WO2007006036A2 - Hydroxylated forms of ergosterols and ergocalciferols, derivatives thereof, methods of production and uses thereof - Google Patents

Abstract

Provided herein is an enzymatic method of producing hydroxylated ergosterols and hydroxylated ergocalciferols using the cytochrome P450scc enzyme and/or the CYP27B1 enzyme and the hydroxylated compounds so produced. Also provided are methods of using the hydroxylated ergosterols and hydroxylated ergocalciferols to treat a tumor or other pathophysiologic condition.

Description

HYDROXYLATED FORMS OF ERGOSTEROLS AND

ERGOCALCIFEROLS, DERIVATIVES THEREOF, METHODS OF

PRODUCTION AND USES THEREFOR

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates generally to the fields of enzymology and secosteroid chemistry and biological activity thereof. More specifically, the present invention relates to an enzymatic production of hydroxy 1 derivatives of ergosterol and ergocalciferol, as well as physicochemical transformation of hydroxy lated ergosterols to corresponding hydroxy lated ergocalciferols, their biological activity and uses therefor.

Description of the Related Art

Cytochrome P450 side chain cleavage (P450scc), product of CYPl IAl locus, is a mitochondrial enzyme whose main function has been purported to be the conversion of cholesterol to pregnenolone. There is a single active site on the cytochrome where successive hydroxylations of the cholesterol side chain occur at positions 22 and 20, followed by cleavage of the side chain to produce pregnenolone and isocapoic aldehyde (1-2). Most recently, 7-dehydrocholesterol has been uncovered as an additional substrate for P450scc, yielding 7- dehydropregnenolone as a final product (3-4). 7-Dehydrocholesterol, besides being a cholesterol precursor, is also a precursor for vitamin D3 through ultraviolet light B photolysis and temperature dependent intramolecular rearrangement (5-6).

Ergosterol, a 5,7-diene sterol, is synthesized by fungi and phytoplankton but not in the animal kingdom (5). Ergosterol serves as a major membrane sterol in fungi (7), and can serve as the precursor for the synthesis of vitamin D2 (5). Ergosterol can act as a membrane antioxidant (8) and a modifier of the effect of cholesterol on human cell cycle progression (9). Antitumor effects of ergosterol have been reported in cell culture (10) and in vivo in rats (11). Anticancerogenic and antimutagenic properties of vitamin D2 (ergocalciferol) are well recognized (5,12) and because of their lower toxicity (minimal hypercalcemic effect), hydroxylated forms of vitamin D2 are considered as potential drugs for treatment of cancer patients (5,13) including melanoma (14). Ergosterol differs from 7-dehydrocholesterol in that its side chain has a

C24-methyl group and C22-C23 double bond. P450scc has been shown to cleave the side chain of other plant sterols, including campesterol, which also has a C24-methyl group (15). Since 7-dehydrocholesterol also serves as a substrate for P450scc (4) and has an identical ring system to ergosterol, cytochrome P450scc may metabolize ergosterol.

The prior art is deficient in the lack of enzymatic production of hydroxylated ergosterol or ergocalciferol derivatives. Specifically, the prior art is deficient in the lack of a cytochrome P450scc enzyme system using ergosterol or ergocalciferol as substrates to produce these compounds and methods of treating neoplastic diseases using the hydroxylated ergosterols or hydroxylated ergocalciferols. The present invention fulfills this long-standing need and desire in the art.

SUMMARY OF THE INVENTION

The present invention is directed to a method of producing an hydroxylated metabolite of ergosterol. The method comprises enzymatically hydroxy lating a substrate of a cytochrome P450scc enzyme system in at least one position, where the substrate, e.g., ergosterol or ergocalciferol is enzymatically convertible to the hydroxylated ergosterol metabolite by the cytochrome P450scc enzyme system. The present invention is directed to a related method of producing hydroxylated ergocalciferols when the substate is ergosterol further comprising thermophotolytically breaking a C9-C10 bond in an hydroxylated ergosterol via UVB radiation and converting the hydroxylated ergosterol to a hydroxylated ergocalciferol via thermal intramolecular rearrangement around the broken bond. The present invention also is directed to another related method of producing a di- or tri- hydroxyergocalfϊcerol from mono- or di-hydroxyergocalciferol precursors via the CYP27B1 enzyme. The present invention also is directed to the hydroxylated ergosterol metabolites and hydroxylated ergocalciferols produced by the en∑ymatic cytochrome P450scc system and/or the CYP27B1 enzyme as described herein. The present invention is directed to a related pharmaceutical composition comprising the hydroxylated ergosterols and hydroxylated ergocalciferols described herein and a pharmaceutically acceptable carrier.

The present invention is directed further to a method of treating in a subject a tumor that has cytochrome P450scc activity. The method comprises administering to the subject an ergosterol for hydroxy lation via the cytochrome P450scc activity of the tumor. An amount of hydroxylated ergosterol so produced is effective to inhibit growth thereof hi the individual thereby treating the tumor.

The present invention is directed further still to a method of inhibiting proliferation of a neoplastic cell. The method comprises administering to the subject a pharmacologically effective amount of a substrate of cytochrome P450scc or a pharmaceutical composition thereof suitable to be enzymatically converted to an hydroxylated metabolite of ergosterol via at least the cytochrome P450scc acitivity of the tumor. The amount of the hydroxylated ergosterol metabolite so produced is effective to inhibit tumor growth in the subject thereby treating the tumor. The present invention is directed to a related method comprising treating a pathophysiologic condition of a cell. The method comprises treating the cell with a pharmacologically effective amount of an hydroxylated ergosterol or hydroxylated ergocalciferol or a pharmaceutical composition thereof.

Other and further aspects, features, benefits, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention are briefly summarized. The above may be better understood by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted; however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

Figures 1A-1D depict the schema for production of 17α,24- dihydroxyergosterol and mono-, di- and trihydroxylated vitamin D2 metabolites. Figure IA is the sequence for the P450scc catalysed transformation of ergosterol showing structures of expected mono- and di-hydroxy reaction products. Figure IB is the sequence for the further photochemical and thermic transformation to 17<x,24- dihydroxy vitamin D2. Figure 1C is the sequence for the P450scc catalysed transformation of vitamin D2 with chemical structures of the reaction products. Figure ID depict the lα-hydroxylated ergocalciferol products of the CYP27B1 catalysed hydroxylation of the precursor mono- and di-hydroxy ergocalciferols.

Figures 2A-2C depict the analysis of products of ergosterol metabolism in vesicle-reconstituted P450scc. Lanes 1 and 4 are controls with all components present except P450scc and lane 5 contains ergosterol (ergo) and pregnenolone (preg) standards (Figure 2A). The arrows labeling OH-ergo and DiOH-ergo indicate the positions of the respective products of ergosterol metabolism, identified by EI mass spectrometry as hydroxyergosterol (Figure 2B) and dihydroxyergosterol (Figure 2C). Figures 3A-3D depict finger print regions in proton NMR spectra of ergosterol COSY (Figure 3A), dihydroxyl metabolite (Figure 3B), ergosterol HSQC methyls (Figure 3C), and dihydroxyl metabolite HSQC methyls (Figure 3D). The corresponding proton ID NMR spectra are shown as projections. The formation of 24-OH is clearly indicated by 1) the missing H23-H24 correlation in the spectrum in Figure 2B with concurrent downfield shift of H22 (5.18 ppm) and H23 (5.22 ppm) to 5.35 ppm, 2) the transition of 28 doublet methyl at 0.92 ppm in the spectrum in Figure 3C to a downfield singlet at 1.35 ppm in the spectrum in Figure 3D.

Figure 3E depicts part of the proton-carbon HSQC spectra of ergosterol standard (left) and its dihydroxyl metabolite (right). The two circled spots in ergosterol HSQC spectrum (left) correspond to correlation between H14 (1.89 ppm) and C14 (54.7 ppm) and H17 (1.27 ppm) and C17 (55.9 ppm). No such spots are visible in those regions for ergosterol metabolite (right spectrum) due to 17- hydroxylation. The shift of HSQC correlation spot between H14 and C14 to new position (2.72 ppm, 58.0 ppm) is caused by interaction of 14a hydrogen with 17α-0H group.

Figure 4 demonstrates the effects of ergosterol and dihydroxyergosterol on the visible absorbance of P450scc. P450scc was incorporated into phospholipids vesicles and spectra recorded against a reference cuvette containing all components except P450scc. Curve 1 is of vesicles containing no substrate, curve 2 is of vesicles containing 0.1 mol cholesterol/mol phospholipids, curve 3 is of vesicles containing both 0.1 mol cholesterol/mol phospholipid and 0.2 mol ergosterol/mol phospholipid; and curve 4 is of vesicles containing both 0.1 mol cholesterol/mol phospholipid and 0.004 mol dihydroxy ergosterol/mol phospholipid.

Figure 5 depicts a double reciprocal plot showing ergosterol binding to P450scc in phospholipid vesicles containing cholesterol. Vesicles contained 0.05 mol cholesterol/mol phospholipid and ergosterol at the indicated ratios. The open symbol on the Y- axis represents the absorbance change expected for complete reversal of cholesterol-induced high spin state back to the substrate-free low spin state. DA₈, absorbance difference (416-392 nm) in the presence of ergosterol; DA₀, absorbance difference (416-392 nm) in the absence of ergosterol with 0.05 mol cholesterol/mol phospholipid present.

