WO1999049027A1 - Methods for prevention and treatment of cancer - Google Patents

Methods for prevention and treatment of cancer Download PDF

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Publication number
WO1999049027A1
WO1999049027A1 PCT/US1999/006491 US9906491W WO9949027A1 WO 1999049027 A1 WO1999049027 A1 WO 1999049027A1 US 9906491 W US9906491 W US 9906491W WO 9949027 A1 WO9949027 A1 WO 9949027A1
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Prior art keywords
vitamin
cells
hydroxylase
gene expressing
nucleotide sequence
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PCT/US1999/006491
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French (fr)
Inventor
Gary G. Schwartz
Balakrishna L. Lokeshwar
Tai C. Chen
Lyman W. Whitlatch
Xiang Fu Kong
Michael F. Holick
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Cutanogen, Inc.
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Priority to JP2000537988A priority Critical patent/JP2002507415A/en
Priority to AU33641/99A priority patent/AU3364199A/en
Priority to CA002323782A priority patent/CA2323782A1/en
Priority to EP99915026A priority patent/EP1066377A1/en
Publication of WO1999049027A1 publication Critical patent/WO1999049027A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/13126—Vitamin D3 24-hydroxylase (1.14.13.126)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to methods for the treatment of cancer in animals.
  • the method comprises the step of increasing the amount of a metabolic precursor of 1 J5-dihydroxy vitamin D available to target cells or tissues in the animal.
  • the method comprises administering the gene expressing the enzyme 25 -hydroxy vitamin D- l ⁇ -hydroxylase to a target cell to increase production of 1,25-dihydroxy vitamin D within the target cell.
  • the present invention also relates to a method for the treatment of hyperproliferative cell disorders and disorders of calcium and bone metabolism in animals, comprising administration of the gene expressing 25-hydroxyvitamin D- 1 ⁇ -hydroxylase to target cells or tissues of the animal.
  • vitamin D has been used in the literature to refer generally to a class of steroid hormones that possess anti-rachitic activity, including ergocalciferol (vitamin D 2 ), cholecalciferol (vitamin D 3 ), calcidiol (25- hydroxy vitamin D), calcitriol (1,25 -dihydroxy vitamin D), and 20 or more other metabolites of vitamin D.
  • vitamin D is used herein in its more restrictive sense to refer only to vitamin D 2 or vitamin D 3 in order to distinguish the useful metabolites which are part of this invention. 1,25-Dihydroxyvitamin D
  • the most biologically active vitamin D metabolite is 1 J5-dihydroxy vitamin D ("l,25(OH) 2 D"), also known as calcitriol.
  • the synthesis of 1J5(0H) 2 D in an animal, e.g., humans, begins with the cutaneous production of vitamin D after exposure to sunlight or ultraviolet (UN) radiation, or after the intestinal absorption of plant- and yeast-derived vitamin D 2 or animal-derived (typically from fish liver oil and fatty fish) vitamin D 3 obtained from the diet.
  • vitamin D must undergo two hydroxylation steps. See Fig. 1 .
  • the first hydroxylation step occurs in the liver by hydroxylation at the 25th carbon position, forming 25-hydroxyvitaminD ("25(OH)D"),the major circulating metabolite of vitamin D.
  • the second hydroxylation occurs in the kidney by hydroxylation at the l ⁇ position, forming 1J5(0H) 2 D, the hormonally active metabolite.
  • the kidney is the major source of 1 J5(OH) 2 D
  • the enzyme l ⁇ -hydroxylase (“l ⁇ -OHase”) that converts 25(OH)D to 1J5(0H) 2 D is also present in several types of non-renal cells, e.g., activated macrophages and keratinocytes (Barbour, G.L., et al., N.
  • the l ⁇ -OHase enzyme has been cloned recently from mouse kidney, human kidney, and human keratinocytes (Takeyama, K., et al. , Science 277: 1827-
  • VDR vitamin D receptor
  • VDR VDR receptor ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 01S-11 IS (1995); Gross, C, et al, in Vitamin D, D. Feldman et al. (eds.), Academic Press: New York (1997), pp.
  • Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells.
  • One frequently-occurring cancer is adenocarcinoma of the prostate gland, which is the most commonly diagnosed malignancy in American males, excluding skin cancer.
  • adenocarcinoma of the prostate gland which is the most commonly diagnosed malignancy in American males, excluding skin cancer.
  • Approximately 317,000 new cases of prostate cancer were diagnosed in the United States in 1996 (Parker, S.L., et al, Cancer J. Clin. 46:5-21 (1996)), accounting for 41% of newly diagnosed malignancies, excluding basal cell and squamous cell carcinomas of the skin. This may actually underestimate the problem because clinically silent prostate cancer is very common.
  • a unique feature of prostate cancer is the high prevalence of "incidental" or "subclinical” cases identified at autopsy.
  • Autopsy data indicate that about 30% of men over the age of 50 have histological prostate cancer, and the prevalence of these incidental cancers reaches 60% in men over the age of 80. Histologically, these incidental cancers are indistinguishable from prostate cancers that are potentially life-threatening and are considered to be an earlier stage in their natural history.
  • incidental prostate cancer is ubiquitous among elderly men regardless of ethnicity or geographic location.
  • prostate carcinoma is generally a slow growing malignancy, mortality from the disease is nonetheless considerable, representing about 15% of all cancer deaths in 1996, which makes prostate cancer the second leading cause of cancer death among U.S. males. (Parker, S.L., et al, Cancer J. Clin. 46:5-21 (1996)).
  • prostate cancer has rapidly become a major public health concern not only in the United States, but worldwide. See Gross, C, et al, in Vitamin D, D. Feldman et al. (eds.), Academic Press: San Diego, CA (1997), pp. 1125-1139.
  • Prostate cancer mortality rates in the contiguous United States are inversely correlated with levels of ultraviolet (UV) radiation, which produces vitamin D in the skin.
  • UV ultraviolet
  • 1J5(0H) 2 D can modulate cell proliferation and differentiation in prostate cells.
  • Supporting evidence includes the ubiquitous presence of receptors for 1 J5(OH) 2 D (vitamin D receptors, or VDRs) in human prostatic cells and the anti-proliferative and pro- differentiating effects of 1 J5(OH) 2 D on these cells in vitro and in vivo (Schwartz, G. G.,AnticancerRes. 74:1077-1082 (1994)).
  • 1 ,25 (OH) 2 D maintains the differentiated phenotype of prostatic cells. Low levels of 1J5(0H) 2 D can therefore increase the risk for life-threatening prostate cancer.
  • 1 J5(OH) 2 D at the cellular level without risking undesired, and potentially unsafe, side effects to the patient.
  • systemic 25(OH)D levels can be increased by in situ production of vitamin D through sun or UV exposure, due to the regulation of 1 J5(OH) 2 D produced from 25(OH)D.
  • serum levels of 1 ,25(OH) 2 D will generally not be elevated to a desired antiproliferative level.
  • reaching a beneficial level of l,25(OH) 2 D was not known to be possible from exposure to the sun or UV radiation, even in amounts that are considered by those in the art to be unsafe or excessive.
  • Certain risks, e.g., skin cancer, inter alia, are associated with such excessive sun or UV exposure.
  • vitamin D dietary supplementation may lower the risk of prostate cancer (Naitoh, J., et al, Prostate Cancer and Prostatic Diseases 7:48-53 (1997)).
  • administering vitamin D as a dietary supplement is recommended to be performed under the supervision of a physician due to the risk of producing vitamin D toxicity, of which the negative side effects can include kidney failure, hypercalcemia, coma, and death.
  • Vitamin D supplementation therapy is also disadvantageous in view of its slow metabolic conversion to 25(OH)D. Therefore, reaching effective levels of 25(OH)D by administering supplemental vitamin D can take weeks or months using a conservative dosing regimen. Conversely, if those vitamin D levels are too high, it can take months to return to normal levels of 25(OH)D after ceasing the administration or reducing the dosage of vitamin D due to the natural storage of vitamin D in body fat. Moreover, administration of 1 ,25(OH) 2 D itself, carries its own risks of producing hypercalcemia, which has been well-documented.
  • l,25(OH) 2 D is considered to the biologically active form of vitamin D. Its principal physiologic function is on calcium and bone metabolism. It stimulates intestinal calcium transport and initiates mobilization of - 7 -
  • Vitamin D dependent rickets type I is an inborn error where there is a defect in the l ⁇ -OHase enzyme.
  • X-linked hypophosphatemic rickets there is a defect in the regulation of the l ⁇ -OHase.
  • administering a vitamin D metabolite and 1J5(0H) 2 D precursor to a patient can prevent cell proliferation, invasive cancer, or metastasis of cancer or tumor cells of an organ, e.g., the prostate, colon, or breast, while reducing the risk of vitamin D toxicity and hypercalcemia.
  • the subject invention concerns novel methods for prevention, treatment or testing for certain types of cancer, in particular prostate cancer, employing the advantages of a metabolite of vitamin D, analogs or derivatives of the vitamin D metabolite, or enzymes involved in the metabolism of vitamin D.
  • the invention provides a method for locally increasing concentrations of 1 ,25 -dihydroxy vitamin D (1 J5(OH) 2 D) in a cell of a target organ so that the anti- proliferative, anti-invasive, antimetastatic and pro-differentiating action of 1 J5(OH) 2 D can be provided to those cells.
  • one embodiment of the subject invention concerns a method for increasing 1J5(0H) 2 D concentrations available to the target organ by administering to a patient a metabolic precursor of 1J5(0H) 2 D wherein the precursor does not have the side effects or risks of skin cancer associated with sun or ultraviolet (UV) radiation exposure, or the side effects or risks of vitamin D toxicity associated with administering high doses of vitamin D or 1J5(0H) 2 D.
  • the invention comprises administering an effective amount of a vitamin D metabolite to a patient in need of therapeutic or prophylactic treatment of a tumor or cancer, wherein the administration of the metabolite provides to a patient a substantially lower risk of skin cancer, vitamin
  • the subject method can be used to prophylactically or therapeutically treat a patient at risk of or having certain subclinical or clinical cancer.
  • the method includes preventing proliferation, invasion, or metastasis of the tumor or cancer cells by administering vitamin D or a metabolite of vitamin D, or an analog, derivative salt, or functional equivalent of the metabolite which can be metabolically converted to 1 ,25 (OH) 2 D or a functional equivalent thereof.
  • the subject treatment preferably comprises administering to a patient having - 9 -
  • Minimal residual cancer refers to detectable tumor cells in post-treatment patients, e.g., post-prostatectomy or post-radiation therapy, wherein the primary tumor has been eliminated or the organ has been removed from the body, but tumor or cancer cells, likely mobilized to a different organ or present in the bloodstream, are still detectable.
  • the subject method for prophylactic or therapeutic treatment of cancer comprises administering an effective amount of 25(OH)D, or an analog, derivative, salt, or functional equivalent thereof, to a patient having subclinical, clinical, or minimal residual cancer of an organ, i.e., the
  • target organ wherein the target organ or cells derived from that target organ can convert 25(OH)D or its analog, derivative, salt, precursor, or functional equivalent, to l,25(OH) 2 D, or its functional equivalent.
  • Organ cells having the enzyme l ⁇ -OHase can locally convert 25(OH)D or an analog thereof to 1 J5(OH) 2 D or its corresponding analog.
  • certain prostatic cells have been shown to have l ⁇ -OHase and the capability to convert 25(OH)D to 1 J5(OH) 2 D.
  • the subject method can be applied to prostate cancer patients having subclinical or clinical disease; patients at risk of developing prostate tumors (e.g., persons with no clinical evidence of prostate cancer), or patients having metastatic disease, e.g., minimal residual cancer, resulting from mobilization of cancerous prostatic cells to other organs or tissues.
  • the subject method can be used for treating a tumor in any organ wherein the cells of that organ can convert a metabolic precursor of 25(OH)D to 1J5(0H) 2 D, or its functional equivalent, and be responsive to the antiproliferative, anti-invasive, antimetastatic, or pro-differentiating effects of
  • VDR vitamin D receptor
  • a cell normally deficient in VDR or l ⁇ -OHase can be genetically engineered, e.g., transgenically altered with an appropriate gene or promotor to induce transcription of the gene and production of the resultant - 10 -
  • the subject invention thus offers a solution to the problem of reducing the risk of invasive (i.e., life-threatening) cancer, including invasive prostate cancer.
  • Therapy with 25(OH)D or an analog, derivative, salt, or functional equivalent thereof can be useful to many men at various stages of the natural history of prostate cancer. These include the millions of men who presently have subclinical prostate cancer: men on "watchful waiting," and men who have been treated for prostate cancer by therapy with curative intent, but who may still have prostatic cancer cells remaining in their bodies. It also may be useful to men without prostate cancer, either subclinical or clinical, to reduce the risk of developing cancer.
  • One form of subclinical prostate cancer is also known as "prostatic intraepithelial neoplasia" or PIN.
  • the invention provides a method of inhibiting proliferation of tumor cells and/or inducing differentiation of tumor cells in an animal, comprising introducing into the cells in need thereof a polynucleotide construct comprising a gene expressing l ⁇ -OHase, whereby 1J5(0H) 2 D or an analog thereof is produced and the proliferation of the cell is inhibited.
  • the invention further comprises introducing into the tumor cells a polynucleotide construct comprising a gene expressing the vitamin D receptor.
  • the gene expressing 1 ⁇ -OHase and the gene expressing the vitamin D receptor can be on either the same DNA construct or on separate DNA constructs.
  • this aspect of the invention provides a method for treating or preventing prostate cancer, breast cancer, skin cancer, colon cancer, lung cancer, leukemia, lymphoma, or other cancers that can respond to the antiproliferative and prodifferentiating actions of endogenously produced 1J5(0H) 2 D or an analog of 1J5(0H) 2 D that is provided by the cell undergoing gene therapy with DNA constructs containing the 1 ⁇ -OHase gene, with or without the VDR gene.
  • an animal being treated by gene therapy as described herein may also be treated substantially simultaneously with 25(OH)D or an analog, derivative, salt or functional equivalent thereof to augment the response to the gene therapy.
  • the invention provides a method of testing for cancer risk of a patient.
  • a method of testing for cancer risk of a patient By measuring levels of certain cellular components in a particular organ wherein the measured component is involved in vitamin D metabolism, vitamin D, a metabolite thereof (e.g., 25(OH)D, or its analogs), an enzyme capable of converting a vitamin D metabolite to 1 J5(OH) 2 D or the activity of that enzyme, - 12 -
  • the l ⁇ -OHase enzyme is capable of converting 25(OH)D to 1J5(0H) 2 D. Therefore, measuring l -OHase levels or the activity of the l ⁇ -OHase enzyme in a cellular sample from a target organ, e.g., prostate, can be used to determine the risk of developing cancer in that organ or an organ comprising a cancerous cell from that organ.
  • Standard molecular biology techniques which are well-known in the art can also be used for measuring genetic polymorphisms in the genes encoding the 1 ⁇ -OHase enzyme or VDRs and correlating allelic differences with risk of tumor development in the individual tested.
  • the invention also provides a method of predicting the rate of successful treatment of a particular cancer in a patient by determining the local levels of certain cellular components in the vitamin D metabolic pathway, such as vitamin D or its metabolites, in an organ, e.g., the prostate, or by determining levels of an enzyme, e.g., l ⁇ -OHase, capable of converting a metabolic precursor into
  • Measuring activity of the enzyme can also be useful for indirectly measuring enzyme levels and thus be predictive of treatment success.
  • the invention also provides a method of treating a hyperproliferative skin disorder in an animal, comprising introducing into skin cells of the animal in need thereof a polynucleotide construct containing a gene expressing l ⁇ -OHase, whereby 1 J5(OH) 2 D or an analog thereof is produced and the proliferation of the cell is inhibited.
  • the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor.
  • the gene expressing 1 ⁇ -OHase and the gene expressing the vitamin D receptor can be on the same
  • DNA molecule of the construct or on separate DNA molecules of said constructs.
  • the invention can be used to treat actinic keratoses, a pre-skin cancer, or ichthyosis.
  • Introduction of the gene coding for l ⁇ -OHase effects an increase of the amount of 1J5(0H) 2 D 3 within the cell, thereby - 13 -
  • the invention is also directed to a method to treat hyperproliferative disorders of the skin such as psoriasis.
  • Patients with psoriasis respond to topical and oral 1J5(0H) 2 D.
  • the invention is a reasonable alternative treatment for psoriasis, whereby the cells are induced to make or boost their production of 1 ,25(OH) 2 D 3 which, in turn, interact with the VDR and decrease the proliferative activity and enhance differentiation, making the skin cells normal.
  • This treatment can also be applied to other hyperproliferative skin disorders such as ichthyosis.
  • the invention also provides a method of treating a disorder in calcium and bone metabolism in an animal, comprising introducing into cells of the animal in need thereof a polynucleotide construct containing a gene expressing 1 ⁇ -OHase, whereby 1 ,25(OH) 2 D or an analog thereof is produced and the disorder is treated.
  • the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor.
  • the gene expressing 1 ⁇ -OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or on separate DNA constructs.
  • This embodiment of the invention provides a method for treating disorders in calcium and bone metabolism such as renal osteodystrophy or a metabolic bone disease.
  • the invention also provides a method of treating vitamin D-dependent rickets type I, X-linked hypophosphatemic rickets, vitamin D-dependent rickets type II, and osteoporosis, as well as such disorders in calcium and bone metabolism as low blood serum calcium due to hypoparathyroidism.
  • the present invention also provides a method of treating or preventing hair loss in an animal, comprising introducing into hair cells in need thereof a - 14 -
  • the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor.
  • the gene expressing l ⁇ -OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs.
  • the invention can therefore be used to treat or prevent hair loss resulting from conditions such as alopecia androgenetica, female pattern baldness, and chemotherapy-induced alopecia.
  • the invention also provides a method of enhancing wound healing in an animal, comprising introducing into cells of the wound in need thereof a polynucleotide construct containing a gene expressing l ⁇ -OHase, whereby 1J5(0H) 2 D or an analog thereof is produced and the healing of the wound is enhanced.
  • the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor.
  • the gene expressing l ⁇ -OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs.
  • the invention also provides a method of treating benign prostatic hyperplasia in an animal, comprising introducing into cells exhibiting benign prostatic hyperplasia in need thereof a polynucleotide construct containing a gene expressing 1 ⁇ -OHase, whereby 1 J5(OH) 2 D or an analog thereof is produced and the benign prostatic hyperplasia is treated.
  • the invention may further comprise introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor.
  • the gene expressing 1 ⁇ -OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs.
  • the invention also provides a method of treating an autoimmune disease in an animal, comprising introducing into cells of the animal in need thereof a - 15 -
