JP2002507415A - Methods for prevention and treatment of cancer - Google Patents

Abstract

(57) SUMMARY Disclosed is a method for inhibiting the growth of cancer cells. In one embodiment, the method, the metabolic precursor of available 1,25-dihydroxyvitamin D to target cells ( "1,25 (OH) ₂ D"), or the amount of an analog or derivative thereof Including the step of increasing. In a preferred embodiment, the precursor is 25-hydroxyvitamin D. In another embodiment, the method comprises providing a gene encoding a vitamin D receptor to increase the production of 1,25 (OH) ₂ D or an analog thereof in a target cell, thereby inhibiting cell growth. Administering, with or without, a gene encoding 25-hydroxyvitamin D-1α-hydroxylase (“1α-OHase”) to target cells. In another embodiment of the present invention, a method of administering a gene encoding 1α-OHase to a target cell or tissue, with or without a gene encoding a vitamin D receptor, comprises administering the gene to a hyperproliferative cell disorder in an animal. Used in the treatment of disorders of hair growth, and disorders of calcium and bone metabolism.

Description

DETAILED DESCRIPTION OF THE INVENTION

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to methods for treating cancer in animals. In one embodiment, the method comprises the steps of obtaining 1,2 target cells or tissues in an animal.
Increasing the amount of metabolic precursor of 5-dihydroxyvitamin D. In another embodiment, the method comprises using 1,25-dihydroxyvitamin D in a target cell.
To increase the production of the target cells, the enzyme 25-hydroxyvitamin D-
Administering a gene that expresses 1α-hydroxylase.

[0002] The present invention also relates to a method for the treatment of hyperproliferative cell disorders and calcium and bone metabolism disorders in animals, which comprises 25-hydroxyvitamin on target cells or tissues of the animals. Including administration of a gene that expresses D-1α-hydroxylase.

Related Art Definitions The term "vitamin D" is commonly used in the literature to refer to a class of steroid hormones that have anti-ricket activity. This includes ergocalciferol (vitamin D _2), cholecalciferol (vitamin D _3), calcidiol (25-hydroxyvitamin D), calcitriol (1,25-dihydroxyvitamin D), and more than 20 other Vitamin D metabolites. However, the term "vitamin D," as used herein, refers only vitamin D ₂ or vitamin D ₃ in order to identify a useful metabolite is a part of the present invention are used in a more limited sense.

(1,25-Dihydroxyvitamin D) The most biologically active vitamin D metabolite is 1,25-dihydroxyvitamin D (“1,25 (OH) ₂ D”), also known as calcitriol. It is. Animals (e.g., human) Synthesis of 1,25 (OH) ₂ D in the sunlight or ultraviolet (UV) after exposure to radiation, or obtained from dietary plant-derived vitamin D ₂ and yeast-derived vitamin D ₂ or animals (typically, from fish topped fish liver oil and fat) derived after the intestinal absorption of vitamin D _3, starting with the skin production of vitamin D. To become biologically active, vitamin D must go through two hydroxylation steps. See FIG.

The first hydroxylation step occurs in the liver by hydroxylation at the 25th carbon and is the major circulating metabolite of vitamin D, 25-hydroxyvitamin D (“25 (OH) D”).
) Is formed. Secondary hydroxylation occurs in the kidney by hydroxylation at the 1α position, forming 1,25 (OH) ₂ D, a hormonally active metabolite. Kidney is a 1,25 (OH) major source of ₂ D, 25 (OH) D and 1,25 (OH) ₂ enzyme 1α- hydroxylase be converted to D ( "1α-OHase") is It is also present on some types of cells other than the kidney, such as activated macrophages and keratinocytes (Barbour, GL et al., N. Engl. J. Med. 3).
05: 440-443 (1981); and Bikle, D .; D. J. et al. Cl
in. Invest. 78: 557-566 (1986)). However, 1α-O
Hase was not previously known to be present in prostate cells.

The enzyme 1α-OHase has been recently cloned from mouse kidney, human kidney, and human keratinocytes (Takeyama K. et al., Science).
277: 1827-1829 (1997); Fu, G .; K. Et al., Mol. End
o. 11: 1961-1970 (1997); Monkawa, T .; Et al., Bio
chem. Biophys. Res. Comm. 239: 527-533 (19
97); K. Et al., DNA and Cell Biology 16
: 1499-1507 (1997); Shinki, T .; Proc. Nat
l. Acad. Sci. USA 94: 12920-12925 (1997);
Brenza, H .; L. Proc. Natl. Acad. Sci. USA
95: 1387-1391 (1998); Murayayama, A .; Et al., Biop
hys. Biochem. Res. Comm. 249: 11-16 (1998)
And Kong, X .; F. Bone 23 (Appendix 5): S186 (199)
8)). This cDNA is derived from 1α-OHa in human kidney and keratinocytes.
and was found to be identical to the se gene (Kitanaka, S. et al., NE
ngl. J. Med. 338: 653-661 (1998); Fu, G .; K. Et al., Mol. Endo. 11: 1961-1970 (1997); and Fu, G.
. K. Et al., DNA and Cell Biology 16: 1499-15.
07 (1997)). 1α-OHase is also a vitamin D-dependent type I rickets (
(Pseudovitamin D deficiency rickets). It was determined that some point mutations occurred in the gene that produces the defective 1α-OHase (Kitanaka, S et al., N. Engl. J. Med. 338: 653-6).
61 (1998).

1,25 (OH)_TwoD can perform most of its biological effects by interacting with certain receptors known as vitamin D receptors (VDRs)
. VDR is present in tissues responsible for regulating calcium and bone metabolism.
This tissue includes the small intestine, kidney and bone. In addition, a wide variety of organizations and
And cells are not involved in calcium and bone metabolism and possess VDR (H
orick, M .; F. , Primer on the Metabolic B
one Diseases and Disorders of Minera
l Metabolism, M .; J. Fabus (ed.), 3rd edition, Lippin
cott-Raven: Philadelphia (1996) 74-81)
. These include, inter alia, brain, heart, gonads, breast, pancreas, skeletal muscle, smooth muscle
, Skin, mononuclear cells and activated T and B lymphocytes
. In addition, promyelocytic leukemia cells, multiple myeloma cells, squamous cell carcinoma cells,
Some tumor cell lines, including prostate, breast and basal cell carcinoma cells,
R (Holick, MF, Bone 17 (supplement l) 107S-1
11S (1995)). The exact function of VDR in these tissues and cells is
Although not well understood, some cells carrying VDRs have reduced their proliferative activity.
1,25 (OH)_TwoResponding to D is known. In addition, some cells, such as keratinocytes, are also induced to differentiate into late stage
. This is for the treatment of hyperproliferative skin diseases such as psoriasis, 1,25 (OH) _Two D and its analogs have been used, and have suggested the potential for treating several cancers, such as breast, colon and prostate cancer (Holick, M .;
F. Bone 17 (supplement): 107S-111S (1995); Gross.
, C et al., Vitamin D, D .; Feldman et al. (Eds.), Academic
 Press: New York (1997), pp. 1125-1139).

Cancer Treatment Cancer is a group of diseases characterized by uncontrolled growth and spread of abnormal cells. One frequently occurring cancer is prostate adenocarcinoma. It is the most commonly diagnosed malignancy in American men, except for skin cancer. About 3
17,000 new cases of prostate cancer were diagnosed in the United States in 1996 (Pa
rker, S.M. L. Et al., Cancer J. et al. Clin. 46: 5-27 (199
6)). This is estimated at 41% of newly diagnosed malignancies, excluding basal and squamous cell carcinomas of the skin. This may actually underestimate the problem, because clinically silent prostate cancer is very common.

[0009] A unique feature of prostate cancer is the high incidence of "accidental" (inc
"identical" or "subclinical" cases. Necropsy data show that approximately 30% of men over the age of 50 have histological prostate cancer, and the incidence of these incidental cancers reaches 60% in men over the age of 80 . Histologically, these incidental cancers are distinguishable from potentially life-threatening prostate cancer and are considered to be an early stage of their natural history. Unlike prostate cancer mortality, incident prostate cancer is ubiquitous among older men, regardless of ethnicity or geographic location.

[0010] Although prostate cancer is generally a slowly growing malignant tumor, nevertheless the mortality of the disease is not negligible, representing about 15% of all cancer deaths in 1996, Prostate cancer is the second leading cause of cancer deaths among men in the United States (Parke
r, S. L. Et al., Cancer J. et al. Clin. 46: 5-27 (1996))
. As a result, prostate cancer has quickly become a major public health problem not only in the United States, but worldwide. Gross, C.I. Vitamin D, D. et al. Feldm
(eds.), Academic Press: San Diego, CA (1
997), pages 1125-1139. Interestingly, the continuous prostate cancer mortality in countries adjacent to the United States is due to the fact that ultraviolet (
UV) inversely correlated with the level of irradiation.

As the above suggests, calcitriol is now known to play an important role in regulating cell proliferation and differentiation (Walter, MR En).
docr. Rev .. 13: 719-764 (1992)). For example, 1,25 (
It has been shown that OH) ₂ D can regulate cell proliferation and cell differentiation in prostate cells. Evidence to support, in vitro and in vivo, uneven distribution of receptors for 1,25 in human prostate cells (OH) ₂ D (vitamin D receptor i.e. V DR), and 1,25 on these cells (OH) ₂ D has an antiproliferative effect and a differentiation promoting effect (Schwartz, GG.
, Anticancer Res. 14 1077-1082 (1994)).
Clearly, 1,25 (OH) ₂ D maintains the differentiated phenotype of prostate cells. Thus, low levels of 1,25 (OH) ₂ D may increase the risk for life-threatening prostate cancer.

1,25 (OH) available for target cells or organs_TwoCreating D appears to reduce the risk of cancer. However, 1,25 (OH)_TwoIncreasing D-level is a challenge for multiple barriers. First, 1,25 (OH) in a normal solid _Two Systemic levels of D are very tightly regulated metabolically. Systemic 1,25 (OH)_TwoElevated D levels are generally due to vitamin D or its specific metabolites (1,25 (OH)_TwoIt does not correlate with elevated systemic levels of D (including 25 (OH) D, a direct metabolic precursor of D). Therefore, UV irradiation or vitamin D is
1,25 (OH)_TwoThe mechanism by which increased exposure to D can occur has not been clear. 1,25 (OH)_TwoDespite being known to have D receptors, prostate cells have traditionally been derived from kidney 1α-OHase using 25 (OH) D as a substrate.
And highly regulated whole body 1,25 (OH)_TwoIt was thought to be regulated only by D. 1,25 (OH) by kidney_TwoWith this highly regulated production of D, an increase in systemic 25 (OH) D results in 1,25 (OH)_TwoIt was well recognized that it did not proportionally produce higher systemic levels of D.