Figures 6A-6F show the RP-HPLC identification of a product of ergosterol metabolism by adrenal mitochondria. Figure 6 A is control incubation without NADPH and isocitrate and Figure 6B is experimental incubation with NADPH and isocitrate. The HPLC elution profiles were monitored by absorbance at 265 nm where the number 1 marks as the metabolite and 2 marks as the ergosterol standard. Figure 6C is the UV spectra of reaction product at RT 14.2 min; Figure 6D is the mass spectra of the reaction product at RT 14.2 min, Figure 6E is the UV spectra of ergosterol (RT 49.5 min) and Figure 6F is the mass spectra of ergosterol.

Figures 7A-7C depict the the analysis of products of vitamin D2 metabolism in vesicle-reconstituted P450scc. Reaction products were analyzed by TLC and visualized by charring. Experimental incubation with NADPH (1); control incubation without NADPH (2); pregnenolone (P) and vitamin D2 standards (3). Ml : metabolites 1 and M2: metabolite 2 are marked by arrows (Figures 7A). EI mass spectrometry of metabolite 1 (Figure 7B) and metabolite 2 (Figure 7C) are shown.

Figures 8A-8D depict the NMR spectra of vitamin D2 metabolite 1 identified as 20-hydroxyvitamin D2. Proton-proton COSY of vitamin D2 standard (Figure 8A), COSY of vitamin D2 metabolite 1 (Figure 8B)₅ proton-carbon HSQC of vitamin D2 standard (Figure 8C), and HSQC of vitamin D2 metabolite 1 (Figure 8D). The separation of 22/23 proton signals in metabolite 1 and the lack of scalar coupling between 20-CH and 22-CH at 5.54 ppm (circle in B) clearly indicates hydroxylation at 20-C. The doublet to singlet transition of proton NMR with concurrent downfield shift of the 21-methyl signal (1.01 ppm and 21.2 ppm to 1.3 ppm and 29.5 ppm) confirms hydroxylation at the 20 position. Impurities in the methyl regions are likely from TLC purification process

Figures 9A-9D depict the NMR spectra of vitamin D2 metabolite 2 identified as 17α,20-dihydroxy vitamin D2. (Figure 9A) proton spectra of metabolite 2; (Figure 9B) proton spectra of vitamin D2; (Figure 9C) COSY of metabolite 2; (Figure 9D) HSQC of methyl regions of metabolite 2. Numbers in Figure 9B indicate proton positions in the vitamin D2 standard. In metabolite 2, the 20-hydroxyl is clearly present and there are no other changes in the side chain as indicated by COSY and HSQC. The large downfield shift of 14-CH from 1.99ρρm to 2.68ρpm with disappearance of the 17-CH signal at 1.32 ppm indicates that hydroxylation has occurred at the 17-C position. Figures 10 A-IOC show the RP-HPLC separation of products of vitamin D2 metabolism by adrenal mitochondria. Incubation of mitochondria in the absence of NADPH and isocitrate (Figure 10A), experimental incubation with NADPH and isocitrate (Figure 10B) and experimental incubation with 200 μM aminoglutethimide (Figure 10C) are shown. The HPLC elution profile was monitored by absorbance at 265 nni. Novel vitamin D2 metabolites are marked 1-6, vitamin D2 is marked 7.

Figure 11 depicts the LC/MS and UV spectra of products of vitamin D2 metabolism in adrenal mitochondria. Products 1, 4, 6, and vitamin D2 - 7. Left panel: UV spectra; right panel: [M+l]⁺.

Figures 12A-12B demonstrate HaCaT keratinocytes (Figure 12A) and SKMEL-188 melanoma cells (Figure 12B) were synchronized and then incubated in Ham's FlO medium containing serum and serial concentrations of dihydroxyergosterol for 24 h. Data is presented as mean ± SEM (n=8). *p<0.05 between control and treatment with dihydroxyergosterol. #p<0.05 between treatment with the solvent (EtOH, 0.2%) and 10^'6 M ergosterol. Solvent at 0.2% concentration did not have an effect on DNA synthesis. Figure 13 demonstrates that dihydroxyergosterol inhibits DNA synthesis in dermal fibroblasts. Fibroblasts were incubated in Ham's FlO medium containing serum and serial concentrations of dihydroxyergosterol for 24 h. Data is presented as mean ± SEM (n=8). *p<0.05 between control and treatment with dihydroxyergosterol.

Figures 14A-14B demonstrate that metabolites of vitamin D2 inhibit DNA synthesis and stimulate differentiation in human HaCaT keratinocytes. HaCaT keratinocytes were synchronized and incubated for 24 h in Ham's FlO medium containing serum and vitamin D2 or its metabolites and [³H]-thymidine (Figure 14A). HaCaT keratinocytes were transfected with a construct containing the involucrin promoter (IVL-Luc) or with empty (promoter-free) construct, synchronized and incubated for 24 h in Ham's FlO medium containing serum and, vitamin D2 or its metabolites (Figure 14B). Data shown as mean ± SEM (n=3-8).

Figures 15A-15B demonstrate that metabolites of vitamin D2 inhibit DNA synthesis in human melanoma cells. Cells were synchronized and then incubated for 24 h in Ham's FlO medium without serum and vitamin D2 metabolite 1, 20-hydroxyvitamin D2, (Figure 15A) or vitamin D2 metaboite 2, 17,20- dihydroxyvitamin D2 (Figure 15B) and [3H]-thymidine (1 μCi/ml). Data presented as mean ± SEM (n=8) and *p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the present invention provides an hydroxylated metabolite of ergosterol, comprising hydroxylating a substrate of a cytochrome P450scc enzyme system in at least one position, where the substrate is enzymatically convertible to the hydroxylated ergosterol metabolite. In one aspect of this embodiment the substrate may be ergosterol. In this aspect P450scc may hydroxylate a Cl 7 sidechain of ergosterol. For example, the Cl 7 sidechain may be hydroxylated at least at position C24 within the Cl 7 chain. An example of such a hydroxylated ergosterol is 24-hydroxy ergosterol. Also, the C17 sidechain may be hydroxylated at Cl 7 and C24. An example of such a hydroxylated ergosterol is 17α,24- dihydroxyergosterol. In addition the Cl 7 sidechain may be hydroxylated in at least at position C20 within the Cl 7 chain. An example of such a hydroxylated ergosterol is 20-hydroxyergosterol. Furthermore, the Cl 7 sidechain may be hydroxylated at C20 and C24. An example of such a hydroxylated ergosterol is 20,24- dihydroxyergosterol.

Further to this aspect, the method may comprise thermophotolytically breaking a C9-C10 bond in the hydroxylated ergosterol via UVB radiation and converting the hydroxylated ergosterol to a hydroxylated ergocalciferol via thermal intramolecular rearrangement around the broken bond. Examples of the hydroxylated ergocalciferols may be 17-hydroxyergocalciferol, 20-hydroxyergocalciferol, 24- hydroxyergocalciferol, 17α,24-dihydroxy ergocalciferol, or 20,24- dihydroxyergocalciferol. Further still to this aspect, the method may comprise hydroxy lating position Cl of the A ring of said hydroxylated ergocalciferol with a CYP27B1 enzyme.

In another aspect of this embodiment the substrate may be ergocalciferol. In this aspect P450scc may hydroxylate a Cl 7 sidechain of ergocalciferol. For example, the Cl 7 sidechain may be hydroxylated at least at position C20 within the Cl 7 chain. An example of an hydroxylated ergocalciferol is 20-hydroxyergocalciferol or 17α,20-dihydroxyergocalciferol. Further to this aspect, the method may comprise hydroxy lating position Cl of the A ring of said hydroxylated ergocalciferol with a CYP27B1 enzyme. In all aspects of this embodiment the cytochrome P450scc enzyme system may be an in vitro system, comprising cytochrome P450scc enzyme; adrenodoxin; adrenodoxin reductase; and NADPH. Furthermore, the in vitro system may comprise a phospholipid vesicle having these components of the enzyme system and the substrate encapsulated therein. Also in all aspects the cytochrome P450scc enzyme system may comprise a eukaryotic cell or a prokaryotic cell. The eukaryotic cell may be a mammalian cell. Examples of a mammalian cell may be an adrenal cell, a gonadal cell, a placental cell, a cell from the gastrointestinal tract, a kidney cell, a brain cell, or a skin cell. Furthermore the mammalian cell may be in vitro or in vivo. The prokaryotic cell may be a yeast cell or a bacterial cell. In these aspects the cytochrome P450scc enzyme system may be a recombinant system in said cell.

