WO2003000023A2 - Method of treating hyperproliferative diseases using active vitamin d analogues - Google Patents

Method of treating hyperproliferative diseases using active vitamin d analogues Download PDF

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Publication number
WO2003000023A2
WO2003000023A2 PCT/US2002/020475 US0220475W WO03000023A2 WO 2003000023 A2 WO2003000023 A2 WO 2003000023A2 US 0220475 W US0220475 W US 0220475W WO 03000023 A2 WO03000023 A2 WO 03000023A2
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Prior art keywords
vitamin
compound
hydrogen
accordance
alkenyl
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PCT/US2002/020475
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French (fr)
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WO2003000023A3 (en
Inventor
Charles W. Bishop
Richard B. Mazess
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Bone Care International, Inc.
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Publication date
Application filed by Bone Care International, Inc. filed Critical Bone Care International, Inc.
Priority to EP02756332A priority Critical patent/EP1408983A4/en
Priority to CA002450942A priority patent/CA2450942A1/en
Priority to MXPA03011307A priority patent/MXPA03011307A/en
Priority to IL15906802A priority patent/IL159068A0/en
Priority to KR10-2003-7016888A priority patent/KR20040015753A/en
Priority to AU2002322346A priority patent/AU2002322346B2/en
Priority to JP2003506479A priority patent/JP2004535429A/en
Publication of WO2003000023A2 publication Critical patent/WO2003000023A2/en
Publication of WO2003000023A3 publication Critical patent/WO2003000023A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/59Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/02Antineoplastic agents specific for leukemia
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • A61P35/04Antineoplastic agents specific for metastasis
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates generally to a method of treating hyperprohferative diseases, and in particular, to the use of active forms of hypocalcemic vitamin D to inhibit the hyperprohferative cellular activity of these diseases and to promote differentiation of the cells.
  • vitamin D the hormonally active form of vitamin D
  • LNCaP human prostatic carcinoma cell line
  • Vitamin D receptors have also been described for many other neoplastic cells, e.g., carcinomas of the breast and the colon.
  • vitamin D compounds and analogues are potent inhibitors of malignant cell proliferation and are inducers/stimulators of cell differentiation.
  • U.S. Patent No. 4,391,802 issued to Suda et al. discloses that l ⁇ -hydroxyvitamin D compounds, specifically l ⁇ ,25-dihydroxyvitamin D and l ⁇ -hydroxyvitamin D 3 , possess potent antileukemic activity by virtue of inducing the differentiation of malignant cells (specifically leukemia cells) to nonmalignant macrophages (monocytes), and are useful in the treatment of leukemia.
  • vitamin D 3 compounds Even though these compounds may indeed be highly effective in promoting differentiation in malignant cells in culture, their practical use in differentiation therapy as anticancer agents is severely limited because of their equally high potency as agents affecting calcium metabolism. At the levels required in vivo for effective use as, for example, antileukemic agents, these same compounds can induce markedly elevated and potentially dangerous blood calcium levels by virtue of their inherent calcemic activity. That is, the clinical use of l ⁇ ,25-dihydroxyvitamin D 3 and other vitamin D analogues as anticancer agents is precluded, or severely limited, by the risk of hypercalcemia. This indicates a need for compounds with greater specific activity and selectivity of action, i.e., vitamin D compounds with antiproliferative and differentiating effects but which have less calcemic activity.
  • the present invention provides a method of treating hyperprohferative disease conditions such as those characterized by hyperproliferative cell growth and/or abnormal cell differentiation.
  • the method includes use of active vitamin D compounds to inhibit abnormal cell growth and promote cell differentiation.
  • a method of inhibiting the hyperproliferative activity of neoplastic or hyperplastic cells comprising treating the cells with an effective amount of a hypocalcemic vitamin D compound.
  • the treating step includes inhibiting proliferation of, and inducing and enhancing differentiation in such cells.
  • the hypocalcemic vitamin D compounds of the present invention include vitamin D compounds having a hydrocarbon moiety substituted at the C-24 position on the sidechain of the molecule and a hydroxy group substituted in at least one of the Ci, C 24 or C 25 positions.
  • the vitamin D compound of the present invention is an active vitamin D and is suitably represented by the formula (I) described hereafter.
  • the compounds of formula (I) suitably include l ⁇ ,24-dihydroxyvitamin D 2 , l ⁇ ,24-dihydroxyvitamin D 4 , l ⁇ ,25- dihydroxyvitamm D , l ⁇ ,25-dihydroxyvitamin D 2 , l ⁇ -hydroxyvitamin D and l ⁇ - hydroxyvitamin D 4 .
  • hypocalcemic vitamin D compounds are valuable for the treatment of breast and colon cancer, as well as other neoplasms such as pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell cancer of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma, retinoblastoma, and sarcomas of the soft tissue and bone.
  • neoplasms such as pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell cancer of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocy
  • the proliferative activity of the abnormal neoplastic cells is inhibited, reduced, or stabilized, and cell differentiation is induced, promoted or enhanced, with significantly less hypercalcemia and hypercalciuria than is observed after the same amount of an activated vitamin D 3 (e.g., l ⁇ -OH D 3 , l ⁇ ,25-(OH) 2 D 3 ) is administered in previously known formulations.
  • an activated vitamin D 3 e.g., l ⁇ -OH D 3 , l ⁇ ,25-(OH) 2 D 3
  • the compound in accordance with the present invention has an improved therapeutic index relative to active forms of vitamin D 3 analogues.
  • another aspect of the invention is a method of treating human cancer comprising administering to a subject who has cancer an effective amount of hypocalcemic vitamin D compound which has or attains through metabolism in vivo, a vitamin D receptor (VDR) binding affinity substantially equivalent to the binding affinity of l ⁇ ,25-dihydroxyvitamin D 3 and a hypercalcemia risk substantially lower that that of l ⁇ ,25-dihydroxyvitamin D 3 , to inhibit, decrease or stabilize the cellular abnormal proliferative activity of the cancer.
  • VDR vitamin D receptor
  • hypocalcemic vitamin D compounds can be suitably administered alone as an active ingredient, as an antiproliferative agent in a pharmaceutical composition, or co- administered with an anticancer agent.
  • vitamin D of formula (I) with a cytotoxic or anticancer agent.
  • agents suitably include antimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine), antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g.
  • cisplatin carboplatin, oxaliplatin, JM-216, CI-973
  • anthracyclines e.g., doxrubicin, daunorubicin
  • antibiolitics e.g., mitomycin, idarubicin, adriamycin, daunomycin
  • topoisomerase inhibitiors e.g., etoposide, camptothecins
  • any other antineoplastic agents estramustine phosphate, prednimustine.
  • hypocalcemic vitamin D compounds used in combination with various anticancer drugs can give rise to a significantly enhanced cytotoxic effect on cancerous cells, thus providing an increased therapeutic effect.
  • a significantly increased growth-inhibitory effect is obtained with the above disclosed combinations utilizing lower concentrations of the anticancer drugs compared to the treatment regimes in which the drugs are used alone, there is the potential to provide therapy wherein adverse side effects associated with the anticancer drugs are considerably reduced than normally observed with the anticancer drugs used alone in larger doses.
  • Possible dose ranges of these co-administered anticancer agents are about 0.1 to 20 mg/kg/day.
  • analogue of formula (I) in conjunction with administration of hormones or other agents, e.g., estrogens, which are known to ameliorate bone diseases or disorders.
  • hormones or other agents e.g., estrogens
  • prostate cancer often metastasizes to bone, causing bone loss and associated pain.
  • bone agents may include conjugated estrogens or their equivalents, calcitonin, bisphosphonates, calcium supplements, cobalamin, pertussis toxin and boron.
  • the invention is a pharmaceutical composition which includes an anticancer agent which is an active vitamin D compound; an agent selected from the group consisting of (i) an anticancer agent, (ii) a bone agent, and combinations thereof; and a physiologically acceptable carrier.
  • the present invention provides an effective method for the treatment of neoplasms and hyperproliferative diseases.
  • the present invention relates to therapeutic methods for inhibiting, reducing or stabilizing the hyperproliferative cellular activity of diseased cells, and inducing, enhancing or promoting cell differentiation in the diseased cells.
  • the present invention provides a novel treatment of a patient suffering from a hyperproliferative disease such as prostatic cancer or prostatic hype ⁇ lasia with a hypocalcemic hydroxyvitamin D analogue.
  • the vitamin D analogue is suitably a l ⁇ -hydroxyvitamin D or a 24-hydroxyvitamin D compound.
  • hypocalcemic hydroxyvitamin D analogue represented by formula (I) as described hereinbelow is provided to the patient without causing dose-limiting hypercalcemia and hypercalciuria, i.e., unphysiologically high and deleterious blood calcium levels and urine calcium levels, respectively. These attributes are achieved through specific chemical properties of the hypocalcemic vitamin D compounds as described.
  • hypocalcemic vitamin D compounds when effective amounts of the hypocalcemic vitamin D compounds are administered to patients with cancer or hype ⁇ lasia, the proliferative activity of the abnormal cells is inhibited, maintained, or alleviated, and cell differentiation is induced, promoted or enhanced, with significantly less hypercalcemia and hypercalciuria than is observed after the same amount of activated vitamin D 3 is administered in previously known formulations.
  • the hypocalcemic vitamin D compounds of the present invention have an improved therapeutic index relative to active forms of vitamin D 3 analogues.
  • vitamin D 3 must be hydroxylated in the C-1 and C-25 positions before it is activated, i.e., before it will produce a biological response.
  • the term "activated vitamin D” or “active vitamin D” is intended to refer to a vitamin D compound or analogue that has been hydroxylated in at least the C-1, C-24 or C-25 position of the molecule and either the compound itself or its metabolites in the case of a prodrug, such as l ⁇ -hydroxyvitamin D 2 , binds the vitamin D receptor (VDR).
  • vitamin D "prodrugs” include compounds which are hydroxylated in the C-1 position. Such compounds undergo further hydroxylation in vivo and their metabolites bind the VDR.
  • hypocalcemic vitamin D compound is in reference to active vitamin
  • D analogs which demonstrate reduced calcemic activity relative to the calcemic activity of l ⁇ ,25-dihydroxyvitamin D 3 .
  • Such compounds include 24-hydroxyvitamin D compounds, 25-hydroxyvitamin D compounds and l ⁇ -hydroxyvitamin D compounds.
  • the calcemic activity of these compounds ranges from 0.001 to 0.5 that of l ⁇ ,25- dihydroxyvitamin D 3 .
  • the term "lower" as a modifier for alkyl, alkenyl acyl, or cycloalkyl is meant to refer to a straight or branched, saturated or unsaturated hydrocarbon radical having 1 to 4 carbon atoms.
  • hydrocarbon radicals are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl, isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl or cyclopropyl.
  • aromatic acyl is meant to refer to a unsubstituted or substituted benzoyl group.
  • hydrocarbon moiety refers to a lower alkyl, a lower alkenyl, a lower acyl group or a lower cycloalkyl, i.e., a straight or branched, saturated or unsaturated C i -C 4 hydrocarbon radial.
  • the compound in accordance with the present invention is an active hypocalcemic vitamin D compound.
  • the active vitamin D in accordance with the present invention may have an unsaturated sidechain, e.g., there is suitably a double bond between C-22 and C-23, between C-25 and C-26 or between C-26 and C-27.
  • a hypocalcemic hydroxyvitamin D of the present invention has the general formula described in formula (I)
  • R 1 and R 2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that both R 1 and R 2 cannot both be an alkenyl, or taken together with the carbon to which they are bonded, form a C 3 -C 8 cyclocarbon ring;
  • R 3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl;
  • X 1 is hydrogen or hydroxyl, X 2
  • a l ⁇ -hydroxyvitamin D compound of formula (I) is characterized by the general formula (II):
  • a 1 and A each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23;
  • R 1 and R 2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that both R 1 and R 2 cannot both be an alkenyl, or taken together with the carbon to which they are bonded, form a C 3 -C 8 cyclocarbon ring;
  • R is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, 1
  • O-lower acyl, O-aromatic acyl or lower cycloalkyl X is hydrogen or hydroxyl, X is hydrogen or hydroxyl, or, may be taken with R 1 or R 2 , to constitute a double bond, and Y is a methylene group if the bond to Y is a double bond or is a methyl group or hydrogen if the bond to Y is a single bond.
  • R 1 and R 2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that both R 1 and R 2 cannot both be an alkenyl, or taken together with the carbon to which they are bonded, form a C 3 -C 8 cyclocarbon ⁇ ng; R is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X 1 is hydrogen or hydroxyl, and X
  • hypocalcemic vitamin D compounds of the present invention are those that have effective antiproliferative and cell differentiation activity (i.e., reversal of malignant transformation), but have a lower tendency or inability to cause the undesired side effects of hypercalcemia and/or hypercalciuria.
  • the compounds of the present invention can be administered at dosages that allow them to act as antiproliferative agents and cell differentiation agents when exposed to malignant or other hype ⁇ roliferative cells without significantly altering calcium metabolism. This selectivity and specificity of action makes the hypocalcemic vitamin D compounds useful and preferred agents for safely inhibiting hype ⁇ roliferation and promoting malignant or hype ⁇ lastic cell differentiation.