[0013] Methods for providing an effective amount of 1,25 (OH) ₂ D at the cellular level without risking undesirable and potentially unsafe side effects for the patient have also heretofore been unknown. For example, systemic 25 (OH) D levels are elevated by in situ production of vitamin D through sun or UV exposure due to regulation of 1,25 (OH) ₂ D produced from 25 (OH) D. I can do it. Thus, excessive exposure to sunlight or UV irradiation is elevated serum levels of 25 (OH) D, 1,25 ( OH) 2 D serum levels are generally not rise to the desired antiproliferative level. Therefore, 1,25
Reaching beneficial levels of (OH) ₂ D was not known to be possible from exposure to the sun or UV radiation, even in amounts deemed dangerous or excessive by those skilled in the art. Certain risks, such as, inter alia, skin cancer, are associated with such excessive sun or UV exposure.

It has now been suggested that dietary supplementation of vitamin D may reduce the risk of prostate cancer (Naitoh, J et al., Prostate Cancer and P
rostatic Diseases 1: 48-53 (1997)). However, it is recommended that administration of vitamin D as a dietary supplement be performed under the supervision of a physician due to the risk of producing vitamin D toxicity. The negative side effects may include renal failure, hypercalcemia, coma and death.

[0015] Vitamin D replacement therapy is also disadvantageous in terms of its slow metabolic conversion to 25 (OH) D. Thus, achieving effective levels of 25 (OH) D by administration of supplemental vitamin D can take weeks or months using conservative dosage regimes. Conversely, if their vitamin D levels are too high, after a vitamin D withdrawal or dose reduction, due to the natural accumulation of vitamin D in body fat,
Restoring 5 (OH) D to normal levels can take several months. In addition, 1,25
Administration of (OH) ₂ D itself bears its own risk of developing hypercalcemia. This is well documented.

Several studies have shown that several cancer cell lines, including colon, melanoma, prostate and breast with VDR, reduced their growth when incubated with 1,25 (OH) ₂ D (Holick, MF, Bone 17 (Supplement 2):
107S-111S (1995); Halline, A .; G. FIG. Endocr
Inology 134: 1710-1717 (1994); Konety, B.
. R. Et al., Cell Growth & Differentiation 7: 1
563-1570 (1996); and Kivineva, M et al. Ster
oid Biochem. Mol. Biol. 66: 121-127 (1998)
)). Furthermore, human skin cells (Bikle, DD, et al., Biochemist).
ry 25: 1545~1548 (1986)) and normal prostate cells and prostate cancer cells, have been shown to have the ability to convert the 25 (OH) D 1,25 (OH ) in ₂ D (Schwartz , GG, et al., Cancer Ep.
idemiol. Biomark. & Prev. 7: 391-395 (1998)
)). Thus, when such cells are exposed to 25 (OH) D, 25 (OH) D
) D is thought to be converted to 1,25 (OH) ₂ D by 1α-OHase of the cells. The internally produced 1,25 (OH) ₂ D then interacts with the cell's VDR and regulates the cell's proliferative activity.

As noted above, 1,25 (OH) ₂ D is considered to be a biologically active form of vitamin D. Its important physiological functions are on calcium metabolism and bone metabolism. It stimulates intestinal calcium transport and initiates fluidization of calcium accumulation from bone. Patients with renal failure that cannot form 1,25 (OH) ₂ D develop hypocalcemia, secondary hyperparathyroidism, and bone disease, renal osteodystrophy. Two rare genetic disorders also cause 1α-OHa in the kidney
It is associated with a genetic deletion of se. Vitamin D-dependent type I rickets is an enzyme 1α-
It is a congenital abnormality with a defect in OHase. In X-related hypophosphatemic rickets, there is a defect in the regulation of 1α-OHase. Elderly people with osteoporosis lack the ability to upregulate 1α-OHase (H
orick, M .; F. , Primer on the Metabolic B
one Diseases and Disorders of Minera
l Metabolism, M .; J. Fabus (eds.) 3rd edition, Lippinc
ott-Raven: Philadelphia (1996) pp. 74-81).

SUMMARY OF THE INVENTION [0018] The administration of a vitamin D metabolite and a 1,25 (OH) ₂ D precursor to a patient may result in cell proliferation, invasive cancer, or cancer of an organ (eg, prostate, colon, or breast). It has not previously been described to prevent metastasis or metastasis of tumor cells, while reducing the risk of vitamin D toxicity and hypercalcemia.

[0019] The administration of the 1α-OHase gene using gene therapy techniques resulted in 1,25 (OH) _Two D_ThreeCancer or any disease (psoriasis or light) that can affect cell growth and maturation
(Including hyperproliferative skin disorders such as actinic keratosis)
Or it can also be 1,25 (OH)_TwoD_ThreeAgainst exogenous administration of
To treat disorders in calcium and bone metabolism as safer alternatives
Has also not been previously described.

In addition, the levels of the components of the cells in the organs (eg, vitamin D, vitamin D metabolites, enzymes that produce those metabolites, conventionally to determine the risk of developing invasive cancer, and also Or the activity of these enzymes) to determine the success rate of a particular treatment.

Such a method is beneficially provided by the present invention as described herein below.

The present invention relates to a novel method of prevention, treatment or testing for certain types of cancer, especially prostate cancer, which comprises a metabolite of vitamin D, an analog or derivative of a metabolite of vitamin D, or The benefits of enzymes involved in vitamin D metabolism are used.

The present invention relates to the use of 1,25-dihydroxyvitamin D (1,
25 (OH) ₂ D) provides a method for local concentration elevation, so that the antiproliferative, anti-invasive, anti-metastatic and pro-differentiation effects of 1,25 (OH) ₂ D Cells. In particular, one embodiment of the present invention comprises 1,25
A method for increasing 1,25 (OH) ₂ D to a concentration available to a target organ by administering a metabolic precursor of (OH) ₂ D to a patient. Here, this precursor may be a side effect or risk of skin cancer associated with exposure to sun or ultraviolet (UV) radiation, or a vitamin associated with administration of high doses of vitamin D or 1,25 (OH) ₂ D. D Has no toxic side effects or dangers.

In a preferred embodiment, the present invention comprises the step of administering an effective amount of a vitamin D metabolite to a patient in need of therapeutic or prophylactic treatment of a tumor or cancer. Here, administration of metabolites may be accomplished by alternative methods (eg, exposure to UV radiation, or administration of excessively high doses of vitamin D or 1,25 (OH) ₂ D, which may result in such undesirable side effects). It also substantially reduces the risk of skin cancer, vitamin D toxicity or hypercalcemia to the patient.

[0025] Advantageously, the method can be used to treat prophylactically or therapeutically patients who have or are at risk for certain potential or clinical cancers.
This method involves the use of vitamin D or a metabolite of vitamin D or an analog, derivative salt or functional equivalent of this metabolite (which is metabolically metabolized to 1,25 (OH) ₂ D or its functional equivalent). (Can be converted) to prevent the growth, invasion or metastasis of tumor or cancer cells. The treatment is preferably 1,25
Administering an effective amount of 25 (OH) D to a patient having a latent, clinical, or minimal residual cancer responsive to (OH) ₂ D. Minimal residual cancer refers to tumor cells detectable in a patient after treatment (eg, after prostatectomy or radiation therapy), where the primary tumor has been removed or an organ has been dissected from the body But the tumor or cancer cells (possibly migrated to a different organ,
Or present in the bloodstream) and still detectable.

[0026] In a further preferred embodiment, the method for prophylactic or therapeutic treatment of cancer comprises a patient having a occult, clinical, or minimal residual cancer of an organ (ie, a "target organ"). Administering an effective amount of 25 (OH) D, or an analog, derivative, salt or functional equivalent thereof. Here, the target organ or cells, can be converted to 25 (OH) D or an analogue, derivative, salt, a precursor or a functional equivalent, 1,25 (OH) ₂ D or a functional equivalent thereof targets Derived from an organisation. Organ cells having the enzyme 1α-OHase can locally convert 25 (OH) D or its analog to 1,25 (OH) ₂ D or its corresponding analog. For example, certain prostate cells are 1α-OHase, and 25 (O
It has been shown to have the ability to convert H) D 1,25 (OH) to ₂ D. Thus, the method may be used to treat prostate cancer patients with a potential or clinical disease; patients at risk of developing a prostate tumor (eg, individuals without clinical evidence of prostate cancer), or other organs or tissues. Can be indicated for patients with metastatic disease (eg, minimal residual cancer) caused by fixation of cancerous prostate cells.

[0027] Alternatively, the method can be used to treat a tumor in any organ. Here, the cells of the organ convert the metabolic precursor of 25 (OH) D into 1,25 (OH) D.
) ₂ D or obtained converted into a functional equivalent thereof, and 1,25 (OH) ₂ D or anti-proliferative effect of a functional equivalent thereof, anti-invasive effect, the anti-metastatic effect or differentiation promoting effect, with respect to Can respond. Thus, cells that have the vitamin D receptor (VDR) and that can produce the enzyme 1α-OHase can respond to such treatment. In addition, cells that are usually deficient in VDR or 1α-OHase are genetically engineered (eg, altered by transformation with a suitable gene or promoter to induce gene transcription and production of the resulting protein). ), So that the cells can respond to the treatment according to the invention.

Accordingly, the present invention provides a solution to the problem of reducing the risk of invasive (ie, life-threatening) cancer, including invasive prostate cancer. Treatment with 25 (OH) D, or an analog, derivative, salt, or functional equivalent thereof, may be useful for many men at various stages of the natural history of prostate cancer. These are the millions of men who currently have asymptomatic prostate cancer ("watchful waiting"
hful waiting), and men who have been treated for prostate cancer by therapy with the intent to treat, but where prostate cancer cells may remain present in the body). This treatment may also be useful for men who do not have prostate cancer (either asymptomatic or in clinical) to reduce the risk of developing cancer. One form of asymptomatic prostate cancer is also known as "prostatic intraepithelial neoplasia" or PIN.

Including VDR and reducing cell proliferation in cancer cells,
, 25 (OH) ₂ D, by introducing a gene encoding the enzyme 1α-O Hase into the cancer cells, and allowing the cells to grow to 1,25
It should also be possible to inhibit (OH) ₂ D by allowing it to be produced internally. Thus, this gene therapy method can be used to treat these cancers. Furthermore, when co-administering a gene encoding VDR with a 1α-OHase gene to cells lacking VDR, the method may confer such cells with a response to 1,25 (OH) ₂ D ₃ , This method is applied to 1,25
It is useful in the treatment of cancers that are normally non-responsive to (OH) ₂ D ₃ .