In a related embodiment there is provided an hydroxylated ergosterol metabolite produced by the cyctochrome P450scc enzyme system described supra or a pharmaceutical composition thereof. In an aspect of these embodiments the hydroxylated ergosterol metabolite may be 20-hydroxyergosterol, 24- hydroxyergosterol, 17α,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol., 17- hydroxyergocalciferol, 20-hydroxyergocalciferol, 24-hydroxyergocalciferol, 17α,20- dihydroxyergocalciferol, 17α,24-dihydroxyergocalciferol, or 20,24- dihydroxyergocalciferol. In another related embodiment there is provided a di- or tri- hydroxylated ergocalciferol produced by the CYP27B1 enzyme. Examples of these hydroxy lated ergocalciferols are lcc,17α-dihydroxy ergocalciferol, lα,20- dihydroxy ergocalciferol, lα,17α,20-trihydroxyergocalciferol or lα,17α,24- trihydroxyergocalciferol. In another embodiment of the present invention, there is provided a method of treating in a subject a tumor that has cytochrome P450scc activity, administering to the subject a pharmacologically effective amount of a substrate of cytochrome P450scc or a pharmaceutical composition thereof suitable to enzymatically convert to an hydroxylated metabolite of ergosterol via at least the cytochrome P450scc activity of the tumor, wherein an amount of an hydroxylated ergosterol metabolite so produced is effective to inhibit tumor growth in the subject thereby treating the tumor. In all aspects of this embodiment the hydroxylated ergosterol metabolite may be 20-hydroxyergosterol, 24-hydroxyergosterol 17α,24- dihydroxyergosterol, 20,24-dihydroxyergosterol, 20-hydroxyergocalciferol or 17α,20-dihydroxy ergocalciferol. Representative examples of tumors which may be treated include adrenal tumors, a gonadal tumor, a tumor of the gastrointestinal tract, a kidney tumor, a brain tumor a melanoma, or other skin tumor.

In yet another embodiment of the present invention, there is provided a method of of treating a pathophysiologic condition of a cell, comprising treating said cell with a pharmacologically effective amount of an hydroxylated ergosterol or hydroxylated ergocalciferol or a pharmaceutical composition thereof. The hydroxylated ergosterol may be 20-hydroxyergosterol, 24-hydroxy ergosterol, 17α,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol. The hydroxylated ergocalciferol may be 17-hydroxy ergocalciferol, 20-hydroxyergocalciferol, 24- hydroxyergocalciferol, 17α,20-dihydroxy ergocalciferol, 20,24- dihydroxy ergocalciferol, lα,17α-dihydroxy ergocalciferol, lα,17α,24- trihydroxyergocalciferol, lα,20-dihydroxy ergocalciferol or lα,17α,20-trihydroxy ergocalciferol. In this embodiment the cell may be a neoplastic cell. Examples of a neoplastic cell may be an adrenal cell, a gonadal cell, a pancreatic cell, a cell from the gastrointestinal tract, a prostate cell, a breast cell, a lung cell, an immune cell, a hematologic cell, a kidney cell, a brain cell, a cell of neural crest origin, or a skin cell. In one aspect the pathophysiologic condition may be a melanoma, a carcinoma, a sarcoma, a leukemia, or a lymphoma. In another aspect the pathophysiologic condition may be a skin disorder. Particularly, the skin disorder may be a hyperproliferative skin disorder, a pigmentary skin disorder, an inflammatory skin disorder, or other skin disorder characterized by hair growth on legs, arms, torso, or face, or induced by exposure to solar radiation.

As used herein, the term, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein "another" or "other" may mean at least a second or more of the same or different claim element or components thereof.

As used herein, the term "neoplastic cell" or refers to a cell or a mass of cells or tissue comprising the neoplastic cells characterized by, inter alia, abnormal cell proliferation. The abnormal cell proliferation results in growth of these cells that exceeds and is uncoordinated with that of the normal cells and persists in the same excessive manner after the stimuli which evoked the change ceases or is removed. Neoplastic cells or tissues comprising the neoplastic cells show a lack of structural organization and coordination relative to normal tissues or cells which usually results in a mass of tissues or cells which can be either benign or malignant. As would be apparent to one of ordinary skill in the art, the term "tumor" refers to a mass of malignant neoplastic cells or a malignant tissue comprising the same.

As used herein, the term "treating" or the phrase "treating a tumor" or "treating a neoplastic cell" or "treating a neoplasm" includes, but is not limited to, halting the growth of the neoplastic cell or tumor, killing the neoplastic cell or tumor, or reducing the number of neoplastic cells or the size of the tumor. Halting the growth refers to halting any increase in the size or the number of neoplastic cells or tumor or to halting the division of the neoplastic cells. Reducing the size refers to reducing the size of the tumor or the number of or size of the neoplastic cells.

As used herein, the term "subject" refers to any target of the treatment.

Provided herein are methods of metabolizing a substrate of the cytochrome P450scc enzyme system to produce hydroxylated metabolites.

Particularly, ergosterol or ergocalciferol, i.e., vitamin D2, as individual substrates, are converted by the cytochrome P450scc enzyme system to hydroxy lated metabolites thereof. The structural features of the ergosterol side chain, e.g., the presence of the C22-C23 double bond, prevent its cleavage by P450scc leading to a hydroxylation of the side chain at C24 and/or C17. Preferably, the P450 enzyme hydroxylates ergosterol first at the C24 position to form an intermediate 24-hydroxyergosterol with subsequent hyrdroxylation at C 17. Additionally, the Cl 7 side chain may be hydroxylated at the C20 position or at both the C20 and C24 positions.

Furthermore, ergosterol is a precursor to ergocalciferol, i.e., vitamin D2. It is contemplated that dihydroxylated vitamin D2 derivatives may be produced from the hydroxylated ergosterols via photochemical and thermic transformation of the C9-C10 bond. Particularly, 17-hydrox ergocalciferol, 20-hydroxy ergocalciferol, 24-hydroxyergocalciferol, 17α,24-dihydroxyergocalciferol, or 20,24- dihydroxyergocalciferol is produced from the photochemical and thermic transformation of 17α,24-dihydroxyergosterol. Alternatively, both 20- dihydroxyergocalciferol and 17α,20-dihydroxyergocalciferol may be produced directly from vitamin D2 as substrate of a purified, reconstituted cytochrome P450scc enzyme system. In addition, it is contemplated that the hydroxylated ergosterols and hydroxylated ergocalciferols of the present invention may be produced using chemical synthetic methods known and standard in the art. The methods of producing hydroxylated ergosterols or hydroxylated ergocalciferols may be utilized in vitro or in vivo. The cytochrome P450scc eiLzyme system may be a reconstituted and purified in vitro system comprising cytochrome P450scc enzyme, adrenodoxin, adrenodoxin reductase, and NADPH. A phospholipid vesicle may be used to encapsulate the P450 enzyme system and the substrate ergosterol. The cytochrome P450scc enzyme system may comprise a eukaryotic cell or a prokaryotic cell, for example, but not limited to a vertebrate cell, an invertebrate cell, a yeast cell or a bacterial cell. Preferably the vertebrate cell is a mammalian cell. The mammalian cell may be in vitro or in vivo. Mammalian cells having the ability to express P450 are, but not limited to, an adrenal cell, a gonadal cell, a placental cell, a cell from the gastrointestinal tract, a kidney cell, a brain cell, or a skin cell.

Alternatively, the cytochrome P450scc enzyme system may be a recombinant system introduced into a cell using well-known and standard molecular biological techniques. It is contemplated that these recombinant systems are suitable for the large scale production of the hydroxylated ergosterols and hydroxylated ergocalciferols presented herein.

Thus, the present invention provides an hydroxylated ergosterol and hydroxylated ergocalciferols enzymatically produced by the cytochrome P450scc system. Also provided are pharmaceutical compositions comprising the hydroxylated ergosterols and a pharmaceutically acceptable carrier. It is particularly contemplated that the hydroxylated ergosterol is a dihydroxyergosterol hydroxylated in the Cl 7 side chain of ergosterol, preferably, 20-hydroxyergosterol, 24-hydroxyergosterol, 17α,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol. Furthermore, the present invention provides hydroxylated ergocalciferols derivatized from ergosterol and hydroxylated ergosterols, particularly 17-hydroxyergocalciferol, 20- hydroxyergocalciferol, 24-hydroxyergocalciferol, 17ct,24- hydroxy ergocalciferol, or 20,24-hydroxyergocalciferol. In addition, the present invention provides the hydroxylated ergocalciferols 20-hydroxyergocalciferol and 17cx,20- hydroxyergocalciferol, including pharmaceutical compositions thereof comprising a pharmaceutically acceptable carrier.

The methods of hydroxylating ergosterol or ergocalciferol are useful in treating a tumor that has cytochrome P450 activity. The cytochrome P450 comprising the tumor cells would, upon administration of ergosterol or ergocalciferol or a pharmaceutical composition thereof to the tumor, hydroxylate the ergosterol or ergocalciferol as described herein. Production of a sufficient or effective amount of a dihydroxyergosterol, e.g., 17α,24-dihydroxyergosterol or 20,24-dihydroxyergosterol, or of a mono- or dihydroxy ergocalciferol, e.g., 20-hydroxyergocalciferol and 17α,20-hydroxyergocalciferol, by the tumor cells, would inhibit tumor growth. Alternatively, a monohydroxylated ergosterol, e.g., 20-hydroxyergosterol or 24- hydroxyergosterol, administered to the subject would exhibit the same effect, as these compounds are produced by the cytochrome P450 enzyme system.

