MXPA97009683A - Use of vitamin d4 derivatives for treatment against the can - Google Patents

Abstract

The present invention relates to the 1a-hydroxy vitamin D4 and its novel analogues, and preferably 1,24 hydroxy vitamin D4, which are useful as active compounds of pharmaceutical compositions for the treatment of disorders of calcium metabolism and colon cancers and pec

Description

USE OF VITAMIN D4 DERIVATIVES FOR TREATMENT AGAINST CANCER Field of the art This invention relates to biologically active compounds of vitamin D4. More specifically, this invention relates to the novel la-hydroxy vitamin D4 and novel intermediates that are used in its synthesis, the novel 1, 25-dihydroxyvitamin D and the novel 1,24-dihydroxy vitamin D4-. This invention also relates to a pharmaceutical composition that includes a pharmaceutically effective amount of the novel 1 a-hydroxy vitamin D compounds and a method for controlling abnormal calcium metabolism by admixing a pharmaceutically effective amount of the novel compounds. D is known to be important in the regulation of calcium metabolism in animals and humans, see Harrison's Principles of Internal Medicine: Part Eleven, "Disorder of Bone and Mineral Metabolism, Chapter 335," E. Braunwald, et al., (eds.) McGraw-Hill, New York, 1987, pp. 1860-1865 The two most commonly known forms of vitamin D are vitamin D and vitamin D. Vitamin D is synthesized endogenously in the skin of animals or humans. , whereas the vitamin that vitamin D is the form of vitamin D provided by plants.Vitamin D, differs from vitamin D-, because it contains a double bond between C22 and C23 and also contains a methyl group in C24. In humans and rats, vitamin D and vitamin D have biopower equivalent. Vitamin D4 also known as 24, 23-dihydro-ergosterol or 22, 23-dihydro vitamin D or 22,23-dihydroergocalciferol irradiated, differs from vitamin D_ > because it contains a methyl group in C24. Vitamin D4 was first described in 1936. See Grab, W., Z. Physiol.Chem. , 243: 63 (1936); McDonald, F. G., J. BiolChe., 247: 185-188 11937). These references report some disagreement in the level of biological activity of the vitamin that suggests that in the rat, vitamin D4 is one-third or three-quarters as active as vitamin D, and, in the chick, one tenth or one fifth as active as vitamin D¿. A more definitive study of the biological activity of vitamin D4 was made by DeLuca et al., Arch. Biochem. Biophys., 124: 122-128 (1968). In this, the authors confirmed that vitamin D4 was less active than vitamin Dj. DeLuca et al. They report that, vitamin D4 is two thirds as active as vitamin D, or vitamin Dz in the rat, and a fifth as active as Dd in the chick. DeLuca et al. It refers to the fact that "the synthesis of vitamin D4 has apparently been little used since it was first discovered by Windhaus and Trautman and they comment," this may be due to the fact that vitamin D "is only of academic interest." Where the applicants know, vitamin D4 has been "of academic interest only" when applicants are not aware of any other study of vitamin D4 since it was reported by DeLuca eta.In fact The Merck Index establishes with respect to the vitamin D4, "their biological activity seems doubtful." Merck Index, S. Bundavari (ed.), 11"'ed., Merck & Co., Rahway, N-J., (1989) pp. 1579, # 9930. Since DeLuca et al., Discovered the active form of vitamin D ?, 1,25-dihydroxy vitamin D ?, (US Patent 3, 697, 559) and its synthetic precursor, la-hydroxy vitamin D (US Patent 3,697,996) , more interest has been focused on developing therapeutic use of these active vitamin D metabolites. Unfortunately, while the metabolites of vitamin D promise much as therapeutic agents, this promise has never been fully realized due to the extreme toxicity of these agents. For example, toxicity limits the efficacy of vitamin D, its active and analogous forms, to prevent bone loss or restore lost bone. Multiple studies indicate that at the doses necessary for these agents to be effective in preventing or restoring bone loss, problems arise such as hypercalcemia and hypercalciuria. It has been reported that la-hydroxy vitamin D, in a daily dose of 2 μg / day (which has been shown in some studies to be effective in preventing bone loss) causes toxicity in approximately 67% of patients. What is needed is a bioactive, low-toxicity vitamin D metabolite, so that the drug is practical as a therapeutic agent.

SUMMARY OF THE INVENTION The novel compounds of the invention, la-hydroxy vitamin D-1, 1, 25-dihydroxy vitamin D4 and 1,24-dihydroxy vitamin D4 are bioactive forms of vitamin D4. The present inventors have discovered that these active forms of vitamin D4 show biopotency much higher than what is predicted based on the previously reported bioassays of vitamin D4. The present inventors have also discovered that novel bioactive compounds are less toxic than predicted on the basis of their biopotency. This combination of high activity with low toxicity makes the compounds of the invention useful as therapeutic agents in the treatment of calcium metabolism disorders. The novel compounds of the invention are advantageously used as the active compounds of pharmaceutical compositions for diseases induced by abnormal calcium metabolism. To study the novel compounds of the invention it was necessary to develop processes for their production. An α-hydroxy vitamin D compound was produced synthetically and during this synthesis the novel intermediates were also produced. Compounds 1, 25-dihydroxy vitamin D4 and 1,24-dihydroxy vitamin D were isolated as biological products from the metabolism of α-hydroxy vitamin D4. Other advantages and a more complete appreciation of the specific adaptations, variations in the compositions and physical and chemical attributes of the present invention will be obtained with the examination of the following detailed description of the invention, taken together with the attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be described in conjunction with the accompanying drawings, in which like denominations refer to the same elements throughout the description and in which: Figure 1 illustrates the preparatory steps for the synthesis of vitamin D4; and Figure 2 illustrates the preparatory steps for the synthesis of la-hydroxy vitamin D4 starting with vitamin D4.

DETAILED DESCRIPTION The present invention provides the synthetic compounds la-hydroxy vitamin D (la-OH-D ") as well as the tosylated and cyclic derivatives of vitamin D4. As used herein, the terms "biologically active" or "biological activity" refer to the biochemical properties of the compounds, such as the affectation of the metabolism, for example, impairment of serum calcium concentration or binding to a suitable receptor protein, for example, binding to the vitamin D receptor protein.

