EP1622455A2 - Composes de la vitamine d3 gemini et methodes d'utilisation de ces composes - Google Patents

Composes de la vitamine d3 gemini et methodes d'utilisation de ces composes

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Publication number
EP1622455A2
EP1622455A2 EP04751195A EP04751195A EP1622455A2 EP 1622455 A2 EP1622455 A2 EP 1622455A2 EP 04751195 A EP04751195 A EP 04751195A EP 04751195 A EP04751195 A EP 04751195A EP 1622455 A2 EP1622455 A2 EP 1622455A2
Authority
EP
European Patent Office
Prior art keywords
compound
vitamin
subject
disorder
dihydroxy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04751195A
Other languages
German (de)
English (en)
Other versions
EP1622455A4 (fr
Inventor
Luciano Adorini
Guiseppe Penna
Milan R. Uskokovic
Hubert Maehr
Yan Chun Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bioxell SpA
University of Chicago
Original Assignee
Bioxell SpA
University of Chicago
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Filing date
Publication date
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Publication of EP1622455A2 publication Critical patent/EP1622455A2/fr
Publication of EP1622455A4 publication Critical patent/EP1622455A4/fr
Withdrawn legal-status Critical Current

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    • C07B2200/05—Isotopically modified compounds, e.g. labelled

Definitions

  • vitamin D cholesterol calcium and phosphorous homeostasis
  • the operation of the vitamin D endocrine system depends on the following: first, on the presence of cytochrome P450 enzymes in the liver (Bergman, T. and Postlind, H. (1991) Biochem. J. 276:427-432; Ohyama, Y and Okuda, K. (1991) J. Biol. Chem. 266:8690-8695) and kidney (Henry, H.L. and Norman, A.W. (1974) J. Biol. Chem.
  • Vitamin D 3 and its hormonally active forms are well-known regulators of calcium and phosphorous homeostasis. These compounds are known to stimulate, at least one of, intestinal absorption of calcium and phosphate, mobilization of bone mineral, and retention of calcium in the kidneys. Furthermore, the discovery of the presence of specific vitamin D receptors in more than 30 tissues has led to the identification of vitamin D 3 as a pluripotent regulator outside its classical role in calcium/bone homeostasis.
  • vitamin D 3 A paracrine role for l ⁇ ,25(OH) 2 D 3 has been suggested by the combined presence of enzymes capable of oxidizing vitamin D 3 into its active forms, e.g., 25-OHD-l ⁇ -hydroxylase, and specific receptors in several tissues such as bone, keratinocytes, placenta, and immune cells. Moreover, vitamin D 3 hormone and active metabolites have been found to be capable of regulating cell proliferation and differentiation of both normal and malignant cells (Reichel, H. et al. (1989) Ann. Rev. Med. 40: 71-78).
  • the invention provides vitamin D 3 compounds having formula T:
  • Ai is a single or double bond
  • a 2 is a single, a double or a triple bond
  • Ri, R 2; R 3 and Rt are each independently C ⁇ -C 4 alkyl, C ⁇ -C deuteroalkyl, hydroxyalkyl, or haloalkyl;
  • the invention also provides methods for treating a subject for a vitamin D 3 associated state, by administering to the subject an effective amount of a vitamin D 3 compound of formula I above or otherwise described herein.
  • Another embodiment of the invention provides a method for treating a subject for a urogenital disorder, comprising administering to the subject an effective amount of a vitamin D 3 compound of formula I above or otherwise described herein, such that said subject is treated for the urogential disorder.
  • the invention provides a method of treating an ILT3- associated disorder in a subject. The method includes administering to the subject a vitamin D 3 compound of formula I above or otherwise described herein, in an amount effective to modulate the expression of an ILT3 surface molecule.
  • the invention provides a method of inhibiting transplant rejection in a subject.
  • the method includes administering to the subject a a vitamin D 3 compound of formula I above or otherwise described herein in an amount effective to modulate the expression of an ILT3 surface molecule.
  • the invention also provides a method for treating a subject for hypertension, comprising administering to the subject an effective amount of a Gemini vitamin D 3 compound of the invention, such that the subject is treated for hypertension.
  • the invention provides a method of suppressing renin expression in a subject comprising administering a to a subject an effective amount of a Gemini vitamin D 3 compound such that renin expression in said subject is suppressed.
  • the invention also provides a method of ameliorating a deregulation of calcium and phosphate metabolism. The method includes administering to a subject a therapeutically effective amount of a vitamin D 3 compound of formula I or otherwise described herein, so as to ameliorate the deregulation of the calcium and phosphate metabolism.
  • the invention provides a method of modulating the expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule in a cell.
  • the method includes contacting the cell with a vitamin D 3 compound of formula I or otherwise described herein, in an amount effective to modulate the expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule in the cell.
  • the invention provides a method of inducing immunological tolerance in a subject, by administering to the subject a vitamin D 3 compound of formula I or otherwise described herein, in an amount effective to modulate the expression of an ILT3 surface molecule, to thereby induce immunological toleranc in the subject.
  • the invention provides a method for modulating immunosuppressive activity by an antigen-presenting cell, by contacting an antigen-presenting cell with a vitamin D 3 compound of formula I or otherwise described herein, in an amount effective to modulate ILT3 surface molecule expression, to thereby modulating immunosuppressive activity by an antigen-presenting cell.
  • the invention also provides a pharmaceutical composition, comprising an effective amount a vitamin D 3 compound of formula I or otherwise described herein and a pharmaceutically acceptable carrier.
  • the invention provides a packaged formulation which includes a pharmaceutical composition comprising a vitamin D 3 compound of formula I or otherwise described herein, and a pharmaceutically-acceptable carrier packaged with instructions for use in the treatment of a for use in the treatment of a vitamin D 3 associated associated state.
  • Figure 1 is a graph depicting the modulation (upregulation) of expression of ILT3 on the cell surface of monocyte-derived immature dendritic cells with various compounds.
  • administration includes routes of introducing the vitamin D 3 compound(s) to a subject to perform their intended function.
  • routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal.
  • the pharmaceutical preparations are, of course, given by forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
  • the injection can be bolus or can be continuous infusion.
  • the vitamin D 3 compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function.
  • the vitamin D 3 compound can be administered alone, or in conjunction with either another agent as described above or with a pharmaceutically-acceptable carrier, or both.
  • the vitamin D 3 compound can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent.
  • the vitamin D 3 compound can also be administered in a proform which is converted into its active metabolite, or more active metabolite in vivo.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C ⁇ -C 30 for straight chain, C 3 -C 30 for branched chain), preferably 26 or fewer, and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.
  • alkyl as used throughout the specification and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino , (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, triflu
  • alkylaryl is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • alkyl also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six, and most preferably from one to four carbon atoms in its backbone structure, which may be straight or branched-chain.
  • lower alkyl groups include methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and so forth.
  • the term "lower alkyl” includes a straight chain alkyl having 4 or fewer carbon atoms in its backbone, e.g., C ⁇ -C alkyl.
  • alkoxyalkyl refers to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.
  • the invention contemplates cyano and propargyl groups.
  • the term "antigen" includes a substance which elicits an immune response.
  • the antigens of the invention to which tolerance is induced may or may not be exogenously derived relative to the host.'
  • the method of the invention may be used to induce tolerance to an "autoantigen.”
  • An autoantigen is a normal constituent of the body that reacts with an autoantibody.
  • the invention also includes inducing tolerance to an "alloantigen.” Alloantigen refers to an antigen found only in some members of a species, for example the blood group substances.
  • An allograft is a graft to a genetically different member of the same species. Allografts are rejected by virtue of the immunological response of T lymphocytes to histocompatibility antigens.
  • the method of the invention also provides for inducing tolerance to a "xenoantigen.”
  • Xenoantigens are substances that cause an immune reaction due to differences between different species.
  • a xenograft is a graft from a member of one species to a member of a different species. Xenografts are usually rejected within a few days by antibodies and cytotoxic T lymphocytes to histocompatibility antigens.
  • antigen-presenting cell includes a cell that is able to present an antigen to, for example, a T helper cell.
  • Antigen-presenting cells include B lymphocytes, accessory cells or non-lymphocytic cells, such as dendritic cells, Langerhans cells, and mononuclear phagocytes that help in the induction of an immune response by presenting antigen to helper T lymphocytes.
  • the antigen-presenting cell of the present invention is preferably of myeloid origin, and includes, but is not limited to, dendritic cells, macrophages, monocytes.
  • APCs of the present invention may be isolated from the bone marrow, blood, thymus, epidermis, liver, fetal liver, or the spleen.
  • the terms "antineoplastic agent” and “antiproliferative agent” are used interchangeably herein and includes agents that have the functional property of ( inhibiting the proliferation of a vitamin D 3 -responsive cells, e.g., inhibit the development or progression of a neoplasm having such a characteristic, particularly a hematopoietic neoplasm.
  • aryl refers to the radical of aryl groups, including 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles," “heteroaryls” or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, s
  • Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
  • autoimmune disease or "autoimmune disorder” refers to the condition where the immune system attacks the host's own tissue(s). In an autoimmune disease, the immune tolerance system of the patient fails to recognize self antigens and, as a consequence of this loss of tolerance, brings the force of the immune system to bear on tissues which express the antigen.
  • Autoimmune disorders include, but are not limited to, type 1 insulin-dependent diabetes mellitus, adult respiratory distress syndrome, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitic, leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever, Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biliary cirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS inflammatory disorder, antigen-antibody complex mediated diseases, autoimmune haemolytic anemia, Hashimoto's thyroiditis, Graves disease
  • vitamin D 3 includes all activities elicited by vitamin D 3 compounds in a responsive cell. It includes genomic and non-genomic activities elicited by these compounds (Gniadecki R. and Calverley MJ. (1998) Pharmacology & Toxicology 82: 173-176; Bouillon, R. et al. (1995) Endocrinology Reviews 16(2):206-207; Norman A.W. et al. (1992) J Steroid Biochem Mol. Biol 41:231-240; Baran D.T. et al. (1991) J Bone Miner Res. 6:1269-1275; Caffrey J.M. and Farach-Carson M.C. (1989) J. Biol. Chem. 264:20265-20274; Nemere I. et al. (1984) Endocrinology 115:1476-1483).
  • bone metabolism includes direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate. This term is also intended to include effects of compounds of the invention in bone cells, e.g., osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration.
  • calcium and phosphate homeostasis refers to the careful balance of calcium and phosphate concentrations, intracellularly and extracellularly, triggered by fluctuations in the calcium and phosphate concentration in a cell, a tissue, an organ or a system. Fluctuations in calcium levels that result from direct or indirect responses to compounds of the invention are intended to be included by these terms.