It is contemplated that the mono- and di-hydroxy products of the cytochrome P450 enzyme system may be hydroxylated at the lα-position by lα- hydroxylase enzyme CYP27B1. For example, 17α-hydroxyergocalciferol, 20- hydroxyergocalciferol, 17α,20-dihydroxyergocalciferol, or 17α,24- dihydroxyergocalciferol, are hydroxylated to form lα,17α-dihydroxyergocalciferol, lcx,20-dihydroxy ergocalciferol, lα,17α,20-trihydroxy ergocalciferol, or lα,17α,24- trihydroxyergocalciferol. Hydroxylation may occur in the skin by topical administration of the precursor(s) expressing CYP27B1 or its homolog, in cultured cells or microorganisms, either in a native state or after transfection by a genetic construct expressing CYP27B1, or in a reconstituted in vitro enzymatic system hydroxy lating the precursor A ring at position 1. Alternatively, the trihydroxylated ergocalciferols may be chemically synthesized. Thus, the present invention also provides trihydroxylated ergocalciferols described herein or pharmaceutical compositions thereof.

It is contemplated that production of mono- or dihydroxyergosterols from ergosterol or mono- or dihydroxyergocalciferols from ergocalciferol occurs in organs expressing cytochrome P450scc, such as, but not limited to, adrenals, gonads, placenta, brain, gastrointestinal tract, kidney, and skin. It also is contemplated that neoplastic cells transformed from these cells would maintain cytochrome P450scc activity. For example, melanoma cells express P450scc. It is further contemplated that production of lcc-di- and trihydroxy ergocalciferols from corresponding mono- and dihydroxy substrates occurs in organs expressing CYP27B1, such as, but not limited to kidney cells and other peripheral tissues, e.g., skin.

More particularly, the hydroxylated ergosterols or hydroxylated ergocalciferols thereof may be used to treat a pathophysiologic condition characterized by, but not limited to, uncontrolled proliferation of a cell such as a neoplastic cell or a cell comprising a skin disorder. The neoplastic cell may be malignant or benign. For example, it is contemplated that the antiproliferative action against HaCaT keratinocytes and human melanoma cells, which are epithelial cells, demonstrated herein is indicative of an antiproliferative action against neoplastic cells comprising the epithelium, a breast, the genitourinary tract, the respiratory tract, the prostate, the endocrine system, the musculoskeletal and connective tissue systems, the vascular system, the hematologic system, the nervous system, the skin, or the immune system. These cells may be adrenal cell, a gonadal cell, a pancreatic cell, a cell from the gastrointestinal tract, a prostate cell, a breast cell, a lung cell, an immune cell, a hematologic cell, a kidney cell, a brain cell, a cell of neural crest origin, or a skin cell. Also, it is contemplated that the antiproliferative action against fibroblasts demonstrated herein is indicative of an action against neoplastic cells comprising a melanoma, a sarcoma, a leukemia, or a lymphoma. The melanoma may be a melanocytic tumor or a melanoma of the skin, the eye or of an undetermined primary site. The sarcoma may be fibrosarcoma, dermatofibrosarcoma protuberans, liposarcoma, osteosarcoma, angioarcoma, or Kaposi sarcoma.

Furthermore, it is contemplated that the antiproliferative action against keratinocytes and fibroblasts is indicative that the cell may comprise a skin disorder, such as, but not limited to, a hyperproliferative skin disorder, a pigmentary skin disorder, an inflammatory skin disorder, or other skin disorder. A hyperproliferative skin disorder may be psoriasis, seborrheic keratosis, actinic keratosis, benign adnexal tumor, fribromatosis, or keloids. A pigmentary skin disorder may be vitiligo, solar lentigo, lentigo simplex, hypermelanosis, or dysplastic melanocytic nevus. An inflammatory skin disorder may be allergic contact dermatitis, mummular dermatitis, atopic dermatitis, irritant contact dermatitis, or seborrheic dermatitis.

Other skin disorders may be alopecia of the scalp or a disorder encompassing overproduction of hair on the legs, arms, torso or face. In addition, a skin disorder may be induce by exposure to solar radiation. For example, aging of the skin is caused by this exposure. It is contemplated that the action of the hydroxy lated ergosterols or hydroxylated ergocalciferols may be useful in controlling, attenuating or preventing aging of the skin.

The hydroxylated ergosterols or hydroxylated ergocalciferols provided herein may be used to treat a subject, preferably a mammal, more preferably a human, having the pathophysiological condition characterized by the presence of neoplastic cells, such as comprising, but not limited, to a malignant or benign tumor, or a phathophysiological condition comprising a skin disorder. Administration of the hydroxylated ergosterols or hydroxylated vitamin D2 derivatives or pharmaceutical compositions thereof is effective to inhibit proliferation of a neoplastic cell or to treat a disorder such as a skin disorder. The hydroxylated ergosterols, hydroxylated ergocalciferols or pharmaceutical compositions thereof can be administered by any method standard in the art and suitable for administration to the subject.

Dosage formulations of these hydroxylated ergosterols or hydroxylated ergocalciferols may comprise conventional non-toxic, physiologically or pharmaceutically acceptable carriers or vehicles suitable for the method of administration. The hydroxylated ergosterols, hydroxylated ergocalciferols or pharmaceutical compositions thereof may be administered independently one or more times to achieve, maintain or improve upon a pharmacologic or therapeutic effect. It is well within the skill of an artisan to determine dosage or whether a suitable dosage comprises a single administered dose or multiple administered doses. An appropriate dosage depends on the subject's health, the progression or remission of the disease or disorder, the route of administration and the formulation used.

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.

EXAMPLE 1

Metabolism of ergosterol and of vitamin D2 by reconstituted P450scc Bovine cytochrome P450scc and adrenododoxin reductase were isolated from adrenals (17-18). Adrenodoxin was expressed in E. coli and purified as described before (33). The reation mixture comprised 510 μM phospholipid vesicles (dioleoyl phosphatidylcholine plus 15 mol% cardiolipin), prepared by sonication (34), with a substrate to phospholipid molar ratio of 0.2. For ergosterol, the reaction mixture further comprised 50 μM NADPH, 2 mM glucose 6-phosphate, 2 U/ml glucose 6-phosphate dehydrogenase, 0.2 μM adrenodoxin reductase, 10 μM adrenodoxin, 2.0 μM cytochrome P450scc and buffer pH 7.4. For vitamin D2, the reaction mixture further comprised 50 μM NADPH, 2 mM glucose 6-phosphate, 2 U/ml glucose 6-phosphate dehydrogenase, 0.3 μM adrenodoxin reductase, 6.5 μM adrenodoxin, 3.0 μM cytochrome P450scc and buffer pH 7.4. After incubation at 35⁰C for 3 h the mixture was extracted with methylene chloride and dried under nitrogen. After incubation at 35⁰C for 3 h the mixture was extracted with methylene chloride and dried under nitrogen.

Products were analyzed and purified by preparative thin layer chromatography on silica gel G with three developments in hexane:ethyl acetate (3:1, v/v). Products in selected lanes were visualized by charring. For nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses they were eluted from the silica gel with chloroform:methanol (1:1, v/v); dried separately under nitrogen and shipped on dry ice. Corresponding products from ergosterol metabolism in other lanes were eluted from the silica gel as before and the amount of products determined from their UV spectrum using an extinction coefficient of 9900 M^-1Cm^"1 at 281 nm, determined for the 5,7 diene structure of ergosterol. Metabolism of ergosterol and vitamin D2 bv adrenal mitochondria

Adrenals were obtained from male Wistar rats aged 3 months, terminated under anesthesia. The animals were housed at the vivarium of the

Department of Biotestings of Bioorganic Chemistry Institute (Minsk, Belarus). The experiments were approved by the Belarus University Animal Care and Use

Committee.

The adrenal mitochondrial fraction was prepared as described previously. The washed mitochondrial fraction was resuspended in 0.25 M sucrose and used for enzymatic reactions as described (17-18). Briefly, isolated mitochondria prepared from the adrenals were preincubated (10 min at 37 C) with the sterols ergosterol or 7-dehydrocholesterol (20 mM) or with vitamin D2 (100 mM) dissolved in 45% 2-hydroxypropyl-cyclodextrin [4] in buffer comprising 0.25 M sucrose, 50 mM HEPES pH 7.4, 20 mM KCl, 5 mM MgSO₄, and 0.2 mM EDTA. The reactions were started by adding NADPH (0.5 mM) and isocitrate (5 mM) to the samples and after 90 min mixtures were extracted with methylene chloride and the organic layers combined and dried. The residues were dissolved in methanol and subjected to liquid chromatography mass spectrometry (LC/MS) analysis as detailed below.

Measurement of visible absorbance spectra for P450scc and ergosterol binding Vesicles were prepared from phosphatidylcholine and bovine heart cardiolipin using a bath-type sonicator in buffer comprising 20 mM Hepes pH 7.4,

100 mM NaCl₅ 0.1 mM dithiothreitol and 0.1 mM EDTA, as before (34). The cardiolipin content was 15 mol% of the total phospholipid. Ergosterol, dihydroxyergosterol and/or cholesterol were included in the vesicles as required. Purified P450scc (1.0 μM) was incorporated into the vesicles (400 μM phospholipid) by incubation at room temperature for 30 min in a final volume of 0.7 ml (34).