In one of its aspects, the invention comprises the biologically active compounds of the general formula (I) (i) where Ri is H or OH and R¿ is H or OH, and the salts, hydrates and solvates thereof. Preferred compounds among those of the formula (I) are those in which Ri and R¿ both are H; Rx = OH and R, = H; and Rx = OH. In another aspect, the invention includes the preparation of the compound of the formula (I). The synthesis of la-hydroxy vitamin D4, that is, the compounds of the formula (I) wherein Ri and R2 are H, is obtained according to the scheme presented in figures 1 and 2. As can be seen in figure 1, the synthesis as the initial material. Ergosterol is subjected to saturation in the side chain in a 6-step process to produce 22,22-dihydroergosterol (VIII), using a procedure similar to that of Barton, et al., JCS Perkin I, 1976, 821-826. Then, the 22, 23-dihydroergosterol is irradiated as described in Windaus, et al., Z. Physol. Chem., 1937, 147: 185, to produce vitamin D [22,23-dihydroergocalciferol] (IX). As seen in Figure 2, vitamin D is then hydroxylated in a 4-step process to produce la-hydroxy vitamin D using a procedure similar to that described in Paaren et al., J. Ora. Chem., 1980, 45: 3253. Specifically, ergosterol is acetylated to form 3β-acetate. This ergosterol acetate is subjected to hydroxyhalogenation in the double bond 5, 6 to form the 5a-chloro-5a-hydroxy derivative. This chlorohydrin is reduced and reacetylated to the 5a-hydroxy derivative (ie, 5a-ol). The 5a-ol is subjected to hydrogenation to saturate the side chain. The resulting 3ß-acetoxiergost-7-en-5a-ol is reduced to 22,23 dehydroergosterol acetate which in turn is reduced to produce 22,23 dehydroergosterol. The 22,23 dehydroergosterol then radiates to form vitamin D4. Vitamin D4 is then tosylated to produce 3ß-tosyl vitamin D4. The tosylate is displaced by solvolysis for r ^ v i r1-methoxy-3,5-cyclo vitamin D4. The vitamin D4 cycle is subjected to ring oxidation to form the la-hydroxy vitamin derived cycle. The subsequently derived la-hydroxy vitamin cycle is solvolized and subjected to a Diels-Alder type reaction that removes the 5-methoxy group and separates the la-hydroxy vitamin D "(5,6-cis) from 5,6-trans la-hydroxy vitamin D 1,24-dihydroxy vitamin D4 and 1,25-dihydroxy vitamin D4 metabolites of α-hydroxy vitamin D4 are synthesized by incubating the hydroxy-derivatives with human hepatic cells, culturing the cells and recovering 1,24-dihydroxy or 1, 25 dihydroxy vitamin D. Using the binding assays for the vitamin D receptor protein, this: - -_-..-_ OS are determined to be biologically active.

It has been found that the compounds of the formula (I) possess valuable pharmacological activity, namely, as control agents for calcium metabolism, especially serum calcium concentrations. Specifically, the compounds of the formula (I) increase the serum calcium concentrations in rats with vitamin D deficiency. It has also been found that the compounds of the formula (I) have little toxicity, which favors their pharmaceutical properties. The compounds of the fórirulí p), -.-. In a? toxicity, when measured by the LDb test, which is similar to that corresponding to the vitamin D compounds and lower than that corresponding to the compounds of the vitamin Dj. In this way, the compounds of the invention are appreciable for the various clinical areas and for veterinary use and particularly useful for the treatment of abnormal calcium and phosphorus metabolism. In another aspect, the invention comprises a method of controlling calcium metabolism, such as to treat abnormal calcium metabolism caused, for example, by liver deficiencies, renal deficiency, gastrointestinal deficiency, etc. The compounds of the formula (I) can be used to treat in a prophylactic or therapeutic way the diseases caused by vitamin deficiency? and related diseases, e.g., renal osteodystrophy, steatorrhea, anticonvulsant osteomalacia, hypophosphatemic vitamin D-resistant rickets, osteoporosis, including postmenopausal osteoporosis, senile osteoporosis, steroid-induced osteoporosis, and other disease states characterized by loss of mass bone, rickets due to pseudo-deficiency (dependent on vitamin D), nutritional rickets and malabsorption, osteomalacia, osteopenia secondary to hypoparathyroidism, post-surgical hi-opatyroidism, idiopathic hypoparathyroidism, pseuodohipoparathyroidism alcoholism. The compounds of the formula (I), preferably those in which R- or R is OH, such as 24-dihydroxy vitamin D4, are valuable for the treatment of hyperproliferative disorders of the skin such as psoriasis, eczema, lack of adequate firmness of the skin, dermal hydration and secretion of bait. Particularly preferred for use in the treatment of these skin disorders is the (R) steroisomer of the 24-dihydroxy vitamin D, ie, the 24 (R) -dihydroxy vitamin D, substantially free of its form (S). ) or in combination with a small amount of the form (S). In this way, the present invention provides a method for treating skin disorders by administering a patient suffering from these, u ^. Therapeutically effective amount of a compound of the formula (I), preferably a compound of the formula (I) in which Ri or R 1 is OH, such as 24-dihydroxy vitamin D. More preferred is the compound of the formula (I) which is the 24 (R) -dihydroxy vitamin D4, substantially without its form (S). The compounds of the formula (I), preferably those in which Ri or R 'is OH, such as 24-dihydroxy-vitamin D4, are additionally of value in inhibiting the hyperproliferative activity of the cells. malignant, that is, cancer cells. In other words, the compounds of the formula (I) particularly, for example, the dihydroxy vitamin D4, act as antiproliferative agents when exposed to the malignant cells. Particularly preferred for use as an antiproliferative agent is the steroisomer (R) of 1α, 24-dihydroxy vitamin D4, ie, the, 24 (R) -dihydroxy vitamin D4, substantially without its (S) form or in combination with a small amount of the form (S). Thus, the present invention provides a method of treating malignant cells, for example, human cancer cells, i.e., inhibiting their hyperproliferative activity) with an effective amount of a cancer ». of the formula (I), preferably a computer of the formula (I) where R. or R_. it is OH, as it can be, 24-dihydroxy vitamin D4. More preferred is the compounds of the formula (I) which is the 24 (R) -dihydroxy vitamin D, substantially without its form (S). The effective amount is in the range from about 1 μg / dose to about 500 μg / dose. Of particular value in the treatment of cancers, is the use of the compounds of formula (I) for the treatment of skin cancers, which use the constituents in another aspect of the invention. In part, the compounds of the? -formula (I) are valuable for the treatment of breast cancer and colon cancer. In another aspect, the invention is a method for the treatment of the hyperproliferative cellular effects of breast cancer and colon cancer by administering to a patient suffering therefrom, a therapeutically effective amount of a compound of the formula (I) , preferably a compound of the formula (I) wherein R ± or R 'is OH, such as 24-dihydroxy vitamin D. More preferred is the compound of the formula (I) la, 24 (R) -dihydroxy vitamin D substantially without its form (S). The compounds of the formula (I), additionally, are useful in the treatment of non-cancerous disorders of the skin such as dermatitis, contact and ectopic. The compounds of the formula (I) are useful as active compounds in pharmaceutical compositions having reduced side effects and low toxicity compared to the known analogues of active forms of vitamin DJ # when applied, for example, in diseases induced by abnormal metabolism of calcium. These pharmaceutical compounds constitute another aspect of the invention. The pharmacologically active compounds of this invention can be processed according to conventional methods of the pharmacy to produce medicinal agents for administration to patients, for example, mammals including humans. For example, the compounds of the formula (I) can be used in mixtures with conventional excipients, for example, the pharmaceutically acceptable carrier substances suitable for enteric (eg, oral), parenteral or topical application that do not react in a harmful manner with the active compounds. Suitable pharmaceutically acceptable carriers include, but are not limited to water, saline solutions, alcohols, gum arabic, vegetable oils (e.g., corn oil, cottonseed oil, peanut oil, olive oil, coconut oil) , fish liver oils, oily esters such as Polysorbate 80, polyethylene glycols, gelatin, carbohydrates (for example lactose, amylose or starch), magnesium stearate talc, silicic acid, viscous paraffin, monoglycerides and diglycerides of fatty acids, acid esters fatty acids of pentaerythritol, hydroxymethyl cellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffer solutions, colorants, flavors and / or one or more than other active compounds, for example, vitamin Dj or D2'and their la-hydroxylated metabolites, conjugated estrogens or their antiestrogenic equivalents, calcitonin, biophosphonates, calcium supplements, cobalamin, pertussis toxin and boron.