There are several advantages to this method. For example, exogenous administration of 1,25 (OH) ₂ D ₃ and its analogs has the potential to produce hypercalcemia (Holick, MF, Primer on the Metaboli).
c Bone Diseases and Disorders of Min
eral Metabolism, M.E. J. Fabus (eds.), 3rd edition, Lip
pincott-Raven: Philadelphia (1996), 74-
81, Holick, M .; F. , Bone 17 (2 supplements): 107S-11
1S (1995); and Gross, C.I. Vitamin D, D. et al. Fe
ldman et al. (eds.), Academic Press: San Diego, C
A (1997), pp. 1125-1139), which suggests that 1α-OH is present in cancer cells.
It is mitigated or eliminated by incorporating the ase gene and allowing the cell to produce 1,25 (OH) ₂ D internally. Co-administration of VDR gene, the cellular response to confer or enhance for anti-proliferative activity of 1,25 (OH) ₂ D produced endogenously. Once produced, 1,25 (OH) ₂ D, the was like are degraded in the cell after use, whereby is to eliminate any potential toxicity.

In a preferred embodiment, the present invention provides a method of inhibiting tumor cell growth and / or inducing tumor cell differentiation in an animal. This method
Introducing a polynucleotide construct comprising a gene that expresses 1α-OHase into a cell in need thereof, thereby producing 1,25 (OH) ₂ D or an analog thereof, and growing the cell. Is inhibited. In a preferred embodiment, the present invention further comprises the step of introducing into the tumor cells a polynucleotide construct comprising a gene that expresses the vitamin D receptor. In this case, the gene expressing 1α-OHase and the gene expressing the vitamin D receptor can be either on the same DNA construct or on another DNA construct.

In a particularly preferred embodiment, this aspect of the invention relates to prostate cancer, breast cancer, skin cancer, colon cancer, lung cancer, leukemia, lymphoma, or other cancers (DNA constructs containing the 1α-OHase gene (VDR gene provided by cells through gene therapy by including or without), 1,25 produced endogenously (OH) ₂ D or 1, 25 (OH) antiproliferative effects of analogues of ₂ D and pro differentiation Methods for treating or preventing cancer that is responsive to prodifacting effects).

In another embodiment, the animal being treated by the gene therapy described herein also has a 25 (OH) to enhance its response to the gene therapy.
)) Can be treated substantially simultaneously with D, or an analog, derivative, salt, or functional equivalent thereof.

Further, the invention provides a method of testing a patient for cancer risk. Measurement of the level of a particular cellular component in a particular organ can determine the relative risk of developing a life-threatening cancer in that organ, where the measured component is vitamin D metabolism, vitamin D, Its metabolites (eg, 25 (OH) D or its analogs), enzymes capable of converting vitamin D metabolites to 1,25 (OH) ₂ D, or involved in the activity of the enzymes. For example, 1α-OHase enzyme is 25 (O
H) can be converted to D 1,25 (OH) to ₂ D. Thus, measuring the level of 1α-OHase or the activity of 1α-OHase enzyme in a cell sample from a target organ (eg, a prostate gland) can be used to measure cancer in that organ or an organ, including cancer cells derived from that organ. May be determined.

Standard molecular biology techniques well known in the art also include 1α-OHas
e Polymorphisms in the gene encoding the enzyme or VDR can be measured and used to correlate allelic differences with the risk of developing a tumor in the tested individual.

The present invention also provides a method for predicting the rate of successful treatment of a particular cancer in a patient. This method determines local levels of specific cellular components (eg, vitamin D or its metabolites) in the vitamin D metabolic pathway in organs (eg, the prostate), or reduces metabolic precursors to 1,25 (OH) enzyme capable of converting into ₂ D (e.g., 1α-OHase) by determining the level of. Measuring the activity of an enzyme can also be useful for measuring enzyme levels indirectly, and thus can predict successful treatment.

The invention also relates to hyperproliferation in animals.
ve) A method for treating a skin disorder is provided. The method involves introducing a polynucleotide construct comprising a gene that expresses 1α-OHase into skin cells of an animal in need thereof, whereby 1,25 (OH) ₂ D or an analog thereof. Is produced and cell growth is inhibited. In another embodiment, the invention further comprises the step of introducing into the cell a polynucleotide construct comprising a gene that expresses the vitamin D receptor. In this embodiment of the invention,
The gene expressing 1α-OHase and the gene expressing the vitamin D receptor can be on the same DNA molecule of the construct or on another DNA molecule of the construct.

More specifically, the present invention relates to actinic keratosis, pre-skin ca
ncer), or to treat ichthyosis. Introduction of the gene encoding 1α-OHase results in an increase in the amount of 1,25 (OH) ₂ D ₃ in the cell, thereby reducing proliferative activity and inducing the cell to differentiate into a normal form. I do.

The present invention also relates to a method for treating a skin hyperproliferative disorder such as psoriasis. Patients with psoriasis responds to 1,25 (OH) 2D local of 1,25 (OH) ₂ D and mouth. However, there are concerns about potential toxicity (Ho
lick, M .; F. , Primer on the Metabolic Bo
ne Diseases and Disorders of Mineral
Metabolism, M .; J. Fabus (ed.), 3rd edition, Lippinc
ott-Raven: Philadelphia (1996), pp. 74-81;
And Holick, M .; F. , Bone 17 (2 supplements): 107S-111.
S (1995)). Thus, the present invention is a rational alternative treatment for psoriasis whereby cells are induced to produce or boost 1,25 (OH) ₂ D ₃ , and then 1 , 25 (OH) ₂ D ₃ interact with VDR,
It then reduces proliferative activity and enhances differentiation to normalize skin cells. This treatment may also be applied to other hyperproliferative skin disorders such as ichthyosis.

The present invention also provides a method of treating a disorder in calcium and bone metabolism in an animal. The method involves introducing a polynucleotide construct comprising a gene that expresses 1α-OHase into cells of an animal in need thereof, thereby producing 1,25 (OH) ₂ D or an analog thereof. And the disorder is treated. In one embodiment, the invention further comprises the step of introducing into the cell a polynucleotide construct comprising a gene that expresses a vitamin D receptor. In this embodiment, the gene expressing 1α-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or on a separate D
It can be anywhere on the NA construct.

This embodiment of the invention provides a method for treating disorders in calcium and bone metabolism (eg, renal osteodystrophy) or metabolic bone disease. The present invention also relates to vitamin D-dependent type I rickets, X-linked hypophosphatemic rickets, vitamin D-dependent type II rickets, and osteoporosis, as well as low serum calcium due to hypoparathyroidism. Methods for treating disorders in calcium and bone metabolism are provided.

The present invention also provides a method of treating or preventing hair loss in an animal.
The method involves introducing a polynucleotide construct comprising a gene that expresses 1α-OHase into a hair cell in need thereof, whereby the 1,25 (
OH) ₂ D or an analog thereof is produced and the hair loss is treated. In one embodiment, the invention further comprises the step of introducing into the cell a polynucleotide construct comprising a gene that expresses a vitamin D receptor. In this embodiment, the gene expressing 1α-OHase and the gene expressing the vitamin D receptor can be either on the same DNA construct or on another DNA construct.

Accordingly, the present invention relates to a male pattern baldness (alopecia androgenet).
ica), female pattern baldness, and chemotherapy-induced alopecia can be used to treat or prevent hair loss.

The invention also provides a method for enhancing wound healing in an animal. This method is
A polynucleotide construct containing a gene that expresses 1α-OHase
Introducing into the cells of the wound of interest, whereby 1,25 (OH) _Two D or an analog thereof is produced and the healing of the wound is enhanced. In one embodiment, the present invention further provides the cell with a vitamin D receptor.
Introducing a polynucleotide construct containing the gene to be expressed. This fruit
In an embodiment, a gene expressing 1α-OHase and a vitamin D receptor
Genes that are expressed on the same DNA construct or on another DNA construct.
It can be.

The present invention also provides a method of treating benign prostate hyperplasia in an animal. The method comprises introducing a polynucleotide construct comprising a gene that expresses 1α-OHase into cells that exhibit the required benign prostatic hyperplasia, thereby providing 1,25 (OH) ₂ D or a derivative thereof. Analogs are produced and the benign prostate hyperplasia is treated. In this embodiment, the invention further comprises the step of introducing into the cell a polynucleotide construct comprising a gene that expresses the vitamin D receptor. In this embodiment of the invention, the gene expressing 1α-OHase and the gene expressing the vitamin D receptor can be on the same DNA construct or on a separate DNA construct.

The present invention also provides a method for treating an autoimmune disease in an animal. The method comprises the step of introducing a polynucleotide construct comprising a gene expressing 25-hydroxyvitamin D-1α-hydroxylase into cells of an animal in need thereof, whereby 1,25-dihydroxyvitamin D Or its analogs are produced and the autoimmune disease is treated. In one embodiment,
The invention can further include the step of introducing into the cells a polynucleotide construct comprising a gene that expresses a vitamin D receptor. In this embodiment, 1
The gene that expresses α-OHase and the gene that expresses the vitamin D receptor can be on the same DNA construct or on a separate DNA construct.

The present invention can be used to treat autoimmune diseases such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis, and psoriatic arthritis.

Further, the present invention provides an isolated polynucleotide molecule comprising a 1α-OHase regulatory sequence or its complement. In one embodiment, the 1α-O
The Hase regulatory sequence or its complement comprises a polynucleotide selected from the group consisting of: (a) at least 9 nucleotides in the nucleotide sequence set forth in SEQ ID NO: 3;
A polynucleotide comprising a nucleotide sequence that is 0% identical; and (b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (a) or a complement thereof. In other embodiments, the 1α-OHase regulatory sequence or its complement comprises a nucleotide sequence that is 95%, 97%, 98%, or 99% identical to the sequence set forth in SEQ ID NO: 3, or 3 comprising the nucleotide sequence shown in FIG. The present invention also relates to the sequence shown in SEQ ID NO: 1
, 97%, 98% or 99% identical, or a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO: 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to a novel method for preventing or treating the potential for cell proliferation, invasion, or metastasis, or for promoting cell differentiation. The invention further relates to methods for testing for cancer, as well as methods for predicting cell proliferation or successful treatment for cancer.