  • carcinoma refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas.
  • Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • chiral refers to molecules which have the property of non- superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • diastereomers refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
  • deuteroalkyl refers to alkyl groups in which one or more of the of the hydrogens has been replaced with deuterium.
  • effective amount includes an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., sufficient treat a vitamin D 3 associated state or to modulate ILT3 expression in a cell.
  • An effective amount of vitamin D 3 compound may vary according to factors such as the disease state, age, and weight of the subject, and the ability of the vitamin D 3 compound to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • An effective amount is also one in which any toxic or detrimental effects (e.g., side effects) of the angiogenesis inhibitor compound are outweighed by the therapeutically beneficial effects.
  • a therapeutically effective amount of vitamin D 3 compound may range from about 0.001 to 30 ⁇ g kg body weight, preferably about 0.01 to 25 ⁇ g/kg body weight, more preferably about 0.1 to 20 ⁇ g/kg body weight, and even more preferably about 1 to 10 ⁇ g/kg, 2 to 9 ⁇ g/kg, 3 to 8 ⁇ g/kg, 4 to 7 ⁇ g/kg, or 5 to 6 ⁇ g/kg body weight.
  • treatment of a subject with a therapeutically effective amount of a vitamin D 3 compound can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with a vitamin D 3 compound in the range of between about 0.1 to 20 ⁇ g/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.
  • the effective dosage of a vitamin D 3 compound used for treatment may increase or decrease over the course of a particular treatment.
  • enantiomers refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.
  • An equimolar mixture of two enantiomers is called a “racemic mixture” or a “racemate.”
  • Vitamin D 3 compounds is intended to include vitamin D 3 compounds and analogs thereof having bis C20 side chains.
  • Vitamin D 3 compounds are characterized by an "A" ring (monocycle) which is connected to a “B” ring (bicycle) which is connected to a side chain at carbon C20 of the side chain.
  • the Gemini compounds of the invention have two side chains and are, therefore, conspicuously distinguishable from vitamin D 3 compounds having a single side chain.
  • Candidate A and B rings for the Gemini compounds of the invention are disclosed in U.S. Patent Nos.
  • vitamin D 3 activities or effects of vitamin D 3 is intended to include those activities mediated by the nuclear receptor for l ⁇ , 25(OH) 2 D 3 (VD 3 R), e.g., transcriptional activation of target genes.
  • halogen designates -F, -Cl, -Br or -I.
  • haloalkyl is intended to include alkyl groups as defined above that are mono-, di- or polysubstituted by halogen, e.g., fluoromethyl and trifluoromethyl.
  • hydroxyl means -OH.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
  • hormone secretion is art-recognized to mean maintenance of static, or constant, conditions in an internal environment.
  • hormone secretion is art-recognized and includes activities of vitamin D 3 compounds that control the transcription and processing responsible for secretion of a given hormone e.g., a parathyroid ho ⁇ none (PTH) of a vitamin D 3 responsive cell (Bouillon, R. et ⁇ l. (1995) Endocrine Reviews 16(2):235-237).
  • PTH parathyroid ho ⁇ none
  • hypercalcemia or “hypercalcemic activity” is intended to have its accepted clinical meaning, namely, increases in calcium serum levels that are manifested in a subject by the following side effects, depression of central and peripheral nervous system, muscular weakness, constipation, abdominal pain, lack of appetite and, depressed relaxation of the heart during diastole. Symptomatic manifestations of hypercalcemia are triggered by a stimulation of at least one of the following activities, intestinal calcium transport, bone calcium metabolism and osteocalcin synthesis (reviewed in Boullion, R. et ⁇ l. (1995) Endocrinology Reviews 16(2): 200-257).
  • hyperproliferative and neoplastic are used interchangeably, and include those cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • “Pathologic hyperproliferative” cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • ILT3 -associated disorder includes a disease, disorder or condition which is associated with an ILT3 molecule.
  • ILT3 associated disorders include disorders in which ILT3 activity is aberrant or in which a non-ILT3 activity that would benefit from modulation of an ILT3 activity is aberrant.
  • the ILT3 -associated disorder is an immune disorder, e.g., an autoimmune disorder, such as type 1 insulin- dependent diabetes mellitus, adult respiratory distress syndrome, inflammatory bowel disease, dermatitis, meningitis, thrombotic thrombocytopenic purpura, Sjogren's syndrome, encephalitis, uveitic, leukocyte adhesion deficiency, rheumatoid arthritis, rheumatic fever, Reiter's syndrome, psoriatic arthritis, progressive systemic sclerosis, primary biliary cirrhosis, pemphigus, pemphigoid, necrotizing vasculitis, myasthenia gravis, multiple sclerosis, lupus erythematosus, polymyositis, sarcoidosis, granulomatosis, vasculitis, pernicious anemia, CNS inflammatory disorder, antigen- antibody complex mediated diseases, autoimmune haemolytic an autoimmune disorder
  • immunoglobulin-like transcript 3 refers to a cell surface molecule of the immunoglobulin superfamily, which is expressed by antigen- presenting cells (APCs) such as monocytes, macrophages and dendritic cells.
  • APCs antigen- presenting cells
  • ILT3 is a member of the immunoglobulin-like transcript (ILT) family and displays a long cytoplasmic tail containing putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs). ILT3 has been shown to behave as an inhibitory receptor when cross-linked to a stimulatory receptor.
  • a cytoplasmic component of the ILT3 -mediated signaling pathway is the SH2-containing phosphatase SHP-1, which becomes associated with ILT3 upon cross-linking.
  • ILT3 is also internalized and ILT3 ligands are efficiently presented to specific T cells (see, e.g., Cella, M. et al. (1997) J Exp. Med. 185:1743).
  • the determination of whether the candidate vitamin D 3 compound modulates the expression of the ILT3 surface molecule can be accomplished, for example, by comparison of ILT3 surface molecule expression to a control, by measuring mRNA expression, or by measuring protein expression.
  • the term " immune response" includes T and/or B cell responses, e.g., cellular and/or humoral immune responses.
  • the claimed methods can be used to reduce both primary and secondary immune responses.
  • the immune response of a subject can be determined by, for example, assaying antibody production, immune cell proliferation, the release of cytokines, the expression of cell surface markers, cyto
  • immunological tolerance or "tolerance to an antigen” or “immune tolerance” include unresponsiveness to an antigen without the induction of a prolonged generalized immune deficiency. Consequently, according to the invention, a tolerant host is capable of reacting to antigens other than the tolerizing antigen. Tolerance represents an induced depression in the response of a subject that, had it not been subjected to the tolerance-inducing procedure, would be competent to mount an immune response to that antigen.
  • immunological tolerance is induced in an antigen-presenting cell, e.g., an antigen-presenting cell derived from the myeloid or lymphoid lineage, dendritic cells, monocytes and macrophages.
  • immunosuppressive activity refers to the process of inhibiting a normal immune response. Included in this response is when T and/or B clones of lymphocytes are depleted in size or suppressed in their reactivity, expansion or differentiation. Immunosuppressive activity may be in the form of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The functions of activated T cells may be inhibited by suppressing immune cell responses or by inducing specific tolerance, or both. Immunosuppression of T cell responses is generally an active, non-antigen-specific, process that requires continuous exposure of the T cells to the suppressive agent.
  • Tolerance which involves inducing non-responsiveness or anergy in T cells, is distinguishable from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent has ceased. Operationally, tolerance can be demonstrated by the lack of a T cell response upon re-exposure to specific antigen in the absence of the tolerizing agent.
  • improved biological properties refers to any activity inherent in a compound of the invention that enhances its effectiveness in vivo. In a preferred embodiment, this term refers to any qualitative or quantitative improved therapeutic property of a vitamin D 3 compound, such as reduced toxicity, e.g. , reduced hypercalcemic activity.
  • the language "inhibiting the growth" of the neoplasm includes the slowing, interrupting, arresting or stopping its growth and metastases and does not necessarily indicate a total elimination of the neoplastic growth.
  • the phrase "inhibition of an immune response” is intended to include decreases in T cell proliferation and activity, e.g., a decrease in IL2, interferon- ⁇ , GM-CSF synthesis and secretion (Lemire, J. M. (1992) J. Cell Biochemistry 49:26-31, Lemire, J. M. et al. (1994) Endocrinology 135 (6): 2813-2821; Bouillon, R. et al. (1995) Endocine Review 16 (2):231-32).
  • isomers or “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • leukemia is intended to have its clinical meaning, namely, a neoplastic disease in which white corpuscle maturation is arrested at a primitive stage of cell development.
  • the disease is characterized by an increased number of leukemic blast cells in the bone marrow, and by varying degrees of failure to produce normal hematopoietic cells.
  • the condition may be either acute or chronic.
  • Leukemias are further typically categorized as being either lymphocytic i.e., being characterized by cells which have properties in common with normal lymphocytes, or myelocytic (or myelogenous), i.e., characterized by cells having some characteristics of normal granulocytic cells.
  • Acute lymphocytic leukemia arises in lymphoid tissue, and ordinarily first manifests its presence in bone marrow.
  • Acute myelocytic leukemia arises from bone marrow hematopoietic stem cells or their progeny.
  • the term acute myelocytic leukemia subsumes several subtypes of leukemia: myeloblastic leukemia, promyelocytic leukemia, and myelomonocytic leukemia.
  • leukemias with erythroid or megakaryocytic properties are considered myelogenous leukemias as well.
  • leukemic cancer refers to all cancers or neoplasias of the hemopoietic and immune systems (blood and lymphatic system).
  • the acute and chronic leukemias together with the other types of tumors of the blood, bone marrow cells (myelomas), and lymph tissue (lymphomas), cause about 10% of all cancer deaths and about 50% of all cancer deaths in children and adults less than 30 years old.
  • Chronic myelogenous leukemia (CML) also known as chronic granulocytic leukemia (CGL)
  • CML chronic myelogenous leukemia
  • CGL chronic granulocytic leukemia
  • leukemia is art recognized and refers to a progressive, malignant disease of the blood-forming organs, marked by distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow.
  • modulate refers to increases or decreases in the activity of a cell in response to exposure to a compound of the invention, e.g., the inhibition of proliferation and/or induction of differentiation of at least a sub-population of cells in an animal such that a desired end result is achieved, e.g., a therapeutic result.
  • this phrase is intended to include hyperactive conditions that result in pathological disorders.
  • Neoplasia refers to "new cell growth” that results as a loss of responsiveness to normal growth controls, e.g. to neoplastic cell growth.
  • a “hyperplasia” refers to cells undergoing an abnormally high rate of growth.