For parenteral application, particularly suitable are sterile injectable solutions, preferably oil or aqueous solution, as well as suspensions, emulsions or implants including suppositories. The ampoules are convenient unit dosages. For the treatment of cancer, ie skin, breast and colon cancers, the parenteral dose of the compounds of the formula (I), preferably the, 24-dihydroxy vitamin D4, and more particularly, the, 24 (R) -dihydroxy vitamin D, substantially without its form (S), is about 0.5 μg to cerr; of 5 μg per unit dose. For the treatment of hyperproliferative disorders of the skin, such as psoriasis, the parenteral dose of the compounds of the formula (I), preferably the, 24-dihydroxy vitamin Dj, and most preferably the, 2 (R) -dihydroxy Vitamin D4, substantially without its form (S) is about 0.5 μg to about 50 μg per unit dose. For enteric application, particularly suitable are tablets, dragees, liquids, drops, suppositories, u-i2a, -. A syrup, elixir or the like may be used if a sweetened vehicle is desired.Suggested or targeted release compositions may also be formulated, for example, liposomes or those in which the active compound is protected with differentially degradable coatings, for example, by micro encapsulation, multiple coatings, etc. For topical application, suitable solid or semi-solid, viscous, non-sprayable forms including a carrier compatible with topical application may be employed. and having a preferred dynamic viscosity greater than water, suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, balms, aerosols, transdermal patches, etc., which , if desired, they are sterilized or mixed with auxiliary agents, for example, preservatives, stabilizers, demulsifiers, wetting agents, etc. Topical preparations of the compound, according to the present invention, useful for the treatment of skin disorders may also include epithelialization-inducing agents such as retinoids (e.g., vitamin A), chromanols such as vitamin E, beta agonists such as isoproterenol or cyclic adenosine monophosphate (cAMP), anti-inflammatory agents such as corticosteroids (e.g., hiarocortisone or cu acetate or dexamethasone) and keratoplastic agents such as tar or anthralin. The effective amounts of these agents are, for example, vitamin A about 0.003 to about 31 by weight of the composition, vitamin E about 0.1 to about 10%; Isoproterenol near 0.1 to about 2 '; cAMP about 0.1 to about 1-; hydrocortisone near Q.25: about 5¿; tar about 0.2 to about 201; and anthralin about 0.05 to about 21. For the topical treatment of skin disorders, the dosage of the compounds of the formula (I), preferably the 24-dihydroxy vitamin D4 and most preferably the 24 (R) -dihydroxy vitamin D substantially without its form (S), in a composition applied locally is about 1 μg to about 100 μg, - / rt ^ 1 • "> composition For the treatment of cancer, ie skin, breast and colon cancers, the dosage of the compounds of the formula (I), preferably the 24-dihydroxy vitamin D4, and most preferably the 24 (R) -dihydroxy vitamin D, substantially without its form (S), in an applied composition Locally it is from about 1 μg to about 100 μg / g of the composition For the treatment of hyperproliferative disorders of the skin, such as psoriasis, oi icac o of the compounds to the formula, 1. , 'preferably, the, 24-dihydroxy vitamin D and most preferably the, 24 (R) -dihydroxy vitamin D, sub substantially without its (S) form, in a topical composition it is about 1 μg to about 100 μg / g of the composition. For rectal administration, the compounds are formed in a pharmaceutical composition containing a suppository base such as cocoa oil or other triglycerides. To prolong shelf life, the composition includes, for convenience, an antioxidant such as ascorbic acid, hydroxyanisole or butylated hydroxyquinone. Oral administration of the pharmaceutical compositions of the present invention is preferred. In general, the compounds of this invention are administered per unit dosage form containing about 0.? μg to about 25 μg in a pharmaceutically acceptable carrier per unit dose. The dose of the compounds according to this invention is generally from 0.01 to about 0.5 μg / kg / day, preferably from about 0.04 to about 0.3 μg / kg / day. It will be appreciated that the preferred actual amounts of the active compound in a specific case will vary according to the efficacy of the specific compound being used, the particular compositions formulated, the mode of application and the particular site and organ being treated. Po¿ - j -, -.-]. i.-, xa specific dose for a particular patient depends on the age, body weight, general state of health, sex, diet, time and mode of administration, rate of excretion and of the medications used in combination and the severity of the particular disorder to which the therapy is applied. The doses for a given host can be determined using conventional considerations, for example, by customary comparison of the differential activities of the subject compounds and a known agent, such as by means of a suitable conventional pharmacological protocol. In yet another aspect, the compounds of the present invention can be advantageously used in compositions for veterinary use, for example, pet food compositions for treating or preventing hypocalcemia. In general, the compounds of the present invention are administered in the animal feed so that the normal consumption of this food provides the animal with about 0.01 to about 0.5 μg / kg / day. The following examples are constructed simply as illustrative and do not limit the rest of the description in any way. In the following examples, all temperatures are set in degrees Celsius; unless otherwise indicated, all parts and percentages are by weight. The nuclear magnetic resonance spectra of the proton (? NMR) were recorded with an IBM Sy-200 (200 MHz and a Bruker Am-400 (400 MHz) with aspect [sic] of 300 Computer in solutions of CDClj with CHC1.- as Internal standard The infrared spectra were recorded with a Courier Tranform (FTIR) using samples such as potassium bromide (KBr) granules or as liquids Mass spectra were recorded with a Finnigan MAT-90 mass spectrometer at 20 eV / CL). The melting points were determined in a Hoo er-Thomas (capillary) and Uni-melt and an apparatus for Fisher Johns melting point (of the cover type).