  • polynucleotide construct comprising a gene expressing 25-hydroxyvitamin D- l ⁇ -hydroxylase, whereby 1,25-dihydroxy vitamin D or an analog thereof is produced and the autoimmune disease is treated.
  • the invention may further comprise introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor.
  • the gene expressing 1 ⁇ -OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs.
  • the invention can be used to treat autoimmune diseases such as diabetes mellitus type 1, multiple sclerosis, rheumatoid arthritis, and psoriatic arthritis.
  • the invention provides for an isolated polynucleotide molecule comprising a 1 ⁇ -OHase regulatory sequence or a complement thereof.
  • the l ⁇ -OHase regulatory sequence or a complement thereof comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence at least 90% identical to the nucleotide sequence depicted in SEQ ID NO. 3; and (b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (a) or the complement thereof.
  • the 1 ⁇ -OHase regulatory sequence or complement thereof comprises a nucleotide sequence that is 95%, 97%, 98%, or 99% identical to the sequence depicted in SEQ ID NO. 3, or comprises the nucleotide sequence depicted in SEQ ID NO. 3.
  • the invention also provides for an isolated polynucleotide molecule further comprising a nucleotide sequence coding for l ⁇ -OHase that is 95%, 97%, 98%, or 99% identical to the sequence depicted in SEQ ID NO. 1, or one that comprises the nucleotide sequence depicted in SEQ ID NO. 1. - 16 -
  • Fig. 1 shows a diagrammatic representation of vitamin D metabolism in the body. Well-known metabolism steps are shown by solid arrows; synthesis of 1J5(0H) 2 D from 25(OH)D by the prostate is shown by open arrows.
  • Figures 2-5 show a high performance liquid chromatography elution profile of tritium activity of lipid extracts from cultured normal human prostate primary cultures or BPH cultures incubated with [ 3 H] -25 (OH)D in the absence or presence of the P450 inhibitor clotrimazole.
  • Fig. 2 shows second passage of normal human prostate cells incubated withnonradioactive25(OH)D 3 (50 nM), [ 3 H]-25(OH)D 3 (0.91 ⁇ Ci/nmol) and 1J- dianilinoethane (DPPD) (10 ⁇ M) at 37 °C for two hours in the absence of clotrimazole.
  • DPPD 1J- dianilinoethane
  • Fig. 3 shows second passage of normal human prostate cells incubated with nonradioactive 25(OH)D 3 (50 nM), [ 3 H]-25(OH)D 3 (0.91 ⁇ Ci/nmol) and DPPD (10 ⁇ M) at 37 °C for two hours in the presence of the P450 enzyme inhibitor, clotrimazole (20 ⁇ M).
  • Fig. 4 shows second passage of prostate cells derived from BPH cultures incubated with nonradioactive 25(OH)D 3 (50 nM), [ 3 H]-25(OH)D 3 (0.91 ⁇ Ci/nmol) and DPPD (10 ⁇ M) at 37 °C for two hours in the absence of clotrimazole.
  • Fig. 5 shows second passage of prostate cells derived from BPH cultures incubated with nonradioactive 25(OH)D 3 (50 nM), [ 3 H]-25(OH)D 3 (0.91 ⁇ Ci/nmol) and DPPD (10 ⁇ M) at 37 °C for two hours in the absence of clotrimazole.
  • Fig. 6 depicts a graph showing the effect of 25(OH)D 3 concentration on the proliferative activity of normal human keratinocytes transfected with l ⁇ -OHase cDNA.
  • the subject invention concerns novel methods for preventing or treating cell proliferation, invasiveness, or metastatic potential, or for promoting cellular differentiation.
  • the subject invention further concerns a method for testing for cancer, as well as a method for predicting the success of treatment for cell proliferation or cancer.
  • One aspect of the subject invention comprises increasing local cellular levels of 1J5(0H) 2 D.
  • one embodiment of the subject invention concerns preventing or treating cell proliferation, invasiveness, or metastasis, or promoting cellular differentiation by administering an effective amount of a vitamin D metabolite which can be metabolically converted by the target cells to l,25(OH) 2 D.
  • the vitamin D metabolite used in accordance with the subject invention does not cause an increased risk of skin cancer (as compared to sun or ultraviolet (UV) ray exposure), vitamin D toxicity (as compared to supplemental excessive vitamin D administration), and does not significantly contribute to hypercalcemia (as compared to administering 1 ,25(OH) 2 D). More preferably, the subject invention comprises administering an effective amount of 25(OH)D, or an analog, derivative, salt, or functional equivalent thereof to prevent the proliferation, invasion, or metastasis of tumor or cancer cells in a particular organ, or to promote differentiation of cells in, or derived from, that organ.
  • the compound 25(OH)D is commercially available. - 18 -
  • the normally observed concentration of 25(OH)D in serum is about 20- 150 nmol/L (8-60 ng/ml). However, circulating concentrations of up to 250 nmol/L (100 ng/ml) 25(OH)D have commonly been observed in lifeguards after a full summer of exposure to sunlight and is considered to be normal (Holick, M.F., J. Nutrition, Suppl. 720:1464-1469 (1990)). Concentrations of about 350 nmol/L or more are considered to be dangerous to the health of the individual. Hypovitaminosis D, i.e., a deficiency in serum 25(OH)D, is a condition defined when serum levels fall below about 25 nmol/L (10 ng/ml).
  • an effective amount of 25(OH)D administered into the target organ would be any amount which, when administered, increases local cellular levels of 25(OH)D, but maintains serum levels of 25(OH)D within this "normal" range.
  • 25(OH)D levels are increased in the target organ substantially above 25 nmol/L but less than 250 nmol/L. More preferably, 25(OH)D levels are increased to between about 50 and 150 nmol/L.
  • Normal serum levels of 1 J5(OH) 2 D range between about 38- 144 pmol/L
  • an alternative determination of an effective amount of 25(OH)D administered in accordance with the method of the subject invention is to administer an amount which raises the level of 25(OH)D toward the high end of its normal range in the target organ, but which does not raise systemic 1J5(0H) 2 D above the high end of its normal range.
  • 25(OH)D levels preferably can be raised to increase intra-organ levels to between about 25 and 150 nmol/L, but where systemic 1J5(0H) 2 D levels remain less than 145 pmol/L. More preferably, 1J5(0H) 2 D levels remain below 125 nmol/L when 25(OH)D is administered to a target cell, organ, or tissue.
  • Useful analogs, derivatives, or salts of 25(OH)D include alkylated, glycosylated, arylated, halogenated, or hydroxylated 25(OH)D, orthoesters of 25(OH)D, or pharmaceutical salts of 25(OH)D.
  • These analogs, derivatives, or salts can be synthesized or otherwise manufactured by chemical procedures which are well-known and readily available to those of ordinary skill in the art.
  • the vitamin D analogs can be obtained according to the methods disclosed in U.S. - 19 -
  • the term "functional equivalent” is used to refer to any compound which can be used as a substrate for l ⁇ -OHase or otherwise can be converted to 1J5(0H) 2 D or converted to a compound which can bind to or activate the 1J5(0H) 2 D receptor (VDR) such that any one of the beneficial properties of 1J5(0H) 2 D, namely inhibition of invasiveness, proliferation, metastasis, or promotion of cell differentiation, are effected. See, for example, U.S. Patent Nos.
  • compositions which comprise a 1 J5(OH) 2 D metabolic precursor or analog or derivative thereof and, e.g., 25(OH)D, a pharmaceutically acceptable carrier.
  • the subj ect method of administering a metabolic precursor of 1 ,25(OH) 2 D to a patient has been shown to be effective in producing 1 ,25(OH) 2 D by prostatic cancer cells.
  • the subject method has been shown to be effective in two well-characterized human prostate cancer cell lines, DU 145 and PC-3, and two primary cultures of cells, NP96-5 and BPH96- 11 , derived from noncancerous human prostates.
  • the NP96-5 culture was derived from the prostate of a 23-year old organ donor; the BPH96-11 culture was derived from a 56-year old with benign prostatic hyperplasia (BPH).
  • Table 1 Synthesis of 1 ,25(OH) 2 D in the presence and absence of the P450 cytochrome inhibitor, clotrimazole.
  • the DU 145 and PC-3 cell lines produced 0J 1 ⁇ 0.06 and 0.07 ⁇ 0.01 pmol of 1J5(0H) 2 D mg protein/hr, respectively. These cells exhibited 1 ⁇ -OHase activity, as detected by thymus receptor binding assay according to well-known procedures.
  • the production of 1J5(0H) 2 D was completely inhibited in the presence of clotrimazole, a commonly known inhibitor of the cytochrome P450 system, of which l ⁇ -OHase is a known component.
  • no measurable 1 ,25(OH) 2 D was detected in LNCaP cells, which did not exhibit l -OHase activity.
  • NP96-5 normal prostate
  • BHP96-11 BPH
  • the NP96-5 andBPH96-l 1 cultures produced3.08 ⁇ 1.56pmol/mg protein/hr and 1.05 ⁇ 0J 1 pmol/mg protein/hr of 1 ,25(OH) 2 D, respectively, in the presence of DPPD and in the absence of cytochrome P450 inhibitor.
  • the l ⁇ - OHase activity found in the two primary cultures of prostatic cells was comparable to that found in the normal human keratinocytes.
  • the enzyme activity detected in the primary cultures of prostatic cells was further supported by high performance liquid chromatography analysis using a solvent system that specifically separates l,25(OH) 2 D from 10-oxo-19-nor-
  • 25(OH)D another metabolite of 25(OH)D.
  • This metabolite which is present in significant quantity in kidney homogenates of rats and chickens, is known to co- migrate with 1 ,25(OH) 2 D on normal phase HPLC with the n-hexane:isopropanol (9:1) solvent system. Therefore, to ensure that 1J5(0H) 2 D was separated from any 10-oxo- 19-nor-25(OH)D present, the methylene chloride:isopropanol (19:1) normal phase solvent system was used. In addition, l -OHase activity was determined by using a radioactive metabolite, [ 3 H]-25(OH)D, as substrate.
  • Typical HPLC chromatograms of the two primary prostate cell cultures in the presence and absence of clotrimazole are illustrated in Figs. 2-5. Second passage of normal human prostate cells incubated with nonradioactive 25(OH)D 3
  • Both primary cultures of non-cancerous prostate cells produced 1J5(0H) 2 D at levels 10-40 fold higher than the cell lines. Very high levels of 1 J5(OH) 2 D were observed from the NP96-5 culture derived from the 23 year old organ donor.
  • the quantities of l,25(OH) 2 D produced by the two primary non- cancerous human prostate cells, 3.08 ⁇ 1.56 and 1.05 ⁇ 0J 1 pmol/mg protein/hr (normal and BPH, respectively), are comparable to those produced in cultured human keratinocytes (2J ⁇ 0.1 pmol/mg protein hr), and to the HEP 62 hepatoma cell line (2.3 pmol/mg protein/hr), and human T-lymphotrophic virus-transformed lymphocytes (1.6 pmol/mg protein hr). These values are at least 10-fold higher than those reported in other extra-renal sites, such as human bone cells (0.068 pmol/mg protein/hr).
  • DU 145 and PC-3 human prostate cancer cells possess l ⁇ -OHase activity and are capable of converting the major circulating metabolite of vitamin D 3 , 25(OH)D 3 , to the hormonally active vitamin D metabolite, 1J5(0H) 2 D 3 .
  • the enzyme activity found in the primary cultures of prostate cells is comparable to that found in the primary cultures of renal proximal tubular cells
  • renal l ⁇ -OHase is the major enzyme responsible for maintaining the circulating concentration of - 23 -
  • 1J5(0H) 2 D under normal physiological conditions, the amount of 1J5(0H) 2 D produced by prostatic cells can be physiologically significant, especially for the micro environment of prostatic cells.
  • Levels of 1 J5(OH) 2 D as low as 10 "1 ' M can significantly inhibit invasiveness of human prostate cancer cells through an artificial basement membrane composed of human amnions (Schwartz, G.G., et al, Cancer Epidemiol. Biomark. Prev. 6:121-132 (1997)).
  • 1 J5(OH) 2 D can be synthesized from 25(OH)D by prostatic cells provides the nexus for using vitamin D metabolites which are comparatively less toxic than high doses of vitamin D or certain of other metabolites, including 1J5(0H) 2 D, in cancer treatment.
  • the method of the subject invention comprising administering to a patient a vitamin D metabolite which can be converted to 1J5(0H) 2 D by the enzyme l ⁇ -OHase is preferably used in chemoprevention of prostate cancer.
  • cancer cells in other organs or tissues which are capable of converting vitamin D metabolite to l,25(OH) 2 D can respond to the antiproliferative, anti-invasive, antimetastatic, or prodifferentiating properties of 1 J5(OH) 2 D.
  • the subject invention can be applicable to other cancer cells, including colon cancer cells, breast cancer cells, leukemia cells, skin cancer cells, lung cancer cells, and lymphoma cancer cells.
  • the product synthesized from the administered metabolite, 1 J5(OH) 2 D exerts anti-proliferative and pro-differentiating effects on normal and cancerous prostate cells.
  • 1 J5(OH) 2 D significantly inhibit the invasiveness of DU 145 cells through an artificial basement membrane, as correlated with a decrease in the secreted levels of type IV collagenase (MMP-2 and MMP-9). It has further been shown that 1 J5(OH) 2 D exhibits antimetastatic - 24 -
  • 1 J5(OH) 2 D has particular disadvantages due to its causative effect in producing hypercalcemia.
  • 1J5(0H) 2 D is not considered by those in the art to be optimal for safe use as a chemopreventive agent because of this risk of hypercalcemia.
  • Our present findings indicate that by increasing the available substrate, e.g., administering or supplementing 25(OH)D to a patient, local synthesis of 1J5(0H) 2 D by prostatic cells can be achieved, making increased levels of the active metabolite 1J5(0H) 2 D available to the prostatic cells, and thereby reducing proliferation, invasiveness, or metastatic activity of cancerous prostate cells, and promoting their differentiation.
  • 25(OH)D and analogs and derivatives thereof can be useful in several clinical settings, e.g., in slowing or preventing the need for prostatectomy or radiation therapy in men on "watchful waiting," and/or slowing or preventing disease recurrence in men with minimal residual disease.
  • the vitamin D metabolite 25(OH)D is advantageously stored in smaller proportions in body fat as compared to vitamin D or other more fat-soluble metabolites or analogs of vitamin D.
  • 25(OH)D bypasses hepatic 25- hydroxylation. Therefore, its onset and offset of action are faster than vitamin D.
  • 25(OH)D can be effective when there is compromise of the hepatic 25 -hydroxylation of vitamin D 2 or D 3 , as in primary biliary cirrhosis or certain cases of neonatal hypocalcemia. Further, 25(OH)D may have some intrinsic agonist activity that may retain some level of efficacy in the absence of the renal 1 ⁇ -OHase system.
  • the subject method including the preferred method of administering 25 (OH)D or an analog, derivative, salt, or functional equivalent thereof, would not be limited to treatment of prostatic cells.
  • Those cells having l ⁇ -OHase can also convert 25(OH)D to l,25(OH) 2 D, thereby increasing local cellular levels of the 1J5(0H) 2 D end product.
  • Those cells which also have VDRs which bind 1J5(0H) 2 D, or its functional equivalent can respond to that compound and benefit from its antiproliferative, anti-invasive, antimetastatic, and - 25 -
  • colon or breast cells can respond to 1 J5(OH) 2 D due to their possessing 1 ⁇ -OHase and VDRs, either inherently or by use of genetic engineering techniques well-known in the art.
  • the dosage administration to a host in the above indications will be dependent upon the identity of the disease or condition, the type of patient involved, its age, weight, health, kind of concurrent treatment, if any, frequency of treatment, and therapeutic ratio.
  • compositions of the subj ect invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention. In general, the compositions of the subj ect invention will be formulated such that an effective amount of the bioactive compound(s) is combined with a suitable carrier in order to facilitate effective administration of the composition.
  • compositions comprising, as active ingredient, an effective amount of one or more of the subject compounds and one or more non-toxic, pharmaceutically acceptable carriers or diluents can be used by persons of ordinary skill in the art.
  • the pharmaceutical composition can comprise one or more of the subject compounds, e.g., 25(OH)D, as a first active ingredient plus a second active ingredient, e.g., an anti-inflammatory, antimicrobial, antiproliferative, or antiviral compound, known in the art.
  • pharmaceutically effective amounts of a known second active ingredient and the vitamin D metabolite, analog or derivative thereof useful in accordance with the subj ect invention are administered sequentially or concurrently to the patient.
  • the most effective mode of administration and dosage regimen of subject compounds will depend upon the type of disease to be treated, the severity and course of that disease, previous - 26 -
  • the subject compositions may be administered to the patient at one time or over a series of treatments.
  • the subject compound and the second agent are administered sequentially to the patient, with the second agent being administered before, after, or both before and after treatment with the subject compound or composition.
  • Sequential administration involves treatment with the second agent at least on the same day (within 24 hours) of treatment with the subj ect vitamin D metabolite and may involve continued treatment with the second agent on days that the subject compound is not administered.
  • the patient may receive concurrent treatments with compositions comprising at least one of the subject compounds, and a second active ingredient.
  • compositions comprising at least one of the subject compounds, and a second active ingredient.
  • local, intra-tissue (or organ) injection of 25(OH)D or analog or derivative thereof is preferred but can be administered by subcutaneous injection, subcutaneous slow-release implant, intravenously, intraperitoneally, topically, intramuscularly, or orally.
  • compositions used in these therapies can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • the subject invention can employ a gene therapy approach utilizing molecular biology techniques or procedures whereby a target cell or tissue is genetically altered to respond to the active vitamin D metabolite or analog and derivative thereof, e.g., l,25(OH) 2 D, by transgenically providing the target cell with a gene which encodes a protein or plurality of proteins which serve as vitamin D receptors (VDRs) that bind 1 ,25(OH) 2 D.
  • VDRs vitamin D receptors
  • the target cells can be genetically engineered to encode and produce l ⁇ -OHase, which is known to convert 25(OH)D to 1J5(0H) 2 D.
  • genes of interest can be identified using standard probe and sequencing techniques, cloned or otherwise amplified, isolated and inserted into appropriate vectors for transfer into the target cells. Further, the target cells can be provided with promoter sequences which activate the appropriate gene sequence in the genome of the target cell to instruct the cell to produce the desired proteins or promoters.
  • cancers include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphomas, acute lymphocytic leukemia, multiple myeloma, breast carcinomas, ovarian carcinomas, lung carcinomas, Wilms' tumor, testicular carcinomas, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, bladder carcinomas, chronic granulocytic leukemia, primary brain carcinomas, malignant melanoma, small-cell lung carcinomas, stomach carcinomas, colon carcinomas, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, rhab
  • carcinomas endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.
  • 1 ⁇ -OHase DNA (e.g., the nucleic acid sequence depicted in SEQ ID NO. 1) can be incorporated into a polynucleotide construct suitable for introducing the nucleic acid molecule into cells of the animal to be treated, to form a transfection vector.
  • the transfection vector is then introduced into selected target tissues of the cells of the animal in vivo using any of a variety of methods known to those skilled in the art.
  • naked DNA may be transfected into the cells, with or without cationic lipids.
  • transfection vectors containing l ⁇ - OHase DNA are well-known in the art, and are generally described in "Working Toward Human Gene Therapy ' Chapter 28 in Recombinant DNA, 2nd Ed, Watson, J.D. etal. (eds.), Scientific American Books: New York (1992), pp.567- 581 , or Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring
  • Gene therapy approaches that may be used to deliver a VDR and/or 1 ⁇ - OHase gene include injection of plasmid DNA (Horton, H.M., et al. , Proc. Natl. Acad. Sci. USA 96( ⁇ :1553-1558 (1999)); transduction using adenoviral vectors (Waugh, J.M., et al, Proc. Natl. Acad. Sci. USA 9tf 5J: 1065 -1070 (1999)); transduction using retrovial vectors (Axelrod, J.H., et a!, Proc. Natl. Acad. Sci.