(Administration of Vitamin D Metabolic Compounds) One aspect of the present invention involves increasing local cellular levels of 1,25 (OH) ₂ D. In particular, one embodiment of the present invention provides for the administration of an effective amount of a vitamin D metabolite that can be metabolically converted to 1,25 (OH) ₂ D by target cells to provide cell proliferation, invasion, Or, preventing or treating metastasis, or promoting cell differentiation.

Preferably, the vitamin D metabolites used according to the invention have an increased risk of skin cancer (when compared to sun or ultraviolet (UV) exposure), an increased risk of vitamin D toxicity (supplementary). And no significant contribution to hypercalcemia (as compared to administering 1,25 (OH) ₂ D). More preferably, the present invention provides a method for preventing the growth, invasion, or metastasis of tumor or cancer cells in a particular organ, or for promoting the differentiation of cells in or from an organ. D, or an analog, derivative, salt, or functional equivalent thereof, of an effective amount. This compound 25 (OH) D is commercially available.

The normally observed concentration of 25 (OH) D in serum is about 20-150 nmol
/ L (8-60 ng / ml). However, 250 nmol / L (100 ng
Circulating concentrations of 25 (OH) D of up to 1 / ml) are commonly observed in observers after exposure to midsummer sunlight and are considered normal (Horick,
M. F. J. Nutrition, Addendum, 120: 1464-1469 (19
90)). Concentrations above about 350 nmol / L are considered dangerous to the health of the individual. Hypovitaminosis D, a deficiency in serum 25 (OH) D, results in serum levels of about 25 nmol / L.
(10 ng / ml). Thus, the effective amount of 25 (OH) D administered to the target organ, when administered, is 25 (OH) D
But increase serum levels of 25 (OH) D to this "normal"
Any amount to keep within the range. Preferably, 25 (OH) D levels are increased to substantially greater than 25 nmol / L but less than 250 nmol / L in the target organ. More preferably, the 25 (OH) D level is between about 50-150 nm
ol / L.

Normal serum levels of 1,25 (OH) ₂ D range from about 38-144 pmol / L (16-60 pg / ml). Thus, another determination of an effective amount of 25 (OH) D administered according to the methods of the invention would increase the level of 25 (OH) D in the target organ toward the upper end of its normal range, but not its normal The dose is such that the 1,25 (OH) ₂ D of the whole body is not increased beyond the upper limit of the above range. For example, 25 (OH) D levels can be preferably raised to increase intra-organ levels to about 25-150 nmol / L, where systemic 1,25 (O
H) ₂ D level is maintained less than 145pmol / L. More preferably, the 1,25 (OH) ₂ D levels maintain less than 125 nmol / L when 25 (OH) D is administered to the target cells, organs or tissues.

Useful analogs, derivatives, or salts of 25 (OH) D are alkylated 25 (O
H) D, glycosylated 25 (OH) D, arylated 25 (OH) D, halogenated 25 (OH) D, or hydroxylated 25 (OH) D, orthoester of 25 (OH) D, or 25 (OH) D. A) a pharmaceutical salt of D; These analogs, derivatives, or salts are well known to those skilled in the art and may be synthesized or otherwise produced by readily available chemical procedures. Vitamin D analogs may be obtained according to the methods disclosed below:

[0055]

(Equation 1) The term “functional equivalent” refers to any compound that can be used as a substrate for 1α-OHase (OHase), or that can be converted to 1,25 (OH) ₂ D or 1,25 (OH) ₂ D receptor (VDR) can bind or activate, so that any of the beneficial properties of 1,25 (OH) ₂ D (ie, inhibition of invasion, proliferation, metastasis, or promotion of cell differentiation). One is used to refer to other compounds that can be converted to the affected compound. For example, US Pat.
Nos. 167,953; 5,422,099; 5,794,248; and 5,395,829.

A pharmaceutical composition comprising a 1,25 (OH) ₂ D metabolic precursor, or an analog or derivative thereof (eg, 25 (OH) D), and a pharmaceutically acceptable carrier It is also understood that this can be implemented by the use of objects.

The method of the present invention for administering a metabolic precursor of 1,25 (OH) ₂ D to a patient is effective in producing 1,25 (OH) ₂ D by prostate cancer cells. It is shown. In particular, the method of the present invention comprises two well-characterized human prostate cancer cell lines, DU145 and PC-3, and two non-cancerous human prostate-derived cells, NP96-5 and BPH96-11. It has been shown to be effective in one primary culture. The NP96-5 culture is a 23 year old organ donor.
BPH96-11 culture is from the age of 56 with benign prostatic hyperplasia (BPH). Two of these cell lines, DU1
45 and PC-3 as well as primary cultures, 1,25 (OH) ₂ D, may be synthesized from the a metabolic precursor 25 (OH) D. Vitamin D metabolite 1
, 25 (OH) ₂ D was also synthesized from 25 (OH) D by cultured normal human keratinocytes. The results are shown in Table 1 below: Table 1: Synthesis of 1,25 (OH) ₂ D in the presence and absence of clotrimazole, a P450 cytochrome inhibitor.

[0058]

[Table 1] As summarized in Table 1, the DU145 and PC-3 cell lines were 0.31 ± 0.06 and 0.07 ± 0.01 per mg protein / hour, respectively.
pmol of 1,25 (OH) ₂ D was produced. These cells exhibited 1α-OHase activity as detected by a thymic receptor binding assay according to well-known procedures. The production of 1,25 (OH) ₂ D is completely inhibited in the presence of clotrimazole, a commonly known inhibitor of the cytochrome P450 system, in which 1α-OHase is a known component. Was. Conversely, no measurable 1,25 (OH) ₂ D was detected in LNCaP cells, which did not show 1α-OHase activity.

Primary cultures of prostate cells from two patients (NP96-5 (normal prostate) and BPH96-11 (BPH) were also expanded and their enzymatic activities were
Measured in the presence of PD and in the presence or absence of clotrimazole. NP96-5 and BPH96-11 cultures were 3.08 ± 1 .0 in the presence of DPPD and in the absence of cytochrome P450 inhibitors, respectively.
56 pmol / mg protein / hour and 1.05 ± 0.31 pmol / mg
1,25 (OH) ₂ D of protein / hour was produced. The 1α-OHase activity found in the two primary cultures of prostate cells was comparable to the activity found in normal human keratinocytes. As in the two cell lines, the production of 1,25 (OH) ₂ D in primary cultures of prostate cells and keratinocytes was completely inhibited in the presence of clotrimazole.

The enzymatic activity detected in primary cultures of prostate cells was 25 (OH) D
From another metabolite, 10-oxo-19-nor-25 (OH) D, to 1,25 (
OH)_TwoThis was further supported by high performance liquid chromatography analysis using a solvent system that specifically separated D. In rat and chicken kidney homogenates
This metabolite, which is present in any amount, is n-hexane: isopropanol (9: 1)
1,25 (OH) by normal phase HPLC using a solvent system_TwoIt is known to co-migrate with D. Therefore, 1,25 (OH)_TwoD is separated from the presence of any 10-oxo-19-nor-25 (OH) D
Normal phase methylene chloride: isopropanol (19: 1) to ensure
A solvent system was used. In addition, 1α-OHase activity is a radioactive metabolite [ ^Three H] -25 (OH) D was used as the substrate.

[0061] Representative HPLC chromatography of two primary prostate cell cultures in the presence and absence of clotrimazole is illustrated in FIGS. In the absence of clotrimazole (20 μM), non-radioactive 25 (OH) D ₃ (50 nM), [ ³ H
] A second passage of normal human prostate cells incubated with -25 (OH) D ₃ (0.91 μCi / nmol) and 1,2-dianilinoethane (DPPD) (10 μM) for 2 hours at 37 ° C. It is shown in FIG. 1,25 active metabolite
(OH) ₂ D was synthesized at detectable levels (about 600 cpm).

A second passage of normal human prostate cells was also prepared in the presence of the cytochrome P450 inhibitor, clotrimazole (20 μM), with non-radioactive 25 (OH) D ₃ (50 nM), [ ³ H] -25. (OH) D ₃ (0.91 μCi / nmol) and DP
Incubated with PD (10 μM) at 37 ° C. for 2 hours. FIG.
Indicates that active metabolites were undetectable due to significant inhibition of 25 (OH) ₂ D synthesis.

The second passage of prostate cells from the BPH culture was clotrimazole (20 μM
In the absence of) non-radioactive _{25 (OH) D 3 (50nM} ), [3 H] -25 (OH
) Incubated with D ₃ (0.91 μCi / nmol) and DPPD (10 μM) at 37 ° C. for 2 hours. In the absence of a cytochrome P450 inhibitor, the active metabolite is detectable, producing a product that is produced at about 500 cpm. See FIG. In contrast, non-radioactive 25 (OH) D ₃ (50 nM), [ ³ H] -25 (OH) D ₃ (0.91 μCi / nmol) and DPPD (10 μM) in the presence of clotrimazole (20 μM). And a second passage of prostate cells from a BPH culture incubated for 2 hours at 37 ° C. with approximately 200 c
It showed significantly reduced levels of 1,25 (OH) ₂ D metabolites, measured below pm (FIG. 5).

Both primary cultures of non-cancerous prostate cells produced 1,25 (OH) ₂ D at levels 10 to 40 times higher than the cell line. Very high levels 1,25 (OH) ₂ D was observed from NP96-5 cultures derived from 23-year-old organ donor. 2. the amount of 1,25 (OH) ₂ D produced by the two primary non-cancerous human prostate cell lines 08 ± 1.56 and 1.05 ± 0.31 pmol / mg protein / hour (
(Normal and BPH, respectively) are the amount produced by cultured human keratinocytes (2.1 ± 0.1 pmol / mg protein / h) and the HEP62 hepatome cell line (2.3 pmol / mg protein / h) and human T lymphotroph. Comparable to lymphocytes transformed with a baculovirus (1.6 pmol / mg protein / hour). These values are at least 10-fold higher than those reported at other extrarenal sites (eg, human bone cells (0.068 pmol / mg protein / hour)).

After incubation with 25 (OH) D ₃ , DU145 and PC-3 prostate carcinoma cell lines also produced detectable levels of 1,25 (OH) ₂ D ₃ . 1,25 () by clotrimazole, a specific cytochrome P450 inhibitor
100% inhibition of OH) ₂ D production indicates that cytochrome P450-dependent 1α-OHase is present in these prostate cells. The addition of P450 inhibitors inhibited substantially all of the conversion into ^{[3 H] -25 (OH)} D 3 of ^{[3 H] -1,25 (OH)} 2 D 3 in these cells. Therefore, DU145 and PC
-3 human prostate cancer cells, as well as two primary cultures from non-cancerous human prostate
25 (OH) D ₃ , which possesses 1α-OHase activity and is the major circulating metabolite of vitamin D ₃ , is converted to 1,25 (OH) ₂ D ₃ , a hormonally active vitamin D metabolite. Can be converted.