Example 1: Synthesis of la-hydroxy vitamin D ^ Ergosterol (II) was converted to ergosterol (II) acetate by dissolving 100 g (0.25 mole) of ergosterol in 600 ml of anhydrous pyridine and 68 ml (0.7 mole) of anhydride acetic. The solution was stirred overnight at room temperature after which time the solution was cooled by adding 1.2 L of ice causing a precipitate to form. The precipitate was washed 5 times with 400 ml portions of water, then once with 400 ml of CH3CN. The resulting product was dried in the air to produce 79 g (71?) Of ergosterol acetate, as a white crystalline solid and had the following characteristics: melting point (p.F.): 169-171 ° C; ? NMR: (400 MHz, CDC13), sppm 2.05 (3H, s, 3β-CH3CO), 4.65-4.75 (lH, m, 3a-H) 5.15-5.25 (2H, m, 22-H and 23-H), 5.4 (1H, d, 6-H), 5.6 (1H, to 7-H); FTIR [KBrl: 1734cm "1 (C = 0 lengthening) 968 cm" (C-H flexion). The ergosterol (III) acetate (26 g, 0.062 M) was dissolved in 2.5 L of freshly distilled dehydrogenated toluene. This solution 9 ml (0.111 mol) of chromium chloride dissolved in 240 ml of anhydrous CHCl3 was added with nitrogen at -78 ° C for a period of 30 minutes. The reaction system was stirred at minus 78 ° C for an additional 15 minutes and then 62 ml of a saturated solution of sodium borohydride in ethanol was added in one portion. After stirring at -78 ° C for an additional 15 minutes, the reaction solution was emptied into a two-phase system of 3N hydrochloric acid (3L) and benzene (3L). The organic layer was separated, then washed with water (2L), twice with brine solution (2 x 1L) and then dried with anhydrous MgSO4. The dried solution was filtered and concentrated in vacuo. The impure crystalline product was then treated with CHCN (280 ml) and the filtration of the suspension thus formed yielded 12.5 g (41 o) of crystalline white 3β-acetoxy-6a-chloroergota-7,22-dien-5a-ol. (IV) and had the following characteristics: mp: 190-192 ° C; lH NMR: (400 MHz, CDC13), sppm 2.05 (3H, s, 3β-OAc), 4.65 (ΔH, d, 6β-H) 5.1 (1H, s, 7-H), 5.1-5.3 (2H, m , 22-H and 23-H); FTIR fKBr \: 1732 cm "1 (C = 0 lengthening) 968 cm" 1 (CH flexion), 3437 cm "1 (OH = elongation) .3ß-Acetoxy-6a-chloroergote-7,22-dien-5a- ol (IV) (21.4 g, 0.044 mol) in anhydrous THF (900 ml) was added slowly to a stirring suspension of lithium aluminum hydride (2.66 g, 0.07 mol) in anhydrous THF (750 ml) at room temperature. The mixture was refluxed for 3 hours and cooled to 0 ° C. The hydride in the cesium was decomposed with saturated NaSO.sub.4 solution, filtration through Na < S04 anhydrous and evaporation of the The filtrate produced a solid, which was treated directly with anhydrous acetic acid (110 ml) and anhydrous pyridine (220 ml) at 0 ° C. Removal of the solvent under reduced pressure produced the acetate (12.75 g, 61 °), 3ß-acetoxytergote-7. , 22-dien-5a-ol (V) and had the following characteristics: mp: 229-232 ° C; FTIR [KBr]: 1736 cm "1 (C = 0 lengthening) 3460 cm" 1 (OH elongation) 972 cm "1 (CH flexion). The compound 3β-acetoxiergota, -7, 22-dien-5a-ol (V) (2.5 g, 0.0055 mol) was stirred for 17 hours with PtO. freshly prepared (0.5 g) in ethyl acetate (820 ml) with H¿ gas (15 psi). The catalyst was removed by filtration and evaporation of the filtrate gave the impure acetate which was dissolved in CH 2 Cl 1 and passed through chromatography on silica gel. The elution with CHC1 gave the substantially pure 3β-acetoxy ^ -gnt-, -7, 22-dien-5a-ol (VI) compound (2.15 g, 85%) as a white crystalline material and had the following characteristics: p. f .: 228-232 ° C; ? NMR: (400 MHz, CDCl, sppm 2.05 (3H, s, 3β-OAc), 5.05-5.20 (2H,, 3a-H and 7H); FTIR [KBr]: 1736 cm "(C = 0 elongation) 3462 cm 1 (OH elongation): Redistilled thionyl chloride (9.7 ml) in anhydrous pyridine (170 ml) was added to the compound 3ß-acetoxiergost, 7-en-5a-cl (VI) (12.0 g, 0.0262 mol) in anhydrous pyridine. (800 ml) at 0 ° C under nitrogen After 2.5 hours, the solution was diluted with cold H. 0 on ice (1.5 L) and extracted with 2 portions of ether (2.5 L + 1.5 L) the ether extracts combined were washed with solution NaHCO ^ (1.0 L x 2), then with IN HCl (1.5 L x 2) and then water (1 L). The solution ^^^ "^ - ^ dried with Mg? 04 and, after filtration, evaporated under reduced pressure to produce an impure product that was converted into a suspension with CHCN (100 ml). by filtration and recrystallized from CH ^ CN to yield 4.5 g (39o) of a white crystalline 22,23-dihydroergosteryl acetate (VII) and had the following characteristics: mp: 144-147 ° C;? NMR: ( 400 MHz, CDC13), sppm 2.05 (3H, s, 3β-OAc), 4.65-4.75 (HH, m, 3a-H), 5.4 (HH, d, 6-H), 5.6 (HH, d, 7- H); FTIR [KBr]: 1734 cm "'(C = 0 elongation). The 22,23-dihydroergosteryl acetate (VII) (4.8 g, 0.011 mol) was added in a single pass, with stirring, to a suspension of lithium aluminum hydride (2.5 g, 0.066 mol) in anhydrous ether (1.1 L). ) at room temperature. To destroy the excess lithium aluminum hydride, 5N NaOH was added and then H¿0 (500 ml) was added. The aqueous solution was then extracted with 4 portions of 250 ml of ether. The combined ether extracts and the combined organic layer were washed with brine solution (1 L), then dried with NaSO4. Evaporation of the ether under reduced pressure gave the compound 22-22-d? H? Drergosterol, (VIII) (4.1 g, 94 o) as a white crystalline material and had the following characteristics: p. f .: 147-150 ° C; H NMR: (400 MHz, CDCl 3), sppm 3.6-3.7 (H, m, 3a-H), 5.4 (H, d, 6H), 5.6 (H, d, 7H); FTIR [KBr]: 3400 cm "'(OH elongation) .22,23-Dihydroergosterol (VIII) (2.0 g, 5.0 