  • the compounds of the present invention overcome the shortcomings of the known active vitamin D 3 compounds described above, and can be considered preferred agents for the control and treatment of malignant diseases such breast, prostate, testicular and colon cancer, as well as other neoplasms such as pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma, retinoblastoma, and sarcomas of the soft tissue and bone, i.e. neoplasms that express a vitamin D receptor.
  • malignant diseases such breast, prostate, testicular and colon cancer
  • other neoplasms such as pancreatic cancer, endometrial cancer, small
  • the present invention provides a method of treating malignant cells as well as other hype ⁇ roliferative cells, (i.e., inhibiting their hype ⁇ roliferative activity and/or inducing and enhancing their differentiation) with an effective amount of a hypocalcemic vitamin D compound.
  • the effective dosage amount on a daily basis per kilogram of body weight of the patient ranges from about 0.01 ⁇ g/kg/day to about
  • the compounds in accordance with the present invention can be given in daily dose or episodic dose, e.g., once every 2-6 days or once a week, the dose in each day can be a single dose or divided into 2-4 subdoses which can be given, e.g., an hour apart until the total dose is given.
  • the compounds in accordance with the present invention are administered in an amount that raises a serum vitamin D level to a supraphysiological level for a sufficient period of time to induce differentiation or regression of a tumor or neoplasm with causing hypercalcemia.
  • the hypocalcemic properties of the compound permit such supraphysiologic levels.
  • the compounds of formula (I) are valuable for the treatment of cancer and neoplasms in a patient suffering therefrom.
  • the invention is a method for treating a patient suffering from the hype ⁇ roliferative cellular effects of cancer and other neoplasms by administering to the patient a therapeutically effective amount of a compound of formula (I), which is suitably l ⁇ ,24-dihydroxyvitamin D 2 , l ⁇ ,24- dihydroxyvitamin D , l ⁇ ,25-dihydroxyvitamin D 2 , l ⁇ ,25-dihydroxyvitamin D 4 , l ⁇ - hydroxyvitamin D 2 , and l ⁇ -hydroxyvitamin D 4.
  • a compound of formula (I) which has a chiral center in the sidechain, such as at C-24, it is understood that both epimers (e.g., R and S) and the racemic mixture are within the scope of the present invention.
  • the compounds of formula (I) can be prepared as described, e.g., in U.S. Patent 5,488,120 issued to Knutson et al., U.S. Patents 4,554,106, 4,670,190 and 5,486,636 issued to DeLuca et al., and Slitnell et al., 310 Biochem. J. (1995) pp. 233-241, all of which are inco ⁇ orated herein by reference.
  • the biopotencies of the compounds of formula (I) have been studied and compared to that of l ⁇ ,25-dihydroxyvitamin D 3 , the active hormonal form of vitamin D and the standard against which all vitamin D compounds and analogues are measured.
  • VDR vitamin D receptor
  • the vitamin D receptor (VDR) binding affinities of the compounds of formula (I), or their active metabolites are substantially equivalent to (i.e., equal to or up to 3 times weaker than) the affinity of l ⁇ ,25 -dihydroxyvitamm D 3 .
  • Such receptor binding affinities are indicative of potent biological activity.
  • Patent 5,104,864 both of which are inco ⁇ orated herein by reference, it has been shown that l ⁇ -hydroxyvitamin D 2 has the same biopotency as l ⁇ -hydroxyvitamin D 3 and 1 ⁇ ,25 -dihydroxyvitamm D 3 but is much less toxic. Even dosages up to lO ⁇ g/day of l ⁇ -hydroxyvitamin D 2 in women with postmenopausal osteoporosis elicited only mild hypercalciuria (U.Ca >300 mg/24 hrs), and no marked hypercalcemia (S. Ca>11.0 mg/dL) solely due to 1 ⁇ -hydroxyvitamin D 2 was evident.
  • the compound did not adversely affect kidney function, as determined by creatinine clearance and BUN; nor did it increase urinary excretion of hydroxyproline, indicating the absence of any stimulatory effect on bone reso ⁇ tion.
  • Administration of l ⁇ -hydroxyvitamin D 2 to healthy adult males in dosages up to 8 ⁇ g/day showed no clinically significant hypercalcemia or other adverse effects.
  • the compounds of formula (I) are useful as active ingredients in pharmaceutical compositions having reduced side effects and low toxicity as compared with the known analogues of active forms of vitamin D 3 .
  • the pharmacologically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.
  • the hypercalcemic vitamin D compounds of the present invention can be employed in admixtures with conventional excipients, e.g., pharmaceutically acceptable carrier substances suitable for enteral (e.g., oral), parenteral or topical application which do not deleteriously react with the active compounds.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils (e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil, coconut oil), mineral oil, fish liver oils, oily esters such as Polysorbate 80, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
  • vegetable oils e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil, coconut oil
  • mineral oil fish liver oils
  • oily esters such as Polysorbate 80
  • polyethylene glycols gelatine
  • carbohydrates e.g., lactose, amylose or starch
  • magnesium stearate
  • the pharmaceutical preparations can be sterilized and, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or one or more other active compounds, for example, vitamin D 3 and its l ⁇ -hydroxylated metabolites, conjugated estrogens or their equivalents, anti-estrogens, calcitonin, biphosphonates, calcium supplements, cobalamin, pertussis toxin and boron.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or one or more other active compounds, for example, vitamin D 3 and its l ⁇ -hydroxylated metabolites, conjugated estrogens or their equivalents, anti-estrogens, calcitonin, bi
  • parenteral administration suitably includes subcutaneous, intramuscular, or intravenous injection, nasopharyngeal or mucosal abso ⁇ tion, or transdermal abso ⁇ tion.
  • the compounds of formula (I) may be given by direct injection into the tumor, e.g., parathyroid adenoma, or by regional delivery, e.g.,by intraarterial delivery or delivery via the portal vein. Regional delivery is especially suitable for treatment of hepatic cancers. Ampoules are convenient unit dosages.
  • Suitable enteral application particularly suitable are tablets, dragees, liquids, drops, suppositories, lozenges, powders, or capsules.
  • a syrup, elixir, or the like can be used if a sweetened vehicle is desired.
  • suitable nonsprayable viscous, semi-solid or solid forms can be employed which include a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, for example, mineral oil, almond oil, self-emulsifying beeswax, vegetable oil, white soft paraffin, and propylene glycol.
  • suitable formulations include, but are not limited to, creams, ointments, lotions, solutions, suspensions, emulsions, powders, liniments, salves, aerosols, transdermal patches, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, demulsifiers, wetting agents, etc.
  • a cream preparation in accordance with the present invention suitably includes, for example, mixture of water, almond oil, mineral oil and self-emulsifying beeswax; an ointment preparation suitably includes, for example, almond oil and white soft paraffin; and a lotion preparation suitably includes, for example, dry propylene glycol.
  • Topical preparations of the compound in accordance with the present invention useful for the treatment of skin disorders may also include epithelialization-inducing agents such as retinoids (e.g., vitamin A), chromanols such as vitamin E, ⁇ -agonists such as isoproterenol or cyclic adenosine monophosphate (cAMP), anti-inflammatory agents such as corticosteroids (e.g., hydrocortisone or its acetate, or dexamethasone) and keratoplastic agents such as coal tar or anthralin.
  • epithelialization-inducing agents such as retinoids (e.g., vitamin A), chromanols such as vitamin E, ⁇ -agonists such as isoproterenol or cyclic adenosine monophosphate (cAMP), anti-inflammatory agents such as corticosteroids (e.g., hydrocortisone or its acetate, or dexamethasone) and kera
  • Effective amounts of such agents are, for example, vitamin A about 0.003 to about 0.3% by weight of the composition; vitamin E about 0.1 to about 10%; isoproterenol about 0.1 to about 2%; cAMP about 0.1 to about 1%; hydrocortisone about 0.25 to about 5%; coal tar about 0.1 to about 20%; and anthralin about 0.05 to about 2%.
  • the compound is formed into a pharmaceutical composition containing a suppository base such as cacao oil or other triglycerides.
  • a suppository base such as cacao oil or other triglycerides.
  • the composition advantageously includes an antioxidant such as ascorbic acid, butylated hydroxyanisole or hydroquinone.
  • the compound of this invention is dispensed by unit dosage form comprising about 0.5 ⁇ g to about 25 ⁇ g in a pharmaceutically acceptable carrier per unit dosage.
  • the dosage of the compound according to this invention generally is about 0.01 to about 1.0 ⁇ g/kg/day, preferably about 0.04 to about 0.3 ⁇ g/kg/day.
  • Oral dosing for the treatment of cancers and neoplasms and other hype ⁇ roliferative diseases generally is about lO ⁇ g to 200 ⁇ g/day.
  • the dosage of the compound of the present invention in a topical composition generally is about 0.01 ⁇ g to about 50 ⁇ g per gram of composition.
  • the dosage of the hypocalcemic vitamin D compound in a locally applied composition generally is about 0.01 ⁇ g to 100 ⁇ g per gram composition.
  • the dosage of the compounds for the treatment of cancer or neoplasms according to this invention generally is about 0.01 to about 2.0 ⁇ g/kg/day, preferably about 0.01 to about 1.0 ⁇ g/kg/day.
  • dosing of the hypocalcemic vitamin D compounds in accordance with the present invention can be done on an episodic basis, in which higher does can be used, generally about 20 ⁇ g to about 200 ⁇ g given once every 2-7 days.
  • the compounds of this invention are dispensed by unit dosage form in a pharmaceutically acceptable carrier.
  • the actual preferred amounts of active compound in a specific case will vary according to the efficacy of the specific compound employed, the particular compositions formulated, the mode of application, and the particular situs and organism being treated.
  • the specific dose for a particular patient depends on age, body weight, general state of health, on diet, on the timing and mode of administration, on the rate of excretion, and on medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, such as by means of an appropriate conventional pharmacological protocol.
  • agents may suitably include antimetabolites (e.g., 5-fluoro- uracil, methotrexate, fludarabine), antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g.
  • antimetabolites e.g., 5-fluoro- uracil, methotrexate, fludarabine
  • antimicrotubule agents e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel
  • an alkylating agent e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea
  • platinum agents e.g.
  • cisplatin carboplatin, oxaliplatin, JM-216, CI-973
  • anthracyc lines e.g., doxrubicin, daunorubicin
  • antibiolitics e.g., mitomycin, idarubicin, adriamycin, daunomycin
  • topoisomerase inhibitiors e.g., etoposide, camptothecins
  • any other antineoplastic agents estramustine phosphate, prednimustine.
  • co-administration is meant to refer to any administration route in which two or more agents are administered to a patient or subject.
  • the agents may be administered together, or before or after each other.
  • the agents may be administered by different routes, e.g., one agent may be administered intravenously while the second agent is administered intramuscularly, intravenously or orally.
  • the agents may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow both agents to achieve effective concentrations in the body.
  • the agents also may be in an admixture, as, for example, in a single tablet.
  • sequential administration one agent may directly follow administration of the other or the agents may be give episodically, i.e., one can be given at one time followed by the other at a later time, typically within a week.
  • An example of a suitable co- administration regimen is where a hypocalcemic vitamin D compound is administered from 0.5 to 7 days prior to administration of a cytotoxic agent.
  • ⁇ ии also included within the scope of the present invention is the co-administration of effective dosages of hypercalcemic vitamin D compounds in conjunction with administration of hormones or other agents, e.g., estrogens, which are known to ameliorate bone diseases or disorders.
  • hormones or other agents e.g., estrogens
  • prostate cancer often metastasizes to bone, causing bone loss and associated pain.
  • bone agents may include conjugated estrogens or their equivalents, calcitonin, bisphosphonates, calcium supplements, cobalamin, pertussis toxin and boron. Possible dose ranges for these co-administered bone agents are provided in Table 1.
  • Cobalamin ( ⁇ g/day) 5-200 20-100 30-50
  • Pertussis Toxin 0.1-2000 10-1500 100-1000
  • Antiestrogens such as Tamoxifen
  • Tamoxifen are also known bone agents and may be suitably used in conjunction with the l ⁇ -hydroxyvitamin D compounds of the present invention.
  • Example 1 1 ⁇ ,24-dihydroxyvitamin D 2 [ 1 ⁇ ,24-(OH) 2 D 2 ]
  • VDR mammalian vitamin D receptor
  • the affinity of l ⁇ ,24-(OH) 2 D 2 for the mammalian vitamin D receptor (VDR) was assessed using a commercially available kit of bovine thymus VDR and standard l,25-(OH) 2 D 3 solutions from Incstar (Stillwater, Minnesota).
  • the half-maximal binding of chemically synthesized l ⁇ ,24-(OH) 2 D 2 was approximately 150 pg/ml whereas that of l ⁇ ,25-(OH) 2 D 3 was 80 pg/ml.
  • the l ⁇ ,24-(OH) 2 D 2 had a very similar affinity for bovine thymus VDR as did l ⁇ ,25-(OH) 2 D 3 , indicating that 1 ⁇ ,24-(OH) 2 D 2 has potent biological activity.