The enzyme activity found in primary cultures of prostate cells is comparable to the activity found in primary cultures of renal proximal tubule cells (5.3-5.6 pmol / h / mg protein. Kidney 1α) -OHase is 1,25 (O (O) under normal physiological conditions.
The amount of 1,25 (OH) ₂ D produced by prostate cells is particularly important in the microenvironment of prostate cells (H) ₂ D, as it has been established that it is the major enzyme responsible for maintaining circulating concentrations of H) ₂ D. It may be physiologically important for the microenvironment. Levels of 1,25 (OH) ₂ D as low as 10 ⁻¹¹ M can significantly inhibit the invasiveness of human prostate cancer cells through an artificial basement membrane consisting of human amniotic membrane (
Schwartz, G .; G. FIG. Et al., Cancer Epidemiol. Biom
ark. Prev. 6: 727-732 (1997).

No detectable 1,25 (OH) ₂ D ₃ was produced by the LNCaP cells. 24,25-hydroxylase activity has also been reported to be low or undetectable in LNCaP. Miller, G .; J. Cancer
Res. 52: 515-520 (1992); Skowronski, R .; J
. Endocr. Rev .. 132: 1952-1960 (1993).

This unusual finding that 1,25 (OH) ₂ D can be synthesized from 25 (OH) D by prostate cells is relatively less toxic in treating cancer than high doses of vitamin D. Vitamin D metabolite, or certain other metabolites (1,25 (
OH) ₂ D). The method of the present invention comprising administering to a patient a vitamin D metabolite that can be converted to 1,25 (OH) ₂ D by the enzyme 1α-OHase is preferably used in chemoprevention of prostate cancer. However, in light of the principles and procedures described herein, cancer cells in other organs or tissues that can convert vitamin D metabolites to 1,25 (OH) ₂ D are identified as 1,25 (OH). ) antiproliferative properties of ₂ D, anti-invasive characteristics, that may respond to anti-metastatic properties, or before differentiation (prodifferentiating) properties are appreciated by those skilled in the art. Thus, it is understood that the present invention may 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 products synthesized from the administered metabolites (1,25 (OH) ₂ D) have antiproliferative and pro-differentiating effects on normal prostate cells and cancerous prostate cells.
Furthermore, 1,25 (OH) ₂ D physiological levels, as correlated with decreased secretion levels of collagenase type IV (MM P-2 and MMP-9), the invasiveness of DU145 cells through artificial basement membrane Inhibits significantly. In addition, 1,25 (OH) ₂ D has been shown to have an anti-metastatic effect on prostate cancer cells in vivo. However, the administration of 1,25 (OH) ₂ D has certain disadvantages due to its causal effects in producing hypercalcemia. Therefore, 1,25 (OH
) ₂ D, this risk because of hypercalcemia and optimal for safe use as a chemopreventive agent, is not considered by those skilled in the art. Our findings suggest that increasing the available substrate (eg, 25 (OH) D
Administering or supplementing) with 1,25 (O
H) Local synthesis is achieved resulting in ₂ D, the active metabolite production thereof available for prostate cells 1,25 (OH) increases the level of ₂ D, thereby cancerous prostate cell proliferation activity, It is shown to reduce invasive or metastatic activity and promote their differentiation.

As described herein, 25 (OH) D, and analogs and derivatives thereof, are useful in several clinical settings, such as “watchful watch”.
slowing or preventing the need for prostatectomy or radiation therapy in humans and / or slowing or preventing disease recurrence in humans with minimal residual disease May be useful in some cases.

The vitamin D metabolite 25 (OH) D preferentially has a lower proportion in body fat when compared to vitamin D or other more fat-soluble metabolites or analogs of vitamin D. Is stored. In addition, 25 (OH) D is the liver 25-
Bypass hydroxylation. Therefore, its action is started and stopped earlier than vitamin D. In addition, if a hepatic 25 hydroxylation of vitamin D ₂ or vitamin D ₃ is present, 25 (OH), as in certain cases of primary biliary cirrhosis or neonatal hypocalcemia, D
Can be effective. In addition, 25 (OH) D may have some endogenous agonist activity that may retain some level of efficacy in the absence of the renal 1α-OHase system.

The method of the present invention (25 (OH) D, or an analog, derivative, salt or organic compound thereof)
Preferred methods of administering functional equivalents) are not limited to the treatment of prostate cells.
No. Cells with 1α-OHase also convert 25 (OH) D to 1,25 (OH) _Two D, whereby 1,25 (OH)_TwoLocal cell level of D end product
Increase. VDR (this is 1,25 (OH)_TwoD or its functional equivalent) can also respond to the compound and its antiproliferative efficacy
As a result, benefits from anti-invasive, anti-metastatic, and pro-differentiated effects may be obtained. example
If colon cells or breast cells have 1α-OHase and VDR
And either inherently or through the use of genetic engineering techniques well known in the art.
1,25 (OH)_TwoD.

For the above indications, dosage administration to a host may involve identifying the disease or condition, the type of patient (its age, weight, health, type of concurrent treatment (if any), frequency of treatment, And treatment ratio).

The compounds of the present invention may be formulated according to known methods for preparing pharmaceutically useful compositions. Formulations are described in detail in many sources that are well known and readily available to those skilled in the art. For example, E. W. Reming by Martin
ton's Pharmaceutical Science describes formulations that can be used in connection with the present invention. In general, the compositions of the present invention are formulated to combine an effective amount of the biologically active compound with a suitable carrier to facilitate effective administration of the composition.

In accordance with the present invention, pharmaceutical compositions comprising an active ingredient in an effective amount of one or more compounds of the present invention and one or more non-toxic pharmaceutically acceptable carriers or diluents are known to those skilled in the art. Can be used by Further, the pharmaceutical compositions may include one or more compounds of the present invention, for example, 25 (OH) D as the first active ingredient and a second active ingredient (eg, an anti-inflammatory compound known in the art). , Antibacterial, antiproliferative, or antiviral compounds).

In accordance with the present invention, a pharmaceutically effective amount of a known second active ingredient and a vitamin D metabolite, analog or derivative thereof useful in accordance with the present invention are administered to a patient sequentially or simultaneously. The most effective mode of administration and dosage regimen of a compound of this invention will depend on the type of disease to be treated, the severity and course of the disease, previous therapies, the patient's health, and vitamin D metabolites. Responsiveness to analogs and derivatives, and the judgment of the treating physician. The compositions of the present invention may be administered to the patient at one time or over a series of treatments.

Preferably, the compound of the present invention and the second agent are administered to the patient continuously.
This involves administering the second agent before, after, or both before and after treatment with the compound or composition of the present invention. Continuous administration refers to treatment with a second agent, at least on the same day (within 24 hours) of treatment with a vitamin D metabolite of the invention, and second treatment on a day without administration of a compound of the invention. For continuous treatment with the drug. Conventional modes of administration and standard dosage regimens for the compounds of the invention, or pharmaceutical compositions comprising the compounds of the invention, can be used (
Gilman, A .; G. FIG. Et al. (Eds.), The Pharmacological
Basis of Therapeutics, pp. 697-713, 1482.
, 1489-91 (1980); Physicians Desk Refer.
ence, 1986), can be titrated similarly to other compounds used for treatment, or for individual patients.

According to one embodiment of the present invention, a patient may receive concurrent treatment with a composition comprising at least one compound of the present invention and a second active ingredient. For example, 25
Local intra-tissue (or intra-organ) injection of (OH) D, or an analog or derivative thereof, is preferred, but these include subcutaneous injections, subcutaneous slow implants, intravenous,
It can be administered intraperitoneally, topically, intramuscularly, or orally.

The compositions used in these therapies may also be in various forms. These include, for example, solid, semi-solid, and liquid dosage forms (eg,
Tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable solutions and insoluble solutions). The preferred form depends on the intended mode of administration and therapeutic application. The compositions also preferably include a conventional pharmaceutically acceptable carrier and adjuvant, which are known to those skilled in the art. Preferably, the compositions of the invention are in unit dosage form and are usually administered to the patient more than once a day.

Gene Therapy In addition, the present invention provides a method for genetically modifying a target cell having a gene encoding one or more proteins that acts as a vitamin D receptor (VDR) that binds 1,25 (OH) ₂ D. By providing the target, the target cell or tissue is genetically modified to provide an active vitamin D metabolite, or analogs and derivatives thereof (
For example, a gene therapy approach utilizing molecular biology techniques and procedures by responding to 1,25 (OH) ₂ D) may be used. Or 1α-
Target cells can be genetically engineered to encode and produce OHase. This 1α-OHase is known to convert 25 (OH) D to 1,25 (OH) ₂ D. The gene of interest can be identified using standard probes and sequencing techniques, cloned or otherwise amplified, isolated, and placed in an appropriate vector for transfer to target cells. Can be inserted. further,
A target cell can be provided with a promoter sequence that activates the appropriate gene sequence in the genome of the target cell to direct the cell to produce a desired protein or promoter.

[0081] The gene therapy approach of the present invention can be used as a method of treating or preventing cancer. As used herein, such cancers include Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic leukemia, multiple myeloma, breast cancer, ovarian cancer, lung cancer,
Wilms tumor, testicular cancer, soft tissue sarcoma, chronic lymphocytic leukemia, primary macroglobulinemia, bladder cancer, chronic granulocytic leukemia, primary brain cancer
rcinoma), malignant melanoma, small cell lung cancer, gastric cancer, colon cancer, malignant pancreatic insulinoma, malignant carcinoid carcinoid, malignant melanoma, choriocarcinoma, mycosis fungoides, head and neck carcinoma
cinoma), osteogenic sarcoma, pancreatic cancer, acute granulocytic leukemia, hairy cell leukemia, rhabdomyosarcoma, kaposi sarcoma, urogenital cancer, thyroid cancer, esophageal cancer, malignant hypercalcemia, renal cell carcinoma, uterus Includes, but is not limited to, intimal cancer, polycythemia vera, essential thrombocytosis, adrenocortical cancer, skin cancer, and prostate cancer.