mmol) was dissolved in a solution of diethyl ether and benzene (4: 1, 600 ml) and irradiated (Hannovia immersion lamp, 450 watts) with agitation with argon and in a quartz vessel cooled with water for 3 hours.The solution was concentrated in vacuo to produce a gummy solid which was redissolved in 100 ml of ethanol and heated to reflux with argon for 8 hours, then the solution was concentrated in vacuo and the residue was absorbed on a column of silica gel and eluted with 30% ethyl acetate in hexane to produce vitamin D4 (22, 22-d? h). ? droergocalc? ferol) (IX) with a yield of 1.2 g (60 ') and with the following characteristics: H NMR: (400 MHz, CDC13), sppm 0.55 (3H, s, 18-HJ 0.78 (6H, dd , 26-HJy 27-HJ 0.87 (3H, d, 21-HJ 0.93 (3H, d, 28-H 3.94 (H, m, 3-H) 4.82 (H,, (acute), 19-H) 5.04 ( HH, m, (acute), 19-H) 6.4 (HH, d, 7-H) 6.24 (HH, d, 6-H). To a stirred solution of vitamin D (IX) (3.0 g, 7.5 mmol) in 10 ml of anhydrous pyridine was added freshly recrystallized p-toluenesulfonyl chloride (3.6 g, 19 mmol) at 0 ° C. The reaction mixture was stirred at 5 ° C for 24 hours and then the reaction was quenched by pouring the mixture onto ice and saturated NaHC0 (100 ml) with stirring. The aqueous suspension was extracted with CH2Cl3 (3 x 300 mL). The combined organic extracts were washed with 10% HCl (3 x 200 mL), saturated NaHCO 3 (3 x 200 mL) and saturated NaCl (2 x 200 mL), dried over Mg 04 and concentrated in vacuo to yield 3.5 g (84%) of the novel intermediate compound vitamin D4 tosylate (X) and had the following characteristics: XH NMR: (400 MHz, CDC13), sppm 0.54 (3H, s, I8-H3), 0.78 (6H, dd, 26-H¿ and 27-H3) 0.87 (3H, d, 21-H) 0.96 (3H, d, 28-H3) 2.45 (3H, s, CHd (tosylate) 4.68 (3H, m, 3-H) 4.82 (ÍH, m, (acute), 19-H) 5.04 (ÍH, m, (acute), 19-H), 5.95 (1H, d, 7-H) 6.09 (1H, d, 6-H) 7.34 and 7.79 (4H, d, aromatic) To a stirred suspension of NaHCOj (17.0 g, 202 mmol) in methanol (200 ml) was added, dropwise, a solution of vitamin D4 tosylate (X) (3.5 g, 6.3 mmol) in anhydrous CHC1 (10 ml) The reaction mixture was refluxed overnight with argon, then cooled to room temperature and concentrated in vacuo to about 50 ml. react The reaction was diluted with ether (600 ml), washed with water (3 x 300 ml), dried over MgSO0 and concentrated in vacuo. The residue was passed through a column of silica gel and eluted with 10% ethyl acetate in hexane to yield the novel intermediate compound 3., 5-cycle vitamin D4 (XI) (dense oil) with a yield of 1.5 g (58%) and had the following characteristics:? NMR: (400 MHz, CDCL3), sppm 0.56 (3H, s, 18-HP, 0.78 (6H, dd, 26-Hj and 27-H 0.87 (3H, d, 21-HJ, 0.94 (3H, d, 28 -H) 3.28 (3H, s, OCH) 4.2 (H, d, 6-H) 4.91 (H, m, (acute), 19-H) 4.98 (H, d, 7-H) 5.08 (H, m, (acute), 19-H). Anhydrous tert-butyl hydroperoxide in toluene (3M) (2.6 mL, 7.8 mmol) was added to a suspension with stirring of selenium dioxide (0.22 g, 2 mmol) in CH¿. Dry C1Z (150 ml) in a three neck flask The mixture was stirred for 3 hours under argon, then pyridine (0.3 ml, 3.7 mmol) and cyclo-vitamin D4 (XI) (1.5 g, 3.6 mmol) were added. It was then introduced as a solution in CHC1 (50 mL) After stirring for 30 minutes, a 10% aqueous solution of NaOH (200 mL) was added.The reaction mixture was then diluted with ether (500 mL). The phases were separated and the organic phase was washed with 10% NaOH (3 x 200 mL), water (2 x 200 mL) and saturated NaCl solution (2 x 200 mL) and dried over MgSO4 and concentrated in vacuo. E The residue was absorbed onto a column of silica gel and eluted with 30% ethyl acetate in ethane to yield 0.45 g (29%) of novel intermediate la-hydroxy 3,5-cyclo-vitamin D (XII) (oil ) and had the following characteristics:] H NMR: (400 MHz, CDC13), sppm 0.54 (3H, s, 18-H, 0.78 (6H, dd, 26-H ^ and 27-H 0.86 (3H, d, 21 -HJ 0.95 (3H, d, 28-H¿) 3.26 (3H, s, OCH3) 4.2 (HH, d, 6-H), 4.22 (HH,, 1-H), 4.95 (HH, d, 7- H), 5.18 MR. d. 19-H) ..25 (ÍH, d, 19-H). A solution of la-hydroxy 3,5-cyclo vitamin D (XII) (0.45 g, 1.05 mmol) in a solution of dimethylsulfoxide (4.5 ml) and glacial acetic acid (3.6 ml) was heated at 50 ° C with argon for 1 hour. The reaction mixture was then poured onto ice and a saturated solution of NaHCO =, (100 mL), and extracted with ether (3 x 200 mL). The combined ether extracts were washed with a NaHCO solution. saturated (3 x 200 mL), water (3 x 200 mL) and saturated NaCl solution (3 x 200 mL), dried over MgS. concentrated in vacuo to give a mixture containing 5,6-cis and 5,6-trans la-hydroxy vitamin D 4 (about 4: 1 per 1 HNMR) with a yield of 0.4 g, (92 1). The mixture of 5,6-cis and 5,6-trans la-hydroxy vitamin D-, (0.4 g, 0.97 mmol) was dissolved in ethyl acetate (25 ml) and treated with freshly recrystallized maleic anhydride (0.08 g, 0.8 mmol). This reaction mixture was heated at 35 ° C with argon for 24 hours. After evaporation of the solvent in vacuo, the impure mixture was treated by chromatography on a column of silica gel using ethyl acetate and hexane (1: 1) as eluent, to produce the novel active form of vitamin D4, 5, 6 -cis la-hydroxy vitamin D-, (XIII) with a yield of 90 mg (23%) and had the following characteristics: p. f .: 128-130 ° C; IR Vraax (Neat); 3400 cm "'(OH elongation); 1H NMR: (400 MHz, CDC1, sppm 0.55 (3H, s, 18-HJ, 0.79 (6H, dd, 26-Ej and 27-HP 0.87 (3H, d, 21- HP 0.94 (3H, d, 28-H 4.24 (H, m, 3-H) 4.44 (H, m, 1 -H) 5.02 (H, m, (acute), 19-H) 5.34 (1H, m, (acute), 19-H) 6.02 (H, d, 7-H), 6.4 (H, d, 6-H); Mass spectrum [Cl] m / e (relative intensity): 415 (M + 1, 4 °.) 397, (M + 1-OH 100%), 379 (27%), 135 (22%).