Spectra were recorded between 350 and 500 nm against a reference cuvette containing all components except P450scc. The K_d for cholesterol was determined by titrating the absorbance change between 316 and 412 nm with cholesterol, as before (19). The Kd for ergosterol was determined from its ability to reverse the absorbance change induced by cholesterol using competitive binding analysis:

Kd = Kd_{> ap}p /(1 + P]ZKi]), where

Kd₁ ap_P is the apparent Kd for ergosterol in the presence of cholesterol, I is the cholesterol concentration and Ki is the Kd for cholesterol. This method has been used previously to determine Kd values for 20a-hydroxycholesterol and 22R- hydroxycholesterol binding to P450scc (19).

Mass spectrometry Products of ergosterol or vitamin D2 metabolism by purified P450scc were eluted from TLC plates, dissolved in ethanol and electron impact (EI) mass spectra recorded with a Micromass VG Autospec Mass Spectrometer operating at 70 eV with scanning from 800 to 50 at 1 sec/decade.

The products of mitochondrial transformation were dissolved in methanol and analysed on a high performance liquid chromatography mass spectrometer, LCMS-QP8000α, (Shimadzu, Japan) equipped with a Restec Allure Cl 8 column (150 x 4.6 mm; 5 mm particle size; and 60 A pore size), UV /VIS photodiode array detector (SPD-Ml OAvp) and quadrupole mass spectrometer. The LC-MS workstation Class-8000 software was used for system control and data acquisition (Shimadzu, Japan).

Elution was carried out at 40⁰C with a flow rate of 0.75 ml/min. The mobile phases consisted of 85% methanol and 0.1% acetic acid from 0 to 25 min, followed by linear gradient to 100% methanol and 0.1% acetic acid from 25 to 35 min; and 100% methanol and 0.1% acetic acid from 35 to 55 min. The MS operated in APCI (atmospheric pressure chemical ionization) positive ion mode and nitrogen was used as the nebulizing gas. The MS parameters were as follows: the nebulizer gas flow rate was 2.5 1/min; probe high voltage was 3.5 kV for dihydroxy ergosterol or 4.5 kV for vitamin D2 metabolites, probe temperature was 300 C for dihydroxy ergosterol or 250 C for vitamin D2 metabolites, and the CDL (curved desolvation line) heater temperature was 250 C for dihydroxyergosterol or 230 C for vitamin D2 metabolites. Analyses were carried out in the scan mode from m/z 320 to 450 for dihydroxyergosterol or m/z 370 to 430 for vitamin D2 metabolites or, for any, in SIM mode at the expected m/z of the standards.

Nuclear magnetic resonance (NMR)

Samples of the TLC purified dihydroxyergosterol or hydroxylated ergosterol metabolites, the mass of the compounds was confirmed by MS, were dissolved in "100% D" CDCl₃ (Cambridge Isotope Laboratories, Inc., Andover, MA) and NMR spectra were acquired using a Varian Inova-500 M NMR equipped with a 4 mm inverse gHX Nanoprobe (Varian NMR, Inc., Palo Alto, CA). The total volume in the NMR rotor was 40 μl, and all spectra were acquired at a temperature of 294 K with a spinning rate of 2500 Hz. Proton ID NMR, proton correlation spectroscopy (COSY) and proton-carbon correlation spectroscopy (HSQC) were acquired and processed with standard parameters. Possible positions of the hydroxyl groups in the metabolite were analyzed by comparing the acquired spectra with those of parent ergosterol or of parent vitamin D2.

Because of the impurities in TLC purified dihydroxyergosterol

(impairing assignment of one of the hydroxyl groups, as discussed below), it was purified by RP-HPLC through Allure Cl 8 column (150 x 4.6 mm; 3 mm particle size;

Restek Corporation, Bellefonte, CA) following the procedure described for LC-MS, and then analyzed by NMR.

Cell lines HaCaT keratinocytes were grown in DMEM medium with 5% FBS and 1% antibiotic solution. SKMEL- 188 melanoma cells were grown in Ham's FlO medium with 5% FBS and 1% antibiotic solution. Dermal fibroblasts were grown in DMEM medium with 5% FBS, insulin (5μg/ml) and 1% antibiotic solution. Dermal fibroblasts were grown in DMEM medium with 5% FBS, insulin (5μg/ml) and 1% antibiotic solution.

DNA synthesis

Cells were seeded 5,000 per well into 96-well plates in growth medium. After 6 h medium was discarded and serum free Ham's FlO medium was added. After 12 h this medium was changed to 5% FBS Ham's FlO medium containing compounds to be tested at 10^"10 and 10^"12 M. After 12 h medium was discarded and replaced with 5% FBS Ham's medium containing test compounds and [³H] -thymidine (1 μCi/ml), and incubated for an additional 12 h. After treatment media were discarded, cells detached with trypsin, harvested on glass fiber filter and radioactivity proportional to methyl-[³H]thymidine incorporated into DNA was counted with a Packard direct beta counter (Packard, Meriden, CA). Reporter gene assay

The effect of vitamin D2 metabolites on the transcriptional activity of the involucrin promoter was assessed with a reporter gene assay. Vitamin D2 metabolites produced by purified P450scc and isolated by TLC, were further purified by RP-HPLC through a Restec Allure Cl 8 column (150 x 4.6 mm; 5 mm particle size; and 60 A pore size) following the procedure described for LC-MS above. Vitamin D2 and its metabolites were dissolved in cyclodextrin, as described (37). Cells were seeded 20,000/well in 24-well plates in growth medium. After 6 h cells were transfected using transfection reagents (sc-29528 and sc-36868) from Santa Cruz Biotechnology Inc., Santa Cruz, CA in serum free FlO medium with firefly luciferase reporter gene plasmid IVL-Luc containing the involucrin gene promoter region (-668 bp to +34 bp; added at 1 μg/well) and with phRL-TK which expresses Renilla luciferase and serves as normalization control (Promega, Madison, WI) added at 1 μg/well. IVL-Luc and p-Luc (control without promoter, empty vector) plasmids were constructed as described previously (38).

Twelve hours after transfection the medium was changed to 5% FBS Ham's FlO medium containing vitamin D2 and its hydroxy-derivatives. Compounds were added again after 12 h. After another 12 h, the entire incubation with compounds lasted 24 h, cells were lysed with passive lysis buffer and luciferase and Renilla luciferase signals were recorded after sequential addition of Luciferase Assay Reagent II and Stop-Glo Reagent (Promega, Madison, WI) using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). After subtraction of background, the specific signal was divided by the Renilla signal. Resulting values were divided by the mean value for controls, i.e., cells transfected with IVL-Luc construct and incubated without compounds. Data are presented as mean ± SEM (n=3-8) and analysed with Student's t-test. Each experiment was performed independently 2 times.

EXAMPLE 2 Metabolites of P450scc catalyzed ergosterol and vitamin D2 transformation

P450scc transforms ergosterol to 17a,24-dihydroxyl ergosterol with 24 hydroxyergosterol serving as an intermediate of the metabolism (Fig. IA). Also 17α,24-dihydroxyergosterol may be converted to 17α,24-dihydroxy vitamin D2 through photochemical and thermic transformation (Fig. IB). Ultraviolet radiation B (UV-B) energy converts 17α,24-dihydroxyergosterol into 17α,24-dihydroxy previtamin D2. Thermal energy at 37 °C converts 17α,24-dihydroxy previtamin D2 into 17α,24-dihydroxy vitamin D2. Alternatively, P450scc transforms vitamin D2 directly to 20-hydroxyergocalciferol (20-hydroxy vitamin D2) or 17cc,20- dihydroxyergocalciferol (17α,20-dihydroxy vitamin D2) (Fig. 1C). Alphal- hydroxylase (CYP27B1) converts 17α-dihydroxyergocalciferol, 17α,24- dihydroxyergocalciferol, 20-dihydroxyergocalciferol or 17cc,20- dihydroxyergocalciferol to lα,17α-dihydroxyergocalciferol, lα,17α,24- trihydroxyergocalciferol, lα,20-dihydroxyergocalciferol and lα,17α,20-trihydroxy ergocalciferol (Fig. ID).

EXAMPLE 3

Characterization of ergosterol metabolites

TLC and MS characteristics of ergosterol metabolites of purified P450scc Incubation of both human and bovine P450scc with ergosterol in a phospholipid- vesicle reconstituted system resulted in one major product and one minor product being observed upon analysis by TLC (Fig. 2A). Both products were absent in the control incubations where P450scc was omitted. The products, eluted from the TLC plate, had UV absorbance spectra identical to that for ergosterol (not shown). A small spot corresponding to pregnenolone (identity confirmed by mass spectra, not shown) was observed in the test sample for bovine P450scc (Fig. 2 A, lane 3). This is produced from a small quantity of cholesterol which co-purifies with P450scc extracted from adrenal glands (16).