In this preferred embodiment of the invention, 1α-OHase DNA (eg, the nucleic acid sequence set forth in SEQ ID NO: 1) is a polynucleotide construct suitable for introducing a nucleic acid molecule into cells of an animal to be treated. To form a transfection vector. This transfection vector is then introduced into cells of the selected target tissue of the animal in vivo using any of a variety of methods known to those of skill in the art. Alternatively, naked DNA can be transfected into cells with or without cationic lipids.

Techniques for the construction of transfection vectors containing 1α-OHase DNA are well known in the art, and are generally described in “Working To
Ward Human Gene Therapy "Recombinant
DNA (Second Edition), Chapter 28, Watson, J .; D. (Ed.), Scient
ific American Books: New York (1992), pp. 567-581, or Sambrook et al., Molecular Clon.
ing: A Laboratory Manual, Cold Spring
Harbor, New York (1989).

A gene therapy approach that can be used to deliver the VDR and / or 1α-OHase gene is the injection of plasmid DNA (Horton, HM et al., Proc. Natl. Acad. Sci. USA 96 (4) : 1553-1
558 (1999)); transduction using adenovirus vectors (Waug)
h. M. Proc. Natl. Acad. Sci. USA 96 (3)
: 1065-1070 (1990)); transduction using retroviral vectors (Axelrod, JH et al., Proc. Natl. Acad. Sci. U.
SA 87: 5173-5177 (1990); Drumm, M .; L. Et Ce
II 62: 1227-1233 (1990); Krueger, G .; G. FIG. Et al.,
J. Invest. Dermatol. 112: 233-239 (1999);
Palmer, T .; D. Et al., Blood 73: 438-445 (1989);
And Rosenberg, S .; A. Et al. Eng. J. Med. 323: 5
70-578 (1990)); and gene transfer using liposomes (Mas
on, C.I. A. E. FIG. Et al., Nature Medicine 5 (2): 176-
182 (1999)). In addition, the construction of gene therapy vectors and general methods for introducing such vectors into mammals for therapeutic purposes can be obtained in the publications referred to above, the disclosure of which is incorporated by reference in their entirety. It is incorporated by reference in the specification in detail. In one such general method, the vector comprising the 1α-OHase DNA of the present invention is directly injected, preferably by injection, inhalation, ingestion, topical application, or solution, into the cell or tissue of the mammal to be treated. Introduced by introduction into the mucous membrane. Such applications are generally referred to as "in vivo".
It is called gene therapy.

[0085] Alternatively, cells or tissues can be removed from the mammal to be treated and placed in culture according to methods well known to those skilled in the art. Then, 1α-OHase D
The transfection vector or naked DNA containing NA can be introduced into these cells or tissues by any of the methods generally described above for introducing isolated polynucleotides into cells or tissues. . 1α-OHase
After a time sufficient to allow uptake of the DNA, the cells or tissues can then be reinserted into the mammal to be treated. Introduction of 1α-OHase gene
Since performed outside the body of a mammal, this approach is commonly referred to as "ex vivo".
It is called gene therapy. See U.S. Patent No. 5,399,346. Gene transfer by transfection of cells ex vivo can be performed by various 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.).

With reference to 1α-OHase, the term “gene” as used herein refers to 1
It is intended to refer to a DNA sequence that encodes an α-OHase enzyme. Therefore,
“1α-OHase gene” or “1α-OHase-expressing gene”
Segment of genomic DNA encoding 1α-OHase enzyme or 1α-OH
Any of the cDNA sequences encoding the ase enzyme may be mentioned.

For both in vivo and ex vivo gene therapy, the 1α-OH of the invention
The ase DNA can be operably linked to a regulatory DNA sequence for human 1α-OHase (shown in SEQ ID NO: 3), ie, a “promoter”, as described above, to form a genetic construct. Human 1α-OHase promoter and 1
This construct, which contains both α-OHase DNA, is constructed using a suitable vector (eg,
Plasmids, adenoviral vectors, retroviral vectors, etc.) and into an animal to be treated with an in vivo gene therapy approach,
Alternatively, they can be introduced into mammalian cells or tissues using an ex vivo approach.

Alternatively, the 1α-OHase DNA of the present invention can be operably linked to a heterologous regulatory DNA sequence, ie, a promoter, to form a genetic construct, as described above. Heterologous regulatory sequences can be tissue-specific. The vector containing the gene construct is then introduced directly into the animal to be treated or into the cells or tissues of the animal, as described above.

As used herein, the term “operably linked” refers to a region between a regulatory region (typically a promoter element but may include an enhancer element) and a gene. Which indicates that the transcription of the gene is under the control of a regulatory region.

The term “heterologous” refers to a DNA sequence that is not found in the natural genome. That is, two nucleic acid elements are said to be "heterologous" if they are from two different genes or from two different species. Therefore, the “heterologous DNA regulatory sequence” is a regulatory sequence of 1α-OHase.
Indicates that it is not naturally linked to the DNA sequence of the gene.

The term “promoter” is used according to its art-recognized meaning. Promoter is intended to mean a DNA region (usually upstream of the coding sequence of the gene), which binds RNA polymerase and directs the enzyme to the correct transcription start site.

In general, a promoter may be functional in various tissue types and several different species of organism, or its function may be restricted to a particular species and / or a particular tissue. In addition, the promoter can be constitutively active or under certain conditions (eg, if present in a genetic construct containing the promoter, if present).
In the presence of an enhancer element) or during a particular developmental stage of an organism (
For example, active in the fetus (fetus) but silent in adults (adults), it can be selectively activated by certain substances (eg, tissue-specific factors).

[0093] Promoters useful in the practice of the present invention are preferably "tissue-specific"-that is, they maintain the ability to be almost "silent" in other tissue types, while maintaining one tissue. Can drive gene transcription. Examples of tissue-specific promoters are provided in Table 2 below.

[0094]

[Table 2]

[0095]

[Table 3] For further examples of tissue-specific promoters, see US Pat. No. 5,834,3.
See Nos. 06 and 5,416,027 and the references cited therein.

In addition to a promoter, a genetic construct can also include other gene regulatory elements, such as enhancers, suppressor sequences, and silencers, that are used to regulate replication of the vector in target cells. obtain. The only prerequisite is that the genetic element is activated, derepressed, enhanced, or otherwise genetically genetically by a factor in the host cell rather than a non-target cell with respect to the method of treatment. Adjusted.

As used in the context of a nucleic acid construct, “element” refers to a region or nucleic acid fragment of a construct that has a defined function. For example, an enhancer element as used herein is a 1α-operably linked promoter.
When related to the OHase gene, it is a region of DNA that enhances transcription of the gene.

The term “enhancer” is used according to its art-recognized meaning. An enhancer is intended to mean a sequence found in eukaryotes that can enhance transcription from a gene when located up to several kilobases (in either orientation) from the gene being studied. These sequences are usually 5 'to the gene in question (
When located upstream, it acts as an enhancer. However, some enhancers are active when located 3 '(downstream) of the gene. In some cases, enhancer elements can activate transcription from a gene without a (known) promoter.

Preferred enhancers include DF3 breast cancer-specific enhancers and viral-derived enhancers and the steroid receptor family.
Other preferred transcription regulatory sequences include NF1, SP1, AP1, and FOS /
JUN.

The transfection vector of the present invention can be introduced into selected target tissue cells using any of a variety of methods known to those of skill in the art. Such methods include, for example, retrovirus, adeno-associated virus (AAV), herpes virus, vaccinia virus, or RNA virus (eg, Grunhaus and Ho).
rowitz, Semin. Virol. 3: 237-252 (1992); H
erz and Gerard, Proc. Natl. Acad. Sci. USA
90: 2812-2816 (1993); and Rosenfeld et al., Ce.
II 68: 143-155 (1992));
Liposome-mediated gene transfer (Morishita et al., J. Clin. Inves)
t. 91: 2580 (1993); Felgner et al., US Pat. No. 5,703.
055 (1997) and 5,858,784 (1990));
Direct injection of NA into target tissues (eg, Felgner et al., US Pat. No. 5,582)
9,466 (1996); Wolff et al., U.S. Patent No. 5,693,622 (
1997)); and receptor-mediated gene transfer (Wu and Wu, Bioc).
hemistry 27: 887-892 (1988); Wagner et al., PN.
AS USA 87: 3410-3414 (1990); Curiel et al., US Pat. No. 5,547,932 (1996); and Beug et al., US Pat.
, 354, 844 (1994)).

In any of these methods, if the vector can be targeted to selectively transfect a specific population of cells, local administration (such as can be achieved by injection into the target tissue). In addition, it is understood that the vector can be administered systemically (eg, intravenously) in a biologically compatible solution or pharmaceutically acceptable delivery vehicle. Vector constructs administered in this way can selectively infect target tissues. According to the present invention, the presence of a target tissue-specific promoter on the construct provides an independent means of limiting the expression of the therapeutic gene.

[0102] The gene therapy embodiments of the present invention provide methods for treating or preventing a number of diseases and disorders. For example, using this embodiment, the vitamin D receptor (VD
R) can treat or prevent cancer in cells that express it. A polynucleotide construct comprising a gene encoding 1α-OHase is introduced into a cancer cell, wherein:
This gene is subsequently expressed and expressed in an amount effective to inhibit cell growth.
Generate OHase. The gene encoding the VDR (eg, the nucleotide sequence shown in SEQ ID NO: 4) can also be co-administered with the 1α-OHase gene. In this embodiment of the invention, the VDR gene is 1α-
Subcloned into either the same polynucleotide construct as the OHase gene or a separate polynucleotide construct. Both genes are then introduced into target cells or tissues, where they are subsequently expressed, producing 1α-OHase and VDR in amounts effective to treat or prevent the condition.

Gene therapy with a gene that expresses 1α-OHase, and optionally a gene that expresses a VDR gene, can be performed substantially simultaneously with the administration of vitamin D or an analog or derivative thereof. Thus, vitamin D or an analog or derivative thereof may be administered to an animal before, during, or after receiving gene therapy.

In particular, the present invention can be used to treat or prevent prostate, breast, skin, colon, leukemia, lymphoma, and lung cancer.

The present invention also provides methods of treating or preventing any disease (eg, actinic keratosis) and non-cancerous hyperproliferative disorders (eg, including precancerous such as psoriasis and ichthyosis). I do. Here, 1,25 (OH) ₂ D ₃ can affect cell growth and maturation. In this embodiment of the invention, a polynucleotide construct comprising a gene encoding 1α-OHase is introduced into a cell, wherein 1,25 (OH) ₂ D ₃ can affect cell growth and maturation. The 1α-OHase gene is subsequently expressed to produce 1α-OHase in amounts sufficient to inhibit cell growth.

