WO2007006036A2 - Formes hydroxylees d'ergosterols et d'ergocalciferols, derives de ceux-ci, procedes de production et utilisations de ceux-ci - Google Patents

Formes hydroxylees d'ergosterols et d'ergocalciferols, derives de ceux-ci, procedes de production et utilisations de ceux-ci Download PDF

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WO2007006036A2
WO2007006036A2 PCT/US2006/026476 US2006026476W WO2007006036A2 WO 2007006036 A2 WO2007006036 A2 WO 2007006036A2 US 2006026476 W US2006026476 W US 2006026476W WO 2007006036 A2 WO2007006036 A2 WO 2007006036A2
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cell
hydroxylated
ergosterol
ergocalciferol
p450scc
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PCT/US2006/026476
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WO2007006036A3 (fr
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Andrzej Slominski
Robert Tuckey
Wei Li
Blazej Zbytek
Jordan Zjawiony
Jacobo Wortsman
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The University Of Tennessee Research Foundation
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Publication of WO2007006036A2 publication Critical patent/WO2007006036A2/fr
Publication of WO2007006036A3 publication Critical patent/WO2007006036A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07J—STEROIDS
    • C07J9/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of more than two carbon atoms, e.g. cholane, cholestane, coprostane

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  • 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.
  • 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).
  • vitamin D2 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
  • 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.
  • 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- hydroxyergocalf ⁇ cerol 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.
  • Figures 1A-1D depict the schema for production of 17 ⁇ ,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 l ⁇ -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.
  • 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 17 ⁇ -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
  • 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.
  • DA 8 absorbance difference (416-392 nm) in the presence of ergosterol
  • DA 0 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 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
  • 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.
  • Figures 9A-9D depict the NMR spectra of vitamin D2 metabolite 2 identified as 17 ⁇ ,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.
  • FIGS. 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 ⁇ M 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] + .
  • Figure 13 demonstrates that dihydroxyergosterol inhibits DNA synthesis in dermal fibroblasts.
  • FIGS 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 [ 3 H]-thymidine ( Figure 14A).
  • 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 ⁇ Ci/ml).
  • Data presented as mean ⁇ SEM (n 8) and *p ⁇ 0.05.
  • 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.
  • the substrate may be ergosterol.
  • P450scc may hydroxylate a Cl 7 sidechain of ergosterol.
  • 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.
  • the C17 sidechain may be hydroxylated at Cl 7 and C24.
  • hydroxylated ergosterol is 17 ⁇ ,24- dihydroxyergosterol.
  • 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.
  • the Cl 7 sidechain may be hydroxylated at C20 and C24.
  • An example of such a hydroxylated ergosterol is 20,24- dihydroxyergosterol.
  • 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.
  • the hydroxylated ergocalciferols may be 17-hydroxyergocalciferol, 20-hydroxyergocalciferol, 24- hydroxyergocalciferol, 17 ⁇ ,24-dihydroxy ergocalciferol, or 20,24- dihydroxyergocalciferol.
  • the method may comprise hydroxy lating position Cl of the A ring of said hydroxylated ergocalciferol with a CYP27B1 enzyme.
  • the substrate may be ergocalciferol.
  • P450scc may hydroxylate a Cl 7 sidechain of ergocalciferol.
  • 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 17 ⁇ ,20-dihydroxyergocalciferol.
  • the method may comprise hydroxy lating position Cl of the A ring of said hydroxylated ergocalciferol with a CYP27B1 enzyme.
  • the cytochrome P450scc enzyme system may be an in vitro system, comprising cytochrome P450scc enzyme; adrenodoxin; adrenodoxin reductase; and NADPH.
  • the in vitro system may comprise a phospholipid vesicle having these components of the enzyme system and the substrate encapsulated therein.
  • 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.
  • the cytochrome P450scc enzyme system may be a recombinant system in said cell.
  • an hydroxylated ergosterol metabolite produced by the cyctochrome P450scc enzyme system described supra or a pharmaceutical composition thereof.