  • OHase DNA of the present invention are directly introduced into the cells or tissues of the mammal to be treated, preferably by injection, inhalation, ingestion, topical application, or introduction into a mucous membrane via solution. Such an approach is generally referred to as "in vivo" gene therapy.
  • cells or tissues may be removed from the mammal to be treated and placed into culture according to methods that are well-known to one of ordinary skill in the art.
  • Transfection vectors or naked DNA containing the 1 ⁇ - OHase DNA may then be introduced into these cells or tissues by any of the methods described generally above for introducing isolated polynucleotides into a cell or tissue. After a sufficient amount of time to allow incorporation of the 1 ⁇ -
  • the cells or tissues may then be re-inserted into the mammal to be treated. Since introduction of the l ⁇ -OHase gene is performed outside of the body of the mammal, this approach is generally referred to as "ex vivo" gene therapy. See U.S. Patent No. 5,399,346.
  • Gene transfer through transfection of cells ex vivo can be performed by a variety of methods, including, for example, calcium phosphate precipitation, diethylaminoethyl dextran, electroporation, lipofection, or viral infection. Such methods are well known in the art (see, for example, Sambrook et al).
  • OHase gene or "gene expressing l ⁇ -OHase” may refer to either a segment of genomic DNA encoding the 1 ⁇ -OHase enzyme or a cDNA sequence encoding the 1 ⁇ -OHase enzyme.
  • the l ⁇ -OHase DNA of the invention may be operatively linked to the regulatory DNA sequence, or
  • promoter for human l ⁇ -OHase (depicted in SEQ ID NO. 3) to form a genetic construct as described above.
  • This construct containing both the human 1 ⁇ - OHase promoter and the 1 ⁇ -OHase DNA, may be subcloned into a suitable vector such as a plasmid, adeno virus vector, retro virus vector, or the like, and introduced - 30 -
  • the 1 ⁇ -OHase DNA of the invention may be operatively linked to a heterologous regulatory DNA sequence, or promoter, to form a genetic construct as described above.
  • the heterologous regulatory sequence may be tissue specific.
  • the vector containing the genetic construct is then directly introduced into the animal to be treated or into the cells or tissues of the animal, as described.
  • operably linked denotes a relationship between a regulatory region (typically a promoter element, but may include an enhancer element) and the gene, whereby the transcription of the gene is under the control of the regulatory region.
  • heterologous means a DNA sequence not found in the native genome. That is, two nucleic acid elements are said to be “heterologous” if the elements are derived from two different genes, or alternatively, two different species. Thus, “heterologous DNA regulatory sequence” indicates that the regulatory sequence is not naturally ligated to the DNA sequence for the 1 ⁇ -
  • promoter is used according to its art-recognized meaning. It is intended to mean the DNA region, usually upstream to the coding sequence of a gene, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site.
  • a promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular tissue. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-specific factor), under certain conditions (e.g., in the presence of an enhancer element, if present, in the genetic construct containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult). - 31
  • Promoters useful in the practice of the present invention are preferably "tissue-specific” ⁇ that is, they are capable of driving transcription of a gene in one tissue while remaining largely “silent” in other tissue types. Examples of tissue-specific promoters are provided in Table 2 below.
  • Neuron Specific Enolase * neuron 25
  • Glial Fibrillary Acidic Protein human astrocytes (CNS) 28 (GFAP)
  • NF-L Light neurofilament
  • Purkinje cell protein-2 mouse Purkinje 39 cells/cerebellum
  • Choline acetyltransferase human cholinergic neurons 42 rat * 43
  • PTHrP Parathyroid hormone-related human Liver and cecum 50 peptide
  • br brain; B, lymphocytes; mu, skeletal muscle; he, cardiac muscle; te, testis; beta, beta cells; th, thymus; lu, lung; Pa, exocrine pancreas; ys, yolk sac; li, liver; ery, erythroid cells; pit, pituitary and lens, eye lens.
  • tissue-specific promoters see U.S. Patent Nos. 5,834,306 and 5,416,027, and references cited therein.
  • the genetic construct may also contain other genetic control elements, such as enhancers, repressible sequences, and silencers, which may be used to regulate replication of the vector in the target cell.
  • enhancers such as a promoter, repressible sequences, and silencers, which may be used to regulate replication of the vector in the target cell.
  • silencers such as a promoter, a promoter, a promoter, a promoter, and repressible sequences, and silencers, which may be used to regulate replication of the vector in the target cell.
  • the only requirement is that the genetic element be activated, derepressed, enhanced, or otherwise genetically regulated by factors in the host cell and, with respect to methods of treatment, not in the non-target cell.
  • an “element,” when used in the context of nucleic acid constructs, refers to a region of the construct or a nucleic acid fragment having a defined function.
  • a enhancer element is a region of DNA that, when associated with the 1 ⁇ -OHase gene operably linked to a promoter, enhances the transcription of that gene.
  • enhancer is used according to its art-recognized meaning. It is intended to mean a sequence found in eukaryotes which can increase transcription from a gene when located (in either orientation) up to several kilobases from the gene being studied. These sequences usually act as enhancers when on the 5 ' side (upstream) of the gene in question. However, some enhancers are active when placed on the 3' side (downstream) of the gene. In some cases, enhancer elements can activate transcription from a gene with no (known) promoter.
  • Preferred enhancers include the DF3 breast cancer-specific enhancer and enhancers from viruses and the steroid receptor family.
  • Other preferred transcriptional regulatory sequences include NF1, SP1, API, and FOS/JUN.
  • transfection vectors of the present invention may be used to introduce transfection vectors of the present invention into selected target tissue cells.
  • Such methods include, for example, viral-mediated gene transfer using retroviruses, adeno-associated virus (AAV), herpes virus, vaccinia virus, or RNA viruses (e.g., Grunhaus and Horowitz, Semin. Virol. 5:237-252 (1992); Herz and Gerard, Proc. Nat. Acad. Sci. USA 90:2812-2816 (1993); and Rosenfeld et al., Cell 55:143-155 (1992)); liposome-mediated gene transfer (Morishita et a!, J. Clin. Invest. 97:2580 (1993); Feigner et al, U.S. Patent Nos. 5,703,055 (1997) and 5,858,784 (1999)); injection of naked DNA directly into a target tissue (e.g.,
  • a vector may be targeted to selectively transfect a specific population of cells
  • the vector in addition to local administration (such as may be achieved by injection into the target tissue), the vector may be administered systemically (e.g., intravenously) in a biologically-compatible solution or pharmaceutically acceptable delivery vehicle .
  • Vector constructs administered in this way may selectively infect the target tissue.
  • the presence of a target tissue-specific promoter on the construct provides an independent means of restricting expression of the therapeutic gene.
  • the gene therapy embodiment of the invention provides a method of treating or preventing a number of diseases and disorders.
  • this embodiment can be used to treat or prevent cancer in cells that express the vitamin D receptor (VDR).
  • VDR vitamin D receptor
  • a polynucleotide construct containing the gene coding for l ⁇ -OHase is introduced into cancerous cells, where the gene is subsequently expressed, producing 1 ⁇ -OHase in an amount effective to inhibit cell proliferation.
  • a gene encoding the VDR (for example, the nucleotide sequence depicted in SEQ ID NO. 4) can also be co-administered with the l ⁇ -OHase gene.
  • the VDR gene is subcloned into either the same polynucleotide construct as the l ⁇ -OHase gene or into a separate polynucleotide construct, as described above. Both genes are then introduced into the target cell or tissue, where they are subsequently expressed, producing 1 ⁇ -OHase and VDR in amounts effective to treat or prevent the condition.
  • Gene therapy with the gene expressing 1 ⁇ -OHase and, optionally, the gene expressing the VDR gene may be practiced substantially simultaneously with administration of vitamin D or analog or derivative thereof.
  • vitamin D or analog or derivative thereof may be administered to the animal before, during or after receiving gene therapy.
  • the invention can be used to treat or prevent prostate cancer, breast cancer, skin cancer, colon cancer, leukemias, lymphomas, and lung cancer. - 38 -
  • the invention also provides a method of treating or preventing any disease in which l,25(OH) 2 D 3 can influence cell growth and maturation, including pre- cancers such as actinic keratoses, and non-cancerous hyperproliferative disorders such as psoriasis and ichthyosis.
  • a polynucleotide construct containing the gene coding for 1 ⁇ -OHase is introduced into cells in which l,25(OH) 2 D 3 can influence cell growth and maturation.
  • the l ⁇ -OHase gene is subsequently expressed, producing l ⁇ -OHase in an amount effective to inhibit cell proliferation.
  • the invention can therefore be used in the treatment of actinic keratoses, which is a pre-skin cancer.
  • Use of the invention would effect an increase of the amount of 1J5(0H) 2 D 3 (or analog or derivative thereof) within the cell, decreasing proliferative activity and inducing the cells to differentiate into normal morphology.
  • the invention can also be used to treat hyperproliferative disorders of the skin such as psoriasis.
  • This gene therapy approach is a reasonable alternative treatment for psoriasis, whereby the cells would produce or boost their production of 1 J5(OH) 2 D 3 (or analog or derivative thereof) which, in turn, would interact with the VDR and decrease the proliferative activity making the skin cells normal.
  • This treatment can also be applied to other hyperproliferative skin disorders such as ichthyosis.
  • the invention can also be used to treat or prevent disorders in calcium and bone metabolism.
  • a polynucleotide construct containing the gene coding for l ⁇ -OHase can be introduced into a group of cells of the animal, where it is subsequently expressed, enabling these cell to produce l,25(OH) 2 D 3 (or analog or derivative thereof) in a manner similar to the kidney.
  • the invention may be used to prevent bone disease associated with kidney failure and treat vitamin D-dependent rickets type I, X-linked hypophosphatemic rickets, vitamin D- dependent rickets type II, and osteoporosis, as well as such disorders in calcium and bone metabolism as low blood serum calcium due to hypoparathyroidism.
  • a further embodiment of the invention provides a method of treating or preventing any disease or condition mentioned above, including cancer, by administering the gene encoding the VDR as well as the gene coding for l ⁇ -OHase to a target cell or tissue of an animal. Both genes may be subcloned into either the same or different polynucleotide constructs, as described above, which is introduced into the target cell or tissue. Both the 1 ⁇ -OHase gene and the VDR gene are subsequently expressed, producing 1 ⁇ -OHase and VDR in amounts effective to treat or prevent the condition.
  • This particular embodiment may be used with cells that either express or do not already express the VDR.
  • the cell will produce additional VDR which will interact with the increased cellular concentrations of l,25(OH) 2 D (or analog or derivative thereof).
  • the cell will become responsive to l,25(OH) 2 D (or analog or derivative thereof).
  • a cancer cell that had either a defective or absent VDR could be treated according to this embodiment of the invention.
  • the treated cell would then produce VDR which would, in turn, interact with the 1 J5(OH) 2 D (or analog or derivative thereof) that was produced from the l ⁇ -OHase.
  • the interaction leads to a biologic response, including inhibiting proliferation and inducing terminal differentiation, thereby treating cancer, pre-cancer, non- cancerous hyperproliferative disorders, enhancing wound healing, and treating and preventing hair loss.
  • the invention also provides a method of treating benign prostatic hype ⁇ lasia in an animal, by introducing into cells exhibiting benign prostatic hype ⁇ lasia a polynucleotide construct containing a gene coding for l ⁇ -OHase.
  • the l ⁇ -OHase gene is subsequently expressed, enabling the cells to produce 1J5(0H) 2 D (or an analog thereof) in an amount effective to treat the benign prostatic hype ⁇ lasia.
  • a gene encoding the VDR can also be co-administered with the l ⁇ -OHase gene, as described above.
  • expressing 1 ⁇ -OHase and the gene expressing the VDR can be on the same DNA construct or on separate DNA constructs.
  • the invention also provides a method of treating autoimmune diseases in an animal, by introducing into any cells of the animal that would produce a beneficial result a polynucleotide construct containing a gene coding for l ⁇ -OHase.
  • the l -OHase gene is subsequently expressed, enabling the cells to produce 1J5(0H) 2 D (or an analog thereof) in an amount effective to treat the autoimmune disease.
  • a gene encoding the VDR can also be co-administered with the l ⁇ -OHase gene, as described above.
  • the gene expressing 1 ⁇ -OHase and the gene expressing the VDR can be on the same DNA construct or on separate DNA constructs.
  • the invention can be used to treat autoimmune diseases such as diabetes mellitus type 1 , multiple sclerosis, rheumatoid arthritis, or psoriatic arthritis.
  • diabetes mellitus type 1 can be treated by introduction of a polynucleotide construct(s) containing the 1 ⁇ -OHase gene (and optionally the VDR gene) into any cell of the animal that would produce a beneficial result, particularly into pancreatic cells, and more particularly pancreatic cells secreting insulin, such as cells of the Islets of Langerhans.
  • the polynucleotide construct(s) containing the 1 ⁇ -OHase gene can be introduced particularly into any neural cells that would produce a beneficial result.
  • the polynucleotide construct(s) can be introduced particularly into any connective tissue cells that would produce a beneficial result, or in or around the joints of the animal affected by either of these diseases.
  • inventions include isolated nucleic acid molecules that are at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical to the above-described isolated nucleic acid molecules of the present invention.
  • the invention is directed to isolated nucleic acid molecules at least 90%, 95%, 97%, 98%, or 99% identical to the nucleotide - 41 -
  • isolated polynucleotide molecule is intended to refer to a nucleic acid molecule which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for pu ⁇ oses of the invention as are recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated polynucleotide molecules also include such compounds produced synthetically.
  • the invention is further related to nucleic acid molecules capable of hybridizing to a nucleic acid molecule having a sequence complementary to or hybridizing directly to one of the nucleic acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 3 under stringent conditions.
  • stringent conditions is intended overnight incubation at 42 °C in a solution comprising: 50% formamide, 5 x SSC (750 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA (ssDNA), followed by washing the filters in 0.1 x SSC at about 65 °C.
  • Prostatic epithelial cells cultured in a serum-free defined medium express luminal epithelium specific cytokeratins (cytokeratins 8 and 18) as detected immunohistochemically using an anti-cytokeratin antibody, CAM 5.2
  • the serum-free medium contains MCDF170 supplemented with Epidermal Growth Factor, 25 ng/ml, hydrocortisone, 0.5 ⁇ g/ml, ethanolamine, 1 X 10 "4 , insulin, 5 ⁇ g/ml, transferrin, 5 ⁇ g/ml, and whole bovine pituitary extract, 70 ⁇ g/ml.
  • Prostatic cells used were at their first passage in vitro and were cultured in the serum-free RPMI medium during incubation with vitamin D metabolites.
  • NP96-5 cultured from the histologically normal prostate of a 23 year old Caucasian organ donor
  • BPH96-11 cultured from an open prostatectomy specimen of a 56 year old Caucasian with BPH.
  • Keratinocyte Culture Because 1 ⁇ -OHase activity has been well- established in keratinocytes, cultured human keratinocytes were used for comparison to the prostatic cultures. Keratinocytes were grown in culture following a modification of the method of Rheinwald and Green, Cell 6:331 -344 (1975). Briefly, keratinocytes were obtained from neonatal foreskin after trypsinization at 4 ° C. Keratinocytes were plated and grown on lethally irradiated 3T3 fibroblast feeder cells in a serum-free basal medium containing 0J5mM calcium and supplemented with gro th factors including bovine pituitary extract
  • cholera toxin 0.1 ⁇ g/ml
  • hydrocortisone 200 ng/ml were added into the medium during the initial plating of the primary culture and the subsequent subcultures. Cells were fed and maintained without cholera toxin and hydrocortisone, and used for enzyme assay.
  • l ⁇ -OHase activity was determined in monolayer cultures of the cell lines and primary cultures described herein. The assays were performed in the presence of 25(OH)D 3 (50 nM) as the enzyme substrate and DPPD (Sigma-Aldrich, Allentown, PA), an antioxidant and a known inhibitor of free-radical generated
  • the methanol extract was transferred to a glass test tube and the cells were washed with an additional 0.5 ml methanol.
  • the extract and wash were combined, dried down with a stream of nitrogen, and redissolved in 1 ml acetonitrile followed by the addition of 1 ml - 44 -
  • the l ⁇ -OHase activity was also determined in the primary cultures of prostatic cells by using 20 ⁇ g nonradioactive 25(OH)D 3 and 0.91 ⁇ Ci of [ 3 H]-25(OH)D 3 instead of only nonradioactive 25(OH)D 3 as a substrate.
  • the retention volume for 25(OH)D 3 , 24,25(OH) 2 D 3 and l,25(OH) 2 D 3 was calibrated by applying standard 25(OH)D 3 , 24,25(OH) 2 D 3 and 1J5(0H) 2 D 3 to the HPLC column prior, during, and after unknown sample application.
  • the protein concentration in each 35 mm dish was determined by standard procedures.
  • the enzyme activity was expressed as pmol l,25(OH) 2 D 3 / mg protein/hr.
  • Keratinocyte transfection Keratinocytes were maintained in MCDB-153 medium. Cells in 24-well dishes at 50%-60% confluence were - 45 -
  • l ⁇ -OHase cDNA was cloned from human keratinocytes using reverse transcription-PCR (RT-PCR).
  • the 2,150 base pair l ⁇ -OHase cDNA (which is a part of SEQ ID NO. 1) contains a 1,527 bp open reading frame, predicted to encode protein of 508 amino acids (SEQ ID NO. 2).
  • This cDNA was directly subcloned into the pCR3J-Uni (INVITROGEN) mammalian cell expression vector. DNA sequence analysis showed the cDNA in keratinocytes was 100%) identical to the renal l ⁇ -OHase.
  • a human genomic DNA library (CLONTECH) was screened by plaque hybridization using a 700bp fragment from the 5' region of l ⁇ -OHase cDNA (SEQ ID NO. 1 ) as a probe.
  • SEQ ID NO. 1 l ⁇ -OHase cDNA
  • a polynucleotide construct containing the l ⁇ -OHase cloned gene was constructed as outlined in the preceding Methods section and used to transfect normal, human cultured keratinocytes.
  • the cultured keratinocytes transfected with l ⁇ -OHase gene were incubated with varying concentrations of 25(OH)D 3 .
  • As a negative control cultured keratinocytes that had not been transfected were exposed to the same concentrations of 25(OH)D under identical conditions at the same time. If the cells transfected with the - 47 -
  • 1 ⁇ -OHase gene increased the l ⁇ -OHase activity in the cells, the cells would be more efficient in converting 25 (OH)D to 1 J 5 (OH) 2 D which, in turn, would result in higher concentrations within the cell, and therefore, have a more dramatic effect on decreasing the cells' proliferative activity.
  • the keratinocytes transfected with the l ⁇ -OHase gene had a more marked decrease in proliferative activity to the same concentration of 25(OH)D compared to the non-transfected control keratinocytes. This occurred at concentrations from 10 "9 to 10 "7 M.