An electron impact mass spectrum of the major product identified it as dihydroxyergosterol (Fig. 2C). It showed the molecular ion at m/z = 428, with fragment ions at m/z = 410 (428 - H₂O), m/z = 395 (410 - CH₃). In comparison ergosterol has m/z = 396. These results show that the major action of P450scc on ergosterol is to add two hydroxyl groups. The minor product was identified as hydroxyergosterol (Fig. IB). The molecular ion had m/z = 412 with fragment ions m/z = 394 (412 - H₂O), m/z = 379 (394 - CH₃) and m/z = 361 (379 - H₂O). In contrast to cholesterol and 7-dehydrocholesterol, the incubation of ergosterol with P450scc did not yield products indicative of cleavage of the sterol side chain. NMR analysis of the dihydroxyergosterol

The NMR analysis of TLC purified dihydroxyergosterol demonstrates that one OH group is located at C24 (Figs. 3A-3D). Although the presences of impurities in the methyl region as well as the expected hydroxyl region around 3-4.5 ppm prevent a definitive analysis, the regions of the protons on the double bonds are relatively clean. Both the chemical shifts and coupling patterns of H6 and H7 are identical in ergosterol and this metabolite. However, H22 and H23 in the side chain are downshifted from 5.18 ppm and 5.22 ppm to 5.35 ppm. In addition, the correlation between H23 and H24 (chemical shift 1.86 ppm in ergosterol) is missing while that of H22 to H20 (chemical shift 2.04 ppm in ergosterol) is still present in this metabolite. This is a strong indication that H24 becomes hydroxylated in this metabolite. In addition, the doublet of 28-methyl protons in ergosterol (0.92 ppm for proton and 17.7 ppm for carbon) became a singlet and shifted downfϊeld (1.3 ppm for proton and 24 ppm for carbon). This again clearly defines the hydroxylation at C24. The presence of the impurities makes definitive assignment of the second hydroxyl location very difficult. Although further HPLC purification of the ergosterol metabolite (RT of 13.7) removed the impurities, its amount was insufficient. Nevertheless, the comparison of ergosterol and dihydroxyergosterol HSQC spectra (Fig. 3E) strongly indicates that second hydroxyl group is located at the C17.

In Figure 3E, the two circled spots in ergosterol HSQC spectrum (left) correspond to correlation between 14-CH (1.89 ppm for proton and 54.7 ppm for carbon) and 17-CH (1.27 ppm for proton and 55.9 ppm for carbon). No such spots are visible in those regions for the dihydroxyl metabolite (right spectrum). The correlation of 14-CH in this metabolite shifted downfield to a new position (2.72 ppm for proton and 58.0 ppm for carbon) as indicated in the circle in the right spectrum, while the original 17-CH correlation disappeared. The shift of the 14-CH is caused by the formation of 17-OH in this metabolite. Hence, this dihydroxyl metabolite is most likely 17α,24-dihydroxyergosterol.

The rate of ergosterol metabolism by purified P450scc

To obtain an estimate of the initial rate of ergosterol metabolism by P450scc, ergosterol at a molar ratio to phospholipid of 0.2, was incubated with P450scc for 5 min at 35⁰C. The dihydroxyergosterol was extracted, purified by TLC and quantitated from its absorbance at 281 nm. This gave a rate of metabolism of 0.7 mol product /min/mol P450scc. Under similar conditions, cholesterol was converted to pregnenolone at a rate of 17.7 mol/min/mol P450scc (4).

The effect of ergosterol and dihvdroxyergosterol on the spin state of cytochrome

Cytochrome P450scc incorporated into phospholipid vesicles prepared from dioleoyl phosphatidylcholine and cardiolipin displays a typical low spin spectrum of the substrate free enzyme (Fig. 4, spectrum 1) with maximum absorbance at 416 nm (17-18). The inclusion of ergosterol in the vesicles at a molar ratio to phospholipid of 0.2 did not alter the spectrum (not shown). In contrast, the presence of cholesterol at a molar ratio to phospholipid of 0.1 caused a transition to the high spin state with maximum absorbance at 392 nm (Fig. 4, spectrum 2). The magnitude of the change in the spin state provides an index of substrate binding and we have previously used this to determine the K_d for cholesterol and hydroxycholesterol reaction intermediates in this system, which bind competitively. (18-20). Ergosterol was able to induce a shift towards the low spin state in the presence of cholesterol (Fig. 4, spectrum 3). Thus unlike cholesterol, ergosterol is a low spin inducer of P450scc. 17a,24-Dihydroxyergosterol also proved to be a low spin inducer (Fig. 4, spectrum 4). This is similar to 22R-hydroxycholesterol but different from 20a,22R- dihydroxycholesterol which is a high spin inducer (18-19).

The reversal of the high-spin shift induced by cholesterol was used to determine the strength of ergosterol binding to P450scc. In the presence of 0.05 mol cholesterol/mol phospholipid an apparent Kj for ergosterol of 0.5 mol/mol phospholipid was obtained (Fig. 5). Extrapolation of the graph to infinite ergosterol concentration (zero on the X-axis of Fig. 5), reveals that as expected for competitive binding, ergosterol can completely reverse the spin-state shift induced by cholesterol.

Analysis of the competitive binding using the Ka for cholesterol determined independently (0.033 mol cholesterol/mol phospholipid) gives 0.2 mol/mol phospholipid as the true Kj for ergosterol. At a molar ratio to phospholipid of only 0.004, dihydroxyergosterol caused a substantial shift towards the low spin state in the presence of 0.1 mol cholesterol/mol phospholipid. The limited availability of this product prevented us from determining its Ka, but from the concentration used and the magnitude of the absorbance change, its K_d is at least 30 times lower than that for cholesterol.

Ergosterol metabolism by adrenal mitochondria In agreement with the above results with purified P450scc, The RH-

HPLC analysis of the reaction mixture resulting from incubation of adrenal mitochondria with ergosterol showed a single reaction product (metabolite 1) at retention time (RT) 14.2 min (ergosterol RT is 49.5.min), that was absent in control samples (Figs. 6A-6B). The absorbance spectrum of this product was similar to that of ergosterol (Figs. 6C-6D) (4). The mass spectrum of metabolite 1, yielded a molecular ion characteristic of the dihydroxyergosterol fragment with [M+H]⁺ at m/z=411 (429-H₂O) and a fragment ion at m/z=393 (411-2H₂O) (Fig. 6E). The mass spectrum of ergosterol standard (RT=49.5 min), in addition to expected ion m/z=397 (minor product), also yielded the same ions at m/z= 393 (major) indicating that ergosterol (theoretical m/z =397) had undergone desaturation during LC/MS. The ions [M+H]⁺ at m/z= 379 and at m/z =425 obtained during MS analysis of ergosterol (absent in the reaction product) represent methyl group (CH2) loss and methanol addition to desaturated ergosterol, respectively (Fig. 6F). Thus, as for purified P450scc, the major product of ergosterol metabolism by P450scc in adrenal mitochondria is dihydroxyergosterol.

EXAMPLE 4

Characterization of vitamin D2 metabolites

Metabolism of vitamin D2 by purified P450scc in a reconstituted system Vesicle-reconstituted P450scc metabolised vitamin D2 to two novel products as shown by TLC; these were not seen in control incubations where the electron source was omitted (Fig. 7A). As expected, there was production of a little pregnenolone (preg) from cholesterol that co-purified with bovine P450scc, confirming the side chain cleaving activity of the enzyme. Following their elution from TLC plates, both vitamin D2 metabolites displayed UV absorbance corresponding to an intact vitamin D chromophore (l_max at 265 nm and l_mi_n at 228 nm). For metabolite 1, the molecular ion had m/z = 412 with fragment ions m/z = 394 (412 - H₂O), m/z = 379 (394 - CH₃), m/z=376 (412 - 2H₂O) and m/z = 361 (379 - H₂O). The molecular ion of metabolite 2 had m/z = 428, with fragment ions at m/z = 410 (428 - H₂O), m/z = 392 (428 - 2H₂O), m/z = 395 (410 - CH₃) and m/z = 377 (428 - 2H₂O - CH₃). Since vitamin D2 has m/z = 396, metabolite 1 was identified as hydroxyvitamin D2, and metabolite 2 as dihydroxyvitamin D2 (Fig. 7B-7C).

Identification of the structure of vitamin D2 metabolites

Incubation of P450scc (2.0 μM) with vitamin D2 in phospholipid vesicles (40 ml) for 1 h r produced 70 μg of TLC-purifϊed hydroxyvitamin D2 (4% yield) and 60 μg TLC-purifϊed dihydroxyvitamin D2 (3.3% yield). Products from two 40 ml incubations were pooled and used for structural analysis by NMR Identification of metabolite 1 was accomplished by analysis of proton

ID, COSY and HSQC spectra of this compound and of parent vitamin D2. The high order pattern in proton NMR of vitamin D2 at 5.19 ppm (22-CH) and 5.20 ppm (23- CH) became separated to 5.54 ppm (22-CH) and 5.42 ppm (23-CH) in metabolite 1 (Fig. 8A, projections on COSY spectra). The scalar coupling between 22-CH and 20- CH did not exist in this metabolite (Fig. 8B). At the same time, the doublet of the 21- methyl in vitamin D2 (proton at 1.01 ppm and carbon at 21.2 ppm, Fig. 8C) became a singlet in metabolite 1 with a downfield shift (proton at 1.30 ppm and carbon at 29.5 ppm, Fig. 8D), also indicating the removal of scalar coupling from 20-CH. Other regions of the spectra are similar between vitamin D2 and metabolite 1. All these changes can be readily explained by the presence of a 20-OH group in metabolite 1. The impurities present in metabolite 1 have strong NMR signals in the low chemical shift region, but not in the high chemical shift region, and they probably derive from the TLC plate used in the purification process.