  • the hydroxylated ergosterol metabolite may be 20-hydroxyergosterol, 24- hydroxyergosterol, 17 ⁇ ,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol., 17- hydroxyergocalciferol, 20-hydroxyergocalciferol, 24-hydroxyergocalciferol, 17 ⁇ ,20- dihydroxyergocalciferol, 17 ⁇ ,24-dihydroxyergocalciferol, or 20,24- dihydroxyergocalciferol.
  • a di- or tri- hydroxylated ergocalciferol produced by the CYP27B1 enzyme.
  • these hydroxy lated ergocalciferols are lcc,17 ⁇ -dihydroxy ergocalciferol, l ⁇ ,20- dihydroxy ergocalciferol, l ⁇ ,17 ⁇ ,20-trihydroxyergocalciferol or l ⁇ ,17 ⁇ ,24- trihydroxyergocalciferol.
  • 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.
  • the hydroxylated ergosterol metabolite may be 20-hydroxyergosterol, 24-hydroxyergosterol 17 ⁇ ,24- dihydroxyergosterol, 20,24-dihydroxyergosterol, 20-hydroxyergocalciferol or 17 ⁇ ,20-dihydroxy ergocalciferol.
  • 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.
  • 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, 17 ⁇ ,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol.
  • the hydroxylated ergocalciferol may be 17-hydroxy ergocalciferol, 20-hydroxyergocalciferol, 24- hydroxyergocalciferol, 17 ⁇ ,20-dihydroxy ergocalciferol, 20,24- dihydroxy ergocalciferol, l ⁇ ,17 ⁇ -dihydroxy ergocalciferol, l ⁇ ,17 ⁇ ,24- trihydroxyergocalciferol, l ⁇ ,20-dihydroxy ergocalciferol or l ⁇ ,17 ⁇ ,20-trihydroxy ergocalciferol.
  • the cell may be a neoplastic cell.
  • 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.
  • the pathophysiologic condition may be a melanoma, a carcinoma, a sarcoma, a leukemia, or a lymphoma.
  • the pathophysiologic condition may be a skin disorder.
  • 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.
  • the term, "a” or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another or “other” may mean at least a second or more of the same or different claim element or components thereof.
  • Neoplastic cell 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.
  • tumor refers to a mass of malignant neoplastic cells or a malignant tissue comprising the same.
  • 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.
  • the term "subject" refers to any target of the treatment.
  • cytochrome P450scc enzyme system Provided herein are methods of metabolizing a substrate of the cytochrome P450scc enzyme system to produce hydroxylated metabolites.
  • ergosterol or ergocalciferol 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.
  • the P450 enzyme hydroxylates ergosterol first at the C24 position to form an intermediate 24-hydroxyergosterol with subsequent hyrdroxylation at C 17.
  • the Cl 7 side chain may be hydroxylated at the C20 position or at both the C20 and C24 positions.
  • 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, 17 ⁇ ,24-dihydroxyergocalciferol, or 20,24- dihydroxyergocalciferol is produced from the photochemical and thermic transformation of 17 ⁇ ,24-dihydroxyergosterol.
  • both 20- dihydroxyergocalciferol and 17 ⁇ ,20-dihydroxyergocalciferol may be produced directly from vitamin D2 as substrate of a purified, reconstituted cytochrome P450scc enzyme system.
  • 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.
  • 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.
  • 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.
  • the present invention provides an hydroxylated ergosterol and hydroxylated ergocalciferols enzymatically produced by the cytochrome P450scc system.
  • 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, 17 ⁇ ,24-dihydroxyergosterol, or 20,24-dihydroxyergosterol.
  • 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.
  • 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.
  • a sufficient or effective amount of a dihydroxyergosterol e.g., 17 ⁇ ,24-dihydroxyergosterol or 20,24-dihydroxyergosterol, or of a mono- or dihydroxy ergocalciferol, e.g., 20-hydroxyergocalciferol and 17 ⁇ ,20-hydroxyergocalciferol, by the tumor cells, would inhibit tumor growth.
  • 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.
  • the mono- and di-hydroxy products of the cytochrome P450 enzyme system may be hydroxylated at the l ⁇ -position by l ⁇ - hydroxylase enzyme CYP27B1.
  • l ⁇ -hydroxyergocalciferol, 20- hydroxyergocalciferol, 17 ⁇ ,20-dihydroxyergocalciferol, or 17 ⁇ ,24- dihydroxyergocalciferol are hydroxylated to form l ⁇ ,17 ⁇ -dihydroxyergocalciferol, lcx,20-dihydroxy ergocalciferol, l ⁇ ,17 ⁇ ,20-trihydroxy ergocalciferol, or l ⁇ ,17 ⁇ ,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.
  • the trihydroxylated ergocalciferols may be chemically synthesized.
  • the present invention also provides trihydroxylated ergocalciferols described herein or pharmaceutical compositions thereof.
  • cytochrome P450scc cytochrome P450scc
  • 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.
  • 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.