Abstract

Methods for inhibiting cancer cell growth are disclosed. In one embodiment, the method comprises the step of increasing the amount of a metabolic precursor of 1,25-dihydroxyvitamin D ('1,25(OH)2)D') or an analog or a derivative thereof available to a target cell. In a preferred embodiment, the precursor is 25-hydroxyvitamin D. In another embodiment, the method comprises administering a gene coding for 25-hydroxyvitamin D-1α-hydroxylase ('1α-OHase') with or without a gene coding for the vitamin D receptor to the target cell to increase production of 1,25(OH)2D or analog thereof within the target cell, thereby inhibiting cellular proliferation. In another embodiment of the invention, the method of administering a gene coding for 1α-OHase with or without a gene coding for the vitamin D receptor to a target cell or tissue is used in the treatment of hyperproliferative cell disorders, disorders of hair growth, and disorders of calcium and bone metabolism in animals.

Description

Methods for Prevention and Treatment of Cancer
Background of the Invention
Field of the Invention
The present invention relates to methods for the treatment of cancer in animals. In one embodiment, the method comprises the step of increasing the amount of a metabolic precursor of 1 J5-dihydroxy vitamin D available to target cells or tissues in the animal. In another embodiment, the method comprises administering the gene expressing the enzyme 25 -hydroxy vitamin D- lα-hydroxylase to a target cell to increase production of 1,25-dihydroxy vitamin D within the target cell.
The present invention also relates to a method for the treatment of hyperproliferative cell disorders and disorders of calcium and bone metabolism in animals, comprising administration of the gene expressing 25-hydroxyvitamin D- 1 α-hydroxylase to target cells or tissues of the animal.
Related Art
Definitions
The term "vitamin D" has been used in the literature to refer generally to a class of steroid hormones that possess anti-rachitic activity, including ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), calcidiol (25- hydroxy vitamin D), calcitriol (1,25 -dihydroxy vitamin D), and 20 or more other metabolites of vitamin D. However, the term "vitamin D" is used herein in its more restrictive sense to refer only to vitamin D2 or vitamin D3 in order to distinguish the useful metabolites which are part of this invention. 1,25-Dihydroxyvitamin D
The most biologically active vitamin D metabolite is 1 J5-dihydroxy vitamin D ("l,25(OH)2D"), also known as calcitriol. The synthesis of 1J5(0H)2D in an animal, e.g., humans, begins with the cutaneous production of vitamin D after exposure to sunlight or ultraviolet (UN) radiation, or after the intestinal absorption of plant- and yeast-derived vitamin D2 or animal-derived (typically from fish liver oil and fatty fish) vitamin D3 obtained from the diet. To become biologically active, vitamin D must undergo two hydroxylation steps. See Fig. 1 .
The first hydroxylation step occurs in the liver by hydroxylation at the 25th carbon position, forming 25-hydroxyvitaminD ("25(OH)D"),the major circulating metabolite of vitamin D. The second hydroxylation occurs in the kidney by hydroxylation at the lα position, forming 1J5(0H)2D, the hormonally active metabolite. Although the kidney is the major source of 1 J5(OH)2D, the enzyme lα-hydroxylase ("lα-OHase") that converts 25(OH)D to 1J5(0H)2D is also present in several types of non-renal cells, e.g., activated macrophages and keratinocytes (Barbour, G.L., et al., N. Engl. J. Med. 505:440-443 (1981); and Bikle, D.D., etal, J. Clin. Invest. 78:551-566 (1986)). However, lα-OHase was not heretofore known to be present in prostatic cells.
The lα-OHase enzyme has been cloned recently from mouse kidney, human kidney, and human keratinocytes (Takeyama, K., et al. , Science 277: 1827-
1829 (1997); Fu, G.K., et al, Mol. Endo. 77:1961-1970 (1997); Monkawa, T., etal., Biochem. Biophys. Res. Comm. 239:521-533 (1997); Fu, G.K., etal., DNA and Cell Biology 75:1499-1507 (1997); Shinki, T., et al., Proc. Natl. Acad. Sci. USA 94:12920-12925 (1997); Brenza, H.L., et al, Proc. Natl. Acad. Sci. USA 95:1387-1391 (1998); Murayama, A., et al., Biophys. Biochem. Res. Comm.
249:11-16 (1998); andKong, X.F., etα/., Ro«e 5 f5, >/?/. :S186 (1998)). The cDΝA was found to be identical for the lα-OHase gene in the human kidney and keratinocytes (Kitanaka, S., et al., N. Engl. J. Med. 338:653-661 (1998); Fu, G.K., et al. , Mol. Endo. 77:1961-1970 (1997); and Fu, G.K., et al. , DNA and Cell Biology 16:1499-1501 (1997)). The lα-OHase was also cloned from skin from patients with vitamin D dependent rickets type I (pseudovitamin D deficiency rickets). It was determined that several point mutations had occurred in the gene resulting in a defective 1 α-OHase (Kitanaka, S., et al. , N. Engl. J. Med. 338:653- 661 (1998). 1 J5(OH)2D is able to carry out most of its biologic effects by interacting with a specific receptor known as the vitamin D receptor (VDR). VDR is present in tissues that are responsible for regulating calcium and bone metabolism including the small intestine, kidney and bone. In addition, a wide variety of tissues and cells not related to calcium and bone metabolism also possess VDR (Holick, M.F., in Primer on the Metabolic Bone Diseases and Disorders of
Mineral Metabolism, M.J. Favus (ed.), 3rd Ed., Lippincott-Raven: Philadelphia (1996), pp. 74-81). These include, among others, brain, heart, gonads, breast, pancreas, skeletal muscle, smooth muscle, skin, monocytes, and activated T and B lymphocytes. In addition, several tumor cell lines, including promyelocytic leukemic cells, multiple myeloma cells, squamous cell carcinoma cells, prostate cancer cells, breast cancer cells, and basal cell carcinoma cells, possess VDR (Holick, M.F., Bone 17 (Suppl.) : 107S- 111 S (1995)). Although the exact function of VDR in these tissues and cells is not well understood, it is known that some cells that possess VDR respond to 1J5(0H)2D by decreasing their proliferative activity. In addition, some cells such as keratinocytes are also induced to terminally differentiate. This has given rise to the use of 1J5(0H)2D and its analogs for treating hyperproliferative skin diseases such as psoriasis, and has suggested the possibility for treating some cancers such as breast, colon, and prostate (Holick, M.F., Bone 17 (Suppl.) Λ01S-11 IS (1995); Gross, C, et al, in Vitamin D, D. Feldman et al. (eds.), Academic Press: New York (1997), pp.
1125-1139).
Treatment of Cancer
Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells. One frequently-occurring cancer is adenocarcinoma of the prostate gland, which is the most commonly diagnosed malignancy in American males, excluding skin cancer. Approximately 317,000 new cases of prostate cancer were diagnosed in the United States in 1996 (Parker, S.L., et al, Cancer J. Clin. 46:5-21 (1996)), accounting for 41% of newly diagnosed malignancies, excluding basal cell and squamous cell carcinomas of the skin. This may actually underestimate the problem because clinically silent prostate cancer is very common.
A unique feature of prostate cancer is the high prevalence of "incidental" or "subclinical" cases identified at autopsy. Autopsy data indicate that about 30% of men over the age of 50 have histological prostate cancer, and the prevalence of these incidental cancers reaches 60% in men over the age of 80. Histologically, these incidental cancers are indistinguishable from prostate cancers that are potentially life-threatening and are considered to be an earlier stage in their natural history. Unlike prostate cancer mortality rates, incidental prostate cancer is ubiquitous among elderly men regardless of ethnicity or geographic location.
Although prostate carcinoma is generally a slow growing malignancy, mortality from the disease is nonetheless considerable, representing about 15% of all cancer deaths in 1996, which makes prostate cancer the second leading cause of cancer death among U.S. males. (Parker, S.L., et al, Cancer J. Clin. 46:5-21 (1996)). As a result, prostate cancer has rapidly become a major public health concern not only in the United States, but worldwide. See Gross, C, et al, in Vitamin D, D. Feldman et al. (eds.), Academic Press: San Diego, CA (1997), pp. 1125-1139. Interestingly, Prostate cancer mortality rates in the contiguous United States are inversely correlated with levels of ultraviolet (UV) radiation, which produces vitamin D in the skin.
As suggested above, calcitriol is now known to play an important role in the regulation of cell growth and differentiation (Walter, M. R., Endocr. Rev. 73:719-764 (1992)). For example, it has been shown that 1J5(0H)2D can modulate cell proliferation and differentiation in prostate cells. Supporting evidence includes the ubiquitous presence of receptors for 1 J5(OH)2D (vitamin D receptors, or VDRs) in human prostatic cells and the anti-proliferative and pro- differentiating effects of 1 J5(OH)2D on these cells in vitro and in vivo (Schwartz, G. G.,AnticancerRes. 74:1077-1082 (1994)). Apparently, 1 ,25 (OH)2D maintains the differentiated phenotype of prostatic cells. Low levels of 1J5(0H)2D can therefore increase the risk for life-threatening prostate cancer.
It would appear that making 1 ,25(OH)2D available to target cells or organs would reduce the risk of cancer. However, increasing 1J5(0H)2D levels is subject to a plurality of obstacles. First, systemic levels of 1 J5(OH)2D in normal individuals are very tightly regulated metabolically. An increase in systemic 1 J5(OH)2D levels is generally not correlated with an increase in systemic levels of vitamin D or certain of its metabolites, including 25(OH)D, the immediate metabolic precursor of 1 J5(OH)2D. Thus, a mechanism by which UV radiation or vitamin D could result in increased exposure to 1 J5(OH)2D by prostatic cells was previously unclear. Prostatic cells, even though known to have 1 J5(OH)2D receptors, were previously thought to be modulated only by systemic 1 ,25 (OH)2D which was produced in and highly regulated by kidney 1 α-OHase using 25(OH)D as a substrate. Because of this highly regulated production of 1 J5(OH)2D by the kidneys, it was well accepted that increasing systemic 25(OH)D would not produce proportionately higher systemic levels of 1J5(0H)2D. It was also previously unknown how to provide effective amounts of
1 J5(OH)2D at the cellular level without risking undesired, and potentially unsafe, side effects to the patient. For example, systemic 25(OH)D levels can be increased by in situ production of vitamin D through sun or UV exposure, due to the regulation of 1 J5(OH)2D produced from 25(OH)D. Thus, although extensive exposure to sunlight or UV radiation will elevate serum levels of 25(OH)D, serum levels of 1 ,25(OH)2D will generally not be elevated to a desired antiproliferative level. Thus, reaching a beneficial level of l,25(OH)2D was not known to be possible from exposure to the sun or UV radiation, even in amounts that are considered by those in the art to be unsafe or excessive. Certain risks, e.g., skin cancer, inter alia, are associated with such excessive sun or UV exposure. - 6 -
It has recently been suggested that vitamin D dietary supplementation may lower the risk of prostate cancer (Naitoh, J., et al, Prostate Cancer and Prostatic Diseases 7:48-53 (1997)). However, administering vitamin D as a dietary supplement is recommended to be performed under the supervision of a physician due to the risk of producing vitamin D toxicity, of which the negative side effects can include kidney failure, hypercalcemia, coma, and death.
Vitamin D supplementation therapy is also disadvantageous in view of its slow metabolic conversion to 25(OH)D. Therefore, reaching effective levels of 25(OH)D by administering supplemental vitamin D can take weeks or months using a conservative dosing regimen. Conversely, if those vitamin D levels are too high, it can take months to return to normal levels of 25(OH)D after ceasing the administration or reducing the dosage of vitamin D due to the natural storage of vitamin D in body fat. Moreover, administration of 1 ,25(OH)2D itself, carries its own risks of producing hypercalcemia, which has been well-documented. Multiple studies that have shown that several lines of cancer cells including colon, melanoma, prostate and breast that have a VDR, when incubated with l,25(OH)2D, decreased their proliferation (Holick, M.F., Bone 17 (2 Suppl.) Λ01S-11 IS (1995); Halline, A.G., et al, Endocrinology 754:1710-1717 (1994); Konety, B.R., etal, Cell Growth & Differentiation 7:1563-1570 (1996); and Kivineva, M., et al, J. Steroid Biochem. Mol. Biol. 66:121-121 91998)).
Furthermore, human skin cells (Bikle, D.D., et al. , Biochemistry 25: 1545- 1548 (1986)) and normal prostate cells and prostate cancer cells have been shown to have the capacity to convert 25(OH)D to 1J5(0H)2D (Schwartz, G.G., et al, Cancer Epidemiol. Biomark. & Prev. 7:391-395 (1998)). Thus, it is believed that when such cells are exposed to 25(OH)D, that the 25(OH)D is converted by the cells' lα-OHase to l,25(OH)2D. The internally produced l,25(OH)2D then will interact with the cell's VDR to regulate the cell's proliferative activity.
As described above, l,25(OH)2D is considered to the biologically active form of vitamin D. Its principal physiologic function is on calcium and bone metabolism. It stimulates intestinal calcium transport and initiates mobilization of - 7 -
calcium stores from bone. Patients with kidney failure who are unable to make 1 J5(OH)2D develop hypocalcemia, secondary hyperparathyroidism, and the bone disease, renal osteodystrophy. Two rare genetic disorders also are associated with an inherited defect in the renal lα-OHase. Vitamin D dependent rickets type I is an inborn error where there is a defect in the lα-OHase enzyme. In X-linked hypophosphatemic rickets, there is a defect in the regulation of the lα-OHase. The elderly, with osteoporosis, have a defect in their ability to upregulate the lα-OHase (Holick, M.F., in Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, M.J. Favus (ed.), 3rd Ed., Lippincott-Raven: Philadelphia (1996), pp. 74-81).
Summary of the Invention
It has not been heretofore described that administering a vitamin D metabolite and 1J5(0H)2D precursor to a patient can prevent cell proliferation, invasive cancer, or metastasis of cancer or tumor cells of an organ, e.g., the prostate, colon, or breast, while reducing the risk of vitamin D toxicity and hypercalcemia.
It has also not been heretofore described that administration of the lα-OHase gene using gene therapy techniques could be used to treat or prevent cancer or any disease in which 1J5(0H)2D3 can influence cell growth and maturation, including hyperproliferative skin disorders such as psoriasis or actinic keratoses, or that it could also be used to treat disorders in calcium and bone metabolism, as a safer alternative to exogenous administration of 1J5(0H)2D3
In addition, there was previously no known method for determining the risk for development of invasive cancer nor to predict the success rate by a particular treatment by measuring in an organ levels of cellular components, e.g., vitamin D, vitamin D metabolites, the enzymes producing those metabolites, or the activity of those enzymes. Such methods are advantageously provided by the subject invention as described hereinbelow.
The subject invention concerns novel methods for prevention, treatment or testing for certain types of cancer, in particular prostate cancer, employing the advantages of a metabolite of vitamin D, analogs or derivatives of the vitamin D metabolite, or enzymes involved in the metabolism of vitamin D.
The invention provides a method for locally increasing concentrations of 1 ,25 -dihydroxy vitamin D (1 J5(OH)2D) in a cell of a target organ so that the anti- proliferative, anti-invasive, antimetastatic and pro-differentiating action of 1 J5(OH)2D can be provided to those cells. Specifically, one embodiment of the subject invention concerns a method for increasing 1J5(0H)2D concentrations available to the target organ by administering to a patient a metabolic precursor of 1J5(0H)2D wherein the precursor does not have the side effects or risks of skin cancer associated with sun or ultraviolet (UV) radiation exposure, or the side effects or risks of vitamin D toxicity associated with administering high doses of vitamin D or 1J5(0H)2D.
In a preferred embodiment, the invention comprises administering an effective amount of a vitamin D metabolite to a patient in need of therapeutic or prophylactic treatment of a tumor or cancer, wherein the administration of the metabolite provides to a patient a substantially lower risk of skin cancer, vitamin
D toxicity, or hypercalcemia than alternative methods, e.g, exposure to UV radiation, or administration of excessively high doses of vitamin D or 1 ,25(OH)2D which can cause such undesired side effects.
Advantageously, the subject method can be used to prophylactically or therapeutically treat a patient at risk of or having certain subclinical or clinical cancer. The method includes preventing proliferation, invasion, or metastasis of the tumor or cancer cells by administering vitamin D or a metabolite of vitamin D, or an analog, derivative salt, or functional equivalent of the metabolite which can be metabolically converted to 1 ,25 (OH)2D or a functional equivalent thereof. The subject treatment preferably comprises administering to a patient having - 9 -
subclinical, clinical, or minimal residual cancer which is responsive to 1 J5(OH)2D, an effective amount of 25(OH)D. Minimal residual cancer refers to detectable tumor cells in post-treatment patients, e.g., post-prostatectomy or post-radiation therapy, wherein the primary tumor has been eliminated or the organ has been removed from the body, but tumor or cancer cells, likely mobilized to a different organ or present in the bloodstream, are still detectable.
In a further preferred embodiment, the subject method for prophylactic or therapeutic treatment of cancer comprises administering an effective amount of 25(OH)D, or an analog, derivative, salt, or functional equivalent thereof, to a patient having subclinical, clinical, or minimal residual cancer of an organ, i.e., the
"target organ," wherein the target organ or cells derived from that target organ can convert 25(OH)D or its analog, derivative, salt, precursor, or functional equivalent, to l,25(OH)2D, or its functional equivalent. Organ cells having the enzyme lα-OHase can locally convert 25(OH)D or an analog thereof to 1 J5(OH)2D or its corresponding analog. For example, certain prostatic cells have been shown to have lα-OHase and the capability to convert 25(OH)D to 1 J5(OH)2D. Accordingly, the subject method can be applied to prostate cancer patients having subclinical or clinical disease; patients at risk of developing prostate tumors (e.g., persons with no clinical evidence of prostate cancer), or patients having metastatic disease, e.g., minimal residual cancer, resulting from mobilization of cancerous prostatic cells to other organs or tissues.
Alternatively, the subject method can be used for treating a tumor in any organ wherein the cells of that organ can convert a metabolic precursor of 25(OH)D to 1J5(0H)2D, or its functional equivalent, and be responsive to the antiproliferative, anti-invasive, antimetastatic, or pro-differentiating effects of
1 J5(OH)2D, or its functional equivalent. Thus, a cell having a vitamin D receptor (VDR) and capable of producing the enzyme, 1 α-OHase, can respond to such treatment. Further, a cell normally deficient in VDR or lα-OHase can be genetically engineered, e.g., transgenically altered with an appropriate gene or promotor to induce transcription of the gene and production of the resultant - 10 -
protein(s), such that the cell is capable of responding to treatment according to the subject invention.
The subject invention thus offers a solution to the problem of reducing the risk of invasive (i.e., life-threatening) cancer, including invasive prostate cancer. Therapy with 25(OH)D or an analog, derivative, salt, or functional equivalent thereof can be useful to many men at various stages of the natural history of prostate cancer. These include the millions of men who presently have subclinical prostate cancer: men on "watchful waiting," and men who have been treated for prostate cancer by therapy with curative intent, but who may still have prostatic cancer cells remaining in their bodies. It also may be useful to men without prostate cancer, either subclinical or clinical, to reduce the risk of developing cancer. One form of subclinical prostate cancer is also known as "prostatic intraepithelial neoplasia" or PIN.
It should also be possible to inhibit cell proliferation in cancer cells which contain the VDR and which respond to l,25(OH)2D by decreasing their proliferation, by introducing into the cancerous cells the gene coding for the enzyme lα-OHase and allowing the cells to produce 1J5(0H)2D internally. This gene therapy method could therefore be used to treat these cancers. Further, if the gene encoding the VDR were co-administered with the lα-OHase gene to cells lacking the VDR, this method might impart to such cells a response to
1J5(0H)2D3, making this method useful in the treatment of cancers normally unresponsive to 1J5(0H)2D3.
There would be several advantages to this method. For example, exogenous administration of 1J5(0H)2D3 and its analogs has the potential for causing hypercalcemia (Holick, M.F., in Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, M J. Favus (ed.), 3rd Ed., Lippincott- Raven: Philadelphia (1996), pp. 74-81; Holick, M.F., Bone 17 (2 Suppl.) Λ01S- 11 IS (1995); and Gross, C, et al, in Vitamin D, D. Feldman et al. (eds.), Academic Press: San Diego, CA (1997), pp. 1125-1139), which would be mitigated or eliminated by incorporating the 1 α-OHase gene into the cancer cell - 11 -
and allowing it to make 1 ,25(OH)2D internally. The co-administration of the VDR gene will either impart or enhance the cell response to the antiproliferative activity of the endogenously produced 1 ,25(OH)2D. Once made, 1 J5(OH)2D is likely to be degraded in the cell after its use thereby eliminating any potential toxicity. In a preferred embodiment, the invention provides a method of inhibiting proliferation of tumor cells and/or inducing differentiation of tumor cells in an animal, comprising introducing into the cells in need thereof a polynucleotide construct comprising a gene expressing lα-OHase, whereby 1J5(0H)2D or an analog thereof is produced and the proliferation of the cell is inhibited. In a preferred embodiment, the invention further comprises introducing into the tumor cells a polynucleotide construct comprising a gene expressing the vitamin D receptor. In this case, the gene expressing 1 α-OHase and the gene expressing the vitamin D receptor can be on either the same DNA construct or on separate DNA constructs. In a particularly preferred embodiment, this aspect of the invention provides a method for treating or preventing prostate cancer, breast cancer, skin cancer, colon cancer, lung cancer, leukemia, lymphoma, or other cancers that can respond to the antiproliferative and prodifferentiating actions of endogenously produced 1J5(0H)2D or an analog of 1J5(0H)2D that is provided by the cell undergoing gene therapy with DNA constructs containing the 1 α-OHase gene, with or without the VDR gene.
