EP1066377A1 - Methodes de prevention et de traitement du cancer - Google Patents

Methodes de prevention et de traitement du cancer

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Publication number
EP1066377A1
EP1066377A1 EP99915026A EP99915026A EP1066377A1 EP 1066377 A1 EP1066377 A1 EP 1066377A1 EP 99915026 A EP99915026 A EP 99915026A EP 99915026 A EP99915026 A EP 99915026A EP 1066377 A1 EP1066377 A1 EP 1066377A1
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EP
European Patent Office
Prior art keywords
vitamin
cells
hydroxylase
gene expressing
nucleotide sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99915026A
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German (de)
English (en)
Inventor
Gary G. Schwartz
Balakrishna L. Lokeshwar
Tai C. Chen
Lyman W. Whitlatch
Xiang Fu Kong
Michael F. Holick
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cutanogen Inc
Original Assignee
Cutanogen Inc
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Publication date
Application filed by Cutanogen Inc filed Critical Cutanogen Inc
Publication of EP1066377A1 publication Critical patent/EP1066377A1/fr
Withdrawn legal-status Critical Current

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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.

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Abstract

L'invention concerne des méthodes permettant d'inhiber la croissance des cellules cancéreuses. Selon un mode de réalisation, la méthode consiste à accroître la quantité de précurseur métabolique de 1,25-dihydroxyvitamine D ('1,25(OH)2D') ou d'un analogue ou d'un dérivé de ladite vitamine pouvant être utilisée par une cellule cible. Selon un mode de réalisation préféré, le précurseur est 25-hydroxyvitamine D. Selon un autre mode de réalisation, la méthode consiste à administrer à une cellule cible un gène codant pour 25-hydroxyvitamine D-1α-hydroxylase ('1α-OHase') avec ou sans gène codant pour le récepteur de la vitamine D de manière à accroître la production de 1,25(OH)2D ou d'un analogue de cette vitamine à l'intérieur de la cellule cible, ce qui permet d'inhiber la prolifération cellulaire. Selon un autre mode de réalisation de l'invention, on utilise la méthode consistant à administrer à une cellule ou à des tissus cible un gène codant pour 1α-OHase avec ou sans gène codant pour le récepteur de la vitamine D, dans le traitement des troubles liés à une hyperprolifération cellulaire, des troubles de la pousse des cheveux, et des troubles du métabolisme osseux et calcique chez les animaux.
EP99915026A 1998-03-25 1999-03-25 Methodes de prevention et de traitement du cancer Withdrawn EP1066377A1 (fr)

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US4791898A 1998-03-25 1998-03-25
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US122270P 1999-03-01
US122268P 1999-03-01
US12367099P 1999-03-09 1999-03-09
US12366999P 1999-03-09 1999-03-09
US123670P 1999-03-09
US123669P 1999-03-09
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US6538037B2 (en) 1991-01-08 2003-03-25 Bone Care International, Inc. Methods for preparation and use of 1α,24(S)-dihydroxyvitamin D2
US6503893B2 (en) 1996-12-30 2003-01-07 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
US6566353B2 (en) 1996-12-30 2003-05-20 Bone Care International, Inc. Method of treating malignancy associated hypercalcemia using active vitamin D analogues
JPH1175863A (ja) 1997-07-10 1999-03-23 Kyowa Hakko Kogyo Co Ltd ２５−ヒドロキシビタミンＤ３−１α−水酸化酵素および該酵素をコードするＤＮＡ
WO2005101022A2 (fr) * 2004-04-15 2005-10-27 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques destinees aux maladies associees au recepteur 1 de la vitamine d (1,25-dihydroxyvitamine d3) (vdr1)
US7094775B2 (en) 2004-06-30 2006-08-22 Bone Care International, Llc Method of treating breast cancer using a combination of vitamin D analogues and other agents
ES2593047T3 (es) 2006-02-03 2016-12-05 Opko Renal, Llc Tratamiento de la insuficiencia y deficiencia de vitamina D con 25-hidroxivitamina D2 y 25-hidroxivitamina D3
ES2670029T3 (es) * 2006-06-21 2018-05-29 Opko Ireland Global Holdings, Ltd. Terapia usando agente de repleción de la vitamina D y agente de reemplazo de la hormona de la vitamina D
US11752158B2 (en) 2007-04-25 2023-09-12 Eirgen Pharma Ltd. Method of treating vitamin D insufficiency and deficiency
KR20190028822A (ko) 2007-04-25 2019-03-19 사이토크로마 인코포레이티드 비타민 ｄ 화합물과 밀랍성 담체를 포함하는 경구 조절성 방출 조성물
WO2011123476A1 (fr) 2010-03-29 2011-10-06 Cytochroma Inc. Procédés et compositions pour réduction de taux d'hormone parathyroïde
KR101847947B1 (ko) 2013-03-15 2018-05-28 옵코 아이피 홀딩스 Ⅱ 인코포레이티드 안정화되고 변형된 비타민 ｄ 방출 제형
US10220047B2 (en) 2014-08-07 2019-03-05 Opko Ireland Global Holdings, Ltd. Adjunctive therapy with 25-hydroxyvitamin D and articles therefor
CN105030791B (zh) * 2015-06-26 2017-12-22 张雷 25羟基维生素d在治疗颅内良性脑肿瘤中的应用
JP7032322B2 (ja) 2016-03-28 2022-03-08 オプコ アイルランド グローバル ホールディングス リミテッド ビタミンｄ治療法

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US5763429A (en) * 1993-09-10 1998-06-09 Bone Care International, Inc. Method of treating prostatic diseases using active vitamin D analogues

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