Example 2: Biological test of la-hydroxy vitamin D4 Freshly weanned rats (Holtzman strain, Lolcz Company, Madison, Wisconsin) were fed a diet deficient in vitamin D with calcium (0.47%) and phosphorus (Q. ) suitable. At 3 or 4 weeks, this diet induces an extreme deficiency in vitamin D characterized by little serum calcium and poor growth. After 4 weeks on this diet, the rats had serum calcium values less than 7 mg / dL. The rats were then separated into 4 groups and were orally administered la-hydroxy vitamin D4 in a vehicle such as coconut oil or vehicle (control) during each of 14 days. 24 hours after the last dose, the rats were sacrificed and the blood calcium was measured by a laboratory technique standard. The results of these determinations are shown in Table 1.

Table 1 Increase in serum calcium concentration Compound Dosage N Numerometer Concentration of calcium (μg / kg / day rat in serum (mg / dl) ± standard deviation control - 1C 6.1 ± 0.48 la-OH-D4 0.042 8 7.1+ 0.80 la-OH-D4 0.250 7 11.6. ± 0.45 la-OH-D4 1,500 9 12.710.37 The utes of Table l that the coptr ..-- 2. í "-Í lo- hydroxy vitamin D is effective in increasing serum calcium in rats deficient in vitamin D and that the response appears to be dose dependent. Surprisingly, the level of response seems to compare favorably to that reported by Wientroub, et al., For 1, 25-d? H? Drox? Da vitamin is administered to rats deficient in vitamin D under similar experimental conditions described in the above. See, Wientroub, S., P.A. Reddi, AH, "The Dichotomy m the Effects for 1,25 dihydroxy vitamin D = and 24,25 dihydroxy vitamin Dd on Bone Gamma Carboxyglutamic Acid-Contaminating Protein m Serum and Bone m vitamm D-Deficient Rat = 'P Calcif. Tissue Int. (1987) 40: 166-172.

Example 3: Toxicity Tests The acute oral toxicity of la-OH-D- in rats was evaluated by determining the mean lethal dose (LD5?) Using a well-known method. The rats were fed a standard laboratory diet for 8-10 weeks. Five animals of each sex were given an oral dose of la-0H-D4. The animals were observed for 14 days and the number of deaths was recorded. The value of LDs? was determined around 1.0 mg / kg in males and 3.0 mg / kg in females. For comparison, the LD value for o-hydroxy vitamin O1 under the same conditions was found by the applicants of 1.7 and 1.8 mg / kg, in male and female rats, respectively. The toxicity of la-hydroxy vitamin D had previously been reported as lower than that of la-hydroxy vitamin D0, Sjoden, G., Smith, C, Lindgren, U., and DeLuca, H.F. Proc 5oc, Experimental Biol, Med., 178: 432-436 (1985).

Example 4: Production and Isolation of 1,25-Dihydroxy Vitamin D4 La-hydroxy vitamin D4 of the present invention is incubated with cultured human liver cells which metabolize the compound to various products including 1,24-dihydroxy vitamin D metabolites. The metabolite 1.25 is isolated and purified by liquid pressure alpha chromatography and is identified by mass spectroscopy-gas chromatography. Linkage studies demonstrate that 1,25 dihydroxy vitamin D4 has good binding affinity for the vitamin D receptor protein in mammals indicating that it is biologically active. The procedures used are similar to those described in Strugnell, et al., Biochem. Pharm. Vol. 40, 333-341 (1990).

Example 5: Production and isolation of 1,24-dihydroxy vitamin D4 The production and isolation of 1,24-dihydroxy vitamin D4 is carried out as described in Example 4 above. The hydroxy vitamin D4 of the present invention is incubated with cultured human liver cells that metabolize the compound to various products including the metabolite 1,24-dihydroxy vitamin D,. Metabolite 1.24 is isolated and purified using high pressure liquid chromatography and identified by gas chromatography-mass spectrometry. Studies of binding with the new metabolite demonstrate that the metabolite has good binding affinity for the vitamin D receptor protein in mammals, which indicates that the drug is biologically active.

Example 6: Hypercalcemia test Female rats were fed a commercial diet with 0.8% calcium (0.8 o) and phosphorus (0.6%). The rats were divided into 4 groups and each group was orally administered la-OHD4 in a vehicle such as coconut oil or only the vehicle (control) for 13 weeks. 24 hours after the last dose the rats were sacrificed and the calcium content in serum was determined by a standard method. This procedure demonstrates that the serum calcium concentration is unaffected or slightly elevated at the dose of -OHD4 up to 2.5 μg / kg / day.

Example 7: Another biological test Freshly weaned male rats were fed a vitamin D deficient patient with ba or calcium content (0.02o). After a period of 4 weeks, the rats were divided into 4 groups and intravenously administered la-OHD4 in a vehicle such as ethanol or only the vehicle (control). 16 hours after the administration, the rats were sacrificed and intestinal calcium transport was measured using the duodenal bags turned from the inside out, following the method of Martín and DeLuca, Arn. J. Physiol. 216: 1352-1359.

The follow-up of this procedure dampens the stimulus of intestinal calcium transport in a dose-dependent manner.