In another embodiment, an animal being treated by gene therapy as described herein may also be treated substantially simultaneously with 25(OH)D or an analog, derivative, salt or functional equivalent thereof to augment the response to the gene therapy.
In addition, the invention provides a method of testing for cancer risk of a patient. By measuring levels of certain cellular components in a particular organ wherein the measured component is involved in vitamin D metabolism, vitamin D, a metabolite thereof (e.g., 25(OH)D, or its analogs), an enzyme capable of converting a vitamin D metabolite to 1 J5(OH)2D or the activity of that enzyme, - 12 -
the relative risk of developing life-threatening cancer in that organ can be determined. For example, the lα-OHase enzyme is capable of converting 25(OH)D to 1J5(0H)2D. Therefore, measuring l -OHase levels or the activity of the lα-OHase enzyme in a cellular sample from a target organ, e.g., prostate, can be used to determine the risk of developing cancer in that organ or an organ comprising a cancerous cell from that organ.
Standard molecular biology techniques which are well-known in the art can also be used for measuring genetic polymorphisms in the genes encoding the 1 α-OHase enzyme or VDRs and correlating allelic differences with risk of tumor development in the individual tested.
The invention also provides a method of predicting the rate of successful treatment of a particular cancer in a patient by determining the local levels of certain cellular components in the vitamin D metabolic pathway, such as vitamin D or its metabolites, in an organ, e.g., the prostate, or by determining levels of an enzyme, e.g., lα-OHase, capable of converting a metabolic precursor into
1J5(0H)2D. Measuring activity of the enzyme can also be useful for indirectly measuring enzyme levels and thus be predictive of treatment success.
The invention also provides a method of treating a hyperproliferative skin disorder in an animal, comprising introducing into skin cells of the animal in need thereof a polynucleotide construct containing a gene expressing lα-OHase, whereby 1 J5(OH)2D or an analog thereof is produced and the proliferation of the cell is inhibited. In another embodiment, the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor. In this embodiment of the invention, the gene expressing 1 α-OHase and the gene expressing the vitamin D receptor can be on the same
DNA molecule of the construct or on separate DNA molecules of said constructs.
More specifically, the invention can be used to treat actinic keratoses, a pre-skin cancer, or ichthyosis. Introduction of the gene coding for lα-OHase effects an increase of the amount of 1J5(0H)2D3 within the cell, thereby - 13 -
decreasing proliferative activity and inducing the cells to differentiate into normal morphology.
The invention is also directed to a method to treat hyperproliferative disorders of the skin such as psoriasis. Patients with psoriasis respond to topical and oral 1J5(0H)2D. However, there is concern about the potential toxicity
(Holick, M.F., in Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism, M J. Favus (ed.), 3rd Ed., Lippincott-Raven: Philadelphia (1996), pp. 74-81; and Holick, M.F., Bone 17 (2 SupplJ VlSA US (1995)). Thus, the invention is a reasonable alternative treatment for psoriasis, whereby the cells are induced to make or boost their production of 1 ,25(OH)2D3 which, in turn, interact with the VDR and decrease the proliferative activity and enhance differentiation, making the skin cells normal. This treatment can also be applied to other hyperproliferative skin disorders such as ichthyosis.
The invention also provides a method of treating a disorder in calcium and bone metabolism in an animal, comprising introducing into cells of the animal in need thereof a polynucleotide construct containing a gene expressing 1 α-OHase, whereby 1 ,25(OH)2D or an analog thereof is produced and the disorder is treated. In one embodiment, the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor. In this embodiment, the gene expressing 1 α-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or on separate DNA constructs.
This embodiment of the invention provides a method for treating disorders in calcium and bone metabolism such as renal osteodystrophy or a metabolic bone disease. The invention also provides a method of treating vitamin D-dependent rickets type I, X-linked hypophosphatemic rickets, vitamin D-dependent rickets type II, and osteoporosis, as well as such disorders in calcium and bone metabolism as low blood serum calcium due to hypoparathyroidism.
The present invention also provides a method of treating or preventing hair loss in an animal, comprising introducing into hair cells in need thereof a - 14 -
polynucleotide construct containing a gene expressing lα-OHase, whereby 1J5(0H)2D or an analog thereof is produced and the hair loss is treated. In one embodiment, the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor. In this embodiment, the gene expressing lα-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs.
The invention can therefore be used to treat or prevent hair loss resulting from conditions such as alopecia androgenetica, female pattern baldness, and chemotherapy-induced alopecia.
The invention also provides a method of enhancing wound healing in an animal, comprising introducing into cells of the wound in need thereof a polynucleotide construct containing a gene expressing lα-OHase, whereby 1J5(0H)2D or an analog thereof is produced and the healing of the wound is enhanced. In one embodiment, the invention further comprises introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor. In this embodiment, the gene expressing lα-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs. The invention also provides a method of treating benign prostatic hyperplasia in an animal, comprising introducing into cells exhibiting benign prostatic hyperplasia in need thereof a polynucleotide construct containing a gene expressing 1 α-OHase, whereby 1 J5(OH)2D or an analog thereof is produced and the benign prostatic hyperplasia is treated. In this embodiment, the invention may further comprise introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor. In this embodiment of the invention, the gene expressing 1 α-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs.
The invention also provides a method of treating an autoimmune disease in an animal, comprising introducing into cells of the animal in need thereof a - 15 -
polynucleotide construct comprising a gene expressing 25-hydroxyvitamin D- lα-hydroxylase, whereby 1,25-dihydroxy vitamin D or an analog thereof is produced and the autoimmune disease is treated. In one embodiment, the invention may further comprise introducing into said cells a polynucleotide construct containing a gene expressing the vitamin D receptor. In this embodiment, the gene expressing 1 α-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or are on separate DNA constructs. The invention can be used to treat autoimmune diseases such as diabetes mellitus type 1, multiple sclerosis, rheumatoid arthritis, and psoriatic arthritis. In addition, the invention provides for an isolated polynucleotide molecule comprising a 1 α-OHase regulatory sequence or a complement thereof. In one embodiment, the lα-OHase regulatory sequence or a complement thereof comprises a polynucleotide selected from the group consisting of: (a) a polynucleotide comprising a nucleotide sequence at least 90% identical to the nucleotide sequence depicted in SEQ ID NO. 3; and (b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (a) or the complement thereof. In other embodiments, the 1 α-OHase regulatory sequence or complement thereof comprises a nucleotide sequence that is 95%, 97%, 98%, or 99% identical to the sequence depicted in SEQ ID NO. 3, or comprises the nucleotide sequence depicted in SEQ ID NO. 3. The invention also provides for an isolated polynucleotide molecule further comprising a nucleotide sequence coding for lα-OHase that is 95%, 97%, 98%, or 99% identical to the sequence depicted in SEQ ID NO. 1, or one that comprises the nucleotide sequence depicted in SEQ ID NO. 1. - 16 -
Brief Description of the Figures
Fig. 1 shows a diagrammatic representation of vitamin D metabolism in the body. Well-known metabolism steps are shown by solid arrows; synthesis of 1J5(0H)2D from 25(OH)D by the prostate is shown by open arrows. Figures 2-5 show a high performance liquid chromatography elution profile of tritium activity of lipid extracts from cultured normal human prostate primary cultures or BPH cultures incubated with [3H] -25 (OH)D in the absence or presence of the P450 inhibitor clotrimazole.
Fig. 2 shows second passage of normal human prostate cells incubated withnonradioactive25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and 1J- dianilinoethane (DPPD) (10 μM) at 37 °C for two hours in the absence of clotrimazole.
Fig. 3 shows second passage of normal human prostate cells incubated with nonradioactive 25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and DPPD (10 μM) at 37 °C for two hours in the presence of the P450 enzyme inhibitor, clotrimazole (20 μM).
Fig. 4 shows second passage of prostate cells derived from BPH cultures incubated with nonradioactive 25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and DPPD (10 μM) at 37 °C for two hours in the absence of clotrimazole.
Fig. 5 shows second passage of prostate cells derived from BPH cultures incubated with nonradioactive 25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and DPPD (10 μM) at 37 °C for two hours in the absence of clotrimazole. Fig. 6 depicts a graph showing the effect of 25(OH)D3 concentration on the proliferative activity of normal human keratinocytes transfected with lα-OHase cDNA. Cultured human keratinocytes transfected with a vector containing the lα-OHase cDNA as described were incubated with 25(OH)D3 at different concentrations (InM, lOnM, lOOnM) for 18 hours. [3H]thymidine - 17 -
incorporation was measured as described. •-•: [3H]thymidine incorporation of cells not transfected ("Control"); o-o: [3H]thymidine inco oration of cells transfected with vector containing lα-OHase cDNA (" lα-OHase transfection").
Detailed Description of the Preferred Embodiments
The subject invention concerns novel methods for preventing or treating cell proliferation, invasiveness, or metastatic potential, or for promoting cellular differentiation. The subject invention further concerns a method for testing for cancer, as well as a method for predicting the success of treatment for cell proliferation or cancer.
Administration of Vitamin D Metabolite Compounds
One aspect of the subject invention comprises increasing local cellular levels of 1J5(0H)2D. Specifically, one embodiment of the subject invention concerns preventing or treating cell proliferation, invasiveness, or metastasis, or promoting cellular differentiation by administering an effective amount of a vitamin D metabolite which can be metabolically converted by the target cells to l,25(OH)2D.
Preferably, the vitamin D metabolite used in accordance with the subject invention does not cause an increased risk of skin cancer (as compared to sun or ultraviolet (UV) ray exposure), vitamin D toxicity (as compared to supplemental excessive vitamin D administration), and does not significantly contribute to hypercalcemia (as compared to administering 1 ,25(OH)2D). More preferably, the subject invention comprises administering an effective amount of 25(OH)D, or an analog, derivative, salt, or functional equivalent thereof to prevent the proliferation, invasion, or metastasis of tumor or cancer cells in a particular organ, or to promote differentiation of cells in, or derived from, that organ. The compound 25(OH)D is commercially available. - 18 -
The normally observed concentration of 25(OH)D in serum is about 20- 150 nmol/L (8-60 ng/ml). However, circulating concentrations of up to 250 nmol/L (100 ng/ml) 25(OH)D have commonly been observed in lifeguards after a full summer of exposure to sunlight and is considered to be normal (Holick, M.F., J. Nutrition, Suppl. 720:1464-1469 (1990)). Concentrations of about 350 nmol/L or more are considered to be dangerous to the health of the individual. Hypovitaminosis D, i.e., a deficiency in serum 25(OH)D, is a condition defined when serum levels fall below about 25 nmol/L (10 ng/ml). Thus, an effective amount of 25(OH)D administered into the target organ would be any amount which, when administered, increases local cellular levels of 25(OH)D, but maintains serum levels of 25(OH)D within this "normal" range. Preferably, 25(OH)D levels are increased in the target organ substantially above 25 nmol/L but less than 250 nmol/L. More preferably, 25(OH)D levels are increased to between about 50 and 150 nmol/L. Normal serum levels of 1 J5(OH)2D range between about 38- 144 pmol/L
(16-60 pg/ml). Thus, an alternative determination of an effective amount of 25(OH)D administered in accordance with the method of the subject invention is to administer an amount which raises the level of 25(OH)D toward the high end of its normal range in the target organ, but which does not raise systemic 1J5(0H)2D above the high end of its normal range. For example, 25(OH)D levels preferably can be raised to increase intra-organ levels to between about 25 and 150 nmol/L, but where systemic 1J5(0H)2D levels remain less than 145 pmol/L. More preferably, 1J5(0H)2D levels remain below 125 nmol/L when 25(OH)D is administered to a target cell, organ, or tissue. Useful analogs, derivatives, or salts of 25(OH)D include alkylated, glycosylated, arylated, halogenated, or hydroxylated 25(OH)D, orthoesters of 25(OH)D, or pharmaceutical salts of 25(OH)D. These analogs, derivatives, or salts can be synthesized or otherwise manufactured by chemical procedures which are well-known and readily available to those of ordinary skill in the art. The vitamin D analogs can be obtained according to the methods disclosed in U.S. - 19 -
Patent Nos. 5,508,392, 5,457,217, 5,414,098, 5,384,313, 5,373,004, 5,371,249, 5,430,196, 5,260,290, 5,393,749, 5,395,830, 5,250,523, 5,247,104, 5,391,115, 5,194,431, 5,281,731, 5,254,538, 5,232,836, 5,185,150, 5,321,018, 5,086,191, 5,036,061, 5,030,772, 5,246,925, 4,973,584, 5,354,744, 4,927,815, 4,857,518, 4,851,401, 4,851,400, 4,847,012, 4,755,329, 4,940,700, 4,619,920, 4,594,192,
4,588,716, 4,564,474, 4,552,698, 4,588,528, 4,719,204, 4,719,205, 4,689,180, 4,505,906, 4,769,181, 4,502,991, 4,481,198, 4,448,726, 4,448,721, 4,428,946, 4,411,833, 4,410,515, 4,367,177, 4,336,193, 4,360,472, 4,360,471, 4,307,231, 4,307,025, 4,358,406, 4,305,880, 4,279,826, and 4,248,791. The term "functional equivalent" is used to refer to any compound which can be used as a substrate for lα-OHase or otherwise can be converted to 1J5(0H)2D or converted to a compound which can bind to or activate the 1J5(0H)2D receptor (VDR) such that any one of the beneficial properties of 1J5(0H)2D, namely inhibition of invasiveness, proliferation, metastasis, or promotion of cell differentiation, are effected. See, for example, U.S. Patent Nos.
5,167,953; 5,422,099; 5,794,248; and 5,395,829.
It would also be understood that the subject invention can be carried out by use of pharmaceutical compositions which comprise a 1 J5(OH)2D metabolic precursor or analog or derivative thereof and, e.g., 25(OH)D, a pharmaceutically acceptable carrier.
The subj ect method of administering a metabolic precursor of 1 ,25(OH)2D to a patient has been shown to be effective in producing 1 ,25(OH)2D by prostatic cancer cells. In particular, the subject method has been shown to be effective in two well-characterized human prostate cancer cell lines, DU 145 and PC-3, and two primary cultures of cells, NP96-5 and BPH96- 11 , derived from noncancerous human prostates. The NP96-5 culture was derived from the prostate of a 23-year old organ donor; the BPH96-11 culture was derived from a 56-year old with benign prostatic hyperplasia (BPH). Two of these cell lines, DU 145 and PC-3, as well as the primary cultures, can synthesize 1J5(0H)2D from its metabolic precursor 25(OH)D. The vitamin D metabolite 1 J5(OH) 2D was also synthesized 20
from 25(OH)D by cultured normal human keratinocytes. The results are shown in Table 1 below:
Table 1 : Synthesis of 1 ,25(OH)2D in the presence and absence of the P450 cytochrome inhibitor, clotrimazole.
l,25(OH)2D Produced (pmol/mg protein/hr)
CELL TYPE P-450 Inhibitor Absent P-450 Inhibitor Present
DU 145 0.31 ± 0.06 (n = 6) undetectable
PC-3 0.07 ± 0.01 (n = 6) undetectable
LNCaP undetectable undetectable
NP96-5 3.08 ± 1.56 (n = 3) undetectable
BPH96-11 1.05 ± 0.31 (n = 3) undetectable
Figure imgf000022_0001
Human keratinocytes 2J ± 0J (n = 3) undetectable n = number of replications, errors are S.E.M.
As summarized in Table 1 , the DU 145 and PC-3 cell lines produced 0J 1 ± 0.06 and 0.07 ± 0.01 pmol of 1J5(0H)2D mg protein/hr, respectively. These cells exhibited 1 α-OHase activity, as detected by thymus receptor binding assay according to well-known procedures. The production of 1J5(0H)2D was completely inhibited in the presence of clotrimazole, a commonly known inhibitor of the cytochrome P450 system, of which lα-OHase is a known component. Conversely, no measurable 1 ,25(OH)2D was detected in LNCaP cells, which did not exhibit l -OHase activity.
Primary cultures of prostatic cells from two patients were also grown, NP96-5 (normal prostate) and BHP96-11 (BPH), and their enzyme activities determined in the presence of DPPD, and in the presence or absence of clotrimazole. TheNP96-5 andBPH96-l 1 cultures produced3.08 ± 1.56pmol/mg protein/hr and 1.05 ± 0J 1 pmol/mg protein/hr of 1 ,25(OH)2D, respectively, in the presence of DPPD and in the absence of cytochrome P450 inhibitor. The lα- OHase activity found in the two primary cultures of prostatic cells was comparable to that found in the normal human keratinocytes. As in the two cell lines, the - 21 -
production of l,25(OH)2D in the primary cultures of prostatic cells and keratinocytes was completely inhibited in the presence of clotrimazole.
The enzyme activity detected in the primary cultures of prostatic cells was further supported by high performance liquid chromatography analysis using a solvent system that specifically separates l,25(OH)2D from 10-oxo-19-nor-
25(OH)D, another metabolite of 25(OH)D. This metabolite, which is present in significant quantity in kidney homogenates of rats and chickens, is known to co- migrate with 1 ,25(OH)2D on normal phase HPLC with the n-hexane:isopropanol (9:1) solvent system. Therefore, to ensure that 1J5(0H)2D was separated from any 10-oxo- 19-nor-25(OH)D present, the methylene chloride:isopropanol (19:1) normal phase solvent system was used. In addition, l -OHase activity was determined by using a radioactive metabolite, [3H]-25(OH)D, as substrate.
Typical HPLC chromatograms of the two primary prostate cell cultures in the presence and absence of clotrimazole are illustrated in Figs. 2-5. Second passage of normal human prostate cells incubated with nonradioactive 25(OH)D3
(50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and IJ-dianilinoethane (DPPD) (10 μM) at 37° C for two hours in the absence of clotrimazole (20 μM) is shown in Fig. 2. The active metabolite, 1J5(0H)2D, was synthesized in detectable levels (about 600 cpm). Second passage of normal human prostate cells were also incubated with nonradioactive 25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and DPPD (10 μM) at 37° C for two hours in the presence of clotrimazole (20 μM) in the presence of the cytochrome P450 inhibitor (clotrimazole). Fig. 3 shows that 1 J5(OH)2D synthesis was significantly inhibited, whereby the active metabolite was undetectable.
Second passage of prostate cells derived from BPH cultures were incubated with nonradioactive 25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and DPPD (10 μM) at 37 °C for two hours in the absence of clotrimazole (20 μM). Without cytochrome P450 inhibitor, active metabolite was detectable, producing product which generated about 500 cpm. See Fig. 4. By - 22 -
contrast, second passage of prostate cells derived from BPH cultures incubated with nonradioactive 25(OH)D3 (50 nM), [3H]-25(OH)D3 (0.91 μCi/nmol) and DPPD (10 μM) at 37° C for two hours in the presence of clotrimazole (20 μM), showed significantly reduced levels of the 1 J5(OH)2D metabolite, measuring less than about 200 cpm (Fig. 5).
Both primary cultures of non-cancerous prostate cells produced 1J5(0H)2D at levels 10-40 fold higher than the cell lines. Very high levels of 1 J5(OH)2D were observed from the NP96-5 culture derived from the 23 year old organ donor. The quantities of l,25(OH)2D produced by the two primary non- cancerous human prostate cells, 3.08 ± 1.56 and 1.05 ± 0J 1 pmol/mg protein/hr (normal and BPH, respectively), are comparable to those produced in cultured human keratinocytes (2J ± 0.1 pmol/mg protein hr), and to the HEP 62 hepatoma cell line (2.3 pmol/mg protein/hr), and human T-lymphotrophic virus-transformed lymphocytes (1.6 pmol/mg protein hr). These values are at least 10-fold higher than those reported in other extra-renal sites, such as human bone cells (0.068 pmol/mg protein/hr).
After incubation with 25(OH)D3, DU 145 and PC-3 prostate cancer cell lines also produced detectable levels of 1J5(0H)2D3. The 100% inhibition of 1 J5(OH)2D production by the specific cytochrome P450 inhibitor, clotrimazole, shows that a cytochrome P450-dependent 1 α-OHase is present in these prostate cells. The addition of the P-450 inhibitor inhibited substantially all the conversion of [3H]-25(OH)D3 to [3H]-lJ5(OH)2D3 in these cells. Thus, DU 145 and PC-3 human prostate cancer cells, as well as two primary cultures derived from non- cancerous human prostates, possess lα-OHase activity and are capable of converting the major circulating metabolite of vitamin D3, 25(OH)D3, to the hormonally active vitamin D metabolite, 1J5(0H)2D3.
The enzyme activity found in the primary cultures of prostate cells is comparable to that found in the primary cultures of renal proximal tubular cells,
53-5.6 pmol/hr/mg protein. Since it is established that renal lα-OHase is the major enzyme responsible for maintaining the circulating concentration of - 23 -