  • neoplasia and hyperplasia can be used interchangably, as their context will reveal, referring to generally to cells experiencing abno ⁇ nal cell growth rates.
  • Neoplasias and hyperplasias include “tumors,” which may be either benign, premalignant or malignant.
  • non-genomic vitamin D 3 activities include cellular (e.g., calcium transport across a tissue) and subcellular activities (e.g., membrane calcium transport opening of voltage-gated calcium channels, changes in intracellular second messengers) elicited by vitamin D 3 compounds in a responsive cell. Electrophysiological and biochemical techniques for detecting these activities are known in the art. An example of a particular well-studied non-genomic activity is the rapid hormonal stimulation of intestinal calcium mobilization, termed “transcaltachia” (Nemere I. et al. (1984)
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • polycyclyl or “polycyclic radical” refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
  • prodrug includes compounds with moieties which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge etal. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
  • the prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid.
  • prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, (e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters (e.g., acetyloxymethyl ester), acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di- lower alkyl amides, and hydroxy amides.
  • a prophylactically effective anti-neoplastic amount of a compound refers to an amount of a vitamin D 3 compound of the formula (I) or otherwise described herein which is effective, upon single or multiple dose administration to the patient, in preventing or delaying the occurrence of the onset of a neoplastic disease state.
  • the term "psoriasis” is intended to have its medical meaning, namely, a disease which afflicts primarily the skin and produces raised, thickened, scaling, nonscarring lesions. The lesions are usually sharply demarcated erythematous papules covered with overlapping shiny scales. The scales are typically silvery or slightly opalescent. Involvement of the nails frequently occurs resulting in pitting, separation of the nail, thickening and discoloration. Psoriasis is sometimes associated with arthritis, and it may be crippling.
  • reduced toxicity is intended to include a reduction in any undesired side effect elicited by a vitamin D 3 compound when administered in vivo, e.g., a reduction in the hypercalcemic activity.
  • sarcoma is art recognized and refers to malignant tumors of mesenchymal derivation.
  • the term "secosteroid" is art-recognized and includes compounds in which one of the cyclopentanoperhydro- phenanthrene rings of the steroid ring structure is broken. l ⁇ ,25(OH) 2 D 3 and analogs thereof are hormonally active secosteroids. In the case of vitamin D 3 , the 9-10 carbon-carbon bond of the B-ring is broken, generating a seco-B- steroid. The official IUPAC name for vitamin D 3 is 9,10-secocholesta-5,7,10(19)-trien- 3B-ol. For convenience, a 6-s-trans conformer of l ⁇ ,25(OH)2D 3 is illustrated herein having all carbon atoms numbered using standard steroid notation.
  • a dotted line ( — ) indicating a substituent which is in the ⁇ -orientation (i.e. , above the plane of the ring)
  • a wedged solid line ( ) indicating a substituent which is in the ⁇ -orientation (i.e. , below the plane of the molecule)
  • a wavy line ( - W - ) indicating that a substituent may be either above or below the plane of the ring.
  • the stereochemical convention in the vitamin D field is opposite from the general chemical field, wherein a dotted line indicates a substituent on Ring A which is in an ⁇ - orientation (i.e. , below the plane of the molecule), and a wedged solid line indicates a substituent on ring A which is in the ⁇ -orientation (i.e. , above the plane of the ring).
  • the A ring of the hormone l ⁇ ,25(OH) D 3 contains two asymmetric centers at carbons 1 and 3, each one containing a hydroxyl group in well-characterized configurations, namely the l ⁇ - and 3 ⁇ - hydroxyl groups.
  • carbons 1 and 3 of the A ring are said to be "chiral carbons" or "carbon centers.”
  • the term "sulfhydryl” or "thiol” means -SH.
  • subject includes organisms which are capable of suffering from a vitamin D 3 associated state or who could otherwise benefit from the administration of a vitamin D 3 compound of the invention, such as human and non-human animals.
  • Preferred human animals include human patients suffering from or prone to suffering from a vitamin D 3 associated state, as described herein.
  • non-human animals of the invention includes all vertebrates, e.g., , mammals, e.g., rodents, e.g., mice, and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.
  • systemic administration means the administration of a vitamin D 3 compound(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • terapéuticaally effective anti-neoplastic amount of a vitamin D 3 compound of the invention refers to an amount of an agent which is effective, upon single or multiple dose administration to the patient, in inhibiting the growth of a neoplastic vitamin D 3 -responsive cells, or in prolonging the survivability of the patient with such neoplastic cells beyond that expected in the absence of such treatment.
  • transplant rejection refers to an immune reaction directed against a transplanted organ(s) from other human donors (allografts) or from other species such as sheep, pigs, or non-human primates (xenografts). Therefore, the method of the invention is useful for preventing an immune reaction to transplanted organs from other human donors (allografts) or from other species (xenografts).
  • tissues for transplantation include, but are not limited to, heart, liver, kidney, lung, pancreas, pancreatic islets, bone marrow, brain tissue, cornea, bone, intestine, skin, and hematopoietic cells.
  • transplant rejection also includes disease symptoms characterized by loss of organ function. For example, kidney rejection would be characterized by a rising creatine level in blood.
  • Heart rejection is characterized by an endomyocardial biopsy, while pancreas rejection is characterized by rising blood glucose levels.
  • Liver rejection is characterized by the levels of transaminases of liver origin and bilirubin levels in blood. Intestine rejection is determined by biopsy, while lung rejection is determined by measurement of blood oxygenation.
  • urogenital urogenital system
  • urogential tract are used interchangeably and are intended to include all organs involved in reproduction and in the formation and voidance of urine. Included with in these terms are the kidneys, bladder and prostate.
  • VDR vitamin D response element
  • VDRE refers to D ⁇ A sequences composed of half-sites arranged as direct repeats. It is known in the art that type II receptors do not bind to their respective binding site as homodimers but require an auxiliary factor, RXR (e.g. RXR ⁇ , RXR ⁇ , RXR ⁇ ) for high affinity binding Yu et al. (1991) Cell 67:1251-1266; Bugge et al. (1992) EMBOJ. 11:1409-1418; Kliewer et ⁇ /. (1992) Nature 355:446-449; Leid et o/. (1992) EMBOJ. 11:1419-1435; Zhang et al. (1992) Nature 355:441-446).
  • RXR e.g. RXR ⁇ , RXR ⁇ , RXR ⁇
  • RXR ⁇ auxiliary factor
  • vitamin D 3 associated state is a state which can be prevented, treated or otherwise ameliorated by administration of one or more compounds of the invention.
  • Vitamin D 3 associated states include ILT3 -associated disorders, disorders characterized by an aberrant activity of a vitamin D 3 -responsive cell, disorders characterized by a deregulation of calcium and phosphate metabolism, and other disorders or states described herein.
  • vitamin D 3 -responsive cell includes any cell which is capable of responding to a vitamin D 3 compound having the formula I or otherwise described herein, or is associated with disorders involving an aberrant activity of hyperproliferative skin cells, parathyroid cells, neoplastic cells, immune cells, and bone cells. These cells can respond to vitamin D 3 activation by triggering genomic and/or non-genomic responses that ultimately result in the modulation of cell proliferation, differentiation survival, and/or other cellular activities such as hormone secretion. In a preferred embodiment, the ultimate responses of a cell are inhibition of cell proliferation and/or induction of differentiation-specific genes.
  • Exemplary vitamin D 3 responsive cells include immune cells, bone cells, neuronal cells, endocrine cells, neoplastic cells, epidermal cells, endodermal cells, smooth muscle cells, among others.
  • the invention provides Gemini vitamin D 3 compounds of formula I:
  • Ai is a single or double bond
  • a 2 is a single, a double or a triple bond
  • R 3 and t are each independently CpC 4 alkyl, C ⁇ -C deuteroalkyl, hydroxyalkyl, or haloalkyl;
  • R5, R ⁇ and R 7 are each independently hydroxyl, OC(O)C ⁇ -C 4 alkyl, OC(O)hydroxyalkyl, or OC(O)haloalkyl; the configuration at C 20 is R or S;
  • X! is H 2 or CH 2 ;
  • Various embodiments of this aspect of the invention include individual compounds of formula I wherein: Ai is a single bond; A is a single bond; A 2 is a triple bond; Ri, R 2 , R 3 , and t are each independently methyl or ethyl; Ri, R , R 3 , and are each independently C ⁇ -C 4 deuteroalkyl or haloalkyl; R 5 is hydroxyl; R 6 and R 7 are hydroxyl; R 6 and R 7 are each OC(O)C ⁇ -C 4 alkyl; Xi is H 2 ; Xi is CH ; Z is hydrogen; or
  • Z O.
  • R5, Re and R 7 are hydroxyl.
  • R ⁇ and R are each acetyloxy.
  • Z is hydrogen when at least one of Ri and R is -C 4 deuteroalkyl and at least one of R 3 and P ⁇ is haloalkyl or when at least one of Ri and R 2 is haloalkyl and at least one of R 3 and R t is C 1 -C 4 deuteroalkyl;
  • Z is O, -SH, or -NH 2 when Xi is H 2 and the configuration at C 20 is R.
  • Z is -OH.
  • Still other embodiments of this aspect of invention include those wherein Xj is CH 2 ; A 2 is a single bond; R ls R 2 , R 3 , and R t are each independently methyl or ethyl; and Z is -OH.
  • X ⁇ is H 2 ; A 2 is a single bond; R l5 R 2 , R 3 , and R 4 are each independently methyl or ethyl; the configuration at C 2 o is S; and Z is -OH.
  • Ri, R 2 , R 3 , and l ⁇ t are advantageously each methyl.
  • the haloalkyl is fluoroalkyl.
  • fluoroalkyl is fluoromethyl or trifluoromethyl.
  • Additional emobidments of this aspect of the invention include compounds Xi is H 2 ; A 2 is a triple bond; Ri and R 2 are each C ⁇ -C deuteroalkyl; R 3 and R t are each haloalkyl; and Z is hydrogen.
  • Xi is CH 2 ; A 2 is a triple bond; Ri and R 2 are each C ⁇ -C 4 deuteroalkyl; R 3 and Rt are each haloalkyl; and Z is hydrogen.
  • Ri and R 2 are advantageously each deuteromethyl and R 3 and R 4 are advantageously each trifluoromethyl.
  • Speicific compounds of the invention include: 1, 25-Dihydroxy-21- (2R,3-dihydroxy-3-methyl-butyl)-20R-cholecalciferol:
  • the structures of some of the compounds of the invention include asymmetric carbon atoms. Accordingly, the isomers arising from such asymmetry (e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and/or by stereochemically controlled synthesis.