  • Example 2 1 ⁇ ,24-dihydroxy vitamin D 4 [ 1 ⁇ ,24-(OH) 2 D 4 ]
  • the VDR affinity binding of l ⁇ ,24-(OH) 2 D 4 was investigated.
  • the l ⁇ ,24-(OH) 2 D 4 was incubated with vitamin D receptor and radiolabeled tracer l ⁇ ,25-(OH) 2 D 3 . After incubation, the amount of radioactivity bound to the receptor was determined and compared with the amount bound after co-incubation of unlabeled and labeled l ⁇ ,25-(OH) 2 D 3 . It was found that 50 pg/tube of l ⁇ ,24-(OH) 2 D 4 was equivalent to approximately 20 pg l ⁇ ,25-(OH) 2 D .
  • Example 3 1 ⁇ ,24-dihydroxyvitamin D 2 [ 1 ⁇ ,24-(OH) 2 D 2 ]
  • VDR binding of vitamin D compounds by prostate cells is demonstrated using the techniques of Skowronski et al., 136 Endocrinology (1995) 20-26, which is inco ⁇ orated herein by reference.
  • Prostate-derived cell lines are cultured to near confluence, washed and harvested by scraping. Cells are washed by centrifugation, and the cell pellet resuspended in a buffered salt solution containing protease inhibitors. The cells are disrupted by sonication while cooling on ice. The supernatant obtained from centrifuging the disrupted cells at 207,000 x g for 35 min at 4EC is assayed for binding.
  • Example 4 1 ⁇ ,24-dihydroxy vitamin D 4 [ 1 ⁇ ,24-(OH) 2 D 4 ]
  • the procedure of Example 3 is repeated using the active vitamin D analogue l ⁇ ,24-(OH) 2 D 4 , and the specific binding is determined.
  • the results demonstrate that l ⁇ ,24-(OH) 2 D has strong affinity for prostate VDR, indicating that l ⁇ ,24-(OH) 2 D 4 has potent biological activity in respect of prostate cells.
  • Example 3 The procedure of Example 3 is repeated using the active vitamin D analogue l ⁇ ,25-(OH) 2 D 4 , and the specific binding is determined.
  • the results demonstrate that l ⁇ ,25-(OH) 2 D 4 has strong affinity for prostate VDR, indicating that l ⁇ ,25-(OH) 2 D has potent biological activity in respect of prostate cells.
  • Example 6 1 ⁇ ,24-dihydroxy vitamin D 4 [ 1 ⁇ ,24-(OH) 2 D 4 ]
  • One plasmid contained the gene for Growth Hormone (GH) under the control of the vitamin D responsive element (VDRE) and the other plasmid contained the structural gene for the vitamin D receptor (VDR).
  • GH Growth Hormone
  • VDRE vitamin D responsive element
  • VDR vitamin D receptor
  • Example 7 l ⁇ ,24(S)-dihydroxyvitamin D 2 and l ⁇ ,24(R)-dihydroxy- vitamin D 2 [l ⁇ ,24(S)-(OH) 2 D 2 and l ⁇ ,24(R)-(OH) 2 D 2 ]
  • Example 6 The gene expression study described in Example 6 was conducted to compare the biological activity in vitro of chemically synthesized l ⁇ ,24(S)-(OH) 2 D 2 and l ⁇ ,24(R)-(OH) 2 D 2 , with l ⁇ ,25-(OH) 2 D 3 and 25-OH-D 3 .
  • the vitamin D-dependent transcriptional activation model system was used in which plasmids pSG5-hVDRl/3 and p(CT4) 4 TKGH were co-transfected into Green monkey kidney, COS-1 cells.
  • Example 8 l ⁇ ,24-dihydroxyvitamin D 2 [l ⁇ ,24-(OH) 2 D 2 ]
  • Example 9 1 ⁇ ,24-dihydroxy vitamin D 4 [ 1 ⁇ ,24-(OH) 2 D 4 ]
  • the procedure of Example 8 is repeated using the active vitamin D analogue l ⁇ ,24-(OH) 2 D 4 , and the cell number is determined. Cultures incubated with l ⁇ ,24- (OH) 2 D 4 have significantly fewer cells than the control cultures.
  • Example 10 l ⁇ ,25-dihydroxyvitamin D 4 [l ⁇ ,25-(OH) 2 D 4 ]
  • Example 8 The procedure of Example 8 is repeated using the active vitamin D analogue l ⁇ ,25-(OH) 2 D 4 , and the cell number is determined. Cultures incubated with l ⁇ ,25-(OH) 2 D 4 , and the cell number is determined. Cultures incubated with l ⁇ ,25-(OH) 2 D 4 , and the cell number is determined. Cultures incubated with l ⁇ ,25-(OH) 2 D 4 , and the cell number is determined. Cultures incubated with l ⁇ ,25-
  • Example 11 1 ⁇ ,24-dihydroxyvitamin D 2 [ 1 ⁇ ,24-(OH) 2 D 2 ]
  • cells of the cell line, LNCaP which is derived from a human metastatic prostate adenocarcinoma and known to express PSA
  • LNCaP which is derived from a human metastatic prostate adenocarcinoma and known to express PSA
  • the medium is replenished with medium containing vehicle or the active vitamin D analogue, l ⁇ ,24-(OH) 2 D 2 , at concentrations from 10 " " M to 10 "7 M. After 6-7 days, the medium is removed and stored at -20EC for prostate specific antigen (PSA) analysis.
  • PSA prostate specific antigen
  • the cells from parallel cultures are rinsed, precipitated, and the amount of DNA determined by standard procedures.
  • PSA is measured by standard known methods. Cultures incubated with l ⁇ ,24-(OH) 2 D 2 have significantly more PSA than control cultures when expressed as mass of PSA/cell.
  • Example 12 1 ⁇ ,24-dihydroxyvitamin D 4 [ 1 ⁇ ,24-(OH) 2 D 4 ]
  • Example 12 The procedure of Example 12 is repeated except the active vitamin D analogue is l ⁇ ,24-(OH) 2 D 4 .
  • the PSA is measured and cultures incubated with l ⁇ ,24-(OH) D 4 have significantly more PSA than control cultures when expressed as mass of PSA/cell.
  • Example 12 The procedure of Example 12 is repeated except the active vitamin D analogue is l ⁇ ,25-(OH) 2 D 4 .
  • the PSA is measured and cultures incubated with l ⁇ ,25-(OH) D have significantly more PSA than control cultures when expressed as mass of PSA/cell.
  • Patients with a known vitamin D receptor positive tumor participate in an open-label study of a hypocalcemic vitamin D compound in accordance with the present invention.
  • Patients are placed on a reduced calcium diet prior to treatment, to help minimize intestinal abso ⁇ tion and allow ever higher doses of the hypocalcemic vitamin D.
  • This reduced calcium diet may be continued for the duration of treatment, and for one week after the last dose of the l ⁇ ,24(S)-dihydroxyvitamin D 2 .
  • the diet ideally restricts daily calcium intake to 400- 500 mg. Patients also discontinue use of any vitamin D supplements or vitamin D replacement therapies. Each patient is also asked to drink 4-6 cups of fluid more than usual intake to assure adequate oral hydration.
  • Each subject is monitored at regular intervals for: (1) hypercalcemia, hype ⁇ hosphatemia, hypercalciuria, hype ⁇ hosphaturia and other toxicity; (2) evidence of changes in the progression of metastatic disease; and (3) compliance with the prescribed test drug dosage.
  • the dosing regimen is typically on a daily dose basis of 10 ⁇ g or 20 ⁇ g per day to about 100 ⁇ g/day for 24 months.
  • a non-daily dosing regimen can be used, e.g., 40 ⁇ g given every other day, 100 ⁇ g given once a week.
  • the route of administration can vary from oral to intravenous to regional delivery (e.g., arterial infusion, via the portal vein). Oral is, of course, the easiest and most cost effective route.
  • Regional delivery permits high dosing and generally avoids any production of hypercalcemia.
  • the compound of the present invention the compound is substantially hypocalcemic.
  • CAT CAT
  • scans X-rays and bone scans used for evaluating the progress of metastatic disease or partial remission in many patients treated at the lower dosage , and stable disease and partial or complete remission in many patients treated at the higher dosage.
  • Example 15 Treatment of prostate cancer with l ⁇ ,24-dihydroxy vitamin D 2 [l ⁇ ,24-(OH) 2 D 2 ]
  • the patients are monitored at regular intervals for: (1) hypercalcemia, hype ⁇ hosphatemia, hypercalciuria, hype ⁇ hosphaturia and other toxicity; (2) evidence of changes in the progression of metastatic disease; and (3) compliance with the prescribed test drug dosage.
  • the maximal tolerated dosage (MTD) of daily oral l ⁇ ,24-(OH) 2 D 2 is determined by administering progressively higher dosages to successive groups of patients. All doses are administered in the morning before breakfast.
  • the first group of patients is treated with 25.0 ⁇ g of l ⁇ ,24-(OH) 2 D 2 .
  • Subsequent groups of patients are treated with 50.0, 75.0 and 100.0 ⁇ g/day.
  • Dosing is continued uninterrupted for the duration of the study unless serum calcium exceeds 11.6 mg/dL, or other toxicity of grade 3 or 4 is observed, in which case dosing is held in abeyance until resolution of the observed toxic effect(s) and then resumed at a level which has been decreased by 10.0 ⁇ g.
  • results from the first phase of the study show that the MTD for l ⁇ ,24-(OH) 2 D 2 is above 20.0 ⁇ g/day, a level which is 10- to 40-fold higher than can be achieved with l ⁇ ,25-(OH) 2 D .
  • Analysis of blood samples collected at regular intervals from the participating patients reveal that the levels of circulating l ⁇ ,24-(OH) 2 D 2 increase proportionately with the dosage administered, rising to maximum levels well above lOO pg/mL at the highest dosages, and that circulating levels of l ⁇ ,25-(OH) 2 D are suppressed, often to undetectable levels. Serum and urine calcium are elevated in a dose responsive manner. Patients treated with the MTD of l ⁇ ,24-(OH) 2 D 2 for at least six months report that bone pain associated with metastatic disease is significantly diminished.
  • Example 14 The study of Example 14 is repeated for the active vitamin D compound, l ⁇ -OH-D 2 .
  • the results of the phase one study indicate that patients treated with the MTD of 1 ⁇ -OH-D 2 for at least six months report that bone pain associated with metastatic disease is significantly diminished.
  • the results of the phase two study indicate that after two years, CAT scans, X-rays and bone scans used for evaluating the progression of metastatic disease show stable disease or partial remission in many patients treated at the lower dosage, and stable disease and partial or complete remission in many patients treated at the higher dosage.
  • Example 14 The method of Example 14 is used to treat patients with metastatic malignant melanoma of, e.g., the jaw. After 18 months of treatment, the progress of the metastatic disease shows stable disease or partial remission.
  • Example 18 Treatment of retinoblastoma
  • Example 14 The method of Example 14 is used is used to treat patients with metastatic retinoblastoma. After 18 months of treatment, the progress of the metastatic disease shows stable disease or partial remission.
  • Example 14 The method of Example 14 is used to treat patients with hepatoma.
  • the regional delivery of the compound in accordance with the present invention i.e., via arterial infusion, is used. After 18 months of treatment, the progress of the metastatic disease shows stable disease or partial remission.

Abstract

Methods for the utilization of hypocalcemic vitamin D analogy to inhibit the hyperproliferation of malignant or neoplastic cells without incidence of hypercalcemia.

Description

METHOD OF TREATING HYPERPROLIFERATIVE DISEASES USING ACTIVE VITAMIN D ANALOGUES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. application Serial No. 09/596,149, filed February 23, 1998, which is a divisional of U.S. application Serial No. 08/781,910, filed December 30, 1996, now U.S. Patent No. 5,763,429, all of which are incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not Applicable
BACKGROUND OF THE INVENTION
This invention relates generally to a method of treating hyperprohferative diseases, and in particular, to the use of active forms of hypocalcemic vitamin D to inhibit the hyperprohferative cellular activity of these diseases and to promote differentiation of the cells.
Extensive research during the past two decades has established important biologic roles for vitamin D apart from its classic role in bone and mineral metabolism. Specific nuclear receptors for lα,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, are present in cells from diverse organs not involved in calcium homeostasis. For example, specific, biologically active vitamin D receptors have been demonstrated in the human prostatic carcinoma cell line, LNCaP, (Miller et al., 52 Cancer Res. (1992) 515-520); Vitamin D receptors have also been described for many other neoplastic cells, e.g., carcinomas of the breast and the colon.
It has been reported that certain vitamin D compounds and analogues are potent inhibitors of malignant cell proliferation and are inducers/stimulators of cell differentiation. For example, U.S. Patent No. 4,391,802 issued to Suda et al. discloses that lα-hydroxyvitamin D compounds, specifically lα,25-dihydroxyvitamin D and lα-hydroxyvitamin D3, possess potent antileukemic activity by virtue of inducing the differentiation of malignant cells (specifically leukemia cells) to nonmalignant macrophages (monocytes), and are useful in the treatment of leukemia. Antiproliferative and differentiating actions of lα,25-dihydroxyvitamin D and other vitamin D3 analogues have been reported with respect to cancer cell lines. More recently, an association between vitamin D receptor gene polymorphism and cancer risk has been reported, suggesting that vitamin D receptors may have a role in the development, and possible treatment, of cancer.