  • a cell such as a neoplastic cell or a cell comprising a skin disorder.
  • the neoplastic cell may be malignant or benign.
  • 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.
  • 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.
  • 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.
  • 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 reaction mixture further comprised 50 ⁇ M NADPH, 2 mM glucose 6-phosphate, 2 U/ml glucose 6-phosphate dehydrogenase, 0.2 ⁇ M adrenodoxin reductase, 10 ⁇ M adrenodoxin, 2.0 ⁇ M cytochrome P450scc and buffer pH 7.4.
  • the reaction mixture further comprised 50 ⁇ M NADPH, 2 mM glucose 6-phosphate, 2 U/ml glucose 6-phosphate dehydrogenase, 0.3 ⁇ M adrenodoxin reductase, 6.5 ⁇ M adrenodoxin, 3.0 ⁇ M cytochrome P450scc and buffer pH 7.4. After incubation at 35 0 C for 3 h the mixture was extracted with methylene chloride and dried under nitrogen. After incubation at 35 0 C for 3 h the mixture was extracted with methylene chloride and dried under nitrogen.
  • Adrenals were obtained from male Wistar rats aged 3 months, terminated under anesthesia. The animals were housed at the vivarium of the
  • 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 MgSO 4 , 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.
  • NADPH 0.5 mM
  • isocitrate 5 mM
  • Vesicles were prepared from phosphatidylcholine and bovine heart cardiolipin using a bath-type sonicator in buffer comprising 20 mM Hepes pH 7.4,
  • 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
  • Kd 1 ap P is the apparent Kd for ergosterol in the presence of cholesterol
  • I is the cholesterol concentration
  • 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.
  • EI electron impact
  • LCMS-QP8000 ⁇ (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.
  • SPD-Ml OAvp UV /VIS photodiode array detector
  • 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 40 0 C 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.
  • 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.
  • 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 (5 ⁇ g/ml) and 1% antibiotic solution.
  • Dermal fibroblasts were grown in DMEM medium with 5% FBS, insulin (5 ⁇ g/ml) and 1% antibiotic solution.
  • 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.
  • 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 ⁇ g/well) and with phRL-TK which expresses Renilla luciferase and serves as normalization control (Promega, Madison, WI) added at 1 ⁇ g/well.
  • IVL-Luc and p-Luc (control without promoter, empty vector) plasmids were constructed as described previously (38).
  • P450scc transforms ergosterol to 17a,24-dihydroxyl ergosterol with 24 hydroxyergosterol serving as an intermediate of the metabolism (Fig. IA). Also 17 ⁇ ,24-dihydroxyergosterol may be converted to 17 ⁇ ,24-dihydroxy vitamin D2 through photochemical and thermic transformation (Fig. IB).
  • Ultraviolet radiation B (UV-B) energy converts 17 ⁇ ,24-dihydroxyergosterol into 17 ⁇ ,24-dihydroxy previtamin D2.
  • Thermal energy at 37 °C converts 17 ⁇ ,24-dihydroxy previtamin D2 into 17 ⁇ ,24-dihydroxy vitamin D2.
  • P450scc transforms vitamin D2 directly to 20-hydroxyergocalciferol (20-hydroxy vitamin D2) or 17cc,20- dihydroxyergocalciferol (17 ⁇ ,20-dihydroxy vitamin D2) (Fig. 1C).
  • Alphal- hydroxylase (CYP27B1) converts 17 ⁇ -dihydroxyergocalciferol, 17 ⁇ ,24- dihydroxyergocalciferol, 20-dihydroxyergocalciferol or 17cc,20- dihydroxyergocalciferol to l ⁇ ,17 ⁇ -dihydroxyergocalciferol, l ⁇ ,17 ⁇ ,24- trihydroxyergocalciferol, l ⁇ ,20-dihydroxyergocalciferol and l ⁇ ,17 ⁇ ,20-trihydroxy ergocalciferol (Fig. ID).
  • 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).
  • 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 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-CH 2 (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.
  • this dihydroxyl metabolite is most likely to be 17 ⁇ , 20-dihydroxyvitamin D2 (Figs. 9A-9D).
  • 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).
  • ergosterol provitamin D2
  • ergosterol provitamin D2
  • the second hydroxylation is at C24 rather than C20 (37).
  • the detected accumulation of 20-hydroxy vitamin D2 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).
  • P450scc vitamin D2 at a molar ratio to phospholipid of 0.4
  • P450scc vitamin D2 at a molar ratio to phospholipid of 0.4
  • 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
  • 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).
  • 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 ⁇ Ci/ml added for 12 h.
  • EtOH the same concentration as treatment with dihydroxyergosterol 10 "6 .
  • 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 ⁇ Ci/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- [ 3 H]thymidine incorporated into DNA was counted with Packard direct beta counter (Packard, Meriden, CA).