  • Naturally occurring or synthetic isomers can be separated in several ways known in the art. Methods for separating a racemic mixture of two enantiomers include chromatography using a chiral stationary phase (see, e.g., "Chiral Liquid Chromatography,” W.J. Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also be separated by classical resolution techniques. For example, formation of diastereomeric salts and fractional crystallization can be used to separate enantiomers.
  • the diastereomeric salts can be formed by addition of enantiomerically pure chiral bases such as brucine, quinine, ephedrine, strychnine, and the like.
  • diastereomeric esters can be formed with enantiomerically pure chiral alcohols such as menthol, followed by separation of the diastereomeric esters and hydrolysis to yield the free, enantiomerically enriched carboxylic acid.
  • the invention also provides methods for treating a subject for a vitamin D 3 associated state, by administering to the subject an effective amount of a vitamin D 3 compound of formula (I) or otherwise described herein.
  • Vitamin D 3 associated states include disorders involving an aberrant activity of a vitamin D 3 - responsive cell, e.g., neoplastic cells, hyperproliferative skin cells, parathyroid cells, immune cells and bone cells, among others. Vitamin D 3 associated states also include ILT3 -associated disorders.
  • the Gemini vitamin D 3 compounds of the invention can provide a less toxic alternative to current methods of treatment.
  • the subject is a mammal, in particular a human.
  • the Gemini vitamin D 3 compound can be administered in combination with a pharmaceutically acceptable carrier.
  • the pharmaceutically-acceptable carrier provides sustained delivery of the Gemini vitamin D 3 compound to a subject for at least four weeks after administration to the subject.
  • the Gemini vitamin D 3 compound is administered orally. In other embodiments, the vitamin D 3 compound is administered intravenously. In yet other embodiments, the vitamin D 3 compound is administered topically. In still other embodiments, the vitamin D 3 compound is administered topically is administered parenterally.
  • the Gemini vitamin D 3 compounds are administered at a concentration of about 0.001 ⁇ g to about 100 ⁇ g/kg of body weight.
  • the present invention provides a method of treating a subject for a disorder characterized by aberrant activity of a vitamin D 3 -responsive cell.
  • the method involves administering to the subject an effective amount of a pharmaceutical composition of a vitamin D 3 compound of fonnula I or otherwise described herein such that the activity of the cell is modulated.
  • the cells to be treated are hyperproliferative cells .
  • the vitamin D 3 compounds of the invention can be used to inhibit the proliferation of a variety of hyperplastic and neoplastic tissues.
  • vitamin D 3 compounds of the invention can be used in the treatment of both pathologic and non-pathologic proliferative conditions characterized by unwanted growth of vitamin D 3 -responsive cells, e.g., hyperproliferative skin cells, immune cells, and tissue having transformed cells, e.g., such as carcinomas, sarcomas and leukemias.
  • the cells to be treated are aberrant secretory cells, e.g., parathyroid cells, immune cells.
  • this invention features a method for inhibiting the proliferation and/or inducing the differentiation of a hyperproliferative skin cell, e.g., an epidermal or an epithelial cell, e.g. a keratinocytes, by contacting the cells with a vitamin D 3 compound of the invention.
  • the method includes a step of contacting a pathological or non-pathological hyperproliferative cell with an effective amount of such vitamin D 3 compound to promote the differentiation of the hyperproliferative cells
  • the present method can be performed on cells in culture, e.g. in vitro or ex vivo, or can be performed on cells present in an animal subject, e.g., as part of an in vivo therapeutic protocol.
  • the therapeutic regimen can be carried out on a human or any other animal subject.
  • the vitamin D 3 compounds of the present invention can be used to treat a hyperproliferative skin disorder.
  • Exemplary disorders include, but are not limited to, psoriasis, basal cell carcinoma, keratinization disorders and keratosis.
  • eczema lupus associated skin lesions
  • psoriatic arthritis rheumatoid arthritis that involves hyperproliferation and inflammation of epithelial-related cells lining the joint capsule
  • dermatitides such as seborrheic dermatitis and solar dermatitis
  • keratoses such as seborrheic keratosis, senile keratosis, actinic keratosis.
  • photo-induced keratosis, and keratosis follicularis acne vulgaris; keloids and prophylaxis against keloid formation; nevi; warts including verruca, condyloma or condyloma acuminatum, and human papilloma viral (HPV) infections such as venereal warts; leukoplakia; lichen planus; and keratitis.
  • HPV human papilloma viral
  • vitamin D 3 compounds of the invention can be used to inhibit the hyperproliferation of keratinocytes in treating diseases such as psoriasis by administering an effective amount of these compounds to a subject in need of treatment.
  • psoriasis is intended to have its medical meaning, namely, a disease which afflicts primarily the skin and produces raised, thickened, scaling, nonscarring lesions.
  • the lesions are usually sharply demarcated erythematous papules covered with overlapping shiny scales.
  • the scales are typically silvery or slightly opalescent. Involvement of the nails frequently occurs resulting in pitting, separation of the nail, thickening and discoloration.
  • Psoriasis is sometimes associated with arthritis, and it may be crippling. Hyperproliferation of keratinocytes is a key feature of psoriatic epidermal hyperplasia along with epidermal inflammation and reduced differentiation of keratinocytes. Multiple mechanisms have been invoked to explain the keratinocyte hyperproliferation that characterizes psoriasis. Disordered cellular immunity has also been implicated in the pathogenesis of psoriasis.
  • the invention also features methods for inhibiting the proliferation and/or reversing the transformed phenotype of vitamin D 3 -responsive hyperproliferative cells by contacting the cells with a vitamin D 3 compound of formula (I) or otherwise described herein.
  • the method includes a step of contacting pathological or non-pathological hyperproliferative cells with an effective amount of a vitamin D 3 compound of the invention for promoting the differentiation of the hyperproliferative cells.
  • the present method can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed on cells present in an animal subject, e.g., as part of an in vivo therapeutic protocol.
  • the therapeutic regimen can be carried out on a human or other subject.
  • the vitamin D 3 compounds of formula I or otherwise described herein can be tested initially in vitro for their inhibitory effects in the proliferation of neoplastic cells.
  • cell lines that can be used are transformed cells, e.g., the human promyeloid leukemia cell line HL-60, and the human myeloid leukemia U-937 cell line (Abe E. et al. (1981) Proc. Natl. Acad. Sci. USA 78:4990-4994; Song L.N. and Cheng T. (1992) Biochem Pharmacol 43:2292-2295; Zhou J.Y. et al (1989) Blood 74:82-93; U.S. Pat. Nos. 5,401,733, U.S. 5,087,619).
  • vitamin D 3 compounds of the invention can be tested in vivo using various animal models known in the art and summarized in Bouillon, R. et al. (1995) Endocrine Reviews 16(2):233 (Table E), which is incorporated by reference herein.
  • SL mice are routinely used in the art to test vitamin D 3 compounds of the invention as models for MI myeloid leukemia (Honma et al. (1983) Cell Biol. 80:201-204; Kasukabe T. et al. (1987) Cancer Res. 47:567-572); breast cancer studies can be performed in, for example, nude mice models for human MX1 (ER) (Abe J. et al.
  • the subject method may also be used to inhibit the proliferation of hyperplastic/neoplastic cells of hematopoietic origin, e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • hyperplastic/neoplastic cells of hematopoietic origin e.g., arising from myeloid, lymphoid or erythroid lineages, or precursor cells thereof.
  • the present invention contemplates the treatment of various myeloid disorders including, but not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) CritRev. in Oncol/Hemotol. 11 :267-97).
  • APML acute promyeloid leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • Lymphoid malignancies which may be treated by the subject method include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • HLL hairy cell leukemia
  • W Waldenstrom's macroglobulinemia
  • malignant lymphomas contemplated by the treatment method of the present invention include, but are not limited to non- Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF) and Hodgkin's disease.
  • the vitamin D 3 compounds of the invention can be used in combinatorial therapy with conventional cancer chemotherapeutics.
  • Conventional treatment regimens for leukemia and for other tumors include radiation, drugs, or a combination of both.
  • the following drugs are often used to treat acute leukemias: vincristine, prednisone, methotrexate, mercaptopurine, cyclophosphamide, and cytarabine.
  • chronic leukemia for example, busulfan, melphalan, and chlorambucil can be used in combination.
  • All of the conventional anti-cancer drugs are highly toxic and tend to make patients quite ill while undergoing treatment. Vigorous therapy is based on the premise that unless every leukemic cell is destroyed, the residual cells will multiply and cause a relapse.
  • the subject method can also be useful in treating malignancies of the various organ systems, such as affecting lung, breast, lymphoid, gastrointestinal, and urogenital tract as well as adenocarcinomas which include malignancies such as most colon ' cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, bladder cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • malignancies of the various organ systems such as affecting lung, breast, lymphoid, gastrointestinal, and urogenital tract as well as adenocarcinomas which include malignancies such as most colon ' cancers, renal-cell carcinoma, prostate cancer and/or testicular tumors, bladder cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.
  • exemplary solid tumors that can be treated according to the method of the present invention include vitamin D 3 -responsive phenotypes of sarcomas and carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, baldder cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocar
  • a therapeutically effective anti-neoplastic amount or a prophylactically effective anti-neoplastic amount of the vitamin D 3 compound of the invention can be readily made by the physician or veterinarian (the "attending clinician"), as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
  • the dosages may be varied depending upon the requirements of the patient in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed.
  • the therapeutically effective antineoplastic amount or dose and the prophylactically effective antineoplastic amount or dose, a number of factors are considered by the attending clinician, including, but not limited to: the specific hyperplastic/neoplastic cell involved; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desirder time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual patient; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the vitamin D 3 compounds of the invention with other co-administered therapeutics); and other relevant circumstances.
  • the specific hyperplastic/neoplastic cell involved pharmacodynamic characteristics of the particular agent and its mode and route of administration
  • the desirder time course of treatment the species of mammal; its size, age, and general health
  • the specific disease involved the degree of or
  • U.S. Patent 5,427,916 describes method for predicting the effectiveness of antineoplastic therapy in individual patients, and illustrates certain methods which can be used in conjunction with the treatment protocols of the instant invention.
  • Treatment can be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage should be increased by small increments until the optimum effect under the circumstances is reached.
  • the total daily dosage may be divided and administered in portions during the day if desired.
  • a therapeutically effective antineoplastic amount and a prophylactically effective anti- neoplastic amount of a vitamin D 3 compound of the invention is expected to vary from about 0.1 milligram per kilogram of body weight per day (mg/kg/day) to about 100 mg kg/day.