These previous studies have focused exclusively on vitamin D3 compounds. Even though these compounds may indeed be highly effective in promoting differentiation in malignant cells in culture, their practical use in differentiation therapy as anticancer agents is severely limited because of their equally high potency as agents affecting calcium metabolism. At the levels required in vivo for effective use as, for example, antileukemic agents, these same compounds can induce markedly elevated and potentially dangerous blood calcium levels by virtue of their inherent calcemic activity. That is, the clinical use of lα,25-dihydroxyvitamin D3 and other vitamin D analogues as anticancer agents is precluded, or severely limited, by the risk of hypercalcemia. This indicates a need for compounds with greater specific activity and selectivity of action, i.e., vitamin D compounds with antiproliferative and differentiating effects but which have less calcemic activity.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method of treating hyperprohferative disease conditions such as those characterized by hyperproliferative cell growth and/or abnormal cell differentiation. The method includes use of active vitamin D compounds to inhibit abnormal cell growth and promote cell differentiation.
The foregoing, and other advantages of the present invention, are realized in one aspect thereof in a method of inhibiting the hyperproliferative activity of neoplastic or hyperplastic cells, comprising treating the cells with an effective amount of a hypocalcemic vitamin D compound. The treating step includes inhibiting proliferation of, and inducing and enhancing differentiation in such cells.
The hypocalcemic vitamin D compounds of the present invention include vitamin D compounds having a hydrocarbon moiety substituted at the C-24 position on the sidechain of the molecule and a hydroxy group substituted in at least one of the Ci, C24 or C25 positions.
The vitamin D compound of the present invention is an active vitamin D and is suitably represented by the formula (I) described hereafter. The compounds of formula (I) suitably include lα,24-dihydroxyvitamin D2, lα,24-dihydroxyvitamin D4, lα,25- dihydroxyvitamm D , lα,25-dihydroxyvitamin D2, lα-hydroxyvitamin D and lα- hydroxyvitamin D4.
Hypocalcemic vitamin D compounds are valuable for the treatment of breast and colon cancer, as well as other neoplasms such as pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell cancer of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma, retinoblastoma, and sarcomas of the soft tissue and bone.
In accordance with the present invention, when effective amounts of hypocalcemic vitamin D compounds are administered to patients with cancer or neoplasms, the proliferative activity of the abnormal neoplastic cells is inhibited, reduced, or stabilized, and cell differentiation is induced, promoted or enhanced, with significantly less hypercalcemia and hypercalciuria than is observed after the same amount of an activated vitamin D3 (e.g., lα-OH D3, lα,25-(OH)2 D3) is administered in previously known formulations. Thus, the compound in accordance with the present invention has an improved therapeutic index relative to active forms of vitamin D3 analogues. Accordingly, another aspect of the invention is a method of treating human cancer comprising administering to a subject who has cancer an effective amount of hypocalcemic vitamin D compound which has or attains through metabolism in vivo, a vitamin D receptor (VDR) binding affinity substantially equivalent to the binding affinity of lα,25-dihydroxyvitamin D3 and a hypercalcemia risk substantially lower that that of lα,25-dihydroxyvitamin D3, to inhibit, decrease or stabilize the cellular abnormal proliferative activity of the cancer.
For treatment for malignant conditions in accordance with the present invention, the hypocalcemic vitamin D compounds can be suitably administered alone as an active ingredient, as an antiproliferative agent in a pharmaceutical composition, or co- administered with an anticancer agent.
Further, included within the scope of the present invention is the co-administration of the vitamin D of formula (I) with a cytotoxic or anticancer agent. Such agents suitably include antimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine), antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g. cisplatin, carboplatin, oxaliplatin, JM-216, CI-973), anthracyclines (e.g., doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin, idarubicin, adriamycin, daunomycin), topoisomerase inhibitiors (e.g., etoposide, camptothecins) or any other antineoplastic agents, (estramustine phosphate, prednimustine).
It is anticipated that the hypocalcemic vitamin D compounds used in combination with various anticancer drugs can give rise to a significantly enhanced cytotoxic effect on cancerous cells, thus providing an increased therapeutic effect. Specifically, as a significantly increased growth-inhibitory effect is obtained with the above disclosed combinations utilizing lower concentrations of the anticancer drugs compared to the treatment regimes in which the drugs are used alone, there is the potential to provide therapy wherein adverse side effects associated with the anticancer drugs are considerably reduced than normally observed with the anticancer drugs used alone in larger doses. Possible dose ranges of these co-administered anticancer agents are about 0.1 to 20 mg/kg/day.
Also included within the scope of the present invention is the co-administration of effective dosages of the analogue of formula (I) in conjunction with administration of hormones or other agents, e.g., estrogens, which are known to ameliorate bone diseases or disorders. For example, prostate cancer often metastasizes to bone, causing bone loss and associated pain. Such bone agents may include conjugated estrogens or their equivalents, calcitonin, bisphosphonates, calcium supplements, cobalamin, pertussis toxin and boron.
In another aspect, the invention is a pharmaceutical composition which includes an anticancer agent which is an active vitamin D compound; an agent selected from the group consisting of (i) an anticancer agent, (ii) a bone agent, and combinations thereof; and a physiologically acceptable carrier.
Other advantages and a fuller appreciation of specific adaptations, compositional variations, and physical attributes will be gained upon an examination of the following detailed description of preferred embodiments, taken in conjunction with the appended claims.
BRIEF DESCRIPTION OF THE DRAWING(S)
Not Applicable
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an effective method for the treatment of neoplasms and hyperproliferative diseases. Particularly, the present invention relates to therapeutic methods for inhibiting, reducing or stabilizing the hyperproliferative cellular activity of diseased cells, and inducing, enhancing or promoting cell differentiation in the diseased cells. The present invention provides a novel treatment of a patient suffering from a hyperproliferative disease such as prostatic cancer or prostatic hypeφlasia with a hypocalcemic hydroxyvitamin D analogue. The vitamin D analogue is suitably a lα-hydroxyvitamin D or a 24-hydroxyvitamin D compound. The hypocalcemic hydroxyvitamin D analogue represented by formula (I) as described hereinbelow is provided to the patient without causing dose-limiting hypercalcemia and hypercalciuria, i.e., unphysiologically high and deleterious blood calcium levels and urine calcium levels, respectively. These attributes are achieved through specific chemical properties of the hypocalcemic vitamin D compounds as described.
In accordance with the present invention, when effective amounts of the hypocalcemic vitamin D compounds are administered to patients with cancer or hypeφlasia, the proliferative activity of the abnormal cells is inhibited, maintained, or alleviated, and cell differentiation is induced, promoted or enhanced, with significantly less hypercalcemia and hypercalciuria than is observed after the same amount of activated vitamin D3 is administered in previously known formulations. Thus, the hypocalcemic vitamin D compounds of the present invention have an improved therapeutic index relative to active forms of vitamin D3 analogues.
It is known that vitamin D3 must be hydroxylated in the C-1 and C-25 positions before it is activated, i.e., before it will produce a biological response. A similar metabolism appears to be required to activate other forms of vitamin D, e.g., vitamin D2 and vitamin D4. Therefore, as used herein, the term "activated vitamin D" or "active vitamin D" is intended to refer to a vitamin D compound or analogue that has been hydroxylated in at least the C-1, C-24 or C-25 position of the molecule and either the compound itself or its metabolites in the case of a prodrug, such as lα-hydroxyvitamin D2, binds the vitamin D receptor (VDR). For example, vitamin D "prodrugs" include compounds which are hydroxylated in the C-1 position. Such compounds undergo further hydroxylation in vivo and their metabolites bind the VDR.
The term "hypocalcemic vitamin D compound" is in reference to active vitamin
D analogs which demonstrate reduced calcemic activity relative to the calcemic activity of lα,25-dihydroxyvitamin D3. Such compounds include 24-hydroxyvitamin D compounds, 25-hydroxyvitamin D compounds and lα-hydroxyvitamin D compounds. The calcemic activity of these compounds ranges from 0.001 to 0.5 that of lα,25- dihydroxyvitamin D3. Also, as used herein, the term "lower" as a modifier for alkyl, alkenyl acyl, or cycloalkyl is meant to refer to a straight or branched, saturated or unsaturated hydrocarbon radical having 1 to 4 carbon atoms. Specific examples of such hydrocarbon radicals are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, ethenyl, propenyl, butenyl, isobutenyl, isopropenyl, formyl, acetyl, propionyl, butyryl or cyclopropyl. The term "aromatic acyl" is meant to refer to a unsubstituted or substituted benzoyl group.
As used herein, the term "hydrocarbon moiety" refers to a lower alkyl, a lower alkenyl, a lower acyl group or a lower cycloalkyl, i.e., a straight or branched, saturated or unsaturated C i -C4 hydrocarbon radial.
The compound in accordance with the present invention is an active hypocalcemic vitamin D compound. Further, the active vitamin D in accordance with the present invention may have an unsaturated sidechain, e.g., there is suitably a double bond between C-22 and C-23, between C-25 and C-26 or between C-26 and C-27.
A hypocalcemic hydroxyvitamin D of the present invention has the general formula described in formula (I)
Figure imgf000008_0001
1 7 wherein A and A each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that both R1 and R2 cannot both be an alkenyl, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, X2 is hydrogen or hydroxyl, or, may be taken with R1 or R2, to constitute a double bond, X3 is hydrogen or hydroxyl provided that at least one of X , X and X is hydroxyl; and Y is a methylene group if the bond to Y is a double bond or is a methyl group or hydrogen if the bond to Y is a single bond.
A lα-hydroxyvitamin D compound of formula (I) is characterized by the general formula (II):
o
Figure imgf000009_0001
if
wherein A1 and A each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that both R1 and R2 cannot both be an alkenyl, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, 1
O-lower acyl, O-aromatic acyl or lower cycloalkyl; X is hydrogen or hydroxyl, X is hydrogen or hydroxyl, or, may be taken with R1 or R2, to constitute a double bond, and Y is a methylene group if the bond to Y is a double bond or is a methyl group or hydrogen if the bond to Y is a single bond.
Specific lα-hydroxyvitamin D compounds in accordance with the present invention are characterized by the general formula (III):
Figure imgf000010_0001
1 9 wherein A and A each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that both R1 and R2 cannot both be an alkenyl, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon πng; R is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, and X2 is hydrogen or hydroxyl, or, may be taken with R1 or R2, to constitute a double bond.
The hypocalcemic vitamin D compounds of the present invention are those that have effective antiproliferative and cell differentiation activity (i.e., reversal of malignant transformation), but have a lower tendency or inability to cause the undesired side effects of hypercalcemia and/or hypercalciuria. In other words, the compounds of the present invention can be administered at dosages that allow them to act as antiproliferative agents and cell differentiation agents when exposed to malignant or other hypeφroliferative cells without significantly altering calcium metabolism. This selectivity and specificity of action makes the hypocalcemic vitamin D compounds useful and preferred agents for safely inhibiting hypeφroliferation and promoting malignant or hypeφlastic cell differentiation. The compounds of the present invention, thus, overcome the shortcomings of the known active vitamin D3 compounds described above, and can be considered preferred agents for the control and treatment of malignant diseases such breast, prostate, testicular and colon cancer, as well as other neoplasms such as pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma, retinoblastoma, and sarcomas of the soft tissue and bone, i.e. neoplasms that express a vitamin D receptor.
Thus, the present invention provides a method of treating malignant cells as well as other hypeφroliferative cells, (i.e., inhibiting their hypeφroliferative activity and/or inducing and enhancing their differentiation) with an effective amount of a hypocalcemic vitamin D compound. The effective dosage amount on a daily basis per kilogram of body weight of the patient ranges from about 0.01 μg/kg/day to about
2.0 μg/kg/day. The compounds in accordance with the present invention can be given in daily dose or episodic dose, e.g., once every 2-6 days or once a week, the dose in each day can be a single dose or divided into 2-4 subdoses which can be given, e.g., an hour apart until the total dose is given. The compounds in accordance with the present invention are administered in an amount that raises a serum vitamin D level to a supraphysiological level for a sufficient period of time to induce differentiation or regression of a tumor or neoplasm with causing hypercalcemia. The hypocalcemic properties of the compound permit such supraphysiologic levels.
The compounds of formula (I) are valuable for the treatment of cancer and neoplasms in a patient suffering therefrom. In particular, the invention is a method for treating a patient suffering from the hypeφroliferative cellular effects of cancer and other neoplasms by administering to the patient a therapeutically effective amount of a compound of formula (I), which is suitably lα,24-dihydroxyvitamin D2, lα,24- dihydroxyvitamin D , lα,25-dihydroxyvitamin D2, lα,25-dihydroxyvitamin D4, lα- hydroxyvitamin D2, and lα-hydroxyvitamin D4. Among those compounds of formula (I) that have a chiral center in the sidechain, such as at C-24, it is understood that both epimers (e.g., R and S) and the racemic mixture are within the scope of the present invention.
