synthesis by incorporation of 3 H-thymidine.

Cultures incubated with la,24(S)-dihydroxy vitamin D 2 have more cornified envelopes, fewer attached cells, higher transglutaminase activity, and lower DNA synthesis than control cultures.

10 While the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate the various modifications, S"including variations, additions, and omissions, that may be made in what has been described. Accordingly, it is 15 intended that these modifications also be encompassed by the present invention and that the scope of the present invention be limited solely by the broadest interpretation that lawfully can be accorded the appended claims.

20 Example 27: Activity of la,24(S)-(OH) 2

D

2 in HL-60 cell differentiation assay.

A dose-response study is conducted with la,24(S)-(OH) 2 D, in the HL-60 cell differentiation assay as described by DeLuca and Ostrom (DeLuca, H. F. and Ostrem, V. Proa. Clin. Biol. Res., vol. 259, pp. 41-55 (1988)). In this study, la,25-(OH) 2

D

3 is used as a positive control and appropriate solvents are used as negative controls. The following variables are evaluated: nonspecific acid esterase activity, nitroblue tetrazolium (NBT) reduction, and thymidine incorporation. The results show that la,24(S)-(OH) 2

D,

has potent activity in promoting differentiation of promyelocytes to monocytes.

-34- Example 28: Antiproliferative activity of la,24(S)-(OH) 2

D

2 in human cancer cell lines.

Dose-response studies are conducted with la,24(S)-(OH) 2 D, in a battery of human cancer cell lines.

These cell lines include, but are not limited to, the following: BCA-1 or ZR-75-1 (breast) and COL-1 (colon), as described by Shieh, H. L. et al. Chem. Biol.

Interact., vol. 81, pp. 35-55 (1982). In this study, 10 appropriate solvents are used as negative controls. The :results show that la,24(S)-(OH),D 2 has potent (and reversible) antiproliferative activity, as judged by inhibition of thymidine incorporation.

e

I.

Claims (6)

1. 2. A prophylactic or therapeutic pharmaceutical composition for vitamin D deficient diseases, comprising a physiologically acceptable vehicle and an effective amount of la,24-dihydroxy vitamin D,.
2. 3. A composition according to claim 2 wherein the vitamin D 2 is la,24(S)-dihydroxy vitamin D,.
3. 4. A composition according to claim 2 or 3 wherein the amount is effective to prevent or 10 treat loss of bone mass or bone mineral content in a human being suffering from or predisposed to a depletive bone disorder or is effective to stabilize radial and spinal bone .o density in a human being suffering from renal osteodystrophy. S" 5. A composition for use in topical treatment of skin disorders, including a carrier suitable for topical application and la,24 (S)-dihydroxy vitamin D,. 15 6. la,24 (S)-dihydroxy vitamin D, for use in therapy, especially for use in treating skin disorders by topical administration or treating vitamin D deficiency-induced diseases.
4. 7. A method of preparing la,24(S)-dihydroxy vitamin D 2 including: tosylating 24-hydroxy vitamin D, to form 24-hydroxy vitamin D 2 tosylate; solvolyzing the 24-hydroxy vitamin D 2 tosylate to form 24-hydroxy 3,5 cyclovitamin D2; oxidizing the 24-hydroxy 3,5 cyclovitamin D, to form la,24-dihydroxy-3, D 2 and sequentially hydrolyzing, subjecting to a Diels-Alder reaction, and purifying the la,24-dihydroxy-3,5 cyclovitamin D 2 to form la,24 (S)-dihydroxy vitamin D 2 -36-
5. 8. A method according to claim 6 wherein step is carried out by methanolyzing the
6. 24-hydroxy vitamin D, tosylate. 9. A method in accordance with claim 6 or claim 7 including: acetylating ergosterol to form its 313-acetate; reacting with a triazoline dione and ozonating to form the 22-oxo-5L, 8c-(4-phenyl-3 ,5-dioxo- 1,2,4-triazoldine-1I,2-diyl)23,24-dinor-6-cholene-33-yl acetate; adding 3-methylbutan-2-one to 22-oxo-So., 8c-(4-phynyl -3,5 -dioxo- 1, 2,4-triazol idi ti- 1 ,2-diyl)23,24-dinor-6-cholenc-33-yI acetate to form (22E) 5oc,8cc-(4-phenyl- 3,5 -dioxo- 1,2,4- triazolidine-1I,2-diyl) cholesta-6,22-diene-=24-one-3 f-yl acetate;, adding methylmagnesium bromide to .(22E) 5cx,8cL-(4-phenyl-3,5-dioxo-1,2,4- do triazolidine- 1,2-diyl) cholesta-6,22-diene-24-one-3 B-yl acetate to form (22E)-Sx, 8o.- (4-phenyl- 3, 5-dioxo- 1,2,4-triazoldine- I ,2-diyl)-6,22-ergostadiene-3 1,24-diol; reducing the (22E)-5a,8a-(4-phenyl-3, 5-dioxo- 1,2,4-triazolidine- 1 ,2-diyl)-6,22- 1 5 ergo stadiene-3 B, 24-d iol to form 24-hydroxy ergosterol; irradiating 24-hydroxyergosterol to form 24-hydroxy vitamin D 2 toxylating 24-hydroxy vitamin D 2 in the presence of dry pyridine to form 24-hydroxy vitamin D 3 3 13-tosylate; solvolyzing 24-hydroxy vitamin D 2 tosylate to form 24-hydroxy-3,5 cyclovitamin D 2 1 allylically oxidizing the 24-hydroxy-3,5 cyclovitarnin D 3 with selenium dioxide to form 1 c,24-dihydroxy cyclovitamin D 2 1- and 1t 1. -37- The method according to claim 9 including the step of: hydrolyzing the la,24-dihyroxy 3,5 cyclovitamin D, with a mixture of dimethylsulfoxide and an organic acid to form an admixture of the 5,6 cis la,24-dihydroxy and 5,6 trans lao,24-dihydroxy vitamin D 2 and forming a Diels-Alder adduct of the 5,6 trans la-hydroxy vitamin D 2 to allow purification to yield la,24(s)-dihydroxy vitamin D 2 1i. A feed for mammals comprising lca,24(s)-dihydroxy vitamin D 2 wherein normal consumption of the feed by the mammals provides 0.01 to 1.0 lg/kg/day of 9 0 lc,24(S)-dihydroxy vitamin D,. 12. The compound of claim 1 for use as a medicament. 13. Use of the compound of claim 1 for the preparation of a medicament for the treatment of abnormal calcium metabolism, hyperproliferative skin disorders, or cancers. 14. A method in accordance with any one of claims 6, 7 and 9 comprising the step of isolating the la,24(S)-dihydroxy vitamin D, using reverse phase high-pressure liquid 15 chromatography. A method of preparing la,24(S)-dihydroxy vitamin D 2 substantially as hereinbefore described with reference to one or more examples and the drawings. Dated this 30th day of November 1999 PATENT ATTORNEY SERVICES Attorneys for BONE CARE INTERNATIONAL, INC.