  • Healthy individuals protect themselves against foreign invaders using many different mechanisms, including physical barriers, phagocytic cells in the blood and tissues, a class of immune cells known as lymphocytes, and various blood-born molecules. All of these mechanisms participate in defending individuals from a potentially hostile environment. Some of these defense mechanisms, known as natural or innate immunity, are present in an individual prior to exposure to infectious microbes or other foreign macromolecules, are not enhanced by such exposures, and do not discriminate among most foreign substances. Other defense mechanisms, known as acquired or specific immunity, are induced or stimulated by exposure of foreign substances, areakily specific for distinct macromolecules, and increase in magnitude and defensive capabilities with each successive exposure to a particular macromolecule.
  • lymphocytes in each individual are able to recognize and respond to many foreign antigens but are normally unresponsive to the potentially antigenic substances present in the individual.
  • Immunology Today 10:53 This immunological unresponsiveness is referred to as immune tolerance (see, e.g., Burt RK et al (2002) Blood 99:768; Coutinho, A. et al. (2001) Immunol. Rev. 182:89; Schwartz, RH (1990) Science 248:1349; Miller, J.F. et al. (1989) Immunology Today 10:53).
  • Self-tolerance is an acquired process that has to be learned by the lymphocytes of each individual. It occurs in part because lymphocytes pass through a stage in their development when an encounter with antigen presented by antigen-presenting cells (APCs) leads to their death or inactivation in a process known as positive and negative selection (see, e.g., Debatin KM (2001) Ann. Hematol. 80 Suppl 3:B29; Abbas, A. (1991), supra). Thus, potentially self-recognizing lymphocytes come into contact with self-antigens at this stage of functional immaturity and are prevented from developing to a stage at which they would be able to respond to self-antigens.
  • APCs antigen-presenting cells
  • the success of a transplant depends on preyenting the immune system of the host recipient from recognizing the transplant as foreign and, in some cases, preventing the graft from recognizing the host tissues as foreign.
  • the transplanted bone marrow may recognize the new host as foreign, resulting in graft versus host disease (GVHD). Consequently, the survival of the host depends on preventing both the rejection of the donor marrow as well as rejection of the host by the graft immune reaction (see, e.g., Waldmann H etal (2001) Int. Arch. Allergy Immunol. 126:11) .
  • Immunosuppressive drugs such as cyclosporin A (CsA), rapamycin, desoxyspergualine and FK-506 are also widely used.
  • Nonspecific immune suppression agents such as steroids and antibodies to lymphocytes, put the host at increased risk for opportunisitc infection and development of tumors.
  • many immunosuppressive drugs result in bone demineralization within the host (see, e.g., Chhajed PN et al. (2002) Indian J. Chest Dis.
  • the invention provides a method for modulating the activity of an immune cell by contacting the cell with a vitamin D 3 compound of formula I or otherwise described herein.
  • the invention provides a method of modulating the expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule in a cell, comprising contacting said cell with a vitamin D3 compound of described herein above in an amount effective to modulate the expression of an immunoglobulin-like transcript 3 (ILT3) surface molecule in said cell.
  • the cell is within a subject.
  • a related embodiment of the invention provides a method of inducing immunological tolerance in a subject, comprising administering to said subject a vitamin D3 compound described herein above in an amount effective to modulate the expression of an ILT3 surface molecule, thereby inducing immunological tolerance in said subject.
  • Another emboidmetn of the invention provides a method for modulating immunosuppressive activity by an antigen-presenting cell, comprising' contacting an antigen-presenting cell with a vitamin D3 compound described herein above in an amount effective to modulate ILT3 surface molecule expression, thereby modulating said immunosuppressive activity by said antigen-presenting cell.
  • the target of the methods is an antigen-presenting cell.
  • Antigen-presenting cells include dendritic cells, monocytes, and macrophages.
  • the expression of said immunoglobulin-like transcript 3 (ILT3) surface molecule is upregulated.
  • the present invention provides a method for suppressing immune activity in an immune cell by contacting a pathological or non-pathological immune cell with an effective amount of a vitamin D 3 compound of the invention to thereby inhibit an immune response relative to the cell in the absence of the treatment.
  • the present method can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed on cells present in an animal subject, e.g., as part of an in vivo therepeutic protocol. In vivo treatment can be carried out on a human or other animal subject.
  • the vitamin D 3 compounds of the invention can be tested initially in vitro for their inhibitory effects on T cell proliferation and secretory activity, as described in Reichel, H. et al, (1987) Proc. Natl. Acad. Sci. USA 84:3385-3389; Lemire, J. M. et al. (1985) J. Immunol 34:2032-2035.
  • the immunosuppressive effects can be tested in vivo using the various animal models known in the art and summarized by Bouillon, R. et al (1995) Endocine Reviews 16(2) 232 (Tables 6 and 7).
  • animal models for autoimmune disorders e.g., lupus, thyroiditis, encephalitis, diabetes and nephritis are described in (Lemire J.M. (1992) J. Cell Biochem. 49:26-31; Koizumi T. et al. (1985) Int. Arch. Allergy Appl Immunol 77:396-404; Abe J. et al (1990) Calcium Regulation and Bone Metabolism 146-151; Fournier C. et al. (1990) Clin. Immunol Immunopathol 54:53-63; Lemire J.M. and Archer D.C. (1991) J. Clin. Invest. 87:1103-1107); Lemire, J. M.
  • test compounds After identifying certain test compounds as effective suppresors of an immune response in vitro, these compounds can be used in vivo as part of a therapeutic protocol. Accordingly, another embodiment provides a method of suppressing an immune response, comprising administering to a subject a pharmaceutical preparation of a vitamin D 3 compounds of the invention, so as to inhibit immune reactions such as graft rejection, autoimmune disorders and inflammation.
  • the subject vitamin D 3 compound of the invention can be used to inhibit responses in clinical situations where it is desirable to downmodulate T cell responses.
  • autoimmune diseases including, for example, diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, diabetes, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sj ⁇ gren's Syndrome, including keratoconjunctivitis sicca secondary to Sj ⁇ gren's Syndrome, alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, kerato
  • the present invention provides methods and compositions for treating immune disorders, such as, for example, autoimmune disorders and transplant rejections, such as graft versus host disease (GVHD).
  • immune disorders such as, for example, autoimmune disorders and transplant rejections, such as graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • a therapeutically effective immunosuppressive amount can be readily made by the attending clinician, as one skilled in the art, by the use of known techniques and by observing results obtained under analogous circumstances.
  • Compounds which are determined to be effective in animals, e.g., dogs, rodents may be extrapolated accordingly to humans by those skilled in the art.
  • Starting dose/regimen used in animals can be estimated based on prior studies.
  • doses of vitamin D 3 compounds of the invention to treat autoimmune disorders in rodents can be initially estimated in the range of 0.1 g/kg/day to 1 g/kg/day, administered orally or by injection.
  • the present invention also relates to a method of treating in a subject a disorder characterized by deregulation of calcium metabolism.
  • This method comprises contacting a pathological or non-pathological vitamin D 3 responsive cell with an effective amount of a vitamin D 3 compound of the invention to thereby directly or indirectly modulate calcium and phosphate homeostasis.
  • Techniques for detecting calcium fluctuation in vivo or in vitro are known in the art.
  • Exemplary Ca "1-1" homeostasis related assays include assays that focus on the intestine where intestinal 45ca2 + absorption is determined either 1) in vivo (Hibberd K.A. and Norman A.W. (1969) Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al.
  • the bone-oriented assays include: 1) assessment of bone resorption as determined via the release of Ca2+ from bone in vivo (in animals fed a zero Ca ⁇ + diet) (Hibberd K.A. and Norman A.W. (1969) Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967) J. Nutr. 91:319-323), or from bone explants in vitro (Bouillon R. et al. (1992) J. Biol. Chem.
  • vitamin D 3 compounds of the invention can be used to modulate bone metabolism.
  • bone metabolism is intended to include direct or indirect effects in the formation or degeneration of bone structures, e.g., bone formation, bone resorption, etc., which may ultimately affect the concentrations in serum of calcium and phosphate.
  • This term is also intended to include effects of vitamin D 3 compounds in bone cells, e.g. osteoclasts and osteoblasts, that may in turn result in bone formation and degeneration.
  • vitamin D 3 compounds exert effects on the bone forming cells, the osteoblasts through genomic and non-genomic pathways (Walters M.R. et al. (1982) J. Biol Chem.
  • vitamin D 3 compounds are known in the art to support different activities of bone resorbing osteoclasts such as the stimulation of differentiation of monocytes and mononuclear phagocytes into osteoclasts (Abe E. et al. (1988) J. Bone Miner Res. 3:635-645; Takahashi N. et al. (1988) Endocrinology 123:1504-1510; UdagawaN. et al. (1990) Proc. Natl Acad. Sci. USA 87:7260-7264). Accordingly, vitamin D 3 compounds of the invention that modulate the production of bone cells can influence bone formation and degeneration.
  • the present invention provides a method for modulating bone cell metabolism by contacting a pathological or a non-pathological bone cell with an effective amount of a vitamin D 3 compound of the invention to thereby modulate bone formation and degeneration.
  • the present method can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed in cells present in an animal subject, e.g., cells in vivo.
  • Exemplary culture systems that can be used include osteoblast cell lines, e.g., ROS 17/2.8 cell line, monocytes, bone marrow culture system (Suda T. et al. (1990) Med. Res. Rev. 7:333-366; Suda T. et al (1992) J Cell Biochem.
  • a method for treating osteoporosis comprising administering to a subject a pharmaceutical preparation of a vitamin D 3 compound of the invention to thereby ameliorate the condition relative to an untreated subject.
  • Vitamin D 3 compounds of the invention can be tested in ovarectomized animals, e.g., dogs, rodents, to assess the changes in bone mass and bone formation rates in both normal and estrogen-deficient animals. Clinical trials can be conducted in humans by attending clinicians to determine therapeutically effective amounts of the vitamin D 3 compounds of the invention in preventing and treating osteoporosis.
  • therapeutic applications of the vitamin D 3 compounds of the invention include treatment of other diseases characterized by metabolic calcium and phosphate deficiencies.
  • diseases are the following: osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis fibrosa cystica, renal osteodystrophy, osteosclerosis, anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta ossium, secondary hyperparathyrodism, hypoparathyroidism, hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced metabolism, medullary carcinoma, chronic renal disease, hypophosphatemic VDRR, vitamin D-dependent rickets, sarcoidosis, glucocorticoid antagonism, malabsorption syndrome, steatorrhea, tropical sprue, idiopathic hypercalcemia and milk fever.