1J5(0H)2D under normal physiological conditions, the amount of 1J5(0H)2D produced by prostatic cells can be physiologically significant, especially for the micro environment of prostatic cells. Levels of 1 J5(OH)2D as low as 10"1 ' M can significantly inhibit invasiveness of human prostate cancer cells through an artificial basement membrane composed of human amnions (Schwartz, G.G., et al, Cancer Epidemiol. Biomark. Prev. 6:121-132 (1997)).
No detectable 1J5(0H)2D3 was produced by LNCaP cells. 24,25- hydroxylase activity has also been reported to be low or undetectable in LNCaP. See Miller, G. j., Cancer Res. 52:515-520 (1992); Skowronski, R. j., Endocr. Rev. 132: 1952-1960 (1993).
The unique discovery that 1 J5(OH)2D can be synthesized from 25(OH)D by prostatic cells provides the nexus for using vitamin D metabolites which are comparatively less toxic than high doses of vitamin D or certain of other metabolites, including 1J5(0H)2D, in cancer treatment. The method of the subject invention comprising administering to a patient a vitamin D metabolite which can be converted to 1J5(0H)2D by the enzyme lα-OHase is preferably used in chemoprevention of prostate cancer. However, it would be understood by those of ordinary skill in the art that, in view of the principles and procedures described herein, cancer cells in other organs or tissues which are capable of converting vitamin D metabolite to l,25(OH)2D can respond to the antiproliferative, anti-invasive, antimetastatic, or prodifferentiating properties of 1 J5(OH)2D. Accordingly, it would be understood that the subject invention can be applicable to other cancer cells, including colon cancer cells, breast cancer cells, leukemia cells, skin cancer cells, lung cancer cells, and lymphoma cancer cells. The product synthesized from the administered metabolite, 1 J5(OH)2D, exerts anti-proliferative and pro-differentiating effects on normal and cancerous prostate cells. Moreover, physiological levels of 1 J5(OH)2D significantly inhibit the invasiveness of DU 145 cells through an artificial basement membrane, as correlated with a decrease in the secreted levels of type IV collagenase (MMP-2 and MMP-9). It has further been shown that 1 J5(OH)2D exhibits antimetastatic - 24 -
effects on prostate cancer cells in vivo. However, administration of 1 J5(OH)2D has particular disadvantages due to its causative effect in producing hypercalcemia. Thus, 1J5(0H)2D is not considered by those in the art to be optimal for safe use as a chemopreventive agent because of this risk of hypercalcemia. Our present findings indicate that by increasing the available substrate, e.g., administering or supplementing 25(OH)D to a patient, local synthesis of 1J5(0H)2D by prostatic cells can be achieved, making increased levels of the active metabolite 1J5(0H)2D available to the prostatic cells, and thereby reducing proliferation, invasiveness, or metastatic activity of cancerous prostate cells, and promoting their differentiation.
As described herein, 25(OH)D and analogs and derivatives thereof can be useful in several clinical settings, e.g., in slowing or preventing the need for prostatectomy or radiation therapy in men on "watchful waiting," and/or slowing or preventing disease recurrence in men with minimal residual disease. The vitamin D metabolite 25(OH)D is advantageously stored in smaller proportions in body fat as compared to vitamin D or other more fat-soluble metabolites or analogs of vitamin D. Moreover, 25(OH)D bypasses hepatic 25- hydroxylation. Therefore, its onset and offset of action are faster than vitamin D. In addition, 25(OH)D can be effective when there is compromise of the hepatic 25 -hydroxylation of vitamin D2 or D3, as in primary biliary cirrhosis or certain cases of neonatal hypocalcemia. Further, 25(OH)D may have some intrinsic agonist activity that may retain some level of efficacy in the absence of the renal 1 α-OHase system.
The subject method, including the preferred method of administering 25 (OH)D or an analog, derivative, salt, or functional equivalent thereof, would not be limited to treatment of prostatic cells. Those cells having lα-OHase can also convert 25(OH)D to l,25(OH)2D, thereby increasing local cellular levels of the 1J5(0H)2D end product. Those cells which also have VDRs which bind 1J5(0H)2D, or its functional equivalent, can respond to that compound and benefit from its antiproliferative, anti-invasive, antimetastatic, and - 25 -
prodifferentiating effects. For example, colon or breast cells can respond to 1 J5(OH)2D due to their possessing 1 α-OHase and VDRs, either inherently or by use of genetic engineering techniques well-known in the art.
The dosage administration to a host in the above indications will be dependent upon the identity of the disease or condition, the type of patient involved, its age, weight, health, kind of concurrent treatment, if any, frequency of treatment, and therapeutic ratio.
The compounds of the subject invention can be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science by E. W. Martin describes formulations which can be used in connection with the subject invention. In general, the compositions of the subj ect invention will be formulated such that an effective amount of the bioactive compound(s) is combined with a suitable carrier in order to facilitate effective administration of the composition.
In accordance with the invention, pharmaceutical compositions comprising, as active ingredient, an effective amount of one or more of the subject compounds and one or more non-toxic, pharmaceutically acceptable carriers or diluents can be used by persons of ordinary skill in the art. In addition, the pharmaceutical composition can comprise one or more of the subject compounds, e.g., 25(OH)D, as a first active ingredient plus a second active ingredient, e.g., an anti-inflammatory, antimicrobial, antiproliferative, or antiviral compound, known in the art. In accordance with this invention, pharmaceutically effective amounts of a known second active ingredient and the vitamin D metabolite, analog or derivative thereof useful in accordance with the subj ect invention are administered sequentially or concurrently to the patient. The most effective mode of administration and dosage regimen of subject compounds will depend upon the type of disease to be treated, the severity and course of that disease, previous - 26 -
therapy , the patient's health status, and response to vitamin D metabolites, analogs and derivatives, and the judgment of the treating physician. The subject compositions may be administered to the patient at one time or over a series of treatments. Preferably, the subject compound and the second agent are administered sequentially to the patient, with the second agent being administered before, after, or both before and after treatment with the subject compound or composition. Sequential administration involves treatment with the second agent at least on the same day (within 24 hours) of treatment with the subj ect vitamin D metabolite and may involve continued treatment with the second agent on days that the subject compound is not administered. Conventional modes of administration and standard dosage regimens for the subject compound or a pharmaceutical composition comprising the subject compound may be used (see Gilman, A.G., et α/.(eds.), The Pharmacological Basis oj 'Therapeutics, pp. 697-713, 1482, 1489- 91 (1980); Physicians Desk Reference, 1986 Edition), similar to other compounds used for the treatments, or can be titrated for the individual patient.
According to one embodiment of this invention, the patient may receive concurrent treatments with compositions comprising at least one of the subject compounds, and a second active ingredient. For example, local, intra-tissue (or organ) injection of 25(OH)D or analog or derivative thereof is preferred but can be administered by subcutaneous injection, subcutaneous slow-release implant, intravenously, intraperitoneally, topically, intramuscularly, or orally.
The compositions used in these therapies can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions. The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art. Preferably, the compositions of the invention - 27 -
are in the form of a unit dose and will usually be administered to the patient one or more times a day.
Gene Therapy
Further, the subject invention can employ a gene therapy approach utilizing molecular biology techniques or procedures whereby a target cell or tissue is genetically altered to respond to the active vitamin D metabolite or analog and derivative thereof, e.g., l,25(OH)2D, by transgenically providing the target cell with a gene which encodes a protein or plurality of proteins which serve as vitamin D receptors (VDRs) that bind 1 ,25(OH)2D. Alternatively, the target cells can be genetically engineered to encode and produce lα-OHase, which is known to convert 25(OH)D to 1J5(0H)2D. The genes of interest can be identified using standard probe and sequencing techniques, cloned or otherwise amplified, isolated and inserted into appropriate vectors for transfer into the target cells. Further, the target cells can be provided with promoter sequences which activate the appropriate gene sequence in the genome of the target cell to instruct the cell to produce the desired proteins or promoters.
The gene therapy approach of the present invention can be used as a method to treat or prevent cancer. As used herein, such cancers include, but are not limited to, Hodgkin's disease, non-Hodgkin's lymphomas, acute lymphocytic leukemia, multiple myeloma, breast carcinomas, ovarian carcinomas, lung carcinomas, Wilms' tumor, testicular carcinomas, soft-tissue sarcomas, chronic lymphocytic leukemia, primary macroglobulinemia, bladder carcinomas, chronic granulocytic leukemia, primary brain carcinomas, malignant melanoma, small-cell lung carcinomas, stomach carcinomas, colon carcinomas, malignant pancreatic insulinoma, malignant carcinoid carcinomas, malignant melanomas, choriocarcinomas, mycosis fungoides, head and neck carcinomas, osteogenic sarcoma, pancreatic carcinomas, acute granulocytic leukemia, hairy cell leukemia, rhabdomyosarcoma, Kaposi's sarcoma, genitourinary carcinomas, thyroid carcinomas, esophageal carcinomas, malignant hypercalcemia, renal cell - 28 -
carcinomas, endometrial carcinomas, polycythemia vera, essential thrombocytosis, adrenal cortex carcinomas, skin cancer, and prostatic carcinomas.
In this preferred embodiment of the invention, 1 α-OHase DNA (e.g., the nucleic acid sequence depicted in SEQ ID NO. 1) can be incorporated into a polynucleotide construct suitable for introducing the nucleic acid molecule into cells of the animal to be treated, to form a transfection vector. The transfection vector is then introduced into selected target tissues of the cells of the animal in vivo using any of a variety of methods known to those skilled in the art. Alternatively, naked DNA may be transfected into the cells, with or without cationic lipids.
Techniques for the construction of transfection vectors containing lα- OHase DNA are well-known in the art, and are generally described in "Working Toward Human Gene Therapy ' Chapter 28 in Recombinant DNA, 2nd Ed, Watson, J.D. etal. (eds.), Scientific American Books: New York (1992), pp.567- 581 , or Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor, New York (1989).
Gene therapy approaches that may be used to deliver a VDR and/or 1 α- OHase gene include injection of plasmid DNA (Horton, H.M., et al. , Proc. Natl. Acad. Sci. USA 96(¥ :1553-1558 (1999)); transduction using adenoviral vectors (Waugh, J.M., et al, Proc. Natl. Acad. Sci. USA 9tf 5J: 1065 -1070 (1999)); transduction using retrovial vectors (Axelrod, J.H., et a!, Proc. Natl. Acad. Sci. USA 57:5173-5177 (1990); Drumm, M.L., et al, Cell 62:1221-1233 (1990); Krueger, G.G., et al, J. Invest. Dermatol. 112:233-239 (1999); Palmer, T.D., et al, Blood 75:438-445 (1989); and Rosenberg, S.A., et al, N Eng. J. Med. 325:570-578 (1990)); and gene transfer using liposomes (Mason, C.A.E., et al,
Nature Medicine 5(2): 176- 182 (1999)). In addition, general methods for construction of gene therapy vectors and the introduction of such vectors into a mammal for therapeutic purposes may be obtained in the above-referenced publications, the disclosures of which are specifically incorporated herein by reference in their entirety. In one such general method, vectors containing 1 α- - 29 -
OHase DNA of the present invention are directly introduced into the cells or tissues of the mammal to be treated, preferably by injection, inhalation, ingestion, topical application, or introduction into a mucous membrane via solution. Such an approach is generally referred to as "in vivo" gene therapy. Alternatively, cells or tissues may be removed from the mammal to be treated and placed into culture according to methods that are well-known to one of ordinary skill in the art. Transfection vectors or naked DNA containing the 1 α- OHase DNA may then be introduced into these cells or tissues by any of the methods described generally above for introducing isolated polynucleotides into a cell or tissue. After a sufficient amount of time to allow incorporation of the 1 α-
OHase DNA, the cells or tissues may then be re-inserted into the mammal to be treated. Since introduction of the lα-OHase gene is performed outside of the body of the mammal, this approach is generally referred to as "ex vivo" gene therapy. See U.S. Patent No. 5,399,346. Gene transfer through transfection of cells ex vivo can be performed by a variety of methods, including, for example, calcium phosphate precipitation, diethylaminoethyl dextran, electroporation, lipofection, or viral infection. Such methods are well known in the art (see, for example, Sambrook et al).
The term "gene," as used herein with reference to lα-OHase, is intended to refer to a DNA sequence that encodes the lα-OHase enzyme. Thus, " lα-
OHase gene" or "gene expressing lα-OHase" may refer to either a segment of genomic DNA encoding the 1 α-OHase enzyme or a cDNA sequence encoding the 1 α-OHase enzyme.
For both in vivo and ex vivo gene therapy, the lα-OHase DNA of the invention may be operatively linked to the regulatory DNA sequence, or
"promoter," for human lα-OHase (depicted in SEQ ID NO. 3) to form a genetic construct as described above. This construct, containing both the human 1 α- OHase promoter and the 1 α-OHase DNA, may be subcloned into a suitable vector such as a plasmid, adeno virus vector, retro virus vector, or the like, and introduced - 30 -
into the animal to be treated in an in vivo gene therapy approach, or into the cells or tissues of the mammal in an ex vivo approach.
Alternatively, the 1 α-OHase DNA of the invention may be operatively linked to a heterologous regulatory DNA sequence, or promoter, to form a genetic construct as described above. The heterologous regulatory sequence may be tissue specific. The vector containing the genetic construct is then directly introduced into the animal to be treated or into the cells or tissues of the animal, as described.
The term "operably linked", as used herein, denotes a relationship between a regulatory region (typically a promoter element, but may include an enhancer element) and the gene, whereby the transcription of the gene is under the control of the regulatory region.
The term "heterologous" means a DNA sequence not found in the native genome. That is, two nucleic acid elements are said to be "heterologous" if the elements are derived from two different genes, or alternatively, two different species. Thus, "heterologous DNA regulatory sequence" indicates that the regulatory sequence is not naturally ligated to the DNA sequence for the 1 α-
OHase gene.
The term "promoter" is used according to its art-recognized meaning. It is intended to mean the DNA region, usually upstream to the coding sequence of a gene, which binds RNA polymerase and directs the enzyme to the correct transcriptional start site.
In general, a promoter may be functional in a variety of tissue types and in several different species of organisms, or its function may be restricted to a particular species and/or a particular tissue. Further, a promoter may be constitutively active, or it may be selectively activated by certain substances (e.g., a tissue-specific factor), under certain conditions (e.g., in the presence of an enhancer element, if present, in the genetic construct containing the promoter), or during certain developmental stages of the organism (e.g., active in fetus, silent in adult). - 31
Promoters useful in the practice of the present invention are preferably "tissue-specific"~that is, they are capable of driving transcription of a gene in one tissue while remaining largely "silent" in other tissue types. Examples of tissue- specific promoters are provided in Table 2 below.
TABLE 2 Tissue Specific Promoters
Gene Species Tissue Specificity Ref. alpha - Actin rat mu, he 1 alpha - Actin rat te, th, lu 1
Elastase - 1 rat Pa 2 alpha - Fetoprotein mouse ys, li 3 beta - Globin human ery 4 beta - Globin rabbit te, mu 5 beta - Globin rabbit ery 6 tau - Globin human ery 7 alpha - Globin mouse br 8
Growth hormone human pit 9
Immunoglobin - kappa mouse B 10
Immunoglobin - mu mouse B, T 11
Insulin human beta - cells 12
Myosin Light Chain-2 rat mu 13
Protamine 1 mouse te 14 alpha - A-crystallin trans lens 15
Prolactin * pit 16
Pro-opiomelanocortin * pit 17
BTSH * 18
MMTV
Figure imgf000033_0001
mouse breast 19 - 32 -
Gene Species Tissue Specificity Ref.
Albumin * li 20
Keratin * skin 21
Osteonectin * bone 22
Prostate * prostate 23
Olfactory Marker Protein * neuron 24
Neuron Specific Enolase (NSE) * neuron 25
L-7 * neuron 26
Opsin * retina 27
Glial Fibrillary Acidic Protein human astrocytes (CNS) 28 (GFAP)
Tyrosine hydroxylase (TH) rat catecholaminergic 29
* neurons human * 30
Amyloid precursor protein (APP) human neurons 31
* * 32
Dopamine beta - hydroxylase human noradrenergic 33 (DBH) neurons
* adrenergic neurons 34
Myelin basic protein (MBP) mouse oligodendrocyte 35
Light neurofilament (NF-L) rat neurons 36
Tryptophan hydroxylase (TH) human serotonin pineal 37 gland mouse * 38
Purkinje cell protein-2 (Pcp-2) mouse Purkinje 39 cells/cerebellum
L7 mouse cerebellar Purkinje 40
Figure imgf000034_0001
retinal bipolar cells - 33 -
Gene Species Tissue Specificity Ref.
Type II sodium channel rat neuron 41
Choline acetyltransferase (ChAT) human cholinergic neurons 42 rat * 43
Neuron specific enolase (NSE) rat neurons 44
Aromatic L-amino acid human catecholaminergic/ 45 decarboxylase (AADC) 5-HT/D-type cells
Protamine 1 (mPl) mouse spermatids 46
Proenkephalin human neuronal/spermato- 47 genic epididymal cells rat * 48 reg (pancreatic stone protein) human colon and rectal 49 tumors, pancreas, kidney
Parathyroid hormone-related human Liver and cecum 50 peptide (PTHrP) tumors, neurilemoma, kidney, pancreas, adrenal
Stromelysin 3 human breast cancer 51
NSE (see nervous system) * small-cell lung 52 cancer, neurons
AADC (see nervous system) * neurectodermal 53 tumors
Albumin * hepatoma 54 c-erbB3 *
Figure imgf000035_0001
breast cancer 34 -
Gene Species Tissue Specificity Ref. c-erbB4 * breast and gastric cancer
Thyroglobulin * thyroid carcinoma alpha - fetoprotein * hepatoma 55 hemoglobin *
Figure imgf000036_0001
erythrocytes 56
Abbreviations: br, brain; B, lymphocytes; mu, skeletal muscle; he, cardiac muscle; te, testis; beta, beta cells; th, thymus; lu, lung; Pa, exocrine pancreas; ys, yolk sac; li, liver; ery, erythroid cells; pit, pituitary and lens, eye lens.
References:
(1) Shani, Mol. Cell. Biol, 6:2624 (1986); (2) Swift et al, Cell 38:639 (1984); (3) Krumlauf et al. , Nature 579:224 (1985); (4) Townes et al. , EMBO J. 4:\ 115 (1985); (5) Lacy et al, Cell 34:343 (1983); (6) Wagner et al, Proc. Natl. Acad. Sci. U.S.A., 75:6376 (1981); (7) Brinster et al, Nature 283:499 (1980); (8) Rusconi et al, in "The Impact of Gene Transfer Techniques in Eukaryotic Cell Biology/' ed. J. S. Schell et al, pp. 134-152, Berlin: Springer Verlag (1984); (9) Behringer et al, Genes Dev. 2:453 (1988); (10) Storb et al, Nature 570:238 (1984); (11) Grosschedl et al, Cell 55:647 (1984); (12) Selden et al, Nature 321:545 (1986); (13) Shani, Nature 574:283 (1985); (14) Peschon et α/. , Ann. N. York Acad, Sci., 564:186 (1989); (15) Breitmanetα/.,7)ev. 106:451 (1989); (16) Crenshaw et al, Genes and Development 3:959 (1989); (17) Tremblay et al, Proc. Natl. Acad. Sci. U.S.A. 55:8890 (1988); (18) Tatsumi etal, Nippon Rinsho 47:2213 (1989); (19) Muller etα/., Cell 54:105 (1988); (20) Palmiter etα/., w?. Rev. Genet. 20:465 (1986); (21) Vassar et al, Proc. Natl. Acad. Sci. U.S.A. 55:8565 (1989); (22) McVey et al, J. Biol. Chem. 263:11 (1988); (23) Allison et al. , Mol. Cell. Biol. 9:2254 (1989); (24) Danciger et al. , Proc. Natl. Acad. Sci. U.S.A. 55:8565 (1989); (25) Forss-Petter et al. , J. Neurosci. Res. 75:141 (1986); (26) Sutcliffe, Trends in Genetics 3:13 (1987); (27) Nathans et al, Proc. Natl. Acad. Sci. U.S.A. 57:4851 (1984); (28) Brenner, M., et al., J. Neurosci. 74:1030 - 35 -
(1994); (29) Kim, . S., etal.,J. Biol. Chem 255:15689 (1993); (30) Kaneda,N., etal, Neuron 5:583 (1991); (31) Salbaum, J. M., etal, EMBOJ.7:2807 (1988); (32) Wirak, D. O., et al, EMBO J. 70:289 (1990); (33) Mercer E. H., et al, Neuron 7:703 (1991); (34) Hcyle, G. W., etal.,J. Neurosci. 14:2455 (1994); (35) Miura, M., et al, Gene 75:31 (1989); (36) Reeben, M., et al, J. Neurosci. Res.
40:111 (1995); (37) Boularand, S., et al., J. Biol. Chem 270:3151 (1995); (38) Stoll, J. and Goldman, D., J. Neurosci. Res. 28:451 (1991); (39) Vandaele, S., et al, Genes & Dev. 5:1136 (1991); (40) Oberdick, J., et al, Science 248:223 (1990); (41) Maue, R. A., et al, Neuron 4:223 (1990); (42) Hersh, L. B., et al, J. Neurochem. 57:306 (1993); (43) Ibanex, C. F. and Persson, H., Eur. J.
Neurosci. 5:1309 (1991); (44) Forss-Petter, S., et al, Neuron 5:187 (1990); (45) Thai, A. L. V., et al, Mol. Brain Res. 17:221 (1993); (46) Peschon, J. J., et al, Proc. Natl. Acad. Sci. U.S.A. 54:5316 (1987); (47) Borsook, D., et al, Mol. Endocrinol. 5:1502 (1992); (48) Joshi, J. and Sabol, S. L., Mol. Endocήnol. 5:1069 (1991); (49) Watanabe, T., et al, J. Biol. Chem. 265:1432 (1990); (50)
Campos, R. V., et al, Mol. Rnfovtinol. 5:1642 (1992); (51) Basset, P., et al, Nature 348: 699 (1990); (52) Bombardieri, E. et al, Eur. J. Cancer 31A:\ (1995); Koh, T. et al, Int. J. Cancer 50:843 (1995); (53) Thai, A. L. V., et al, Mol. Brain Res. 17:221 (1993); (54) Huber, B. E., RN4S 55:8099 (1991); (55) Zuibel, I., etal.,J. Cell Physio! 162:36 (1995); (56) Watanabe, T., eta!,J. Bio!
Chem. 265:1432 (1990).
For additional examples of tissue-specific promoters, see U.S. Patent Nos. 5,834,306 and 5,416,027, and references cited therein.
In addition to a promoter, the genetic construct may also contain other genetic control elements, such as enhancers, repressible sequences, and silencers, which may be used to regulate replication of the vector in the target cell. The only requirement is that the genetic element be activated, derepressed, enhanced, or otherwise genetically regulated by factors in the host cell and, with respect to methods of treatment, not in the non-target cell. - 36 -