Thus, the present invention can be used for the treatment of actinic keratosis, a pre-skin cancer. The use of the present invention results in an increase in the amount of 1,25 (OH) ₂ D ₃ (or an analog or derivative thereof) in the cell, reduces proliferative activity and induces the cell to differentiate into a normal form I do.

Further, the present invention can also be used to treat skin hyperproliferative disorders such as psoriasis. This gene therapy approach is a reasonable alternative treatment for psoriasis, whereby cells cause or boost the production of 1,25 (OH) ₂ D ₃ (or their analogs or derivatives), and then by this,
Interacts with VDR and reduces proliferative activity that normalizes skin cells. This treatment can also be applied to other hyperproliferative skin disorders such as ichthyosis.

The present invention can also be used to treat or prevent disorders in calcium and bone metabolism. Polynucleotide constructs, again containing the gene encoding 1α-OHase, can be introduced into a cell population of an animal, where it is subsequently expressed and these cells are expressed in a similar manner to the kidney at 1,25 (OH). ) makes it possible to produce a ₂ D ₃ (or an analog or derivative thereof). Therefore, the present invention
Prevents bone disease associated with renal failure and reduces vitamin D-dependent type I rickets, X-linked hypophosphatemic rickets, vitamin D-dependent type II rickets, and osteoporosis, and hypoparathyroidism Can be used to treat disorders in calcium and bone metabolism, such as low serum calcium, caused by E. coli.

A further embodiment of the present invention provides a gene encoding VDR as well as 1α-OH
A method is provided for treating or preventing any of the above diseases or conditions, including cancer, by administering a gene encoding ase to target cells or tissues of an animal. Both genes can be subcloned into either the same or different polynucleotide constructs, as described above, and introduced into a target cell or tissue. 1α
Both the -OHase gene and the VDR gene are subsequently expressed, producing 1α-OHase and VDR in amounts effective to treat or prevent the condition.

Certain embodiments can be used with cells that either express VDR or do not already express it. In the former case (where the target cell already expresses VDR), it produces additional VDR that interacts with an increased cell concentration of 1,25 (OH) ₂ D (or an analog or derivative thereof). I do. In the latter case (where the cell does not already express VDR), the cell becomes responsive to 1,25 (OH) ₂ D (or an analog or derivative thereof). For example, cancer cells having either a defective VDR or the absence of a VDR can be treated according to this embodiment of the invention. The treated cells are then
To produce a VDR, which is then derived from 1,25 produced from 1α-OHase.
Interact with (OH) ₂ D (or their analogs or derivatives) in turn. This interaction is dependent on inhibition of growth and terminal differentiation (terminal dif).
elicits biological responses, including induction of fermentation, whereby cancer, precancerous, non-cancerous hyperproliferative disorders (non-cancerous hyperprr)
Treats olifiable disorders, enhances wound healing, and treats and prevents hair loss.

The present invention also relates to the use of 1α-OHa in cells that show benign prostate hyperplasia in animals.
Methods for treating benign prostate hyperplasia by introducing a polynucleotide construct comprising a gene encoding se are provided. The 1α-OHase gene is subsequently expressed, allowing the cells to produce 1,25 (OH) ₂ D (or an analog thereof) in an amount effective to treat benign prostate hyperplasia. . In this embodiment, the gene encoding the VDR also contains 1α-
It can be co-administered with the OHase gene. The gene expressing 1α-OHase and the gene expressing VDR can be on the same DNA construct or on separate DNA constructs.

The present invention also provides a method for treating an autoimmune disease in an animal. This method relies on introducing a polynucleotide construct comprising a gene encoding 1α-OHase into any cell of the animal that produces a beneficial result. This 1α-O
The Hase gene is subsequently expressed, allowing the cells to produce 1,25 (OH) ₂ D (or an analog thereof) in an amount effective to treat an autoimmune disease. In this embodiment, the gene encoding the VDR can also be co-administered with the 1α-OHase gene, as described above. The gene expressing 1α-OHase and the gene expressing VDR can be on the same DNA construct or on separate DNA constructs.

The present invention can be used to treat autoimmune diseases such as type 1 diabetes, multiple sclerosis, rheumatoid arthritis, or psoriatic arthritis. In this embodiment, for example, type 1 diabetes can produce beneficial results in any cell of the animal, especially pancreatic cells,
And more particularly, it can be treated by introducing a polynucleotide construct comprising a 1α-OHase gene (and, optionally, a VDR gene) into insulin-secreting pancreatic cells (eg, Langerhans islet cells). For use of the present invention in the treatment of multiple sclerosis, the 1α-OHase gene (and optionally,
Polynucleotide constructs comprising the (VDR gene) can be introduced, inter alia, into any neuronal cell that produces a beneficial result. Similarly, for use of the invention in the treatment of rheumatoid arthritis or psoriatic arthritis, the polynucleotide construct may be used in any connective tissue cells that produce beneficial results, or in animals afflicted with any of these diseases. It can be specifically introduced into or around the joint.

In a further embodiment of the present invention, the isolated nucleic acid molecule of the present invention is at least 90% identical to said isolated nucleic acid molecule, and more preferably at least 95%, 97%, 98%.
%, Or isolated nucleic acid molecules that are 99% identical. In particular, the present invention provides that the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 have at least 90%
, 95%, 97%, 98%, or 99% identical. The “% identity” between two nucleic acid sequences is determined using the default parameters.
fastA "computer algorithm (Pearso
n, W. R. And Lipman, D .; J. Proc. Natl. Acad.
Sci. USA 85: 2444 (1988)).

The term “isolated polynucleotide molecule” is intended to refer to a nucleic acid molecule that has been removed from its native environment. For example, a recombinant DNA molecule contained in a vector may be a recombinant DNA molecule maintained in a heterologous host cell or a (partially or substantially) purified DNA molecule in solution, as well as a DNA molecule of the present invention. It is considered isolated for purpose. Isolated polynucleotide molecule also includes such compounds that are produced synthetically.

The present invention further relates to the use of SEQ ID NO: 1 and SEQ ID NO: 3 under stringent conditions.
Can hybridize to a nucleic acid molecule having a sequence complementary to one of the nucleic acid sequences shown in SEQ ID NO: 1 or can hybridize directly to one of the nucleic acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 Related to nucleic acid molecules. "Stringent conditions"
With 50% formamide, 5 × SSC (750 mM NaCl, 15 mM
Overnight at 42 ° C. in a solution containing 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA (ssDNA). Incubation is followed by washing the filters in 0.1 × SSC at about 65 ° C.

The following are examples that illustrate procedures for practicing the present invention. These examples should not be construed as limiting. All percentages are by weight
All solvent mixing ratios are by volume unless otherwise indicated.

Example 1 Materials and Methods Used in the Cell Culture Synthesis of 1,25 (OH) ₂ D from 25 (OH) D by Cell Culture (A) Cell lines. The DU145, PC-3, and LNCaP cell lines were purchased from the American Type Culture Collection, R
Ockville, Maryland. All cell lines were tested and found to be free of Mycoplasma contaminants.

(B) Culture conditions. Prostate cancer cell lines were cultured after several passages in vitro. Cells were cultured in complete medium (fetal calf serum (10%, HyClone Labs.L).
ogan UT) and gentamicin (10 μg / ml, Life Tech)
noologies, Inc. , Gaithersburg, MD)
PMI1640 medium). Cells were plated in 35 mm culture dishes (Corn
ing-Costar, Cambridge, Mass.) at 1 × 10 ⁵ cells / dish in complete medium. The medium was spiked with RPMI containing serum-free medium (insulin (10 μg / ml), transferrin (10 μg / ml) and selenous acid (1 ng / ml) 24 hours before the addition of 25 (OH) D _3.
11640).

[0120] Primary cultures of human prostate epithelial cells were established and were described by Lookeshwar et al.
cancel Res. 53: 4493-4498 (1993). Serum-free defined medium (Mammary Epithelial)
Growth Medium (MEGM), Clontech, San Die
go, CA), prostate epithelial cells were cultured with anti-cytokeratin antibodies (CAM).
5.2) (Becton-Dickinson, Mountain View,
Expresses luminal epithelium-specific cytokeratins (cytokeratins 8 and 18) as detected immunohistochemically using CA). This serum-free medium is Epid
erm Growth Factor (25 ng / ml), hydrocortisone (
0.5 μg / ml), ethanolamine (1 × 10 ⁻⁴ ), insulin (5 μg / ml)
ml), transferrin (5 μg / ml), and bovine whole pituitary extract (70
μg / ml).

Prostate cells at their first passage in vitro were used and cultured in serum-free RPMI medium during incubation with vitamin D metabolites. Two primary cultures were studied: NP96-5 cultured from a histologically normal prostate of a 23-year-old Caucasian organ donor, and an open-look prostatectomy specimen of a 56-year-old Caucasian with BPH.
BPH96-11 cultured from T. SPECIMEN. Histological examination of adjacent tissue sections taken from specimens used for culture confirmed their identity as normal cultures or BPH cultures.

(C) Keratinocyte culture. Since 1α-OHase activity is well established in keratinocytes, cultured human keratinocytes were used for comparison to prostate culture. Keratinocytes were grown in culture according to 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 plated and 0
. Containing 15 mM calcium and bovine pituitary extract (3 μg / ml), EG
Proliferation in lethally irradiated 3T3 fibroblast feeders in serum-free basal medium supplemented with growth factors including F (25 ng / ml), insulin (5 μg / ml) and prostaglandin E ₁ (50 ng / ml) I let it. To enhance plating efficiency, cholera toxin (0.1 μg / ml) and hydrocortisone (200 n
g / ml) was added to the medium during the initial plating of the primary culture and subsequent subcultures. Cells were fed and maintained without cholera toxin and hydrocortisone, and used for enzyme assays.