  • Hormone Secretion is the following: osteoporosis, osteodystrophy, osteo
  • the present invention provides a method for modulating hormone secretion of a vitamin D 3 - responsive cell, e.g., an endocrine cell.
  • Ho ⁇ none secretion includes both genomic and non-genomic activities of vitamin D 3 compounds of the invention that control the transcription and processing responsible for secretion of a given hormone e.g., parathyroid hormone (PTH), calcitonin, insulin, prolactin (PRL) and TRH in a vitamin D 3 responsive cell (Bouillon, R. et al. (1995) Endocrine Reviews 16(2):235-237).
  • PTH parathyroid hormone
  • PRL prolactin
  • the present method can be performed on cells in culture, e.g. in vitro or ex vivo, or on cells present in an animal subject, e.g., in vivo.
  • Vitamin D 3 compounds of the invention can be initially tested in vitro using primary cultures of parathyroid cells.
  • Other systems that can be used include the testing by prolactin secretion in rat pituitary tumor cells, e.g., GH4C1 cell line (Wark J.D. and Tashjian Jr. A.H. (1982) Endocrinology 111:1755-1757; Wark J. D. and Tashjian Jr. A.H. (1983) J. Biol. Chem. 258:2118-2121; Wark J.D. and Gurtler V.
  • vitamin D 3 compounds of the invention can be characterized in vivo using animals models as described in Nko M. et al (1982) Miner Electrolyte Metab. 5:67-75; Oberg F. et al. (1993) J. Immunol. 150:3487-3495; Bar-Shavit Z. et al. (1986) Endocrinology 118:679-686; Testa U. et al. (1993) J. Immunol. 150:2418-2430; Nakamaki T. et al (1992) Anticancer Res.
  • the vitamin D 3 compounds of the present invention can be used to inhibit parathyroid hormone (PTH) processing, e.g., transcriptional, translational processing, and/or secretion of a parathyroid cell as part of a therapeutic protocol.
  • PTH parathyroid hormone
  • Therapeutic methods using these compounds can be readily applied to all diseases, involving direct or indirect effects of PTH activity, e.g., primary or secondary responses.
  • vitamin D 3 compounds of the invention include treating diseases such as secondary hyperparathyroidism of chronic renal failure (Slatopolsky E. et al. (1990) Kidney Int. 38:S41-S47; Brown A.J. et al (1989) J. Clin. Invest. 84:728-732).
  • diseases such as secondary hyperparathyroidism of chronic renal failure (Slatopolsky E. et al. (1990) Kidney Int. 38:S41-S47; Brown A.J. et al (1989) J. Clin. Invest. 84:728-732).
  • Dete ⁇ nination of therapeutically affective amounts and dose regimen can be performed by the skilled artisan using the data described in the art.
  • the present invention provides a method of protecting against neuronal loss by contacting a vitamin D 3 responsive cell, e.g., a neuronal cell, with a vitamin D 3 compound of the invention to prevent or retard neuron loss.
  • the language "protecting against” is intended to include prevention, retardation, and/or termination of deterioration, impairment, or death of a neurons.
  • Neuron loss can be the result of any condition of a neuron in which its normal function is compromised.
  • Neuron deterioration can be the result of any condition which compromises neuron function which is likely to lead to neuron loss.
  • Neuron function can be compromised by, for example, altered biochemistry, physiology, or anatomy of a neuron.
  • Deterioration of a neuron may include membrane, dendritic, or synaptic changes which are detrimental to normal neuronal functioning.
  • the cause of the neuron deterioration, impairment, and/or death may be unknown. Alternatively, it may be the result of age- and/or disease-related changes which occur in the nervous system of a subject.
  • neuron loss When neuron loss is described herein as “age-related”, it is intended to include neuron loss resulting from known and unknown bodily changes of a subject which are associated with aging. When neuron loss is described herein as “disease-related”, it is intended to include neuron loss resulting from known and unknown bodily changes of a subject which are associated with disease. It should be understood, however, that these terms are not mutually exclusive and that, in fact, many conditions that result in the loss of neurons are both age- and disease-related. Exemplary age-related diseases associated with neuron loss and changes in neuronal morphology include, for example, Alzheimer's Disease, Pick's Disease, Parkinson's Disease, Vascular Disease, Huntington's Disease, and Age-Associated Memory Impairment.
  • Alzheimer's Disease patients neuron loss is most notable in the hippocampus, frontal, parietal, and anterior temporal cortices, amygdala, and the olfactory system.
  • the most prominently affected zones of the hippocampus include the CA1 region, the subiculum, and the entorhinal cortex.
  • Memory loss is considered the earliest and most representative cognitive change because the hippocampus is well known to play a crucial role in memory.
  • Pick's Disease is characterized by severe neuronal degeneration in the neocortex of the frontal and anterior temporal lobes which is sometimes accompanied by death of neurons in the striatum.
  • Parkinson's Disease can be identified by the loss of neurons in the substantia nigra and the locus ceruleus. Huntington's Disease is characterized by degeneration of the intrastriatal and cortical cholinergic neurons and GABA-ergic neurons. Parkinson's and Huntington's Diseases are usually associated with movement disorders, but often show cognitive impairment (memory loss) as well.
  • Age- Associated Memory Impairment is another age-associated disorder that is characterized by memory loss in healthy, elderly individuals in the later decades of life.
  • the neural basis for AAMI has not been precisely defined.
  • neuron death with aging has been reported to occur in many species in brain regions implicated in memory, including cortex, hippocampus, amygdala, basal ganglia, cholinergic basal forebrain, locus ceruleus, raphe nuclei, and cerebellum.
  • Vitamin D 3 compounds of the invention can protect against neuron loss by genomic or non-genomic mechanisms.
  • Nuclear vitamin D 3 receptors are well known to exist in the periphery but have also been found in the brain, particularly in the hippocampus and neocortex.
  • Non-genomic mechanisms may also prevent or retard neuron loss by regulating intraneuronal and/or peripheral calcium and phosphate levels.
  • vitamin D 3 compounds of the invention may protect against neuronal loss by acting indirectly, e.g., by modulating serum PTH levels. For example, a positive correlation has been demonstrated between serum PTH levels and cognitive decline in Alzheimer's Disease.
  • the present method can be performed on cells in culture, e.g. in vitro or ex vivo, or on cells present in an animal subject, e.g., in vivo.
  • Vitamin D 3 compounds of the invention can be initially tested in vitro using neurons from embryonic rodent pups (See e.g. U.S. Patent No. 5,179,109-fetal rat tissue culture), or other mammalian (See e.g. U.S. Patent No. 5,089,517-fetal mouse tissue culture) or non-mammalian animal models.
  • the present invention provides a method of modulating the activity of a vascular smooth muscle cell by contacting a vitamin D 3 -responsive smooth muscle cell with a vitamin D 3 compound of the invention to activate or, preferably, inhibit the activity of the cell.
  • activity of a smooth muscle cell is intended to include any activity of a smooth muscle cell, such as proliferation, migration, adhesion and/or metabolism.
  • the vitamin D 3 compounds of the invention can be used to treat diseases and conditions associated with aberrant activity of a vitamin D 3 - responsive smooth muscle cell.
  • the present invention can be used in the treatment of hyperproliferative vascular diseases, such as hypertension induced vascular remodeling, vascular restenosis and atherosclerosis.
  • the compounds of the present invention can be used in treating disorders characterized by aberrant metabolism of a vitamin D 3 -responsive smooth muscle cell, e.g., arterial hypertension.
  • the present method can be performed on cells in culture, e.g. in vitro or ex vivo, or on cells present in an animal subject, e.g., in vivo.
  • Vitamin D 3 compounds of the invention can be initially tested in vitro as described in Catellot et al. (1982), J. Biol. Chem. 257(19): 11256.
  • Renin- angiotensin regulatory cascade plays a significant role in the regulation of blood pressure, electrolyte and volume homeostasis (Y.C. Li, Abstract, DeLuca Symposium on Vitamin D 3 , Tauc, New Mexico, June 15 - June 19, 2002, p. 18).
  • the invention provides a method of treating a subject for for hypertension.
  • the method comprises administering to said subject an effective amount of a Gemini vitamin D 3 compound, such that said subject is treated for hpertension
  • the Gemini vitamin D 3 compound suppresses expression of renin, thereby treating the subject for hypertension.
  • Gemini vitamin D3 compounds useful in the treatment of hypertesion are compounds having formula II:
  • Ai is a single or a double bond
  • a 2 is a single, a double or a triple bond
  • a 3 is a single bond, an E-double bond, a Z-double bond or a triple bond, provided Z is absent when A 3 is a triple bond;
  • Ri, R 2; R 3 and R t are each independently C ⁇ -C 4 alkyl, C ⁇ -C 4 deuteroalkyl, hydroxyalkyl, or haloalkyl; or Ri and R 2 together with C 5 form a C ⁇ -C 4 cycloalkyl or cyclohaloalkyl; or R 3 and together with C 25 form a C ⁇ -C 4 cycloalkyl or cyclohaloalkyl; R 5 , R 7 and R 8 are each independently hydroxyl, OC(O)C ⁇ -C 4 alkyl,
  • Re is hydrogen, hydroxyl, halogen, OC(O)C ⁇ -C 4 alkyl, OC(O)hydroxyalkyl, or OC(O)haloalkyl;
  • Xi is H 2 or CH 2 ;
  • the haloalkyl, the cyclohaloalkyl and the halogen recited in formula II are fluoroalkyl, cyclofluoroalky and fluorine, respectively.
  • the method further comprises obtaining the Gemini vitamin D 3 compound of formula II.
  • Specific compounds of formula II include the following Gemini vitamin D 3 compounds:
  • Particularly advantageous compounds for use in the method include 1, 25- Dihydroxy-21-(2R,3-dihydroxy-3-methyl-butyl)-20R-cholecalciferol, or 1, 25- Dihydroxy-21 - (2R,3-dihydroxy-3-methyl-butyl)-20S-cholecalciferol.
  • the invention provides a method of suppressing renin expression in a subject comprising administering to a subject an effective amount of a Gemini vitamin D 3 compound such that renin expression in said subject is suppressed.
  • the Gemini vitamin D 3 compounds include the compounds of formula II described above.
  • the invention also provides a method for for treating a subject for a urogenital disorder.
  • the method comprises administering to the subject an effective amount of a vitamin D 3 compound of formula I above, such that the subject is treated for the urogential disorder.
  • the urogenital disorder comprises bladder dysfunction, especially bladder dysfunction related to morphological bladder changes.
  • bladder dysfuction as used in this embodiment does not include cancer of the bladder and associated urogenital organs.
  • Morphological bladder changes including a progressive de-nervation and hypertrophy of the bladder wall are frequent histological findings in patients with different bladder disorders such as overactive bladder and clinical BPH.