An "element," when used in the context of nucleic acid constructs, refers to a region of the construct or a nucleic acid fragment having a defined function.
For example, a enhancer element, as used herein, is a region of DNA that, when associated with the 1 α-OHase gene operably linked to a promoter, enhances the transcription of that gene.
The term "enhancer" is used according to its art-recognized meaning. It is intended to mean a sequence found in eukaryotes which can increase transcription from a gene when located (in either orientation) up to several kilobases from the gene being studied. These sequences usually act as enhancers when on the 5 ' side (upstream) of the gene in question. However, some enhancers are active when placed on the 3' side (downstream) of the gene. In some cases, enhancer elements can activate transcription from a gene with no (known) promoter.
Preferred enhancers include the DF3 breast cancer-specific enhancer and enhancers from viruses and the steroid receptor family. Other preferred transcriptional regulatory sequences include NF1, SP1, API, and FOS/JUN.
Any of a variety of methods known to those skilled in the art may be used to introduce transfection vectors of the present invention into selected target tissue cells. Such methods include, for example, viral-mediated gene transfer using retroviruses, adeno-associated virus (AAV), herpes virus, vaccinia virus, or RNA viruses (e.g., Grunhaus and Horowitz, Semin. Virol. 5:237-252 (1992); Herz and Gerard, Proc. Nat. Acad. Sci. USA 90:2812-2816 (1993); and Rosenfeld et al., Cell 55:143-155 (1992)); liposome-mediated gene transfer (Morishita et a!, J. Clin. Invest. 97:2580 (1993); Feigner et al, U.S. Patent Nos. 5,703,055 (1997) and 5,858,784 (1999)); injection of naked DNA directly into a target tissue (e.g.,
Feigner et al., U.S. Patent No. 5,589,466 (1996); Wolff et al., U.S. Patent No. 5,693,622 (1997)); and receptor-mediated gene transfer (Wu and Wu, Biochemistry 27:887-892 (1988); Wagner et al., PNAS USA 57:3410-3414 (1990); Curiel et al., U.S. Patent 5,547,932 (1996); and Beug et al., U.S. Patent No. 5,354,844 (1994)). - 37 -
In any of these methods, where a vector may be targeted to selectively transfect a specific population of cells, it will be understood that in addition to local administration (such as may be achieved by injection into the target tissue), the vector may be administered systemically (e.g., intravenously) in a biologically-compatible solution or pharmaceutically acceptable delivery vehicle .
Vector constructs administered in this way may selectively infect the target tissue. According to the present invention, the presence of a target tissue-specific promoter on the construct provides an independent means of restricting expression of the therapeutic gene. The gene therapy embodiment of the invention provides a method of treating or preventing a number of diseases and disorders. For example, this embodiment can be used to treat or prevent cancer in cells that express the vitamin D receptor (VDR). A polynucleotide construct containing the gene coding for lα-OHase is introduced into cancerous cells, where the gene is subsequently expressed, producing 1 α-OHase in an amount effective to inhibit cell proliferation.
A gene encoding the VDR (for example, the nucleotide sequence depicted in SEQ ID NO. 4) can also be co-administered with the lα-OHase gene. In this embodiment of the invention, the VDR gene is subcloned into either the same polynucleotide construct as the lα-OHase gene or into a separate polynucleotide construct, as described above. Both genes are then introduced into the target cell or tissue, where they are subsequently expressed, producing 1 α-OHase and VDR in amounts effective to treat or prevent the condition.
Gene therapy with the gene expressing 1 α-OHase and, optionally, the gene expressing the VDR gene may be practiced substantially simultaneously with administration of vitamin D or analog or derivative thereof. Thus, vitamin D or analog or derivative thereof may be administered to the animal before, during or after receiving gene therapy.
Specifically, the invention can be used to treat or prevent prostate cancer, breast cancer, skin cancer, colon cancer, leukemias, lymphomas, and lung cancer. - 38 -
The invention also provides a method of treating or preventing any disease in which l,25(OH)2D3 can influence cell growth and maturation, including pre- cancers such as actinic keratoses, and non-cancerous hyperproliferative disorders such as psoriasis and ichthyosis. In this embodiment of the invention, a polynucleotide construct containing the gene coding for 1 α-OHase is introduced into cells in which l,25(OH)2D3 can influence cell growth and maturation. The lα-OHase gene is subsequently expressed, producing lα-OHase in an amount effective to inhibit cell proliferation.
The invention can therefore be used in the treatment of actinic keratoses, which is a pre-skin cancer. Use of the invention would effect an increase of the amount of 1J5(0H)2D3 (or analog or derivative thereof) within the cell, decreasing proliferative activity and inducing the cells to differentiate into normal morphology.
Further, the invention can also be used to treat hyperproliferative disorders of the skin such as psoriasis. This gene therapy approach is a reasonable alternative treatment for psoriasis, whereby the cells would produce or boost their production of 1 J5(OH)2D3 (or analog or derivative thereof) which, in turn, would interact with the VDR and decrease the proliferative activity making the skin cells normal. This treatment can also be applied to other hyperproliferative skin disorders such as ichthyosis.
The invention can also be used to treat or prevent disorders in calcium and bone metabolism. Again, a polynucleotide construct containing the gene coding for lα-OHase can be introduced into a group of cells of the animal, where it is subsequently expressed, enabling these cell to produce l,25(OH)2D3 (or analog or derivative thereof) in a manner similar to the kidney. Thus, the invention may be used to prevent bone disease associated with kidney failure and treat vitamin D-dependent rickets type I, X-linked hypophosphatemic rickets, vitamin D- dependent rickets type II, and osteoporosis, as well as such disorders in calcium and bone metabolism as low blood serum calcium due to hypoparathyroidism. - 39 -
A further embodiment of the invention provides a method of treating or preventing any disease or condition mentioned above, including cancer, by administering the gene encoding the VDR as well as the gene coding for lα-OHase to a target cell or tissue of an animal. Both genes may be subcloned into either the same or different polynucleotide constructs, as described above, which is introduced into the target cell or tissue. Both the 1 α-OHase gene and the VDR gene are subsequently expressed, producing 1 α-OHase and VDR in amounts effective to treat or prevent the condition.
This particular embodiment may be used with cells that either express or do not already express the VDR. In the former case (in which the target cell already expresses the VDR), the cell will produce additional VDR which will interact with the increased cellular concentrations of l,25(OH)2D (or analog or derivative thereof). In the latter case (in which the cells do not already express VDR), the cell will become responsive to l,25(OH)2D (or analog or derivative thereof). For example, a cancer cell that had either a defective or absent VDR could be treated according to this embodiment of the invention. The treated cell would then produce VDR which would, in turn, interact with the 1 J5(OH)2D (or analog or derivative thereof) that was produced from the lα-OHase. The interaction leads to a biologic response, including inhibiting proliferation and inducing terminal differentiation, thereby treating cancer, pre-cancer, non- cancerous hyperproliferative disorders, enhancing wound healing, and treating and preventing hair loss.
The invention also provides a method of treating benign prostatic hypeφlasia in an animal, by introducing into cells exhibiting benign prostatic hypeφlasia a polynucleotide construct containing a gene coding for lα-OHase.
The lα-OHase gene is subsequently expressed, enabling the cells to produce 1J5(0H)2D (or an analog thereof) in an amount effective to treat the benign prostatic hypeφlasia. In this embodiment, a gene encoding the VDR can also be co-administered with the lα-OHase gene, as described above. The gene - 40 -
expressing 1 α-OHase and the gene expressing the VDR can be on the same DNA construct or on separate DNA constructs.
The invention also provides a method of treating autoimmune diseases in an animal, by introducing into any cells of the animal that would produce a beneficial result a polynucleotide construct containing a gene coding for lα-OHase. The l -OHase gene is subsequently expressed, enabling the cells to produce 1J5(0H)2D (or an analog thereof) in an amount effective to treat the autoimmune disease. In this embodiment, a gene encoding the VDR can also be co-administered with the lα-OHase gene, as described above. The gene expressing 1 α-OHase and the gene expressing the VDR can be on the same DNA construct or on separate DNA constructs.
The invention can be used to treat autoimmune diseases such as diabetes mellitus type 1 , multiple sclerosis, rheumatoid arthritis, or psoriatic arthritis. In this embodiment, for example, diabetes mellitus type 1 can be treated by introduction of a polynucleotide construct(s) containing the 1 α-OHase gene (and optionally the VDR gene) into any cell of the animal that would produce a beneficial result, particularly into pancreatic cells, and more particularly pancreatic cells secreting insulin, such as cells of the Islets of Langerhans. For use of the invention in the treatment of multiple sclerosis, the polynucleotide construct(s) containing the 1 α-OHase gene (and optionally the VDR gene) can be introduced particularly into any neural cells that would produce a beneficial result. Similarly, for use of the invention in the treatment of rheumatoid arthritis or psoriatic arthritis, the polynucleotide construct(s) can be introduced particularly into any connective tissue cells that would produce a beneficial result, or in or around the joints of the animal affected by either of these diseases.
Further embodiments of the invention include isolated nucleic acid molecules that are at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical to the above-described isolated nucleic acid molecules of the present invention. In particular, the invention is directed to isolated nucleic acid molecules at least 90%, 95%, 97%, 98%, or 99% identical to the nucleotide - 41 -
sequences depicted in SEQ ID NO. 1 and SEQ ID NO. 3. The "% identity" between two nucleic acid sequences can be determined using the "fastA" computer algorithm (Pearson, W.R. & Lipman, D J., Proc. Nat! Acad. Sci. USA 55:2444 (1988)) with the default parameters. The term "isolated polynucleotide molecule" is intended to refer to a nucleic acid molecule which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for puφoses of the invention as are recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated polynucleotide molecules also include such compounds produced synthetically.
The invention is further related to nucleic acid molecules capable of hybridizing to a nucleic acid molecule having a sequence complementary to or hybridizing directly to one of the nucleic acid sequences shown in SEQ ID NO. 1 and SEQ ID NO. 3 under stringent conditions. By "stringent conditions" is intended overnight incubation at 42 °C in a solution comprising: 50% formamide, 5 x SSC (750 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA (ssDNA), followed by washing the filters in 0.1 x SSC at about 65 °C.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. - 42 -
Example 1
Materials and Methods Used in Cell Culture Synthesis of 1,2 '5 (OH) _D from
25(OH)D by Cell Cultures
(A) Cell Lines. DU 145, PC-3, and LNCaP cell lines were obtained from the American Type Culture Collection, Rockville, Maryland. All cell lines were tested and found to be free of Mycoplasma contamination.
(B) Culture Conditions. Prostate cancer cell lines were cultured following several passages in vitro. Cells were routinely cultured in complete medium (RPMI 1640 medium supplemented with fetal bovine serum (10%, HyClone Labs, Logan UT) and gentamicin (10 μg/ml, Life Technologies, Inc.,
Gaithersburg, MD)). Cells were seeded in 35 mm culture dishes (Corning-Costar, Cambridge, MA) at 1 X 105 cells/dish in complete medium. Medium was changed to a serum-free medium (RPMI 1640 containing insulin (10 μg/ml), transferrin ( 10 μg/ml) and selenous acid (1 ng/ml)) 24 hours before adding 25(OH)D3. Primary cultures of human prostatic epithelial cells were established and characterized as described by Lokeshwar et a!, Cancer Res. 55:4493-4498 (1993). Prostatic epithelial cells cultured in a serum-free defined medium (Mammary Epithelial Growth Medium (MEGM), Clontech, San Diego, CA) express luminal epithelium specific cytokeratins (cytokeratins 8 and 18) as detected immunohistochemically using an anti-cytokeratin antibody, CAM 5.2
(Becton-Dickinson, Mountain View, CA). The serum-free medium contains MCDF170 supplemented with Epidermal Growth Factor, 25 ng/ml, hydrocortisone, 0.5 μg/ml, ethanolamine, 1 X 10"4, insulin, 5 μg/ml, transferrin, 5 μg/ml, and whole bovine pituitary extract, 70 μg/ml. Prostatic cells used were at their first passage in vitro and were cultured in the serum-free RPMI medium during incubation with vitamin D metabolites. Two primary cultures were investigated: NP96-5, cultured from the histologically normal prostate of a 23 year old Caucasian organ donor, and BPH96-11 , cultured from an open prostatectomy specimen of a 56 year old Caucasian with BPH. - 43 -
Histological examination of adj acent tissue sections taken from specimens used for cultures confirmed their identity as normal or BPH cultures.
(C) Keratinocyte Culture. Because 1 α-OHase activity has been well- established in keratinocytes, cultured human keratinocytes were used for comparison to the prostatic cultures. Keratinocytes were grown in culture following a modification of the method of Rheinwald and Green, Cell 6:331 -344 (1975). Briefly, keratinocytes were obtained from neonatal foreskin after trypsinization at 4 ° C. Keratinocytes were plated and grown on lethally irradiated 3T3 fibroblast feeder cells in a serum-free basal medium containing 0J5mM calcium and supplemented with gro th factors including bovine pituitary extract
(3 μg/ml), EGF (25 ng/ml), insulin (5 μg/ml) and prostaglandin E, (50 ng/ml). To enhance the plating efficiency, cholera toxin (0.1 μg/ml) and hydrocortisone (200 ng/ml) were added into the medium during the initial plating of the primary culture and the subsequent subcultures. Cells were fed and maintained without cholera toxin and hydrocortisone, and used for enzyme assay.
(D) 25-hydroxyvitamin D-lα-hydroxylase (lα-OHase) assay. lα- OHase activity was determined in monolayer cultures of the cell lines and primary cultures described herein. The assays were performed in the presence of 25(OH)D3 (50 nM) as the enzyme substrate and DPPD (Sigma-Aldrich, Allentown, PA), an antioxidant and a known inhibitor of free-radical generated
1J5(0H)2D. Assays were also performed in the presence and absence of the cytochrome P450 inhibitor, clotrimazole (20 μM) (Sigma, St. Louis, MO). After two hours of incubation at 37°C, cultures were placed on ice and media were removed. Immediately afterward, 1 ml methanol was added to extract 25(OH)D3 and 1J5(0H)2D3. A 10 μl aliquot of [3H]-l,25(OH)2D3 containing 1,000 cpm radioactivity in ethanol was also added to each well for calculating the recovery. After extraction at room temperature for fifteen minutes, the methanol extract was transferred to a glass test tube and the cells were washed with an additional 0.5 ml methanol. The extract and wash were combined, dried down with a stream of nitrogen, and redissolved in 1 ml acetonitrile followed by the addition of 1 ml - 44 -
0.4M K2HPO4, pH 10.0. The mixture was then applied to a C-18-OH reversed phase cartridge. The fraction containing 1J5(0H)2D was then dried down under a stream of nitrogen and reconstituted in 200 μl ethanol. Two 40 μl aliquots were taken for 1 J5(OH)2D analysis by thymus receptor binding assay as described by Chen, et al, J. Nutr. Biochem. 7:320-327 (1990). The lα-OHase activity was also determined in the primary cultures of prostatic cells by using 20 μg nonradioactive 25(OH)D3 and 0.91 μCi of [3H]-25(OH)D3 instead of only nonradioactive 25(OH)D3 as a substrate. The incubation medium, time, temperature, extraction procedure, and C-l 8-OH cartridge chromatography were the same as described for the thymus receptor method, except that the fraction eluted from C- 18-OH cartridge with 10% methylene chlorine in hexane (25(OH)D fraction) and with 6% isopropanol in n-hexane (l,25(OH)2D and 24J5(OH)2D fraction) was dried down under nitrogen and redissolved in methylene chloride: isopropanol (19:1) for high performance liquid chromatographic analysis as described below.
(E) High Performance Liquid Chromatography (HPLC). A 30 μl aliquot was mixed with 10 μl each of standard nonradioactive 25(OH)D and l,25(OH)2D and was applied to an Econosphere silica column (5 μ particle size, 250 mm x 4.6 mm) with a flow rate of 0.5 ml/min using methylene chloride: isopropanol (19:1) solvent system as the mobile phase. Thirty fractions were collected at one minute intervals from each HPLC. Fractions were allowed to evaporate by air to dryness, followed by adding scintillation fluid and counting with a beta counter. The retention volume for 25(OH)D3, 24,25(OH)2D3 and l,25(OH)2D3 was calibrated by applying standard 25(OH)D3, 24,25(OH)2D3 and 1J5(0H)2D3 to the HPLC column prior, during, and after unknown sample application. The protein concentration in each 35 mm dish was determined by standard procedures. The enzyme activity was expressed as pmol l,25(OH)2D3/ mg protein/hr.
(F) Keratinocyte transfection. Keratinocytes were maintained in MCDB-153 medium. Cells in 24-well dishes at 50%-60% confluence were - 45 -
transfected with 1 mg/ml of lα-OHase cDNA which had been subcloned into pCR3J vector (INVITROGEN). Empty vector was used as a control. For each transfection, 1.0 μg was diluted in 100 μl of medium containing 6-8 μl Plus Reagent. This mixture was incubated for 15 minutes at room temperature. LIPOFECTAMINE (4-6 μl) was added, and the DNA-LIPOFECTAMINE mixture was incubated at room temperature for an additional 15 minutes. Cells were incubated in the DNA-LIPOFECTAMINE mixture for 3 hours, after which the medium was changed. Cells were then treated with 25(OH)D3 at different concentrations ( 1 nM, 1 OnM, 1 OOnM) for 18 hours. [3H]thymidine was added to the cultures and [3H]thymidine incoφoration into the DNA (interpreted as a measure of the DNA synthetic activity, i.e. proliferation of the cells) was measured as previously described (Chen, T.C., et a!, JNutr. Biochem. 4:49-51 (1993)).
Example 2
Cloning, Sequencing, and Expression of Human Keratinocyte l -OHase cDNA
lα-OHase cDNA was cloned from human keratinocytes using reverse transcription-PCR (RT-PCR). The 2,150 base pair lα-OHase cDNA (which is a part of SEQ ID NO. 1) contains a 1,527 bp open reading frame, predicted to encode protein of 508 amino acids (SEQ ID NO. 2). This cDNA was directly subcloned into the pCR3J-Uni (INVITROGEN) mammalian cell expression vector. DNA sequence analysis showed the cDNA in keratinocytes was 100%) identical to the renal lα-OHase. When this construct was transfected in COS-1 cells, which cannot synthesize 1 J5(OH)2D3, lα-OHase activity was demonstrated by a high performance liquid chromatography (HPLC) which separated [3H]- 1 ,25(OH)2D3 produced from the enzyme substrate, [3H]-25(OH)D3. - 46 -
Example 3 Cloning and Partial Characterization of the Human lcc-OHase Gene
A human genomic DNA library (CLONTECH) was screened by plaque hybridization using a 700bp fragment from the 5' region of lα-OHase cDNA (SEQ ID NO. 1 ) as a probe. Four positive clones were identified. Restriction enzymes were used to digest the four genomic clones which were designated PI, P2, P3, P4 respectively. (BamH I, Sac-I) PI was 13kb, and P2 was 7.5kb. These two clones included some portion of the 5' region as identified by PCR. The PI clone was digested with Sac-I and generated four fragments of 5.5kb, 3.5kb, 3.0kb and OJkb in length. These four fragments were first subcloned into the pGem7 cloning vector, and southern blot analysis was applied to identify the subclone containing the 5'-flanking region. Sequence analysis using T7 and Sp6 promoter primers mapped the fragment order. The 5.5kb fragment was found to encode intron 8 to exon 9. The OJkb fragment encoded exon 6 to intron 8, the 3.5kb encoded exon 6 to the 5' flanking region, and the 3.0kb subclone could not be hybridized with any probes made from lα-OHase cDNA.
Example 4
Proliferative Activity as a Function ofl,25(OH)_D of Keratinocytes Transf ected with the Humanl -OHase Gene
To test the ability of a cell to produce 1,25 (OH)2D3 after transfection with lα-OHase, a polynucleotide construct containing the lα-OHase cloned gene (SEQ ID NO. 1) was constructed as outlined in the preceding Methods section and used to transfect normal, human cultured keratinocytes. The cultured keratinocytes transfected with lα-OHase gene were incubated with varying concentrations of 25(OH)D3. As a negative control, cultured keratinocytes that had not been transfected were exposed to the same concentrations of 25(OH)D under identical conditions at the same time. If the cells transfected with the - 47 -
1 α-OHase gene increased the lα-OHase activity in the cells, the cells would be more efficient in converting 25 (OH)D to 1 J 5 (OH)2D which, in turn, would result in higher concentrations within the cell, and therefore, have a more dramatic effect on decreasing the cells' proliferative activity. As can be seen in Fig. 6, the keratinocytes transfected with the lα-OHase gene had a more marked decrease in proliferative activity to the same concentration of 25(OH)D compared to the non-transfected control keratinocytes. This occurred at concentrations from 10"9 to 10"7M. There was a significant (p<0.01) 53.7% reduction in the proliferative activity of the cells transfected with the lα-OHase gene and incubated with 25(OH)D at 10"7 M compared to untransfected keratinocytes.
It should be understood that the examples and embodiments described herein are for illustrative puφoses only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. All publications, patents and patent applications cited herein are incoφorated by reference herein in the entirety.