The compounds of formula (I) can be prepared as described, e.g., in U.S. Patent 5,488,120 issued to Knutson et al., U.S. Patents 4,554,106, 4,670,190 and 5,486,636 issued to DeLuca et al., and Strugnell et al., 310 Biochem. J. (1995) pp. 233-241, all of which are incoφorated herein by reference.
The biopotencies of the compounds of formula (I) have been studied and compared to that of lα,25-dihydroxyvitamin D3, the active hormonal form of vitamin D and the standard against which all vitamin D compounds and analogues are measured. For example, it has been found that the vitamin D receptor (VDR) binding affinities of the compounds of formula (I), or their active metabolites, are substantially equivalent to (i.e., equal to or up to 3 times weaker than) the affinity of lα,25 -dihydroxyvitamm D3. Such receptor binding affinities are indicative of potent biological activity.
At the same time, it has been found that compounds of formula (I) are significantly less toxic than their corresponding vitamin D analogues. For example, in parent co-pending application, Ser. No. 08/265,438, the disclosure of which is incoφorated herein by reference, the LD50 for lα-hydroxyvitamin D was found to be 1.0 mg/kg in males and 3.0 mg/kg in females, i.e., substantially less toxic than lα-hydroxyvitamin D3 (LD50 ~ 0.2 mg/kg). Further, in the parent U.S. Patent No. 5,403,831, and its grandparent U.S. Patent 5,104,864, both of which are incoφorated herein by reference, it has been shown that lα-hydroxyvitamin D2 has the same biopotency as lα-hydroxyvitamin D3 and 1 α,25 -dihydroxyvitamm D3 but is much less toxic. Even dosages up to lO μg/day of lα-hydroxyvitamin D2 in women with postmenopausal osteoporosis elicited only mild hypercalciuria (U.Ca >300 mg/24 hrs), and no marked hypercalcemia (S. Ca>11.0 mg/dL) solely due to 1 α-hydroxyvitamin D2 was evident. Additionally, the compound did not adversely affect kidney function, as determined by creatinine clearance and BUN; nor did it increase urinary excretion of hydroxyproline, indicating the absence of any stimulatory effect on bone resoφtion. Administration of lα-hydroxyvitamin D2 to healthy adult males in dosages up to 8 μg/day showed no clinically significant hypercalcemia or other adverse effects.
The compounds of formula (I) are useful as active ingredients in pharmaceutical compositions having reduced side effects and low toxicity as compared with the known analogues of active forms of vitamin D3.
The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans. For example, the hypercalcemic vitamin D compounds of the present invention can be employed in admixtures with conventional excipients, e.g., pharmaceutically acceptable carrier substances suitable for enteral (e.g., oral), parenteral or topical application which do not deleteriously react with the active compounds.
Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils (e.g., almond oil, corn oil, cottonseed oil, peanut oil, olive oil, coconut oil), mineral oil, fish liver oils, oily esters such as Polysorbate 80, polyethylene glycols, gelatine, carbohydrates (e.g., lactose, amylose or starch), magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
The pharmaceutical preparations can be sterilized and, if desired, be mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or one or more other active compounds, for example, vitamin D3 and its lα-hydroxylated metabolites, conjugated estrogens or their equivalents, anti-estrogens, calcitonin, biphosphonates, calcium supplements, cobalamin, pertussis toxin and boron.
For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solution, as well as suspensions, emulsions, or implants, including suppositories. Parenteral administration suitably includes subcutaneous, intramuscular, or intravenous injection, nasopharyngeal or mucosal absoφtion, or transdermal absoφtion. Where indicated, the compounds of formula (I) may be given by direct injection into the tumor, e.g., parathyroid adenoma, or by regional delivery, e.g.,by intraarterial delivery or delivery via the portal vein. Regional delivery is especially suitable for treatment of hepatic cancers. Ampoules are convenient unit dosages.
For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, lozenges, powders, or capsules. A syrup, elixir, or the like can be used if a sweetened vehicle is desired.
For topical application, suitable nonsprayable viscous, semi-solid or solid forms can be employed which include a carrier compatible with topical application and having a dynamic viscosity preferably greater than water, for example, mineral oil, almond oil, self-emulsifying beeswax, vegetable oil, white soft paraffin, and propylene glycol. Suitable formulations include, but are not limited to, creams, ointments, lotions, solutions, suspensions, emulsions, powders, liniments, salves, aerosols, transdermal patches, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, demulsifiers, wetting agents, etc. A cream preparation in accordance with the present invention suitably includes, for example, mixture of water, almond oil, mineral oil and self-emulsifying beeswax; an ointment preparation suitably includes, for example, almond oil and white soft paraffin; and a lotion preparation suitably includes, for example, dry propylene glycol.
Topical preparations of the compound in accordance with the present invention useful for the treatment of skin disorders may also include epithelialization-inducing agents such as retinoids (e.g., vitamin A), chromanols such as vitamin E, β-agonists such as isoproterenol or cyclic adenosine monophosphate (cAMP), anti-inflammatory agents such as corticosteroids (e.g., hydrocortisone or its acetate, or dexamethasone) and keratoplastic agents such as coal tar or anthralin. Effective amounts of such agents are, for example, vitamin A about 0.003 to about 0.3% by weight of the composition; vitamin E about 0.1 to about 10%; isoproterenol about 0.1 to about 2%; cAMP about 0.1 to about 1%; hydrocortisone about 0.25 to about 5%; coal tar about 0.1 to about 20%; and anthralin about 0.05 to about 2%.
For rectal administration, the compound is formed into a pharmaceutical composition containing a suppository base such as cacao oil or other triglycerides. To prolong storage life, the composition advantageously includes an antioxidant such as ascorbic acid, butylated hydroxyanisole or hydroquinone.
For treatment of calcium metabolic disorders, oral administration of the pharmaceutical compositions of the present invention is preferred. Generally, the compound of this invention is dispensed by unit dosage form comprising about 0.5 μg to about 25 μg in a pharmaceutically acceptable carrier per unit dosage. The dosage of the compound according to this invention generally is about 0.01 to about 1.0 μg/kg/day, preferably about 0.04 to about 0.3 μg/kg/day. Oral dosing for the treatment of cancers and neoplasms and other hypeφroliferative diseases generally is about lOμg to 200μg/day.
For topical treatment of skin disorders, the dosage of the compound of the present invention in a topical composition generally is about 0.01 μg to about 50 μg per gram of composition. For treatment of cancers, the dosage of the hypocalcemic vitamin D compound in a locally applied composition generally is about 0.01 μg to 100 μg per gram composition.
Oral administration of the pharmaceutical compositions of the present invention is preferred. The dosage of the compounds for the treatment of cancer or neoplasms according to this invention generally is about 0.01 to about 2.0 μg/kg/day, preferably about 0.01 to about 1.0 μg/kg/day. As noted above, dosing of the hypocalcemic vitamin D compounds in accordance with the present invention can be done on an episodic basis, in which higher does can be used, generally about 20 μg to about 200 μg given once every 2-7 days. Generally, the compounds of this invention are dispensed by unit dosage form in a pharmaceutically acceptable carrier.
Those of ordinary skill in the art will readily optimize effective doses and coadministration regimens as determined by good medical practice and the clinical condition of the individual patient. Regardless of the manner of administration, it will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the efficacy of the specific compound employed, the particular compositions formulated, the mode of application, and the particular situs and organism being treated. For example, the specific dose for a particular patient depends on age, body weight, general state of health, on diet, on the timing and mode of administration, on the rate of excretion, and on medicaments used in combination and the severity of the particular disorder to which the therapy is applied. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, such as by means of an appropriate conventional pharmacological protocol.
Further, included within the scope of the present invention is a method of co-administration of hypercalemic vitamin D compounds with an anticancer or antineoplastic agent. Such agents may suitably include antimetabolites (e.g., 5-fluoro- uracil, methotrexate, fludarabine), antimicrotubule agents (e.g., vincristine, vinblastine, taxanes such as paclitaxel, docetaxel), an alkylating agent (e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea), platinum agents (e.g. cisplatin, carboplatin, oxaliplatin, JM-216, CI-973), anthracyc lines (e.g., doxrubicin, daunorubicin), antibiolitics (e.g., mitomycin, idarubicin, adriamycin, daunomycin), topoisomerase inhibitiors (e.g., etoposide, camptothecins) or any other antineoplastic agents, (estramustine phosphate, prednimustine). It is anticipated that hypercalcemic vitamin D compounds used in combination with various anticancer drugs can give rise to a significantly enhanced cytotoxic effect on cancerous cells, thus providing an increased therapeutic effect. Specifically, as a significantly increased growth-inhibitory effect is obtained with the above disclosed combinations utilizing lower concentrations of the anticancer drugs compared to the treatment regimes in which the drugs are used alone, there is the potential to provide therapy wherein adverse side effects associated with the anticancer drugs are considerably reduced than normally observed with the anticancer drugs used alone in larger doses. Possible dose ranges of these co- administered anticancer agents are about 0.1 to 20 mg/kg/day. The term "co-administration" is meant to refer to any administration route in which two or more agents are administered to a patient or subject. For example, the agents may be administered together, or before or after each other. The agents may be administered by different routes, e.g., one agent may be administered intravenously while the second agent is administered intramuscularly, intravenously or orally. The agents may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow both agents to achieve effective concentrations in the body. The agents also may be in an admixture, as, for example, in a single tablet. In sequential administration, one agent may directly follow administration of the other or the agents may be give episodically, i.e., one can be given at one time followed by the other at a later time, typically within a week. An example of a suitable co- administration regimen is where a hypocalcemic vitamin D compound is administered from 0.5 to 7 days prior to administration of a cytotoxic agent.
Also included within the scope of the present invention is the co-administration of effective dosages of hypercalcemic vitamin D compounds in conjunction with administration of hormones or other agents, e.g., estrogens, which are known to ameliorate bone diseases or disorders. For example, prostate cancer often metastasizes to bone, causing bone loss and associated pain. Such bone agents may include conjugated estrogens or their equivalents, calcitonin, bisphosphonates, calcium supplements, cobalamin, pertussis toxin and boron. Possible dose ranges for these co-administered bone agents are provided in Table 1.
TABLE 1
Possible Oral Dose Ranges for Various Bone Agents Co-Administered With lα-Hydroxyvitamin D of Formula (I)
Agent Dose Ranges
Broad Preferred Most Preferred
Conjugated Estrogens or Equivalent (mg/day) 0.3-5.0 0.4-2.4 0.6-1.2
Sodium Fluoride (mg/day) 5-150 30-75 40-60
Calcitonin (IU/day) 5-800 25-500 50-200
Bisphosphonates (mg/day) 0.5-20 1-15 5-10
Calcium Supplements (mg/day) 250-2500 500-1500 750-1000
Cobalamin (μg/day) 5-200 20-100 30-50
Pertussis Toxin (mg/day) 0.1-2000 10-1500 100-1000
Boron (mg/day) 0.10-3000 1-250 2-100
Antiestrogens, such as Tamoxifen , are also known bone agents and may be suitably used in conjunction with the lα-hydroxyvitamin D compounds of the present invention.
The present invention is further explained by the following examples which should not be construed by way of limiting the scope of the present invention.
VDR BINDING ANALYSES
Example 1 : 1 α,24-dihydroxyvitamin D2 [ 1 α,24-(OH)2D2]
The affinity of lα,24-(OH)2D2 for the mammalian vitamin D receptor (VDR) was assessed using a commercially available kit of bovine thymus VDR and standard l,25-(OH)2D3 solutions from Incstar (Stillwater, Minnesota). The half-maximal binding of chemically synthesized lα,24-(OH)2D2 was approximately 150 pg/ml whereas that of lα,25-(OH)2D3 was 80 pg/ml. Thus, the lα,24-(OH)2D2 had a very similar affinity for bovine thymus VDR as did lα,25-(OH)2D3, indicating that 1 α,24-(OH)2D2 has potent biological activity.
Example 2 : 1 α,24-dihydroxy vitamin D4 [ 1 α,24-(OH)2D4]
The VDR affinity binding of lα,24-(OH)2D4 was investigated. The lα,24-(OH)2D4 was incubated with vitamin D receptor and radiolabeled tracer lα,25-(OH)2D3. After incubation, the amount of radioactivity bound to the receptor was determined and compared with the amount bound after co-incubation of unlabeled and labeled lα,25-(OH)2D3. It was found that 50 pg/tube of lα,24-(OH)2D4 was equivalent to approximately 20 pg lα,25-(OH)2D .
These results show that lα,24-(OH)2D4 binds slightly less tightly to the vitamin D receptor than does lα,25-(OH)2D3. Such data mean that lα,24-(OH)2D4 has high affinity for the VDR and significant biological activity, similar to that of lα,25- (OH)2D3. These data are consistent with gene expression studies done (described below) with lα,24-(OH)2D4 which demonstrate that lα,24-(OH)2D4 is only slightly less active than is lα,25-(OH)2D3.
These results are suφrising and unexpected in view of the prior art. They are contrary to the normative wisdom in the vitamin D art regarding the very low degree of biological activity of vitamin D4 compounds.