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Abstract

Cette invention se rapporte à un procédé enzymatique de production d'ergostérols hydroxylés et d'ergocalciférols hydroxylés utilisant l'enzyme du cytochrome P450scc et/ou l'enzyme CYP27B1 et aux composés hydroxylés ainsi produits. Cette invention concerne également des procédés d'utilisation des ergostérols hydroxylés et des ergocalciférols hydroxylés pour traiter une tumeur ou un autre état pathologique.
PCT/US2006/026476 2005-07-05 2006-07-05 Formes hydroxylees d'ergosterols et d'ergocalciferols, derives de ceux-ci, procedes de production et utilisations de ceux-ci WO2007006036A2 (fr)

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EP2386634A1 (fr) * 2009-01-07 2011-11-16 Mitsubishi Chemical Corporation Protéine enzymatique de clivage de chaîne latérale de stérol et utilisation de celle-ci

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US20040115629A1 (en) * 2002-01-09 2004-06-17 Panzer Scott R Molecules for diagnostics and therapeutics
US20040131610A1 (en) * 2002-07-15 2004-07-08 Thorpe Philip E. Methods for treating viral infections using antibodies to aminophospholipids
US20040186082A1 (en) * 2003-03-20 2004-09-23 Hartman Raymond A. Enhanced phototherapy for the treatment of cancer and autoimmune disease

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US20040115629A1 (en) * 2002-01-09 2004-06-17 Panzer Scott R Molecules for diagnostics and therapeutics
US20040131610A1 (en) * 2002-07-15 2004-07-08 Thorpe Philip E. Methods for treating viral infections using antibodies to aminophospholipids
US20040186082A1 (en) * 2003-03-20 2004-09-23 Hartman Raymond A. Enhanced phototherapy for the treatment of cancer and autoimmune disease

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TUCKEY ET AL.: 'Side-chain specificities of human and bovine cytochromes P-450scc' EUR. J. BIOCHEM. vol. 217, no. 1, 01 October 1993, pages 209 - 215 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2386634A1 (fr) * 2009-01-07 2011-11-16 Mitsubishi Chemical Corporation Protéine enzymatique de clivage de chaîne latérale de stérol et utilisation de celle-ci
EP2386634A4 (fr) * 2009-01-07 2012-07-18 Mitsubishi Chem Corp Protéine enzymatique de clivage de chaîne latérale de stérol et utilisation de celle-ci

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