Example 8 A clinical study was carried out with external osteoporotic patients, in post-menopausal period, in ages between 55 and 75 years. The study included up to 120 patients divided randomly into 3 treatment groups, and continued for 12 to 24 months. Two of the treatment groups received constant doses of la-v tamine Di (u., I.d, two different dose levels above 3.0 μg / day) and the other group received a placebo. All patients maintain a normal intake of calcium in the diet (500 to 800 mg / day) and avoid using calcium supplements. Efficacy is assessed by comparisons before and after treatment of the groups of patients in relation to (a) the total weight, the mineral density of radial, femoral and / or spinal bone determined by X-ray absorptiometry (DEXA). (b) bone biopsies of the iliac crest and (c) determinations of osteocalcin in serum. Safety was assessed by comparisons of urinary hydroxyproline excretion, serum and urine calcium levels, creatinine clearance, blood urea nitrogen and other routine determinations. This study demonstrates that patients treated with vitamin D4 have densities in total body bone, radial, femoral and / or spinal significantly higher in relation to patients treated with placebo. The treated patients also present significant elevations of osteocalcin in serum. Bone biopsies of treated patients show that la-vitamin D4 stimulates normal bone formation. The monitored safety parameters confirm an insignificant incidence of hypercalcemia or hypercalciuria or any other metabolic disorder with vitamin D4 therapy.

Example 9 A clinical study was carried out with healthy women in the period after menopause, with ages between 55 and 60 years. The study includes up to 80 patients divided randomly into two treatment groups and continues for 12 to 14 months. One treatment group receives a constant dose of vitamin D4 (u., D., A dose level above 3.0 μg / day) and the other receives a corresponding placebo. The study was carried out as indicated in Example 2 above. This study shows that patients treated with vitamin D4 have a reduced loss in total body, radial, femoral and / or spinal bone densities in relation to baseline values. In contrast, patients treated with placebo show significant losses in these parameters in relation to baseline values. The monitored safety parameters confirm the safety of long-term administration of vitamin-D4 at this dose level.

Example 10 A double-blind, placebo-controlled clinical trial for 12 months was performed with 30 men and / or women with renal impairment who were undergoing chronic hemodialysis. All patients entered a control period of 8 weeks, during this time they received a maintenance dose of vitamin D3 (400 IU / day). After this control period, patients were randomly divided into two treatment groups: one group received a constant dose of vitamin D (uid, a dose greater than 3.0 μg / day) and the other group received a placebo correspondent. Both treatment groups receive a maintenance dose of vitamin Dj, maintain a normal intake of calcium in the diet and avoid the use of calcium supplements. Efficacy was assessed by comparisons before and after treatment of groups of patients with respect to (a) direct measurements of intestinal calcium absorption, ib) mineral density of total, radial, femoral and / or spinal body bone, and (c) ) calcium and osteocalcin determinations in serum. Safety was assessed by regular monitoring of serum calcium. The analysis of clinical data shows that vitamin D significantly increases osteocalcin levels in serum and intestinal calcium absorption, as determined by measurements using a simple or double isotope technique. Patients treated with this compound show normalized levels of serum calcium, stable values for body, total, radial, femoral and / or spinal bone densities in relation to baseline values. In contrast, patients treated with placebo showed frequent hypocalcemia, significant reductions in total body density, radial, femoral and / or spinal. A negligible incidence of hypercalcemia was observed in the treated group.

Example 11: Drug preparations A topical cream is prepared by dissolving 1.0 mg of the, 24-dihydroxy vitamin D4 in 1 g of almond oil. To this solution are added 400 g of mineral oil and 20 g of beeswax autoemul = ificante. The mixture is heated until liquefied. After the addition of 40 ml of hot water, the mixture is stirred well. The resulting cream contains approximately 10 μg of the, 24-dihydroxy "itamine D4 per gram of cream.

Example 12 An ointment is prepared by dissolving 1.0 mg of the, 24-dihydroxy vitamin D4 in 30 g of almond oil. To this solution 700 g of white, soft paraffin which has been heated to be liquefied is added. The ointment is mixed well and allowed to cool. This ointment contains approximately 10 μg of the, 24-dihydroxy vitamin D4 per gram of ointment.

Example 13: The ointment of Example 12 is added by mixing 0.5 g of adenosine and 2.0 g of papaverine base, both dissolved in a minimum amount of dimethyl sulfoxide. Additional ingredients are present in a measure up to 0.51-. in weight (adenosine) and 2% in weight (papaverine base).

Example 14: The ointment of Example 12 is added with perfect mixing of 10,000 U of vitamin A dissolved in a minimum amount of vegetable oil. The resulting ointment contains about 100 U of vitamin A per gram of ointment.

Step 15: A dermatological lotion is prepared by dissolving 1.0 mg of the, 24-dihydroxy vitamin D in 100 g of dry propylene glycol. The lotion is stored in a refrigerator, packed in a brown bottle and contains about 10 μg of the, 24-dihydroxy vitamin D4 per gram of lotion.

Example 16: In lg of almond oil 0.2 mg of the, 24-dihydroxy vitamin D is dissolved. To the solution are added 40 q of mineral oil and 20 g of self-emulsifying beeswax, followed by 40 ml of hot water. The mixture combines well to produce a cosmetic cream containing about 2.0 μg of the, 24-dihydroxy vitamin D per gram of cream.

Example 17: To a cosmetic cream prepared according to Example 16 is added 100 mg of adeno = ina. The cream mixes well and contains about 0.1% by weight of adenosine.

Example 18: An ointment is prepared by dissolving 100 μg of the, 24-dihydroxy vitamin D in 30 g of almond oil. To this solution thus produced, 70 g of soft white paraffin which has been heated to be liquefied is added. The ointment is mixed well and allowed to cool. The ointment thus produced contains about 1.0 μg of the, 24-dihydroxy vitamin D4 per gram of ointment.

Example 19: To the cosmetic ointment of Example 18 is added, with mixing, 200 U / g of vitamin A dissolved in a minimum amount of vegetable oil.

Example 20: A cosmetic lotion is prepared by dissolving 300 μg of the, 24-dihydroxy vitamin D4 in 100 g of dry propylene glycol. The lotion is stored in a refrigerator in a brown bottle and contains about 3.0 μg of the, 24-dihydroxy vitamin D- per gram of lotion.