  • the increase in tension and/or strain on the bladder observed in these conditions has been shown to be associated with cellular and molecular alterations, e.g., in cytoskeletal and contractile proteins, in mitochondrial function, and in various enzyme activities of the smooth muscle cells.
  • the growth of the bladder wall also involves alterations in its extracellular matrix and non-smooth muscle components.
  • BPH benign prostatic hyperplasia
  • the invention also provides a method for treatment of BPH comprising administering to a subject an effective amount of a vitamin D 3 compound of formula I above, such that the subject is treated for BPH.
  • BPH is commonly associated with enlargement of the gland (prostate) leading to bladder outlet obstruction (BOO) and symptoms secondary to BOO.
  • BPH is also associated with morphological bladder changes, including a progressive denervation and hypertrophy of the bladder wall, the latter possibly as a consequence of increased functional demands.
  • the compounds of the invention are useful for the treatment of storage (irritative) symptoms of BPH, as well as for bladder outlet obstruction caused by BPH.
  • Urorgenital disorders in accordance with the invention also include interstitial cystitis.
  • the invention also provides a method for treatment of interstitial cystitis comprising administering to a subject an effective amount of a vitamin D 3 compound of formula I above, such that the subject is treated for interstitial cystitis.
  • Interstitial cystitis is a chronic inflammatory bladder disease characterized by pelvic pain, urinary urgency and frequency. Unlike other bladder dysfunction conditions, IC is characterized by chronic inflammation of the bladder wall which is responsible for the symptomatology. In other words, the cause of the abnormal bladder contractility is the chronic inflammation and as a consequence the treatment should target this etiological component. In fact, the traditional treatment of bladder dysfunctions, like overactive bladder, with smooth muscle relaxant agents, is not effective in patients with IC.
  • the invention also provides a pharmaceutical composition, comprising an effective amount a vitamin D 3 compound of formula I or otherwise described herein and a pharmaceutically acceptable carrier.
  • the effective amount is effective to treat a vitamin D 3 associated state, as described previously.
  • the vitamin D 3 compound is administered to the subject using a pharmaceutically-acceptable formulation, e.g., a pharmaceutically-acceptable formulation that provides sustained delivery of the vitamin D 3 compound to a subject for at least 12 hours, 24 hours, 36 hours, 48 hours, one week, two weeks, three weeks, or four weeks after the pharmaceutically-acceptable formulation is administered to the subject.
  • these pharmaceutical compositions are suitable for topical or oral administration to a subject.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the methods of the invention further include administering to a subject a therapeutically effective amount of a vitamin D 3 compound in combination with another pharmaceutically active compound.
  • pharmacuetically active compounds include compounds known to treat autoimmune disorders, e.g., immunosuppressant agents such as cyclosporin A, rapamycin, desoxyspergualine, FK 506, steroids, azathioprine, anti-T cell antibodies and monoclonal antibodies to T cell subpopulations.
  • Other pharmaceutically active compounds that may be used can be found in Harrison's Principles of Internal Medicine, Thirteenth Edition, Eds. T.R.
  • angiogenesis inhibitor compound and the pharmaceutically active compound may be administered to the subject in the same pharmaceutical composition or in different pharmaceutical compositions (at the same time or at different times).
  • pharmaceutically acceptable is refers to those vitamin D 3 compounds of the present invention, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier includes pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • a liquid or solid filler such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (I3) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • compositions containing a vitamin D 3 compound(s) include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • compositions include the step of bringing into association a vitamin D 3 compound(s) with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a vitamin D 3 compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a vitamin D 3 compound(s) as an active ingredient.
  • a compound may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example,
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the vitamin D 3 compound(s) include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3- butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, so
  • the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active vitamin D 3 compound(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more vitamin D 3 compound(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate
  • Dosage forms for the topical or transdermal administration of a vitamin D 3 compound(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active vitamin D 3 compound(s) may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to vitamin D 3 compound(s) of the present invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a vitamin D 3 compound(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • the vitamin D 3 compound(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • Transdermal patches have the added advantage of providing controlled delivery of a vitamin D 3 compound(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the active ingredient across the skin.
  • the rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel.
  • Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more vitamin D 3 compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutan
  • Injectable depot forms are made by forming microencapsule matrices of vitamin D 3 compound(s) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • vitamin D 3 compound(s) When administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically-acceptable carrier.
  • the vitamin D 3 compound(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • An exemplary dose range is from 0.1 to 10 mg per day.
  • a preferred dose of the vitamin D 3 compound for the present invention is the maximum that a patient can tolerate and not develop serious hypercalcemia.
  • the vitamin D 3 compound of the present invention is administered at a concentration of about 0.001 ⁇ g to about 100 ⁇ g per kilogram of body weight, about 0.001 - about 10 ⁇ g/kg or about 0.001 ⁇ g - about 100 ⁇ g/kg of body weight. Ranges intermediate to the above-recited values are also intended to be part of the invention.
  • Schemes 1-5 below graphically depict the reaction steps for the synthesis of the 24- hydroxyl Gemini vitamin D 3 compounds of the invention.
  • 24- hydroxyl geminal vitamin D 3 compounds 2, 3 and 38 the convergent and Wittig-Horner reaction using the Lythgoe phosphine oxide coupling protocol was used (Scheme 1).
  • Two elaborated ketones 34 and 27 were each linked to the functionalized (2- cyclohexylethenyl)diphenylphosphine oxide 28.
  • a single step removed all five silyl protecting groups in 35 and 29 and lead to the target compounds 2 and 3.
  • the diols 6 and 7 were then converted to the iodo alcohols 8 and 9 which served not only as key intermediates for the synthesis of 2 and 3, but also for the stereochemical elucidation of the two hydroboration products from which they were derived.
  • the iodo alcohol 8 derived from the diol with the shorter chromatographic retention time, was reacted with lithium acetylide to furnish an acetylene derivative that was identical with 14 and different from its 6(R) epimer. Both 14, representing the 6(S) configuration, and the corresponding 6(R)-epimer, were previously synthesized.
  • alkeneol 5 was subjected to an ene- reaction with formaldehyde, the resulting mixture of alkenediols 10a and 10b was hydrogenated to furnish the epimeric diol pair 11 and 12 that also could be separated by chromatography.
  • the two protective silyl groups were then removed to produce 15b.
  • This triol was oxidized to the ketone 16 whose configuration was determined by crystal analysis and shown to have the S-configuration at the stereocenter equivalent to C-20 in the vitamin D series.
  • the alkynol derived from 8 was identified as 14.
  • the hydroboration product 6 was regarded as the R-isomer with respect to the stereocenter in the side-chain assembly.
  • Scheme 6 graphically depicts the reaction steps for the synthesis of the 24-keto Gemini vitamin D 3 compounds of the invention, namely l,25-dihydroxy-20S- 21(3-hydroxy-3-methyl-butyl)-24-keto-19-nor-cholecalciferol (12) and 1,25-dihydroxy- 20S-21(3-hydroxy-3-methyl-butyl)-24-keto-cholecalciferol (14).
  • Reaction schemes 7, 8 and 9 below graphically depict the reaction steps for the synthesis of the the non-deuterated compounds 6b and 6a (Maehr, H. and Uskokovic, M., Eur. J. Org. Chem. 1703-1713 (2004)) that correspond to the deuteromethyl Gemini vitamin D 3 compounds of the invention, namely l,25-Dihydroxy-21(3-hydroxy-3- trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero-19-nor-20S-cholecalciferol (39) and 1,25 -Dihydroxy-21(3 -hydroxy-3 -trifluoromethyl-4-trifluoro-butynyl)-26,27- hexadeutero-20S-cholecalciferol (40), respectively.
  • the non-deuterated final product 6b corresponds to 1,25- Dihydroxy-21 (3-hydroxy-3 -trifluoromethyl-4-trifluoro-butynyl)-26,27-hexadeutero- 19- nor-20S-cholecalciferol (39).
  • the non-deuterated final product 6a corresponds to Dihydroxy-21(3-hydroxy-3-trifluoromethyl-4-trifluoro-butynyl)-26,27- hexadeutero-20S-cholecalciferol (40).
  • Chiral synthesis can result in products of high stereoisomer purity. However, in some cases, the stereoisomer purity of the product is not sufficiently high.
  • the skilled artisan will appreciate that the separation methods described herein can be used to further enhance the stereoisomer purity of the vitamin D 3 -epimer obtained by chiral synthesis.
  • 19b l-(Trimethylsilyl)imidazole (1 mL) was added to a solution of 19a (0.8 g) in cyclohexane (10 mL) and stirred overnight then flash-chromatographed using a stepwise gradient of hexane, 1:39 and 1:19 ethyl acetate - hexane.
  • the mixture was diluted with methanol (20 mL), stirred for 3 min, then ice (20 g) was added, stirred for 2 min and the supernatant decanted into a mixture containing saturated ammonium chloride (50 mL).
  • the residue was repeatedly washed with small amounts of tetrahydrofuran that was also added to the salt solution, which was then equilibrated with ethyl acetate (80 mL).
  • the aqueous layer was re-extracted once with ethyl acetate (20 mL), the combined extracts were washed with brine (10 mL) then dried and evaporated.
  • the aqueous phase was re-extracted with ethyl acetate (2x20 mL), the combined extracts were washed with water (5 mL) and brine (10 mL), then 1 : 1 brine - saturated sodium hydrogen carbonate solution and dried.
  • the residue was purified by flash-chromatography using a step-wise gradient from 1:1 to 2:1 ethyl acetate - hexane and neat ethyl acetate to give a residue that was taken up in 1:1 dichloromethane - hexane, filtered and evaporated to furnish amorphous solids, 0.3039 g (85%): [ ⁇ ] D + 42.6° (methanol, c 0.48); !
  • 17b l-(Trimethylsilyl)imidazole (0.146 mL) was added to a solution of 17a (0.145 g, 0.27 mmol) in cyclohexane (2 mL). After 17 h the product was purified by flash chromatography using a stepwise gradient of 1 : 79 and 1:39 ethyl acetate - hexane to give 17b as colorless residue, 0.157 g 0.258 mmol, TLC (1:9 ethyl acetate - hexane) Rf 0.14.
  • the ketone 32 was stirred in a 1 N oxalic acid solution in 90 % methanol. The mixture became homogeneous after a few min. TLC (ethyl acetate) suggested complete reaction after 75 min (Rf 0.24 for 33). Thus, calcium carbonate (0.60 g) was added and the suspension stirred overnight, then filtered.
  • the deprotection reaction of 37 was carried out in IM solution of tetrabutylammonium fluoride in tetrahydrofuran to give 38.