The HSQC spectrum of the methyl region in metabolite 2 was cleaner and similar to that of metabolite 1, indicating the presence of 20-OH and no other hydroxyl group on the side chain (Fig. 8D).

The A-ring and double bond linker was also intact in this metabolite, indicating the second hydroxylation is either at the B-ring or C-ring. The well isolated proton NMR signals of 9-CH₂ (1.68 ppm and 2.82 ppm) have very similar position and coupling patterns in vitamin D2 and metabolite 2, indicating that B-ring stays intact. Therefore, the second hydroxylation must occur in the C-ring. The 14-CH in this metabolite has a large downfield shift in its proton NMR (1.99 ppm in vitamin D2 and 2.68 ppm in metabolite 2, (Figs. 9A-9B), while the proton NMR of the 17-CH in the vitamin D2 standard at 1.32 ppm disappeared. The shift of the 14-CH is caused by the formation of 17-OH in this metabolite. Hence, this dihydroxyl metabolite is most likely to be 17α, 20-dihydroxyvitamin D2 (Figs. 9A-9D).

Thus, it is demonstrated that P450scc hydroxylates vitamin D2, and generates hydroxy- and dihydroxyvitamin D2 as main products in approximately equivalent amounts. NMR analysis further showed that these products correspond to 20-hydroxy vitamin D2 and 17a, 20-dihydroxyvitamin D2, and also reveals that the initial hydroxylation occurs at positions 20 followed by a second hydroxylation at Cl 7. The explanation for hydroxylation in these positions lies in the structure of vitamin D2, which has a C22-C23 double bond that both prevents hydroxylation at C22 and apparently limits hydroxylation of the side chain to C20. Hydroxylation of the C ring at position 17 indicates a shift in substrate orientation in the active site, as compared to cholesterol, vitamin D3, or 24a-methylcholesterol (campesterol) where P450scc is free to hydroxylate at C20 and C22 (3,21,39).

Interestingly, ergosterol (provitamin D2) is hydroxy lated at Cl 7 similar to vitamin D2, but the second hydroxylation is at C24 rather than C20 (37). The detected accumulation of 20-hydroxy vitamin D2 (Fig. 7A) suggests that it can be released from the active site of P450scc, with only a portion remaining bound or rebinding for subsequent hydroxylation at C 17. This is again in contrast to the P450scc-mediated metabolism of ergosterol where the accumulation of monohydroxy product is only minor, and also in contrast to the conversion of cholesterol into pregnenolone where hydroxy cholesterol intermediates are not normally released (21- 22).

The rate of vitamin D2 metabolism by purified P450scc To obtain an estimate of the initial rate of vitamin D2 metabolism by

P450scc, vitamin D2 at a molar ratio to phospholipid of 0.4, was incubated with P450scc for 5 min at 35⁰C. The 20-hydroxyvitamin D2 and 17, 20-dihydroxyvitamin D2 products were extracted, purified by TLC and quantitated from their absorbance at 264 nm. 20-Hydroxyvitamin D2 was produced at a rate of 0.34 mol/min/mol P450scc and 17,20-dihydroxy vitamin D2 was produced at 0.13 mol/min/mol P450scc Under similar conditions, this preparation of P450scc converted cholesterol to pregnenolone at a rate of 14.4 mol/min/mol P450scc. The rate of hydroxylation of vitamin D2 by P450scc is slightly lower than the rate of hydroxylation of its precursor, ergosterol (37). Vitamin D2 metabolism by adrenal mitochondria (AM)

To evaluate the biological relevance of the above findings we incubated purified adrenal mitochondria, which contain a high concentration of P450scc, with vitamin D2. Tests were performed in the presence (experimental) or absence (control) of NADPH and isocitrate. When the reaction products were subjected to LC/MS or LC with UV spectral analysis, we detected six new products by monitoring at 265 nm of HPLC separated fractions. Six new products were detected by UV monitoring (at 265 nm) of HPLC separated fractions (Fig. 10A- 10C) and had UV absorbance spectra characteristic of the vitamin D triene (Fig. 11). AU metabolites displayed a molecular ion [M+l]⁺ at m/z = 413 and a major fragment ion at m/z = 395 (413 - H₂O) suggesting that they represent isomers of hydroxyvitamin D2 (Fig. 1OA shows metabolites 1,4 and 6). The molecular ion [M+ 1]⁺ for vitamin D2 had m/z = 397, as expected (Fig. 11).

To further study the possible involvement of P450scc in the formation of the vitamin D2 metabolites, aminoglutethimide, a known inhibitor for P450scc (40), was added to the reaction mixture. The formation of the unknown metabolites 1,2,3,5 and 6 decreased in a parallel fashion (Fig. 10C). More profound inhibition was observed in the case of metabolite 4. This provides further evidence that vitamin D2 hydroxylation in adrenal mitochondria is catalyzed by P450scc, especially for production of metabolite 4.

EXAMPLE 5

Biological activity of ergosterol and vitamin D2 metabolites

Dihydroxyergosterol inhibits DNA synthesis in epithelial cells and cells of neural crest origin

HaCaT keratinocytes and SKMEL-188 melanoma cells were seeded 5,000 per well into 96-well plates in growth medium. After 6 h medium was discarded and serum free Ham's FlO medium was added. After 12 h this medium was changed to 5% FBS Ham's FlO medium containing serial dilutions of dihydroxyergosterol. Solvent (EtOH) control contained 0.2% EtOH (the same concentration as treatment with dihydroxyergosterol 10^"6). After 12 h medium was discarded and 5% FBS Ham's medium with serial dilutions of dihydrozyergosterol and [3H]-thymidine 1 μCi/ml added for 12 h. Whole incubation with dihydroxyergosterol lasted 24 h. After treatment media discarded, cells detached with trypsin, harvested on fiber glass filter, and radioactivity proportional to methyl- [³H]thymidine incorporated into DNA was counted with Packard direct beta counter (Packard, Meriden, CA).

Addition of 17a,24-dihydroxyergosterol (HPLC purified) to the culture media inhibited DNA synthesis in human epidermal HaCaT keratinocytes (Fig. 12A) and SKMEL-188 melanoma cells with an EC₅₀ of 4.3 x 10^'8 M (Fig. 12B). The hiatus in activity suggests that 17a,24-dihydroxyergosterol could act both directly, and indirectly through a more potent metabolite generated from 17a,24- dihydroxyergosterol during the incubation of the cells. Data is presented as mean ± SEM (n=8), and is analyzed with one-way analysis of variance with appropriate post- hoc tests using Prism 4.00 (GraphPad Software, San Diego). The dose-response curve fitting and EC₅₀ calculation were performed using Prism 4.0 software.

Dihydroxyergosterol inhibits DNA synthesis in fibroblasts Dermal fibroblasts (7^th passage) were seeded 5,000 per well into 96- well plates in growth medium. After 24 h medium was changed to 5% FBS Ham's FlO medium containing 5% FBS and serial dilutions of dihydroxyergosterol. After 12 h medium was discarded and Ham's medium with 5% FBS and serial dilutions of dihydroxyergosterol and [3H]-thymidine 1 μCi/ml added for 12 h. Whole incubation with dihydroxyergosterol lasted 24 h. After treatment media discarded, cells detached with trypsin, harvested on fiber glass filter, and radioactivity proportional to methyl- [³H]thymidine incorporated into DNA was counted with Packard direct beta counter (Packard, Meriden, CA).

Addition of 17a,24-dihydroxyergosterol (HPLC purified) to the culture media inhibited DNA synthesis in dermal fibroblasts with an EC₅₀ of 3.7 x 10^"11 M (Fig. 13). Data is presented as mean ± SEM (n=8), and is analyzed with Student's t tests using Prism 4.00 (GraphPad Software, San Diego). The dose-response curve fitting and EC_5O calculation were performed using Prism 4.0 software.

Biological activity of vitamin D2 metabolites on HaCaT keratinocvtes

Cultured human epidermal HaCaT keratinocytes were incubated with HPLC purified 20-hydroxy vitamin D2 or 17α, 20-dihydroxy vitamin D2 added to the culture media at a concentration of 10^"10 M. This caused inhibition of DNA synthesis, significantly greater than that seen with vitamin D2 itself (Fig. 14A). A similar inhibitory effect of both hydroxy-metabolites was also seen in an additional independent experiment. The effect of hydroxy vitamin D2 products on keratinocyte differentiation, with vitamin D2 and 5 mM Ca²⁺ as positive controls, also was examined. This was done using the firefly luciferase reporter gene plasmid IVL-Luc containing the involucrin gene promoter region (-668 bp to +34 bp) (Fig. 14B). Since involucin expression is characteristically proportional to keratinocyte differentiation (41-45), these assays are typically used in models testing for keratinocyte differentiation (41).