Claims

- 48 -Whatls Claimed Is:
1. A method for inhibiting a tumor cell, said method comprising increasing levels of a metabolic precursor of 1 ,25 -dihydroxy vitamin D available to a target cell while reducing the risks of UV radiation exposure or vitamin D toxicity, wherein said target cell has a hydroxylase enzyme for synthesizing 1 ,25- dihydroxyvitamin D from said metabolic precursor.
2. The method of claim 1, wherein said hydroxylase enzyme is 25- dihydroxy vitamin D-l ╬▒-hydroxylase.
3. The method of claim 1, wherein said target cell is transgenically altered to produce and respond to an active vitamin D metabolite.
4. The method of claim 1 , wherein said target cell is a prostatic cell.
5. The method of claim 1 , wherein said method comprises the step of: administering to a patient having cancerous cells in a target organ, an effective amount of a metabolic precursor of 1,25-dihydroxyvitamin D wherein the precursor is metabolized by a cell of said target organ to 1 ,25 -dihydroxy vitamin
D, or a functional equivalent thereof.
6. The method of claim 5, wherein said metabolic precursor is 25- hydroxy vitamin D or an analog, derivative, salt, or functional equivalent thereof.
7. The method of claim 6, wherein an effective amount of said precursor is an amount which results in intra-organ levels of said metabolic precursor between about 25 and 250 nmol/L. - 49 -
8. The method of claim 6, wherein said analog or derivative of 25- hydroxyvitamin D is alkylated, arylated, halogenated, glycosylated, or 25- hydroxy vitamin D further hydroxylated at any carbon position except carbon- 1.
9. The method of claim 1, wherein said 1,25-dihydroxyvitamin D metabolic precursor is administered as a composition comprising said metabolic precursor or a salt thereof and a pharmaceutically acceptable carrier.
10. The method of claim 1, wherein said method promotes differentiation of the target cells.
11. A method for preventing tumor cell proliferation, said method comprising increasing levels of a metabolic precursor of 1 ,25 -dihydroxy vitamin
D or an analog or a derivative thereof available to a target cell while reducing the risks of UV radiation exposure or vitamin D toxicity, wherein said target cell has a hydroxylase enzyme for synthesizing 1,25-dihydroxy vitamin D from said metabolic precursor.
12. A method for inhibiting invasion of cancer cells, said method comprising increasing levels of a metabolic precursor of 1,25-dihydroxy vitamin D or an analog or a derivative thereof available to a target cell while reducing the risks of UV radiation exposure or vitamin D toxicity, wherein said target cell has a hydroxylase enzyme for synthesizing 1,25-dihydroxy vitamin D from said metabolic precursor.
13. A method for inhibiting metastasis of cancer cells, said method comprising increasing levels of a metabolic precursor of 1,25 -dihydroxy vitamin D or an analog or a derivative thereof available to a target cell while reducing the risks of UV radiation exposure or vitamin D toxicity, wherein said target cell has - 50 -
a hydroxylase enzyme for synthesizing 1,25-dihydroxy vitamin D from said metabolic precursor.
14. The method of claim 1, wherein said inhibiting is inhibiting the proliferation of the tumor cell.
15. A method of inhibiting proliferation of tumor cells and/or inducing differentiation of said cells in an animal, comprising introducing into said cells in need thereof a polynucleotide construct comprising a gene expressing 25- hydroxy vitamin D-l╬▒-hydroxylase, whereby 1,25-dihydroxy vitamin D or an analog thereof is produced and the proliferation of the cell is inhibited.
16. The method of claim 15, wherein said construct comprises a homologous regulatory sequence.
17. The method of claim 16, wherein said homologous regulatory sequence comprises a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence that is at least 90% identical to the nucleotide sequence depicted in SEQ ID NO. 3; and
(b) a nucleotide sequence of a polynucleotide that hybridizes under stringent conditions to a polynucleotide having the nucleotide sequence of (a) or its complement.
18. The method of claim 17, wherein said homologous regulatory sequence comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence depicted in SEQ ID NO. 3.
19. The method of claim 17, wherein said homologous regulatory sequence comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence depicted in SEQ ID NO. 3. - 51 -
20. The method of claim 17, wherein said homologous regulatory sequence comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence depicted in SEQ ID NO. 3.
21. The method of claim 17, wherein said homologous regulatory sequence comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence depicted in SEQ ID NO. 3.
22. The method of claim 17, wherein said homologous regulatory sequence comprises the nucleotide sequence depicted in SEQ ID NO. 3.
23. The method of claim 15, wherein said construct comprises a heterologous regulatory sequence.
24. The method of claim 23, wherein said heterologous regulatory sequence is a tissue specific promoter.
25. The method of claim 15 , further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
26. The method of claim 25, wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D-1 ╬▒-hydroxylase are on the same DNA construct.
27. The method of claim 25 , wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate DNA constructs. - 52 -
28. The method of claim 15, wherein said tumor cells are selected from the group consisting of prostate cancer cells, breast cancer cells, skin cancer cells, colon cancer cells, lung cancer cells, leukemia cells, and lymphoma cells.
29. A method for detecting risk of cancer, said method comprising: measuring in a target organ levels of vitamin D, a vitamin D metabolite, or a vitamin D metabolizing enzyme; and correlating the measurement with cancer risk level.
30. The method of claim 29, wherein said vitamin D metabolite is 25- hydroxyvitamin D, or an analog or derivative thereof.
31. The method of claim 29, wherein said vitamin D metabolite is 1,25- dihydroxy vitamin D.
32. The method of claim 29, wherein said vitamin D metabolizing enzyme is 25 -hydroxy vitamin D-l╬▒-hydroxylase.
33. The method of claim 32, wherein 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase levels are indirectly measured from their activity.
34. A method for predicting cancer treatment success, said method comprising: measuring in a target organ levels of vitamin D, a vitamin D metabolite, or a vitamin D metabolizing enzyme; and correlating the measurement with treatment success.
35. A composition for inhibiting proliferation, invasion, or metastasis of a cancerous cell, said composition comprising a vitamin D metabolite which is - 53 -
a 1 J5-dihydroxyvitamin D metabolic precursor, and a pharmaceutically acceptable carrier.
36. A method of treating benign prostatic hypeφlasia in an animal, comprising introducing into cells exhibiting benign prostatic hypeφlasia in need thereof a polynucleotide construct comprising a gene expressing 25- hydroxy vitamin D-lα-hydroxylase, whereby 1,25-dihydroxy vitamin D or an analog thereof is produced and said hypeφlasia is treated.
37. The method of claim 36, wherein said construct comprises a homologous regulatory sequence.
38. The method of claim 37, wherein said homologous regulatory sequence comprises a nucleotide sequence depicted in SEQ ID NO. 3.
39. The method of claim 36, wherein said construct comprises a heterologous regulatory sequence.
40. The method of claim 39, wherein said heterologous regulatory sequence is a tissue specific promoter.
41. The method of claim 36, further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
42. The method of claim 41 , wherein said gene expressing the vitamin D receptor and said gene expressing 25-hydroxy vitamin D- 1 ╬▒-hydroxylase are on the same DNA construct. - 54 -
43. The method of claim 41 , wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate DNA constructs.
44. A method of treating a hypeφroliferative skin disorder in an animal, comprising introducing into skin cells of the animal in need thereof a polynucleotide construct comprising a gene expressing 25 -hydroxy vitamin D- 1 α-hydroxylase, whereby 1,25 -dihydroxy vitamin D or an analog thereof is produced and the proliferation of the cell is inhibited.
45. The method of claim 44, wherein said construct comprises a homologous regulatory sequence.
46. The method of claim 45, wherein said homologous regulatory sequence comprises the nucleotide sequence depicted in SEQ ID NO. 3.
47. The method of claim 44, wherein said construct comprises a heterologous regulatory sequence.
48. The method of claim 47, wherein said heterologous regulatory sequence is a tissue specific promoter.
49. The method of claim 44, further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
50. The method of claim 49, wherein said gene expressing the vitamin
D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on the same DNA construct. - 55 -
51. The method of claim 49, wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate DNA constructs.
52. The method of claim 44, wherein said hypeφroliferative skin disorder is psoriasis.
53. The method of claim 44, wherein said hypeφroliferative skin disorder is selected from the group consisting of ichthyosis and actinic keratoses.
54. A method of treating a disorder in calcium and bone metabolism in an animal, comprising introducing into cells of the animal in need thereof a polynucleotide construct comprising a gene expressing 25-hydroxyvitamin D-
1 ╬▒-hydroxylase, whereby 1,25 -dihydroxy vitamin D or an analog thereof is produced and said disorder is treated.
55. The method of claim 54, wherein said construct comprises a homologous regulatory sequence.
56. The method of claim 55, wherein said homologous regulatory sequence comprises a nucleotide sequence depicted in SEQ ID NO. 3.
57. The method of claim 54, wherein said construct comprises a heterologous regulatory sequence.
58. The method of claim 57, wherein said heterologous regulatory sequence is a tissue specific promoter. - 56 -
59. The method of claim 54, further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
60. The method of claim 59, wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on the same DNA construct.
61. The method of claim 59, wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate constructs.
62. The method of claim 54, wherein said disorder in calcium and bone metabolism is renal osteodystrophy.
63. The method of claim 54, wherein said disorder in calcium and bone metabolism is a metabolic bone disease.
64. The method of claim 54, wherein said disorder in calcium and bone metabolism is selected from the group consisting of vitamin D-dependent rickets type I, x-linked hypophosphatemic rickets, vitamin D-dependent rickets type II, and osteoporosis.
65. The method of claim 54, wherein said disorder in calcium and bone metabolism is low blood serum calcium due to hypoparathyroidism.
66. A method of treating or preventing hair loss in an animal, comprising introducing into hair cells in need thereof a polynucleotide construct comprising a gene expressing 25 -hydroxy vitamin D-l╬▒-hydroxylase, whereby - 57 -
1,25-dihydroxy vitamin D or an analog thereof is produced and said hair loss is treated or prevented.
67. The method of claim 66, wherein said construct comprises a homologous regulatory sequence.
68. The method of claim 67, wherein said homologous regulatory sequence comprises a nucleotide sequence depicted in SEQ ID NO. 3.
69. The method of claim 66, wherein said construct comprises a heterologous regulatory sequence.
70. The method of claim 69, wherein said heterologous regulatory sequence is a tissue specific promoter.
71. The method of claim 66, further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
72. The method of claim 71 , wherein said gene expressing the vitamin D receptor and said gene expressing 25-hydroxy vitamin D- 1 ╬▒-hydroxylase are on the same DNA construct.
73. The method of claim 71 , wherein said gene expressing the vitamin D receptor and said gene expressing 25-hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate DNA constructs.
74. The method of claim 66, wherein said hair loss is a result of a condition selected from the group consisting of alopecia androgenetica, female pattern baldness, and chemotherapy-induced alopecia. - 58 -
75. A method of enhancing wound healing in an animal, comprising introducing into cells of the wound in need thereof a polynucleotide construct comprising a gene expressing 25 -hydroxy vitamin D-l╬▒-hydroxylase, whereby 1 J5-dihydroxy vitamin D or an analog thereof is produced and the healing of the wound is enhanced.
76. The method of claim 75, wherein said construct comprises a homologous regulatory sequence.
77. The method of claim 76, wherein said homologous regulatory sequence comprises a nucleotide sequence depicted in SEQ ID NO. 3.
78. The method of claim 75, wherein said construct comprises a heterologous regulatory sequence.
79. The method of claim 78, wherein said heterologous regulatory sequence is a tissue specific promoter.
80. The method of claim 75, further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
81. The method of claim 80, wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on the same DNA construct.
82. The method of claim 80, wherein said gene expressing the vitamin
D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate DNA constructs. - 59 -
83. A method of treating an autoimmune disease in an animal, comprising introducing into cells of the animal in need thereof a polynucleotide construct comprising a gene expressing 25 -hydroxy vitamin D-l╬▒-hydroxylase, whereby 1,25-dihydroxyvitamin D or an analog thereof is produced and the autoimmune disease is treated.
84. The method of claim 83, wherein said construct comprises a homologous regulatory sequence.
85. The method of claim 84, wherein said homologous regulatory sequence comprises a nucleotide sequence depicted in SEQ ID NO. 3.
86. The method of claim 83, wherein said construct comprises a heterologous regulatory sequence.
87. The method of claim 86, wherein said heterologous regulatory sequence is a tissue specific promoter.
88. The method of claim 83 , further comprising introducing into said cells a polynucleotide construct comprising a gene expressing a vitamin D receptor.
89. The method of claim 88, wherein said gene expressing the vitamin D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on the same DNA construct.
90. The method of claim 88, wherein said gene expressing the vitamin
D receptor and said gene expressing 25 -hydroxy vitamin D- 1 ╬▒-hydroxylase are on separate DNA constructs. - 60 -
91. The method of claim 83, wherein said autoimmune disease is selected from the group consisting of diabetes mellitus type 1 , multiple sclerosis, rheumatoid arthritis, and psoriatic arthritis.
92. An isolated polynucleotide molecule comprising a 25- hydroxy vitamin D- 1 ╬▒-hydroxylase regulatory sequence or a complement thereof.
93. The polynucleotide molecule according to claim 92, wherein said regulatory sequence comprises a polynucleotide selected from the group consisting of:
(a) a polynucleotide comprising a nucleotide sequence at least 90% identical to the nucleotide sequence depicted in SEQ ID NO. 3; and
(b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (a) or the complement thereof.
94. A polynucleotide molecule of claim 93 , wherein said polynucleotide comprises a nucleotide sequence at least 95% identical to the nucleotide sequence depicted in SEQ ID NO. 3.
95. A polynucleotide molecule of claim 93 , wherein said polynucleotide comprises a nucleotide sequence at least 97% identical to the nucleotide sequence depicted in SEQ ID NO. 3.
96. A polynucleotide molecule of claim 93 , wherein said polynucleotide comprises a nucleotide sequence at least 98% identical to the nucleotide sequence depicted in SEQ ID NO. 3. - 61 -
97. A polynucleotide molecule of claim 93, wherein said polynucleotide comprises a nucleotide sequence at least 99% identical to the nucleotide sequence depicted in SEQ ID NO. 3.
98. A polynucleotide molecule of claim 93 , wherein said polynucleotide comprises the nucleotide sequence depicted in SEQ ID NO. 3.
99. The polynucleotide molecule according to claim 92, further comprising a nucleotide sequence coding for 25 -hydroxy vitamin D-l╬▒- hydroxylase.
100. The polynucleotide molecule according to claim 99, wherein said nucleotide sequence coding for 25-hydroxyvitamin D- 1 ╬▒-hydroxylase is at least
90% identical to the sequence depicted in SEQ ID NO. 1.
101. The polynucleotide molecule according to claim 99, wherein said nucleotide sequence coding for 25 -hydroxy vitamin D-l╬▒-hydroxylase is at least 95% identical to the sequence depicted in SEQ ID NO. 1.
102. The polynucleotide molecule according to claim 99, wherein said nucleotide sequence coding for 25 -hydroxy vitamin D-l╬▒-hydroxylase is at least 97% identical to the sequence depicted in SEQ ID NO. 1.
103. The polynucleotide molecule according to claim 99, wherein said nucleotide sequence coding for 25 -hydroxy vitamin D-1 ╬▒-hydroxylase is at least 98% identical to the sequence depicted in SEQ ID NO. 1.
104. The polynucleotide molecule according to claim 99, wherein said nucleotide sequence coding for 25 -hydroxy vitamin D-l╬▒-hydroxylase is at least 99% identical to the sequence depicted in SEQ ID NO. 1. - 62 -
105. The polynucleotide molecule according to claim 99, wherein said nucleotide sequence coding for 25 -hydroxy vitamin D-1 ╬▒-hydroxylase comprises the sequence depicted in SEQ ID NO. 1.
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1074627A2 (en) * 1997-07-10 2001-02-07 Kyowa Hakko Kogyo Co., Ltd. 25-hydroxyvitamin D3-1a-hydroxylase and DNA encoding the hydroxylase
US6503893B2 (en) 1996-12-30 2003-01-07 Bone Care International, Inc. Method of treating hyperproliferative diseases using active vitamin D analogues
US6538037B2 (en) 1991-01-08 2003-03-25 Bone Care International, Inc. Methods for preparation and use of 1α,24(S)-dihydroxyvitamin D2
US6566353B2 (en) 1996-12-30 2003-05-20 Bone Care International, Inc. Method of treating malignancy associated hypercalcemia using active vitamin D analogues
US6573256B2 (en) 1996-12-30 2003-06-03 Bone Care International, Inc. Method of inhibiting angiogenesis using active vitamin D analogues
WO2005101022A2 (en) * 2004-04-15 2005-10-27 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with vitamin d (1,25-dihydroxyvitamin d3) receptor 1 (vdr1)
US20180296576A1 (en) * 2006-06-21 2018-10-18 Opko Ireland Global Holdings, Ltd. Method of Treating and Preventing Secondary Hyperparathyroidism
US10220047B2 (en) 2014-08-07 2019-03-05 Opko Ireland Global Holdings, Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
US10300078B2 (en) 2013-03-15 2019-05-28 Opko Ireland Global Holdings, Ltd. Stabilized modified release vitamin D formulation and method of administering same
US11007204B2 (en) 2006-02-03 2021-05-18 Opko Renal, Llc Treating vitamin D insufficiency and deficiency with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
US11154509B2 (en) 2007-04-25 2021-10-26 Eirgen Pharma Ltd. Methods for controlled release oral dosage of a vitamin D compound
US11173168B2 (en) 2016-03-28 2021-11-16 Eirgen Pharma Ltd. Methods of treating vitamin D insufficiency in chronic kidney disease
US11672809B2 (en) 2010-03-29 2023-06-13 Eirgen Pharma Ltd. Methods and compositions for reducing parathyroid levels
US11752158B2 (en) 2007-04-25 2023-09-12 Eirgen Pharma Ltd. Method of treating vitamin D insufficiency and deficiency

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105030791B (en) * 2015-06-26 2017-12-22 张雷 Application of the 25(OH)VD in Intracranial Benign Tumor brain tumor is treated

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763429A (en) * 1993-09-10 1998-06-09 Bone Care International, Inc. Method of treating prostatic diseases using active vitamin D analogues

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763429A (en) * 1993-09-10 1998-06-09 Bone Care International, Inc. Method of treating prostatic diseases using active vitamin D analogues

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE DIALOG MEDLINE 1 January 1900 (1900-01-01), GLORIEUX F H, ST-ARNAUD R: "Molecular Cloning of (25-OH d)-1 alpha-Hydroxylase: An Approach to the Understanding of Vitamin D Pseudo-Deficiency", XP002921182, Database accession no. 1998441850 *
K. TAKEYAMA ET AL.,: "25-hydroxyvitamin D3 1alpha-hydroxylase and vitamin D synthesis", SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE, US, vol. 277., 19 September 1997 (1997-09-19), US, pages 1827 - 1830., XP002092699, ISSN: 0036-8075, DOI: 10.1126/science.277.5333.1827 *

Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6538037B2 (en) 1991-01-08 2003-03-25 Bone Care International, Inc. Methods for preparation and use of 1α,24(S)-dihydroxyvitamin D2
US6566353B2 (en) 1996-12-30 2003-05-20 Bone Care International, Inc. Method of treating malignancy associated hypercalcemia using active vitamin D analogues
US6680309B2 (en) 1996-12-30 2004-01-20 Bone Care International, Inc. Method of treating hyperproliferative diseases using active vitamin D analogues
US6573256B2 (en) 1996-12-30 2003-06-03 Bone Care International, Inc. Method of inhibiting angiogenesis using active vitamin D analogues
US6503893B2 (en) 1996-12-30 2003-01-07 Bone Care International, Inc. Method of treating hyperproliferative diseases using active vitamin D analogues
EP1074627A2 (en) * 1997-07-10 2001-02-07 Kyowa Hakko Kogyo Co., Ltd. 25-hydroxyvitamin D3-1a-hydroxylase and DNA encoding the hydroxylase
US6274359B1 (en) 1997-07-10 2001-08-14 Kyowa Hakko Kogyo Co., Ltd. 25-hydroxyvitamin D3-1α-hydroxylase and DNA encoding the hydroxylase
EP1074627A3 (en) * 1997-07-10 2001-04-04 Kyowa Hakko Kogyo Co., Ltd. 25-hydroxyvitamin D3-1a-hydroxylase and DNA encoding the hydroxylase
WO2005101022A2 (en) * 2004-04-15 2005-10-27 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with vitamin d (1,25-dihydroxyvitamin d3) receptor 1 (vdr1)
WO2005101022A3 (en) * 2004-04-15 2006-02-09 Bayer Healthcare Ag Diagnostics and therapeutics for diseases associated with vitamin d (1,25-dihydroxyvitamin d3) receptor 1 (vdr1)
US11911398B2 (en) 2006-02-03 2024-02-27 Opko Renal, Llc Treating Vitamin D insufficiency and deficiency with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
US11007204B2 (en) 2006-02-03 2021-05-18 Opko Renal, Llc Treating vitamin D insufficiency and deficiency with 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3
US20180296576A1 (en) * 2006-06-21 2018-10-18 Opko Ireland Global Holdings, Ltd. Method of Treating and Preventing Secondary Hyperparathyroidism
US10668089B2 (en) * 2006-06-21 2020-06-02 Opko Ireland Global Holdings, Ltd. Method of treating and preventing secondary hyperparathyroidism
US11752158B2 (en) 2007-04-25 2023-09-12 Eirgen Pharma Ltd. Method of treating vitamin D insufficiency and deficiency
US11154509B2 (en) 2007-04-25 2021-10-26 Eirgen Pharma Ltd. Methods for controlled release oral dosage of a vitamin D compound
US11672809B2 (en) 2010-03-29 2023-06-13 Eirgen Pharma Ltd. Methods and compositions for reducing parathyroid levels
US10300078B2 (en) 2013-03-15 2019-05-28 Opko Ireland Global Holdings, Ltd. Stabilized modified release vitamin D formulation and method of administering same
US11253528B2 (en) 2013-03-15 2022-02-22 Eirgen Pharma Ltd. Stabilized modified release Vitamin D formulation and method of administering same
US10357502B2 (en) 2013-03-15 2019-07-23 Opko Ireland Global Holdings, Ltd. Stabilized modified release vitamin D formulation and method of administering same
US10350224B2 (en) 2013-03-15 2019-07-16 Opko Ireland Global Holdings, Ltd. Stabilized modified release vitamin D formulation and method of administering same
US11007205B2 (en) 2014-08-07 2021-05-18 Eirgen Pharma Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
US10493084B2 (en) 2014-08-07 2019-12-03 Opko Ireland Global Holdings, Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
US11738033B2 (en) 2014-08-07 2023-08-29 Eirgen Pharma Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
US10220047B2 (en) 2014-08-07 2019-03-05 Opko Ireland Global Holdings, Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
US11173168B2 (en) 2016-03-28 2021-11-16 Eirgen Pharma Ltd. Methods of treating vitamin D insufficiency in chronic kidney disease

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EP1066377A1 (en) 2001-01-10
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CA2323782A1 (en) 1999-09-30

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