Example 21: Dermatological test For the therapeutic efficacy of the composition in the topical treatment of dermatitis (contact and ectopic) compositions with a content of the, 24-dihydroxy vitamin D were evaluated. The evaluated composition is an ointment containing 10 μg of the, 24-d? Hydroxy vitamin D per gram of ointment in a base of almond-petrolatum oil. The control composition is identical, except that ^? the active agent contains the dihydroxy vitamin D. Patients are treated in an outpatient clinic. These are instructed to use the preparation twice a day. The ointment is applied as much as possible to a single lesion, or to one area of the disease. The ointment and its container are weighed before starting the treatment and when returning it with any content not used to re-weigh it at the end of the treatment. The area of the treated lesion is calculated and recorded and the lesion is photographed as required. -p.?r.tc < ~? V adequate "control" injuries. The latter are preferably lesions of similar size and stage of development, close to the lesion treated or symmetrically contralateral. The important details of the photographic procedure are recorded so that they can be reproduced when the injuries are re-photographed (distance, opening, angle, background, etc.). The ointment is applied twice a day and, preferably, is left uncovered. The "control" lesions are left untreated, but if this is not possible, the treatment used is noted. Evaluations of erythema, scaling and thickness are performed at weekly intervals by a doctor, with the severity of the lesion classified from 0 to 3. The final evaluation is usually carried out at the end of 4 to 6 weeks of treatment. Those lesions treated with the, 24- (OH) ¿D4 have lower registers than the control lesions. A negligible incidence of hypercalcemia was also observed.

Example 22: Epidermal cell proliferation and differentiation test Human keratinocytes are cultured according to known modifications of the system originally described by Rheinwand and Green (Cell, vol.6, p.331 (1975)). La, 24-dihydroxy vitamin D, isolated in ethanol, is added to cells to produce a series of concentrations between 0.05 and 5 μg / ml with the ethanol concentration not exceeding 0.5-, v / v. The control cultures are complemented with ethanol at a final concentration of 0.5 v / v. The differentiation and proliferation of the epidermal cells in the culture were examined by: 1. Quantification of the cornified tunics; 2. Quantification of cell density of cells bound to discs; 3. Monitoring of the activity of transglutaminase; or 4. Monitoring of DNA synthesis by incorporation of H-imidine. The cultures incubated with the, 24-dihydroxy vitamin D4 have more cornified tunics, fewer adherent cells, higher transglutaminase activity and lower DNA synthesis than the control cultures.

Example 23: Activity of the, 24- (OH) D in HL-60 cell differentiation assay A dose-response study with the, 24-OH) ¿D4 was performed in the cell differentiation assay HL-60 as describes in DeLuca and Ostrem (DeLuca, HF and Ostrem, VK, Prog. Clin Biol. Res., vol 259, pp. 41-55, (1988)). In this study s <; = 1. ?? 'i 7 1 (X' 25- (0H) 2D as a positive control and the appropriate solvents are used as negative controls.The following variables are evaluated: nonspecific acid esterase activity, nitro blue reduction of tetrazolium (NBT) and incorporation of thymidine The results show that 24- (OH) _Di has potent activity to favor the differentiation of promyelocytes HL-60 to monocytes.

Example 24: Antiproliferative activity of the, 24- (OH) _D4 in human cancer cell lines Dose-response studies were performed with the, 24- (OH) _D in a battery of cell lines with human cancer. These cell lines include, but are not limited to, the following: BCA-1 ~ ZR-75-J '~ v ^ - cOL-i (colon), as described in Shieh, H.L. et al., Chem. Biol. Interact., vol. 81, pp.35-55 (1982). In this study, suitable solvents are used as negative controls. The results show that la, 24- (OH) D4 has potent (and reversible) antiproliferative activity, judging by the inhibition of thymidine incorporation.

Example 25: Treatment of psoriasis An oral dose formulation containing the 24-dihydroxy vitamin D < T - ~ • :: .- bl blind for the therapeutic efficacy of the formulation in the treatment of dermatitis (contact and ectopic). The evaluated formulation contains 1.0 to 10.0 μg of the, 24-dihydrox? vitamin D . The control formulation is identical, except that it does not contain the la, 24-d? H? Drox? vitamin D-, . Patients were treated in an outpatient clinic and divided into experimental and control population. They were instructed to take the medication once a day, in the morning before breakfast. In each patient (experimental and control) an area of the skin containing the lesion is selected, which is ordinarily covered by clothing and patients are instructed not to expose the area of the pial selected for sunlight to sunlight. study. The area of the lesion is calculated and recorded and the lesion (s) is photographed. The important details of the photographic procedure are recorded so that they can be reproduced when the lesions are photographed later (distance, opening, angle, background, etc.). The evaluations of the subject, desquamation and thickness are made at weekly intervals by a doctor. The final evaluation is usually carried out after 4 to 6 weeks of treatment. The results of the study show that daily oral administration of 1,24-dihydroxy vitamin D4 significantly reduces the degree of erythema, desquamation and thickness compared to control patients.

Although the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate that various modifications, including variations, additions and omissions, can be made in what is described. Accordingly, it is proposed that these modifications also be included in the present invention and that the scope of the present invention be limited only by the broad interpretation that may be legally agreed upon in the appended claims.

Claims (12)

1. A method for inhibiting the hyperproliferative activity of human cancer cells, which is to treat the cancer cells with an effective amount of the compound of the formula (I):

(I) where Ri is H or OH and R¿ is H or OH and the salts hydrates and solvates thereof. 2. The method of claim 1, wherein the compound of the formula (I) is the, 24-dihydroxy vitamin Di

3. The method of claim 2, wherein the compound of the formula (I) is lor. 4-di b-5 ^ r ^ xi vitamin D4, substantially without its form (S). 4. The method of claim 1, wherein the amount is about 1 μg / dose to about 500 μg / dose.

A method of treatment, to a human, to alleviate the hyperproliferative cellular activity of breast cancer and colon cancer, which involves administering human being a therapeutically effective amount of composed of the formula (I):

(I) where Ri is H or OH and R is H or OH and the salts, hydrates and solvates of this one.

6. The method of claim 5, wherein the compound of the formula (I) is the, 24-dihydroxy vitamin Di.

7. The method of claim 6, wherein the compound of the formula (I) is the, 24- (R dihidrn -f ^ -. t mine D¡, substantially without its form (S).

8. The method of claim 5, wherein the therapeutic amount is about 1 μg / dose to about 500 μg / dose.

9. A pharmaceutical composition containing a compound of the formula (I): (I) wherein Ri is H or OH and R¿ is H or OH, in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 9, wherein the composition is an oral composition and wherein the compound of the formula (I) is present at a concentration of about 1 μg / a about 100 μg / g of the composition . 11. The composition of claim 10, wherein the ü-a ueüLü Je Pa fórir í * (I) is 1 a,? -dihydroxy vitamin D4. The composition of claim 11, wherein the compound of the formula (I) is the, 24- (R) -dihydroxy vitamin D, substantially without its (S) form. The pharmaceutical composition of claim 9, wherein the composition is a topical composition and wherein the compound of the formula (I) is present in a concentration of about lμg / g to about 100μg / g of the composition. The composition of claim 13, wherein the compound of the formula (I) is the, 24-dihydroxy vitamin D, The composition of claim 14, wherein the compound of the formula (I) is the, 24- (R ) -dihydroxy vitamin D, substantially without its form (S).