Example 3 : 1 α,24-dihydroxyvitamin D2 [ 1 α,24-(OH)2D2]
VDR binding of vitamin D compounds by prostate cells is demonstrated using the techniques of Skowronski et al., 136 Endocrinology (1995) 20-26, which is incoφorated herein by reference. Prostate-derived cell lines are cultured to near confluence, washed and harvested by scraping. Cells are washed by centrifugation, and the cell pellet resuspended in a buffered salt solution containing protease inhibitors. The cells are disrupted by sonication while cooling on ice. The supernatant obtained from centrifuging the disrupted cells at 207,000 x g for 35 min at 4EC is assayed for binding. 200 TL of soluble extract, (1-2 mg protein ml supernatant) is incubated with a 1 nM 3H-lα,25-(OH)2D3 and increasing concentrations of lα,24-(OH)2-D2 (0.01-100 nM) for 16-20 hr at 4EC. Bound and free hormones are separated with hydroxylapatite using standard procedures. Specific binding is calculated by subtracting nonspecific binding obtained in the presence of a 250-fold excess of nonradioactive lα,25-(OH)2D3 from the total binding measured. The results demonstrate that lα,24-(OH)2D2 has strong affinity for prostate VDR, indicating that lα,24-(OH)2D2 has potent biological activity in respect of prostate cells.
Example 4 : 1 α,24-dihydroxy vitamin D4 [ 1 α,24-(OH)2D4] The procedure of Example 3 is repeated using the active vitamin D analogue lα,24-(OH)2D4, and the specific binding is determined. The results demonstrate that lα,24-(OH)2D has strong affinity for prostate VDR, indicating that lα,24-(OH)2D4 has potent biological activity in respect of prostate cells.
Example 5: lα,25-dihydroxyvitamin D [lα,25-(OH)2D4]
The procedure of Example 3 is repeated using the active vitamin D analogue lα,25-(OH)2D4, and the specific binding is determined. The results demonstrate that lα,25-(OH)2D4 has strong affinity for prostate VDR, indicating that lα,25-(OH)2D has potent biological activity in respect of prostate cells.
GENE EXPRESSION
Example 6 : 1 α,24-dihydroxy vitamin D4 [ 1 α,24-(OH)2D4]
Using the plasmids p(CT4)4TKGH, a vitamin D receptor (VDR)-expressing plasmid, and pSG5-hVDRl/3, a plasmid containing a Growth Hormone (GH) gene, under the control of a vitamin D-responsive element (VDRE), experiments were conducted to explore the ability of lα,24-(OH)2D4 to induce vitamin D-dependent growth hormone acting as a reporter gene compared to that of lα,25-(OH)2D3. Cells in culture were transfected with these two plasmids. One plasmid contained the gene for Growth Hormone (GH) under the control of the vitamin D responsive element (VDRE) and the other plasmid contained the structural gene for the vitamin D receptor (VDR). These transfected cultures were incubated with lα,24-(OH)2D4 or lα,25-(OH)2D3, and the production of growth hormone was measured. Table 2 below shows the results of this assay:
TABLE 2
Induction of Growth Hormone by Vitamin D Compounds
Compound Concentration Growth Hormone Used (M) Induction (ng/ml) l,25-(OH)2D3 1 x 10'10 39 l,25-(OH)2D3 5 x 10"10 248 l,24-(OH)2D4 5 x 10"10 165 l,24-(OH)2D4 1 x 10"9 628 l,24-(OH)2D4 5 x 10"9 1098
These data show that the ability of lα,24-(OH)2D4 to stimulate vitamin D- dependent growth hormone is nearly equivalent to that of lα,25-(OH)2D3. Such results are truly suφrising and would not have been expected by following the teachings of the prior art.
Example 7: lα,24(S)-dihydroxyvitamin D2 and lα,24(R)-dihydroxy- vitamin D2 [lα,24(S)-(OH)2D2 and lα,24(R)-(OH)2D2]
The gene expression study described in Example 6 was conducted to compare the biological activity in vitro of chemically synthesized lα,24(S)-(OH)2D2 and lα,24(R)-(OH)2D2, with lα,25-(OH)2D3 and 25-OH-D3. The vitamin D-dependent transcriptional activation model system was used in which plasmids pSG5-hVDRl/3 and p(CT4)4TKGH were co-transfected into Green monkey kidney, COS-1 cells.
Transfected cells were incubated with vitamin D metabolites and growth hormone production was measured. As shown in Table 3, both lα,24(S)-(OH)2D2 and its epimer, lα,24(R)-(OH) D2, had significantly more activity in this system than 25-OH-D3, with lα,24(S)-(OH)2D2 having nearly the same activity as lα,25-(OH)2D3.
TABLE 3
Vitamin D-Inducible Growth Hormone Production In Transfected COS-1 Cells
Vitamin DCInducible Growth Hormone
Production
Net
Total GH vitamin DCinducible
Molar Production* GH-production
Inducer Concentration (ng/ml) (nε/ml)
Ethanol 44 0
25-OH-D3 lxlO"7 245 201 lxlO"6 1100 1056 lxlO"5 775 731 lα,25-(OH)2D3 lxlO'10 74 30 lxlO"9 925 881 lxlO"8 1475 1441 lα,24(S)-(OH)2D2 5x10-'° 425 381 5xl0"9 * 1350 1306 5xl0-8 1182 1138 lα,24(R)-(OH)2D lxlO"9 80 36
2 lxlO"8 1100 1056 lxlO"7 1300 1256
* Averages of duplicate determinations
INHIBITION OF CELL PROLIFERATION
Example 8: lα,24-dihydroxyvitamin D2 [lα,24-(OH)2D2]
Inhibition of cell proliferation is demonstrated using the techniques of Skowronski et al., 132 Endocrinology (1993) 1952-1960 and 136 Endocrinology (1995)
20-26, both of which are incoφorated herein by reference. The cell lines, LNCaP and PC-3, which are derived from human prostate adenocarcinoma, are seeded in six-well tissue culture plates at a density of about 50,000 cells/plate. After the cells have attached and stabilized, about 2-3 days, the medium is replenished with medium containing vehicle or the active vitamin D analogue lα,24-(OH)2D2, at concentrations
1 1 7 from 10" M to 10" M. Medium containing test analogue or vehicle is replaced every three days. After 6-7 days, the medium is removed, the cells are rinsed, precipitated with cold 5% trichloroacetic acid, and washed with cold ethanol. The cells are solubilized with 0.2 N sodium hydroxide, and the amount of DNA determined by standard procedures. The results show that cultures incubated with lα,24-(OH)2D in accordance with the present invention have significantly fewer cells than the control cultures.
Example 9 : 1 α,24-dihydroxy vitamin D4 [ 1 α,24-(OH)2D4] The procedure of Example 8 is repeated using the active vitamin D analogue lα,24-(OH)2D4, and the cell number is determined. Cultures incubated with lα,24- (OH)2D4 have significantly fewer cells than the control cultures.
Example 10: lα,25-dihydroxyvitamin D4 [lα,25-(OH)2D4]
The procedure of Example 8 is repeated using the active vitamin D analogue lα,25-(OH)2D4, and the cell number is determined. Cultures incubated with lα,25-
(OH)2D4 have significantly fewer cells than the control cultures.
STIMULATION OF CELL DIFFERENTIATION
Example 11: 1 α,24-dihydroxyvitamin D2 [ 1 α,24-(OH)2D2]
Using the techniques of Skowronski et al., 132 Endocrinology (1993) 1952-1960 and 136 Endocrinology (1995) 20-26, both of which are incoφorated herein by reference, cells of the cell line, LNCaP, which is derived from a human metastatic prostate adenocarcinoma and known to express PSA, are seeded in six-well tissue culture plates at a density of about 50,000 cells/plate. After the cells have attached and stabilized, about 2-3 days, the medium is replenished with medium containing vehicle or the active vitamin D analogue, lα,24-(OH)2D2, at concentrations from 10"" M to 10"7 M. After 6-7 days, the medium is removed and stored at -20EC for prostate specific antigen (PSA) analysis.
The cells from parallel cultures are rinsed, precipitated, and the amount of DNA determined by standard procedures. PSA is measured by standard known methods. Cultures incubated with lα,24-(OH)2D2 have significantly more PSA than control cultures when expressed as mass of PSA/cell.
Example 12 : 1 α,24-dihydroxyvitamin D4 [ 1 α,24-(OH)2D4]
The procedure of Example 12 is repeated except the active vitamin D analogue is lα,24-(OH)2D4. The PSA is measured and cultures incubated with lα,24-(OH) D4 have significantly more PSA than control cultures when expressed as mass of PSA/cell.
Example 13: lα,25-dihydroxyvitamin D4 [lα,24-(OH)2D4]
The procedure of Example 12 is repeated except the active vitamin D analogue is lα,25-(OH)2D4. The PSA is measured and cultures incubated with lα,25-(OH) D have significantly more PSA than control cultures when expressed as mass of PSA/cell.
CLINICAL STUDIES
Example 14: General Treatment of Cancers
Patients with a known vitamin D receptor positive tumor (e.g., adenocarcinoma of the prostate, breast, lung, colon or pancreas, or transitional cell carcinoma of the bladder, or melanoma) participate in an open-label study of a hypocalcemic vitamin D compound in accordance with the present invention. Patients are placed on a reduced calcium diet prior to treatment, to help minimize intestinal absoφtion and allow ever higher doses of the hypocalcemic vitamin D. This reduced calcium diet may be continued for the duration of treatment, and for one week after the last dose of the lα,24(S)-dihydroxyvitamin D2. The diet ideally restricts daily calcium intake to 400- 500 mg. Patients also discontinue use of any vitamin D supplements or vitamin D replacement therapies. Each patient is also asked to drink 4-6 cups of fluid more than usual intake to assure adequate oral hydration.
Each subject is monitored at regular intervals for: (1) hypercalcemia, hypeφhosphatemia, hypercalciuria, hypeφhosphaturia and other toxicity; (2) evidence of changes in the progression of metastatic disease; and (3) compliance with the prescribed test drug dosage.
The dosing regimen is typically on a daily dose basis of 10 μg or 20 μg per day to about 100 μg/day for 24 months. Alternatively, a non-daily dosing regimen can be used, e.g., 40 μg given every other day, 100 μg given once a week. The route of administration can vary from oral to intravenous to regional delivery (e.g., arterial infusion, via the portal vein). Oral is, of course, the easiest and most cost effective route. Regional delivery permits high dosing and generally avoids any production of hypercalcemia. Although, in the case of the compound of the present invention, the compound is substantially hypocalcemic.
After 18 months of treatment, CAT, scans, X-rays and bone scans used for evaluating the progress of metastatic disease or partial remission in many patients treated at the lower dosage , and stable disease and partial or complete remission in many patients treated at the higher dosage.
Example 15: Treatment of prostate cancer with lα,24-dihydroxy vitamin D2 [lα,24-(OH)2D2]
Patients with advanced androgen-independent prostate cancer participate in an open-labeled study of lα,24-(OH)2D . Qualified patients are at least 40 years old, exhibit histologic evidence of adenocarcinoma of the prostate, and present with progressive disease which had previously responded to hormonal intervention(s). On admission to the study, patients begin a course of therapy with oral lα,24-(OH)2D2 lasting 26 weeks, while discontinuing any previous use of calcium supplements, vitamin D supplements, and vitamin D hormone replacement therapies. During treatment, the patients are monitored at regular intervals for: (1) hypercalcemia, hypeφhosphatemia, hypercalciuria, hypeφhosphaturia and other toxicity; (2) evidence of changes in the progression of metastatic disease; and (3) compliance with the prescribed test drug dosage.
The study is conducted in two phases. During the first phase, the maximal tolerated dosage (MTD) of daily oral lα,24-(OH)2D2 is determined by administering progressively higher dosages to successive groups of patients. All doses are administered in the morning before breakfast. The first group of patients is treated with 25.0 μg of lα,24-(OH)2D2. Subsequent groups of patients are treated with 50.0, 75.0 and 100.0 μg/day. Dosing is continued uninterrupted for the duration of the study unless serum calcium exceeds 11.6 mg/dL, or other toxicity of grade 3 or 4 is observed, in which case dosing is held in abeyance until resolution of the observed toxic effect(s) and then resumed at a level which has been decreased by 10.0 μg.
Results from the first phase of the study show that the MTD for lα,24-(OH)2D2 is above 20.0 μg/day, a level which is 10- to 40-fold higher than can be achieved with lα,25-(OH)2D . Analysis of blood samples collected at regular intervals from the participating patients reveal that the levels of circulating lα,24-(OH)2D2 increase proportionately with the dosage administered, rising to maximum levels well above lOO pg/mL at the highest dosages, and that circulating levels of lα,25-(OH)2D are suppressed, often to undetectable levels. Serum and urine calcium are elevated in a dose responsive manner. Patients treated with the MTD of lα,24-(OH)2D2 for at least six months report that bone pain associated with metastatic disease is significantly diminished.
During the second phase, patients are treated with lα,24-(OH)2D2 for 24 months at 0.5 and 1.0 times the MTD. After one and two years of treatment, CAT scans, X-rays and bone scans used for evaluating the progression of metastatic disease show stable disease or partial remission in many patients treated at the lower dosage, and stable disease and partial or complete remission in many patients treated at the higher dosage. Example 16: Treatment of prostate cancer with lα-hydroxyvitamin D2 [lα-OH-D2]
The study of Example 14 is repeated for the active vitamin D compound, lα-OH-D2. The results of the phase one study indicate that patients treated with the MTD of 1 α-OH-D2 for at least six months report that bone pain associated with metastatic disease is significantly diminished. The results of the phase two study indicate that after two years, CAT scans, X-rays and bone scans used for evaluating the progression of metastatic disease show stable disease or partial remission in many patients treated at the lower dosage, and stable disease and partial or complete remission in many patients treated at the higher dosage.