All of the tested compounds stimulated transcriptional activity of the involucrin promoter; the most significants effect being shown by Ca^+"1" and 17a, 20- dihydroxy vitamin D2 that simulated luciferase activity by 25 and 12 fold, respectively (Fig. 14B). The stimulatory effect of 17a,20-dihydroxy vitamin D2 was significantly higher than that of vitamin D2 (p<0.05), while the effect of 20- hydroxyvitamin D2 on involucrin promoter activity was statistically insignificant (p>0.05). Thus, the data above indicate that vitamin D2 can be converted to product(s) of higher biological activity by P450scc.

Hydroxy and dihydrovyvitamin D2 metabolites inhibit DNA synthesis

Cells were synchronized and then incubated for 24 h in Ham's FlO medium without serum and vitamin D2 metabolites and [3H]-thymidine (1 μCi/ml) added for last 12 h of incubation. Cultured human melanoma cells were incubated with HPLC purified 20-hydroxy vitamin D2 or 17,20-dihydroxy vitamin D2 added to the culture media at increasing concentrations (Figs. 15A- 15B). This caused inhibition of cell proliferation, i.e., DNA synthesis, in a dose dependent manner, demonstrating a bell-shaped response, with maximal inhibition at concentrations 10^{" 12}-10^"10 M for 20-hydroxyvitamin D2 and 10^"10-10^"9 M for 17, 20-dihydroxyvitamin D2. The inhibitory effects were statistically significant (p<0.05).

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Any publications mentioned in this specification are indicative of the levels of those skilled in the art to which the invention pertains. Further, these publications are incorporated by reference herein to the same extent as if each individual publication was specifically and individually incorporated by reference. One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The present examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention as defined by the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1. A method of producing an hydroxylated metabolite of ergosterol, comprising: hydroxylating a substrate of a cytochrome P450scc enzyme system in at least one position, said substrate enzymatically convertible to said hydroxylated ergosterol metabolite.

2. The method of claim 1, wherein said substrate is ergosterol.

3. The method of claim 2, wherein P450scc hydroxylates a C17 sidechain of ergosterol.

4. The method of claim 3, wherein said ergosterol Cl 7 sidechain is hydroxylated in at least at position C24 within the Cl 7 chain.

5. The method of claim 4, wherein said hydroxylated ergosterol is 24-hydroxyergosterol or 17α,24-dihydroxy ergosterol.

6. The method of claim 2, wherein said Cl 7 sidechain is hydroxylated at least at position C20 within the C 17 chain.

7. The method of claim 6, wherein said hydroxylated ergosterol is 20-hydroxyergosterol.

8. The method of claim 2, wherein said C17 sidechain is hydroxylated at C20 and C24.

9. The method of claim 8, wherein said hydroxylated ergosterol is 20,24-dihydroxyergosterol.

10. The method of claim 2, further comprising: thermophotolytically breaking a C9-C10 bond in said hydroxylated ergosterol via UVB radiation; and converting said hydroxylated ergosterol to a hydroxylated ergocalciferol via thermal intramolecular rearrangement around the broken bond.

11. The method of claim 10, wherein said hydroxylated vitamin D2 is 17-hydroxy ergocalciferol, 20-hydroxyergocalciferol, 24-hydroxy ergocalciferol,

17α,24-dihydroxyergocalciferol, or 20,24- dihydroxy ergocalciferol.

12. The method of claim 10, further comprising hydroxylating position Cl of the A ring of said hydroxylated ergocalciferol with a CYP27B1 enzyme.

13. The hydroxylated ergocalciferol of claim 12, wherein said hydroxylated ergocalciferol is lα,17α-dihydroxy ergocalciferol, lα,20- dihydroxy ergocalciferol, lα,17α,20-trihydroxyergocalciferol, or lα,17α,24- trihydroxy ergocalciferol.

14. An hydroxylated ergocalciferol produced by the CYP27B1 enzyme of claim 12.

15. The method of claim 1, wherein said substrate is ergocalciferol.

16. The method of claim 15, wherein P450scc hydroxylates a C17 sidechain of ergocalciferol.

17. The method of claim 16, wherein said ergocalciferol Cl 7 sidechain is hydroxylated in at least at position C20 within the Cl 7 chain.

18. The method of claim 17, wherein said hydroxylated ergocalciferol is 20-hydroxyergocalciferol or 17α,20-dihydroxy ergocalciferol.

19. The method of claim 16, further comprising hydroxylating position Cl of the A ring of said hydroxylated ergocalciferol with a CYP27B1 enzyme.

20. The method of claim 19, wherein said hydroxylated ergocalciferol is lα,20~dihydroxyergocalciferol or lα,17α,20-trihydroxy ergocalciferol.

21. An hydroxylated ergocalciferol produced by the CYP27B1 enzyme of claim 19.

22. The method of claim 1, wherein said cytochrome P450scc enzyme system is an in vitro system, comprising: cytochrome P450scc enzyme; adrenodoxin; adrenodoxin reductase; and NADPH.

23. The method of claim 22, further comprising a phospholipid vesicle having components of the enzyme system and said substrate encapsulated therein.

24. The method of claim 1, wherein said cytochrome P450scc enzyme system comprises a eukaryotic cell or a prokaryotic cell.

25. The method of claim 24, wherein said eukaryotic cell is a mammalian cell.

26. The method of claim 24, wherein said mammalian cell is an adrenal cell, a gonadal cell, a placental cell, a cell from the gastrointestinal tract, a kidney cell, a brain cell, or a skin cell.

27. The method of claim 24, wherein said mammalian cell is in vitro or in vivo.

28. The method of claim 24, wherein said prokaryotic cell is a yeast cell or a bacterial cell.

29. The method of claim 24, wherein said cytochrome P450scc enzyme system is a recombinant system in said cell.

30. An hydroxy lated ergosterol metabolite produced by the cyctochrome P450scc enzyme system of claim 1 or a pharmaceutical composition thereof.

31. The hydroxylated ergosterol metabolite of claim 30, wherein said metabolite is 20-hydroxyergosterol, 24-hydroxyergosterol, 17α,24- dihydroxy ergosterol, or 20,24-dihydroxyergosterol, 17-hydroxyergocalciferol, 20- hydroxyergocalciferol, 24-hydroxyergocalciferol, 17α,20-dihydroxyergocalciferol, 17α,24-dihydroxyergocalciferol, or 20,24-dihydroxyergocalciferol.

32. A method of treating in a subject a tumor that has cytochrome P450scc activity, comprising: administering to the subject a pharmacologically effective amount of a substrate of cytochrome P450scc or a pharmaceutical composition thereof suitable to enzymatically convert to an hydroxylated metabolite of ergosterol via at least the cytochrome P450scc acitivity of the tumor, wherein an amount of an hydroxylated ergosterol metabolite so produced is effective to inhibit tumor growth in the subject thereby treating the tumor.

33. The method of claim 32, wherein said hydroxylated ergosterol metabolite is 20-hydroxyergosterol, 24-hydroxyergosterol 17<x,24- dihydroxyergosterol, 20,24-dihydroxyergosterol, 20-hydroxyergocalciferol or 17α,20-dihydroxyergocalciferol.

34. The method of claim 32, wherein said tumor is an adrenal tumor, a gonadal tumor, a tumor of the gastrointestinal tract, a kidney tumor, a brain tumor, an epithelial tumor, a melanoma, or other skin tumor.

35. A method of treating a pathophysiological condition of a cell, comprising: treating said cell with a pharmacologically effective amount of an hydroxylated ergosterol or hydroxylated ergocalciferol or a pharmaceutical composition thereof.

36. The method of claim 35, wherein said hydroxylated ergosterol is 20-hydroxyergosterol, 24-hydroxy ergosterol 17α,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol.

37. The method of claim 35, wherein said hydroxylated ergocalciferol is 17-hydroxy ergocalciferol, 20-hydroxyergocalciferol, 24- hydroxyergocalciferol, 17oc,20-dihydroxyergocalciferol, 17α,24- dihydroxyergocalciferol, 20,24-dihydroxyergocalciferol, lα,17α- dihydroxyergocalciferol, 1 α, 17α,24-trihydroxyergocalciferol, 1 α,20- dihydroxy ergocalciferol or lα,17α,20-trihydroxy ergocalciferol.

38. The method of claim 35, wherein said cell is a neoplastic cell.

39. The method of claim 38, wherein said neoplastic cell is an adrenal cell, a gonadal cell, a pancreatic cell, a cell from the gastrointestinal tract, a prostate cell, a breast cell, a lung cell, an immune cell, a hematologic cell, a kidney cell, a brain cell, a cell of neural crest origin or a skin cell.

40. The method of claim 35, wherein said pathophysiological condition is a melanoma, a sarcoma, a leukemia, or a lymphoma.

41. The method of claim 35, wherein said pathophysiological condition is a skin disorder.

42. The method of claim 40, wherein said skin disorder is a hyperproliferative skin disorder, a pigmentary skin disorder, an inflammatory skin disorder, or other skin disorder characterized by hair growth on legs, arms, torso, or face, or induced by exposure to solar radiation.