  • the mixture was diluted with brine after 25 h, stirred for 5 min and then equilibrated with ethyl acetate and water.
  • the aqueous layer was re-extracted once with ethyl acetate, the combined extracts were washed with water and brine, and then dried and evaporated.
  • the residue was flash-chromatographed to give a residue that was taken up in methyl formate and evaporated to yield 38.
  • the aqueous layer was re- extracted once with 25 mL of 1 : 1 dichloromethane - hexane.
  • the organic layers were combined then washed once with 15 mL of brine, dried and evaporated.
  • the resulting material was chromatographed on silica gel using hexane, 1 :39, 1:19 and 1 :9 ethyl acetate - hexane as stepwise gradients.
  • the main band was eluted with 1 :9 ethyl acetate - hexane to provide 1.2611 g of 6 as a colorless syrup.
  • the column was eluted with dichloromethane followed by 1 : 1 ethyl acetate - hexane until no solute was detectable in the effluent.
  • the effluent was evaporated and the colorless oil.
  • This oil was then chromatographed on a silica gel using 1:4, 1:3, 1:2, 1:1 and 2:1 ethyl acetate - hexane as stepwise gradients to furnish 0.2077 g of the diketone 9.
  • This material was chromatographed on a flash column, 15x150 mm using hexane and 1:100 ethyl acetate - hexane as stepwise gradients to yield 0.1572 g of the title compound 11 as a colorless syrup.
  • the light-tan solution was the diluted with 5 mL of brine, stirred for 5 min and transferred to a separatory funnel with 50 mL of ethyl acetate and 5 mL of water then re-extraction with 5 mL of ethyl acetate.
  • the organic layers were combined, washed with 5x10 mL of water, 10 mL of brine, dried and evaporated.
  • Compound 13 was prepared as described for 11 in Example 4 but by reacting 10 with [(2Z)-2-[(3S,5R)-3,5-bis(tert-butyldimethylsilanyloxy) methylenecyclohexylidene]- ethyl]diphenylphosphine oxide (17).
  • the maximum tolerated dose of the vitamin D 3 compounds of the invention were determined in eight week-old female C57BL/6 mice (3 mice/group) dosed orally (0.1 ml/mouse) with various concentrations of Vitamin D 3 analogs daily for four days. Analogs were formulated in miglyol for a final concentration of 10, 30, 100 and 300 ⁇ g/kg when given at 0.1 ml/mouse p.o. daily. Blood for serum calcium assay was drawn by tail bleed on day five, the final day of the study. Serum calcium levels were determined using a colorimetric assay (Sigma Diagnostics, procedure no. 597). The highest dose of analog tolerated without inducing hypercalcemia (serum calcium >10.7 mg/dl) was taken as the maximum tolerated does (MTD). Table 1 shows the relative MTD for four vitamin D 3 compounds.
  • Immature dendritic cells were prepared as described in Romani, N. et al, J. Immunol. Meth. 196:137.
  • IFN- ⁇ production by allogeneic T cell activation in the mixed leukocyte response (MLR) was determined as described in Penna, G., et al, J. Immunol., 164: 2405-2411 (2000).
  • peripheral blood mononuclear cells PBMC
  • peripheral blood mononuclear cells PBMC
  • PBMC peripheral blood mononuclear cells
  • IFN- ⁇ production in the MLR assay was measured by ELISA and the results expressed as amount (nM) of test compound required to induce 50% inhibition of IFN- ⁇ production (IC 50 ).
  • IC 50 amount of test compound required to induce 50% inhibition of IFN- ⁇ production
  • Table 1 '*' represents good down regulation of INF- ⁇ (e.g., less than 100 ICso pM), and '**' represents very good down regulation of INF- ⁇ (e.g., greater than 100 IC 50 pM).
  • vitamin D 3 supplementation reduces blood pressure in patients with essential hypertension (Lind, L., et al, Am. J. Hypertens., 2: 20-25 (1989); Pfeifer, M., et al, J.Clin. Endocrinol. Metab. 86: 1633-1637 (2001)) and treatment with 1,25 dihydroxy vitamin D 3 [l,25(OH) 2 D 3 ] reduces blood pressure, plasma renin activity and Angiotensin II levels in patients with hyperparathyroidism (Kimura, Y., et al, Intern. Med. 38: 31-35 (1999); Park, C.W., et al, Am. J. Kidney Dis.
  • l,25(OH) 2 D 3 is a negative regulator of the renin- angiotensin system (Li, Y.C, et al, J. Clin. Invest. 110(2): 229-238 (2002)). In other words, l,25(OH) 2 D 3 suppresses expression of renin.
  • a renin suppression assay (Li, Y.C, et al, J. Clin. Invest. 110(2): 229-238 (2002)) was used to compare the renin suppression activity of certain Gemini vitamin D 3 compounds of the invention with that of l,25(OH) 2 D 3 .
  • 12 Gemini compounds according to the invention were used to treat As4.1hVDR cells at 10 "10 , 10 "9 and 10 "8 M for 24 hours. Renin mRNA was quantified by Northern blots. The same cells were treated with l,25(OH) 2 D 3 under the same considitions as a control.
  • * - indicates no activity; +/- and + indicate less active than 1,25-dihydroxyvitamin D 3 ; ++ indicates as active as 1,25-dihydroxyvitamin D3; and +++ indicates more active that 1,25-dihydroxyvitamin D 3 .
  • Bladder cancer cell lines (T24, RT112, HT1376 and RT4 are human bladder cancer cell lines; NHEK are normal human keratinocytes) were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were plated at 3 x 103 per well, in flat bottomed 96-well plates in 100 ⁇ l of DMEM medium containing: 5% Fetal Clone I, 50 ⁇ g/1 gentamicin, 1 mM sodium pyruvate and 1% non-essential amino acids.
  • VDR ligands compounds 2, 3, 12, 14, 38, 39 and 40
  • concentrations ranging from 100 ⁇ M to 0.3 ⁇ M in 100 ⁇ l of above-mentioned complete medium.
  • cell proliferation was measured using a fluorescence-based proliferation assay kit. (CyQuant Cell Proliferation Assay Kit, Molecular Probes, Eugene, OR, USA). The IC50 was calculated from the regression curve of the tifration data. The results are shown in Table 3.
  • PBMCs peripheral blood mononuclear cells
  • monocytes were cultured for 6 to 7 days at a cell density of 1-2 x 106 in RPMI medium supplemented with 5% FetalClone I (Hyclone Laboratories, Logan, Utah) 2 mM L-glutamine, 50 mg/ml gentamicin, 1 mM sodium pyruvate and 1% nonessential amino acids, containing 800 U/ml GM-CSF (Mielogen 300, Schering-Plough) and 10 ng/ml interieukin (IL)-4 (PharMingen, San Diego, California). Every other day, approximately 20% of the medium was removed and replaced by the same volume of fresh medium containing GM-CSF and IL-4.
  • FetalClone I Hyclone Laboratories, Logan, Utah
  • gentamicin 1 mM sodium pyruvate
  • nonessential amino acids containing 800 U/ml GM-CSF (Mielogen 300, Schering-Plough) and 10 ng
  • non-adherent cells (representing immature Dendritic Cells) were harvested and cultured for 24 hours in the presence of graded doses of vitamin D compounds - l,25(OH) 2 D 3 (i), l,24R,25-trihydroxy-20R-21-(3-hydroxy-3- methylbutyl) cholecalciferol (ix) (compound 2 herein) and other vitamin D compounds (x) to (xv); and the compounds Mycophenolate Mofetil (MMF) and Dexamethasone (DEX).
  • Immunoglobulin-like transcript-3 upregulation was evaluated by flow cytometric analysis. Briefly, cells were preincubated with 200 mg/ml human IgG (Sigma Chemical, St. Louis, Missouri) and subsequently stained with anti-human ILT3 antibody (see, e.g., Cella, M. et al (1997) J. Exp. Med. 185:1743) followed by anti-mouse IgG- phycoerithryn (Jackson). Cells were then analyzed with a FACScanR flow cytometer using a Cell QuestR software program (both from Beckton Dickinson, Mountain View, California).
  • BHT and BHA is suspended in Miglyol 812 and warmed to about 50 °C with stirring, until dissolved.
  • a Gemini vitamin D 3 compound of the invention is dissolved in the solution from step 1 at 50 °C
  • Di- ⁇ -Tocopherol is suspended in Miglyol 812 and warmed to about 50 °C with stirring, until dissolved.

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Abstract

L'invention concerne des composés de la vitamine D3 Gemini, des méthodes utilisant lesdits composés pour traiter des états associés à la vitamine D3, et des compositions pharmaceutiques contenant ces composés.
EP04751195A 2003-04-30 2004-04-30 Composes de la vitamine d3 gemini et methodes d'utilisation de ces composes Withdrawn EP1622455A4 (fr)

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US20050192255A1 (en) * 2003-07-30 2005-09-01 Jin Tian Use of Vitamin Ds or Vitamin D analogs to treat cardiovascular disease
CA2540325A1 (fr) * 2003-09-24 2005-04-07 Bioxell Spa Traitement du dysfonctionnement de la vessie
CA2588453A1 (fr) * 2004-12-02 2006-06-08 Abbott Laboratories Methodes pour reduire une hyperplasie intime, une proliferation cellulaire de muscle lisse et une restenose chez des mammiferes
EP1898924A1 (fr) * 2005-03-23 2008-03-19 Bioxell S.p.a. Methodes de traitement de l'osteoporose et d'un hyperparathyroidisme secondaire, faisant appel a des composes jumeaux 20-methyle de la vitamine d3
CA2601914A1 (fr) * 2005-03-23 2006-09-28 Bioxell S.P.A. Vitamine d3 gemini 20-alkyle, composes et procedes d'utilisation
US7286295B1 (en) 2005-11-30 2007-10-23 Sandia Corporation Microoptical compound lens
US20100093675A1 (en) * 2006-09-22 2010-04-15 Bioxell S.P.A. Novel method
US20100144684A1 (en) * 2007-04-25 2010-06-10 Proventiv Therapeutics, Inc. Method of Safely and Effectively Treating and Preventing Secondary Hyperparathyroidism in Chronic Kidney Disease
US20110274743A1 (en) * 2009-01-16 2011-11-10 Pharmaceutical Compounding Nz Limited Medicament for the Treatment of Pain and Inflammation
CN104758300A (zh) * 2014-01-02 2015-07-08 上海泽生科技开发有限公司 维生素d及其组合物的抗菌用途

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JP2006525367A (ja) 2006-11-09
EP1622455A4 (fr) 2009-12-16

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