Example 17: Treatment of Melanoma
The method of Example 14 is used to treat patients with metastatic malignant melanoma of, e.g., the jaw. After 18 months of treatment, the progress of the metastatic disease shows stable disease or partial remission.
Example 18: Treatment of retinoblastoma
The method of Example 14 is used is used to treat patients with metastatic retinoblastoma. After 18 months of treatment, the progress of the metastatic disease shows stable disease or partial remission.
Example 19: Treatment of liver cancer
The method of Example 14 is used to treat patients with hepatoma. The regional delivery of the compound in accordance with the present invention, i.e., via arterial infusion, is used. After 18 months of treatment, the progress of the metastatic disease shows stable disease or partial remission.
While the present invention has now been described and exemplified with some specificity, those skilled in the art will appreciate the various modifications, including variations, additions, and omissions, that may be made in what has been described. Accordingly, it is intended that these modifications also be encompassed by the present invention and that the scope of the present invention be limited solely by the broadest inteφretation lawfully accorded the appended claims.

Claims

CLAΓM(S)What is claimed is:
1. A method of inhibiting hypeφroliferation of malignant or neoplastic cells, comprising treating the cells with an antiproliferative amount of a hypocalcemic hydroxyvitamin D compound having a hydrocarbon moiety at the C24 position, the cells expressing a vitamin D receptor.
2. The method of claim 1, wherein the cells are cancers of the breast, colon, lung, neck and head, pancreas, endometrium, bladder, cervix, testes, ovaries, squamous cell carcinoma, myeloid and lymphocytic leukemia, lymphoma, medullary thyroid carcinoma, melanoma, multiple myeloma, retinoblastoma or sarcomas of the soft tissues and bone.
3. The method of claim 1, wherein the hypocalcemic vitamin D is a compound represented by formula (I)
Figure imgf000028_0001
wherein A1 and A2 each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R1 and R2 cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, or, taken with R3, constitutes a bond when R3 is an alkenyl group, and X2 is hydrogen or hydroxyl, or, taken with R1 or R2, constitutes a double bond, and X3 is hydrogen or hydroxyl provided that at least one of X , X and X is hydroxyl; and Y is a methylene group if the bond to Y is a double bond or is a methyl group or hydrogen if the bond to Y is a single bond.
4. A method in accordance with claim 1 wherein the hypocalcemic vitamin D compound is a compound of formula II
Figure imgf000029_0001
wherein A and A each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R1 and R2 cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, or, taken with R3, constitutes a bond when R3 is an alkenyl group, and X2 is hydrogen or hydroxyl, or, taken with R1 or R2, constitutes a double bond, , and Y is a methylene group if the bond to Y is a double bond or is a methyl group or hydrogen if the bond to Y is a single bond.
5. A method in accordance with claim 1, wherein the hypocalcemic vitamin D compound is a compound of formula III:
Figure imgf000030_0001
wherein A1 and A2 each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R1 and R2 cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, or, taken with R3, constitutes a bond when R3 is an alkenyl group, and X2 is hydrogen or hydroxyl, or, taken with R1 or R2, constitutes a double bond.
6. A method of inhibiting the hypeφroliferative activity of malignant or neoplastic cells, comprising administering to a patient suffering therefrom, an anitproliferarive amount of a hypocalcemic hydroxyvitamin D compound.
7. A method in accordance with claim 6, wherein the hypocalcemic vitamin D compound is administered in a daily regimen or an episodic regimen.
8. A method in accordance with claim 7, wherein the espisodic regimen is a dose once every 2 to 7 days.
9. A method in accordance with claim 7, wherein the hypocalcemic vitamin D compound is administered daily at a dose of about 10 to 100 μg/day.
10. A method in accordance with claim 6, wherein the hypocalcemic vitamin D compound is administered orally, is administered intravenously, is directly injected to a cancer site or is regionally delivered to a cancer site.
11. A method in accordance with claim 10, wherein the hypocalcemic vitamin D compound is administered orally.
12. A method in accordance with claim 6, wherein the hypocalcemic vitamin D compound is co-administered with a cytotoxic agent.
13. A method in accordance with claim 12, wherein the cytotoxic agent is an antimetabolite, and antimicrotubule agent, an alkyating agent, a platinum agent, an anthracycline, a topoisomase inhibitor, or an antibiotic.
14. A method in accordance with claim 13, wherein the antimetabolite is 5-fluoro-uracil, methotrexate or fludarabine.
15. A method in accordance with claim 13, wherein the antimicrotubule agent is vincristine, vinblastine or a taxane.
16. A method in accordance with claim 14, wherein the taxane is paclitaxel or docetaxel.
17. A method in accordance with claim 12, wherein the alkylating agent is cyclophasphamide, melphalan, biochoroethylnitrosurea or hydroxyurea.
18. A method in accordance with claim 12, wherein the platinum agent is cisplatin, carboplatin, oxaliplatin, JM-216 or CI-973.
19. A method in accordance with claim 12, wherein the anthracycline is doxrubicin or daunorubicin.
20. A method in accordance with claim 12, wherein the antibiotic is mitomycin, idarubicin, adriamycin or daunomycin.
21. A method in accordance with claim 12, wherein the topoisomerase inhibitior is etoposide or camptothecins.
22. A method in accordance with claim 12, wherein the cytotoxic agent is estramustine phosphate or prednimustine.
23. A method in accordance with claim 11, wherein an antiproliferative effective amount of the cytotoxic agent is lower than the antiproliferative effective amount of the cytotoxic agent when administered alone.
24. The method of claim 5, wherein the compound of formula (III) is lα,24- dihydroxyvitamm D2, lα,24-dihydroxyvitamin D4, lα,25-dihydroxyvitamin D2, lα,25- dihydroxyvitamin D4, lα-hydroxyvitamin D2 or lα-hydroxyvitamin D .
25. A method of treating a human to alleviate the pathological effects of breast cancer, colon cancer, testicular cancer, pancreatic cancer, endometrial cancer, small cell and non-small cell cancer of the lung (including squamous, adneocarcinoma and large cell types), squamous cell of the head and neck, bladder, ovarian and cervical cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid carcinoma, multiple myeloma, melanoma, retinoblastoma or sarcomas of the soft tissue and bone, comprising administering to the human an therapeutic amount of a hypocalcemic hydroxyvitamin D compound.
26. A method of claim 25, wherein said hypocalcemic vitamin D is a lα-hydroxyvitamin D compound represented by formula (III)
Figure imgf000033_0001
wherein A and A each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R1 and R2 cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, or, taken with R3, constitutes a bond when R3 is an alkenyl group, and X2 is hydrogen or hydroxyl, or, taken with R or R , constitutes a double bond.
27. The method of claim 26, wherein said therapeutic amount is 0.01 μg/kg/day to 2.0 μg/kg/day.
28. The method of claim 26, wherein the compound of formula (III) is lα,24- dihydroxyvitamin D2, lα,24-dihydroxyvitamin D4, 1 α,25 -dihydroxyvitamm D2, lα,25- dihydroxyvitamin D4, lα-hydroxyvitamin D2 or lα-hydroxyvitamin D4.
29. A method of enhancing the antiproliferative effect of a cytotoxic agent in a patient with a disease in need of treatment by a cytotoxic agent, comprising administering to the patient a therapeutic amount of hypocalcemic vitamin D compound and the cytotoxic agent.
30. A method in accordance with claim 29, wherein the hypocalcemic vitamin D compound is administered from 0.5 to 7 days prior to administration of the cytotoxic agent.
31. A method in accordance with claim 29, wherein the hypocalcemic vitamin D compound is administered 2 to 4 days prior to administration of the cytotoxic agent.
32. A method of claim 29, wherein said hypocalcemic vitamin D is a lα-hydroxyvitamin D compound represented by formula (III)
Figure imgf000034_0001
wherein A1 and A2 each are hydrogen or a carbon-carbon bond, thus forming a double bond between C-22 and C-23; R1 and R2 are identical or different and are hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl with the proviso that R1 and R2 cannot both be an alkenyl group, or taken together with the carbon to which they are bonded, form a C3-C8 cyclocarbon ring; R3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower cycloalkyl; X1 is hydrogen or hydroxyl, or, taken with R3, constitutes a bond when R3 is an alkenyl group, and X2 is hydrogen or hydroxyl, or, taken with R1 or R2, constitutes a double bond.
33. The method of claim 32, wherein said therapeutic amount of the vitamin D compound is 0.01 μg/kg/day to 2.0 μg/kg/day.
34. The method of claim 32, wherein the compound of formula (III) is lα,24- dihydroxyvitamin D2, lα,24-dihydroxyvitamin D4, lα,25-dihydroxyvitamin D2, lα,25- dihydroxyvitamm D4, lα-hydroxyvitamin D2 or lα-hydroxyvitamin D .
35. A method in accordance with claim 32, wherein the cytotoxic agent is an antimetabolite, and antimicrotubule agent, an alkyating agent, a platinum agent, an anthracycline, a topoisomase inhibitor, or an antibiotic.
36. A method of inducing differentiation in malignant or neoplastic cells, comprising treating to the cells with a prodifferentiative amount of a hypocalcemic vitamin D compound.
37. A method of treating in a subject tumor or neoplasm that expresses a vitamin D receptor, comprising administering to the subject an effective amount of hypocalcemic vitamin D compound sufficient to raise a blood level of vitamin D to a sufficiently supraphysiological level for a sufficient period of time to inhibit growth of the tumor or neoplasm without causing hypercalcemia in the subject.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1448150A2 (en) * 2001-11-28 2004-08-25 Bone Care International, Inc. Treatment of hyperproliferative diseases using active vitamin d analogues

Families Citing this family (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040009958A1 (en) * 1991-01-08 2004-01-15 Bone Care International, Inc. Methods for preparation and use of 1alpha,24(S)-dihydroxyvitamin D2
US5763429A (en) * 1993-09-10 1998-06-09 Bone Care International, Inc. Method of treating prostatic diseases using active vitamin D analogues
US6566353B2 (en) * 1996-12-30 2003-05-20 Bone Care International, Inc. Method of treating malignancy associated hypercalcemia using active vitamin D analogues
US6392071B1 (en) * 1997-03-17 2002-05-21 Wisconsin Alumni: Research Foundation 26,27-homologated-20-EPI-2-alkylidene-19-nor-vitamin D compounds
US6316642B1 (en) * 1997-03-17 2001-11-13 Wisconsin Alumni Research Foundation 26,27-Homologated-20-EPI-2alkyl-19-nor-vitamin D compounds
US6087350A (en) * 1997-08-29 2000-07-11 University Of Pittsburgh Of The Commonwealth System Of Higher Education Use of pretreatment chemicals to enhance efficacy of cytotoxic agents
WO1999049870A1 (en) * 1998-03-27 1999-10-07 Oregon Health Sciences University Vitamin d and its analogs in the treatment of tumors and other hyperproliferative disorders
GB9920548D0 (en) * 1999-08-31 1999-11-03 Rhone Poulenc Rorer Sa Treatment of hepatocellular carcinoma
EP1461044A4 (en) * 2001-12-03 2007-06-13 Novacea Inc Pharmaceutical compositions comprising active vitamin d compounds
US20080249068A1 (en) * 2002-09-05 2008-10-09 Deluca Hector F Method of Extending the Dose Range of Vitamin D Compounds
US7259143B2 (en) * 2002-09-05 2007-08-21 Wisconsin Alumni Research Foundation Method of extending the dose range of vitamin D compounds
US20040053895A1 (en) * 2002-09-18 2004-03-18 Bone Care International, Inc. Multi-use vessels for vitamin D formulations
US7148211B2 (en) * 2002-09-18 2006-12-12 Genzyme Corporation Formulation for lipophilic agents
US20040058895A1 (en) * 2002-09-18 2004-03-25 Bone Care International, Inc. Multi-use vessels for vitamin D formulations
US20050026877A1 (en) * 2002-12-03 2005-02-03 Novacea, Inc. Pharmaceutical compositions comprising active vitamin D compounds
US20050020546A1 (en) * 2003-06-11 2005-01-27 Novacea, Inc. Pharmaceutical compositions comprising active vitamin D compounds
WO2005097128A1 (en) * 2004-03-30 2005-10-20 Novacea, Inc. 1,4-bis-n-oxide azaanthracenediones and the use thereof
US20060003950A1 (en) * 2004-06-30 2006-01-05 Bone Care International, Inc. Method of treating prostatic diseases using a combination of vitamin D analogues and other agents
US7094775B2 (en) * 2004-06-30 2006-08-22 Bone Care International, Llc Method of treating breast cancer using a combination of vitamin D analogues and other agents
US20060189543A1 (en) * 2005-02-23 2006-08-24 Rosenbloom Richard A Compositions and methods for the treatment of leukemia
US7914810B2 (en) * 2005-05-06 2011-03-29 Synthes Usa, Llc Methods for the in situ treatment of bone cancer
LT3095447T (en) 2006-02-03 2022-02-10 Opko Renal, Llc Treating vitamin d insufficiency and deficiency with 25-hydroxyvitamin d2 and 25-hydroxyvitamin d3
US8329677B2 (en) 2006-06-21 2012-12-11 Cytochroma, Inc. Method of treating and preventing secondary hyperparathyroidism
TW200833345A (en) * 2006-11-01 2008-08-16 Novacea Inc Use of vitamin D compounds and mimics thereof to enhance delivery of therapeutics and oxygen to tumors and other tissues
US8501717B2 (en) * 2007-02-09 2013-08-06 Merck, Sharp & Dohme Corp. Methods to treat and/or prevent mucositis
EP2148683A4 (en) * 2007-04-25 2012-09-12 Proventiv Therapeutics Llc Method of safely and effectively treating and preventing secondary hyperparathyroidism in chronic kidney disease
WO2009047644A2 (en) 2007-04-25 2009-04-16 Cytochroma Inc. Method of treating vitamin d insufficiency and deficiency
WO2008134512A1 (en) 2007-04-25 2008-11-06 Cytochroma Inc. Oral controlled release compositions comprising vitamin d compound and waxy carrier
WO2009124210A1 (en) 2008-04-02 2009-10-08 Cytochroma Inc. Methods, compositions, uses, and kits useful for vitamin d deficiency and related disorders
WO2009151569A2 (en) * 2008-06-09 2009-12-17 Combinatorx, Incorporated Beta adrenergic receptor agonists for the treatment of b-cell proliferative disorders
EA201691980A1 (en) 2009-01-27 2017-07-31 БЕРГ ЭлЭлСи WAYS TO REDUCE SIDE EFFECTS ASSOCIATED WITH CHEMOTHERAPY
WO2011019617A2 (en) 2009-08-14 2011-02-17 Cytotech Labs, Llc Vitamin d3 and analogs thereof for treating alopecia
PT2552484T (en) 2010-03-29 2020-04-03 Opko Ireland Global Holdings Ltd Methods and compositions for reducing parathyroid levels
KR101847947B1 (en) 2013-03-15 2018-05-28 옵코 아이피 홀딩스 Ⅱ 인코포레이티드 Stabilized modified release vitamin d formulation
SG10201709894RA (en) 2013-05-29 2018-01-30 Berg Llc Preventing or mitigating chemotherapy induced alopecia using vitamin d
US20180085381A1 (en) 2014-08-07 2018-03-29 Opko Ireland Global Holdings, Ltd. Adjunctive Therapy With 25-Hydroxyvitamin D
CN106674306A (en) * 2015-01-13 2017-05-17 南京理工大学 Vitamin D2 glycoside analogues as well as synthesis and application thereof
JP7032322B2 (en) 2016-03-28 2022-03-08 オプコ アイルランド グローバル ホールディングス リミテッド Vitamin D treatment

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5403831A (en) * 1988-08-02 1995-04-04 Bone Care International, Inc. Method of treating and preventing loss of bone mass using 1α-hydroxy-vitamin D2
US5602116A (en) * 1988-08-02 1997-02-11 Bone Care International, Inc. Method for treating and preventing secondary hyperparathyroidism
US5763427A (en) * 1995-03-31 1998-06-09 Hamilton Civic Hospitals Research Development Inc. Compositions and methods for inhibiting thrombogenesis
US5798345A (en) * 1990-09-21 1998-08-25 Bone Care International, Inc. Method of inhibiting the hyperproliferation of malignant cells

Family Cites Families (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2383446A (en) 1941-06-04 1945-08-28 Du Pont Antirachitic materials and processes for their production
US3697559A (en) 1971-02-25 1972-10-10 Wisconsin Alumni Res Found 1,25-dihydroxycholecalciferol
US3741996A (en) 1971-12-02 1973-06-26 Wisconsin Alumni Res Found 1{60 -hydroxycholecalciferol
US4670190A (en) 1973-01-10 1987-06-02 Hesse Robert H 1-α-hydroxy vitamin D compounds and process for preparing same
US3907843A (en) 1974-06-14 1975-09-23 Wisconsin Alumni Res Found 1{60 -Hydroxyergocalciferol and processes for preparing same
US4202829A (en) 1978-01-05 1980-05-13 Wisconsin Alumni Research Foundation Process for preparing 1α-hydroxylated compounds
US4195027A (en) 1978-01-16 1980-03-25 Wisconsin Alumni Research Foundation Process for preparing 1α-hydroxylated compounds
BE877356R (en) 1978-01-16 1979-10-15 Wisconsin Alumni Res Found PROCESS FOR PREPARING 1ALPHA-HYDROXYL COMPOUNDS
US4260549A (en) 1979-05-21 1981-04-07 Wisconsin Alumni Research Foundation Process for preparing 1α-hydroxylated compounds
US4225596A (en) 1978-10-13 1980-09-30 Wisconsin Alumni Research Foundation Method for treating calcium imbalance and improving calcium absorption in mammals
US4234495A (en) 1979-09-10 1980-11-18 Wisconsin Alumni Research Foundation Process for preparing 1α-hydroxyvitamin D compounds from 1α-hydroxy-3,5-cyclovitamin D compounds
US4362710A (en) 1980-07-04 1982-12-07 Nissan Gosei Kogyo Co., Ltd. Feeds for baby pigs, process for preparing the same and method of breeding baby pigs
JPS57149224A (en) 1981-03-13 1982-09-14 Chugai Pharmaceut Co Ltd Tumor-suppressing agent
CH655396B (en) 1981-11-11 1986-04-15
US4508651A (en) 1983-03-21 1985-04-02 Hoffmann-La Roche Inc. Synthesis of 1α,25-dihydroxyergocalciferol
DE3490215C2 (en) 1983-05-09 1991-07-25 Wisconsin Alumni Res Found
US4689180A (en) 1984-01-30 1987-08-25 Wisconsin Alumni Research Foundation 1α,25-dihydroxy-22Z-dehydroxyvitamin D compound
US4588716A (en) 1984-05-04 1986-05-13 Wisconsin Alumni Research Foundation Method for treating metabolic bone disease in mammals
US4555364A (en) 1984-11-01 1985-11-26 Wisconsin Alumni Research Foundation Method for preparing 1-hydroxyvitamin D compounds
US4554106A (en) 1984-11-01 1985-11-19 Wisconsin Alumni Research Foundation Method for preparing 1α-hydroxyvitamin D compounds
US4661294A (en) 1985-03-18 1987-04-28 The General Hospital Corporation Biologically active 1-thio derivatives of vitamin D
IL78342A (en) 1985-04-04 1991-06-10 Gen Hospital Corp Pharmaceutical composition for treatment of osteoporosis in humans comprising a parathyroid hormone or a fragment thereof
EP0227826B1 (en) 1985-08-02 1989-10-25 Leo Pharmaceutical Products Ltd. A/S (Lovens Kemiske Fabrik Produktionsaktieselskab) Novel vitamin d analogues
US4833125A (en) 1986-12-05 1989-05-23 The General Hospital Corporation Method of increasing bone mass
JP2550391B2 (en) 1988-06-30 1996-11-06 日清製粉株式会社 Method for producing 1β-hydroxyvitamin D 2 below and D 3 below
CA1333616C (en) 1989-03-09 1994-12-20 Hector F. Deluca 19-nor-vitamin d compounds
US5372996A (en) 1989-03-10 1994-12-13 Endorecherche, Inc. Method of treatment of androgen-related diseases
JP2645130B2 (en) 1989-03-31 1997-08-25 日清製粉株式会社 Steroid derivatives
GB2229921B (en) 1989-04-05 1992-12-16 Chugai Pharmaceutical Co Ltd Treatment for hyperparathyroidism with use of vitamin d derivatives
US6025346A (en) 1990-09-21 2000-02-15 Bone Care International, Inc. 1α-hydroxy vitamin D4 and novel intermediates and analogues
US5801164A (en) 1990-09-21 1998-09-01 Bone Care International, Inc. Methods of treating osteoporosis prophylactically or therapeutically
AU650286B2 (en) 1990-09-21 1994-06-16 Bone Care International, Inc. Novel 1alpha-hydroxy vitamin D4 and novel intermediates and analogues
US5763428A (en) 1990-09-21 1998-06-09 Bone Care International, Inc. Methods of treating skin disorders with novel 1a-hydroxy vitamin D4 compounds and derivatives thereof
US6166000A (en) 1991-01-08 2000-12-26 Bone Care International, Inc. Methods for preparation and use of 1α,24(S)-Dihydroxy vitamin . D.sub2
US5786348A (en) 1991-01-08 1998-07-28 Bone Care International, Inc. Methods for preparation and use of 1α,24(S)-dihydroxy vitamin D2
ATE240736T1 (en) 1991-01-08 2003-06-15 Bone Care Int Inc METHOD FOR PRODUCING 1-ALPHA-24-DIHYDROXY-VITAMIN D2
US6538037B2 (en) * 1991-01-08 2003-03-25 Bone Care International, Inc. Methods for preparation and use of 1α,24(S)-dihydroxyvitamin D2
EP0503630B1 (en) 1991-03-13 1995-12-27 Kuraray Co., Ltd. Cyclohexanetriol derivatives
WO1992021355A1 (en) 1991-05-28 1992-12-10 The Procter & Gamble Company Calcium, trace mineral, vitamin d and drug therapy combinations
AU650751B2 (en) 1991-05-28 1994-06-30 Wisconsin Alumni Research Foundation Novel synthesis of 19-nor vitamin D compounds
DE69125836T2 (en) 1991-11-14 1997-12-18 Quantum Corp Spindle and hub equipment
DE69318142T2 (en) 1992-01-29 1998-09-17 Bone Care Int Inc 1 alpha-HYDROXY-24- (EPI) -VITAMIN D4
EP0562497A1 (en) 1992-03-27 1993-09-29 Nisshin Flour Milling Co., Ltd. 1 alpha-hydroxy vitamins D7 and D4' processes for the preparation thereof and pharmaceutical compositions
CA2121689C (en) * 1992-08-28 2008-03-18 Ronald L. Horst 1.alpha.,24(s)-dihydroxy vitamin d2, its formation and use
US5350745A (en) 1993-01-29 1994-09-27 Lunar Corporation Treatment of myocardial failure
US5763429A (en) 1993-09-10 1998-06-09 Bone Care International, Inc. Method of treating prostatic diseases using active vitamin D analogues
DK0664287T3 (en) 1994-01-20 1999-04-19 Duphar Int Res Vitamin D compounds and methods for preparing them
DE19549243A1 (en) 1995-12-21 1997-06-26 Schering Ag Pharmaceutical preparations containing clathrates of cyclodextrins and unnatural vitamin D analogues
US6034074A (en) 1996-09-13 2000-03-07 New Life Pharmaceuticals Inc. Prevention of ovarian cancer by administration of a Vitamin D compound
WO1998020123A2 (en) * 1996-11-04 1998-05-14 Institut Curie Stable cell lines expressing the cftr protein or a mutant of this protein, tool for selecting molecules having an effect on the intracellular transport of these proteins
AUPO727097A0 (en) 1997-06-10 1997-07-03 Unisearch Limited Method of treatment of hepatoma and pharmaceutical compositions for use therein
US6087350A (en) 1997-08-29 2000-07-11 University Of Pittsburgh Of The Commonwealth System Of Higher Education Use of pretreatment chemicals to enhance efficacy of cytotoxic agents
CA2323782A1 (en) 1998-03-25 1999-09-30 Cutanogen, Inc. Methods for prevention and treatment of cancer
WO1999049870A1 (en) 1998-03-27 1999-10-07 Oregon Health Sciences University Vitamin d and its analogs in the treatment of tumors and other hyperproliferative disorders
US20010002396A1 (en) 1998-07-16 2001-05-31 Charles Achkar Compositions and methods of treating skin conditions
DK1220676T3 (en) 1999-09-29 2005-09-05 Colotech As Prevention of colorectal cancer
EP1276482B1 (en) * 2000-03-02 2008-01-16 University Of Pittsburgh Of The Commonwealth System Of Higher Education Combination chemotherapy

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5403831A (en) * 1988-08-02 1995-04-04 Bone Care International, Inc. Method of treating and preventing loss of bone mass using 1α-hydroxy-vitamin D2
US5602116A (en) * 1988-08-02 1997-02-11 Bone Care International, Inc. Method for treating and preventing secondary hyperparathyroidism
US5798345A (en) * 1990-09-21 1998-08-25 Bone Care International, Inc. Method of inhibiting the hyperproliferation of malignant cells
US5763427A (en) * 1995-03-31 1998-06-09 Hamilton Civic Hospitals Research Development Inc. Compositions and methods for inhibiting thrombogenesis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1408983A2 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1448150A2 (en) * 2001-11-28 2004-08-25 Bone Care International, Inc. Treatment of hyperproliferative diseases using active vitamin d analogues
EP1448150A4 (en) * 2001-11-28 2006-07-05 Bone Care Int Inc Treatment of hyperproliferative diseases using active vitamin d analogues

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