(D) 25-hydroxyvitamin D-1α-hydroxylase (1α-OHa
se) Assay. 1α-OHase activity was measured in monolayer cultures of the cell lines and primary cultures described herein. This assay was performed using 2
5 (OH) D ₃ (50 nM), and DPPD (Sigma-Aldrich, Allentown, PA), antioxidants and free radical producing 1,25 (
Performed in the presence of a known inhibitor of OH) ₂ D. The assay was also performed with the cytochrome P450 inhibitor, clotrimazole (20 μM) (Sigma, St.
. (Louis, MO). After a 2 hour incubation at 37 ° C., the culture was placed on ice and the medium was removed. Immediately thereafter, 1 ml of methanol was added to extract 25 (OH) D ₃ and 1,25 (OH) ₂ D ₃ . A 10 μl aliquot of [ ³ H] -1,25 (OH) ₂ D ₃ containing 1000 cpm radioactivity in ethanol was also added to each well to calculate recovery. After extraction at room temperature for 15 minutes, the methanol extract was transferred to a glass tube and the cells were washed with an additional 0.5 ml of methanol.
The combined the extracts and washings were dried under a stream of nitrogen and redissolved in 1ml of acetonitrile, followed by addition of 0.4M K ₂ HPO ₄ (pH10.0) in 1ml. This mixture was then applied to a C-18-OH reverse phase cartridge.
The fractions containing 1,25 (OH) ₂ D were then dried under a stream of nitrogen and reconstituted in 200 μl ethanol. Two 40 μl aliquots were prepared according to Chen et al. Nutr. Biochem. 1: were taken for 1,25 (OH) ₂ D analyzed by thymus receptor binding assays as described in 320 through 327 (1990). In addition, using 1α-OHase activity as a substrate, a simple non-radioactive 25
20 μg instead of (OH) D ₃ Non-radioactive 25 (OH) D ₃ and 0.91 μC
It was determined in primary cultures of prostate cells by using the ^{[3 H] -25 (OH)} D 3 of i. Incubation medium, time, temperature, extraction procedure, and C-
18-OH cartridge chromatography was similar to that described for the thymus receptor method (10% methylene chloride in hexane).
fraction eluted from a C-18-OH cartridge with ene chlorine (25 (OH) D fraction) and a fraction eluted from a C-18-OH cartridge with 6% isopropanol in n-hexane ( The 1,25 (OH) ₂ D and 24,25 (OH) ₂ D fractions) were dried under nitrogen and methylene chloride: isopropanol (19:19) for high performance liquid chromatography analysis as described below. 1) except that it is redissolved during).

(E) High performance liquid chromatography (HPLC). Aliquot 30 μl
Mix with 10 μl each of standard non-radioactive 25 (OH) D and 1,25 (OH) ₂ D, and use methylene chloride: isopropanol (19: 1) solvent system as mobile phase, flow rate 0.5 ml. / Min, an Ecosphere silica column (5 μl)
Particle size, 250 mm x 4.6 mm). 30 fractions were collected from each HPLC
And collected every other minute. Fractions were evaporated with air for dryness, followed by addition of scintillation fluid and counting on a beta counter. The retention capacities for 25 (OH) D ₃ , 24,25 (OH) ₂ D ₃ , and 1,25 (OH) ₂ D ₃ were determined before, during, and after unknown sample application by HPL.
For the C column, correction was made by applying standard 25 (OH) D ₃ , 24,25 (OH) ₂ D ₃ , and 1,25 (OH) ₂ D ₃ . The protein concentration in each 35 mm dish was determined by standard procedures. This enzymatic activity is expressed in pmol
, 25 (OH) ₂ D ₃ / mg protein / hour.

(F) Keratinocyte transfection. Keratinocytes were purchased from MCDB-
153 medium. Cells in 24-well dishes at 50% -60% confluence were transfected with 1 mg / ml 1α-OHase cDNA subcloned into the pCR3.1 vector (INVITROGEN). An 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 at room temperature for 15 minutes. LIP
OFECTAMINE (4-6 μl) is added and DNA-LIPOFEC
The TAMINE mixture was incubated at room temperature for another 15 minutes. Cells, 3
Incubated for a time in the DNA-LIPOFECTAMINE mixture, after which the medium was changed. The cells were then incubated at different concentrations (1 nM, 10 nM, 100 nM).
18 hours nM), it was treated with 25 (OH) D _3. [ ³ H] thymidine was added to the culture and [ ³ H] thymidine incorporation into DNA (interpreted as a measure of DNA synthesis activity, ie, cell proliferation) was measured as previously described. (Che
n, T. C. Et al., J Nutr. Biochem. 4: 49-57 (1993)
).

Example 2 Cloning, Sequencing, and Expression of Human Keratinocyte 1α-OHase cDNA The 1α-OHase cDNA was prepared using reverse transcription-PCR (RT-PCR).
Cloned from human keratinocytes. 2,150 base pairs of 1α-OHas
The e cDNA, which is part of SEQ ID NO: 1, contains a 1,527 bp open reading frame predicted to encode a 508 amino acid (SEQ ID NO: 2) protein. This cDNA was cloned into pCR3.1-Uni (INVITRO
GEN) Subcloned directly into mammalian cell expression vector. DNA sequence analysis indicated that the cDNA in keratinocytes was 100% identical to kidney 1α-OHase. This construct was transferred to COS-1 cells (1,25 (OH
) ₂ D ₃ not be synthesized) when transfected, the 1α-OHase activity was demonstrated by high performance liquid chromatography (HPLC), which is an enzyme substrate ^{[3 H] -25 (OH)} D 3 the ^{[3 H] -1,25 (OH)} 2 D 3 produced from released min.

Example 3 Cloning and Partial Characterization of Human 1α-OHase Gene A human genomic DNA library (CLONETECH) was prepared using 1α-OHase.
Screening was performed by plaque hybridization using a 700 bp fragment from the 5 'region of the cDNA (SEQ ID NO: 1) as a probe. 4
One positive clone was identified. Using restriction enzymes, P1, P2, P3 respectively
Four genomic clones, designated P4, were digested. (BamHI, Sac-
I) P1 was 13 kb and P2 was 7.5 kb. These two clones contained some parts of the 5 'region as identified by PCR. The P1 clone was digested with Sac-I and 5.5 kb long, 3.5 k
Four fragments of b length, 3.0 kb length and 0.7 kb length were generated. These four fragments were first subcloned into the pGem7 cloning vector and Southern blot analysis was applied to identify subclones containing the 5 'flanking region. Sequence analysis using T7 and Sp6 promoter primers mapped the order of the fragments. The 5.5 kb fragment was found to encode exon 9 from intron 8. The 0.7 kb fragment encodes exon 6 to intron 8, the 3.5 kb fragment encodes the exon 6 to 5 'flanking region, and the 3.0 kb subclone was made from 1α-OHase cDNA. Could not be hybridized with any probe.

Example 4 Proliferative Activity of Keratinocytes Transfected with the Human 1α-OHase Gene as a Function of 1,25 (OH) ₂ D 1,25 (OH) After Transfection with 1α-OHase to test the ability of cells to produce ₂ D _3, the polynucleotide construct comprising 1α-OHase cloning gene (SEQ ID NO: 1), it was constructed as summarized in the section of the aforementioned methods, and normal human Cultured keratinocytes were used to transfect. The 1α-OHase gene were cultured and transfected with keratinocytes were incubated with 25 (OH) D ₃ in various concentrations. As a negative control, untransfected cultured keratinocytes were simultaneously exposed to the same concentration of 25 (OH) D under the same conditions. 1α
When cells transfected with -OHase gene increased the 1α-OHase activity in a cell, the cell is from 25 (OH) D, converts 1,25 (OH) to ₂ D, then more in cells It is more efficient in producing high concentrations. Thus, it has a more dramatic effect on reducing the proliferative activity of the cells. As can be seen in FIG. 6, keratinocytes transfected with the 1α-OHase gene have more pronounced proliferative activity for the same concentration of 25 (OH) D as compared to untransfected control keratinocytes. Significant reduction. This occurred at concentrations between 10 ^{-9 and} 10 ^-7 M. 1α-OHa
A significant decrease in proliferation activity of cells transfected with the se gene and incubated with 10 ( ^-7 ) M with 25 (OH) D of 53.7% compared to non-transfected keratinocytes (p < 0.01).

The examples and embodiments described herein are for illustrative purposes only, and various modifications or alterations in view of them will be suggested to those skilled in the art, and the spirit of the present application It is to be understood that they are to be included within the scope and scope of the appended claims. All publications cited herein,
Patents and patent applications are incorporated herein by reference in their entirety.

[Sequence list]

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of vitamin D metabolism in the body. Known metabolic steps are indicated by black arrows; synthesis of 1,25 (OH) ₂ D from 25 (OH) D by the prostate is indicated by white arrows.

FIG. 2 shows the presence or absence of the P450 inhibitor clotrimazole [ ^Three [H] -25 (OH) D, high-performance liquid of tritium activity of lipid extracts from cultured normal human prostate primary cultures or BPH cultures, incubated with H-25D
3 shows a body chromatography elution profile. FIG. 2 shows non-radioactive 25 (OH) D in the absence of clotrimazole._Three(50 nM), [^ThreeH] -25 (OH) D_Three(0.91 μCi / nmol), and 1,2
-Ink for 2 hours at 37 ° C with dianilinoethane (DPPD) (10 μM)
3 shows a second passage of normal human prostate cells that have been passaged.

FIG. 3 shows that [P450 inhibitor clotrimazole in the absence or presence of [ ^Three [H] -25 (OH) D, high-performance liquid of tritium activity of lipid extracts from cultured normal human prostate primary cultures or BPH cultures, incubated with H-25D
3 shows a body chromatography elution profile. FIG. 3 shows that in the presence of the P450 enzyme inhibitor, clotrimazole (20 μM
), Non-radioactive 25 (OH) D_Three(50 nM), [^ThreeH] -25 (OH) D_Three(0.91 μCi / nmol) and DPPD (10 μM) at 37 ° C. for 2 hours
2 shows a second passage of normal human prostate cells incubated for a second time.

FIG. 4 shows that [P450 inhibitor clotrimazole in the absence or presence of [ ^Three [H] -25 (OH) D, high-performance liquid of tritium activity of lipid extracts from cultured normal human prostate primary cultures or BPH cultures, incubated with H-25D
3 shows a body chromatography elution profile. FIG. 4 shows non-radioactive 25 (OH) D in the absence of clotrimazole._Three(50 nM), [^ThreeH] -25 (OH) D_Three(0.91 μCi / nmol), and DPP
D (10 μM), incubated at 37 ° C. for 2 hours, from BPH culture
Shows the second passage of the coming prostate cells.

FIG. 5 shows that in the absence or presence of P450 inhibitor clotrimazole [ ^Three [H] -25 (OH) D, high-performance liquid of tritium activity of lipid extracts from cultured normal human prostate primary cultures or BPH cultures, incubated with H-25D
3 shows a body chromatography elution profile. FIG. 5 shows the non-radioactive 25 (OH) D in the absence of clotrimazole._Three(50 nM), [^ThreeH] -25 (OH) D_Three(0.91 μCi / nmol), and DPP
D (10 μM), incubated at 37 ° C. for 2 hours, from BPH culture
Shows the second passage of the coming prostate cells.

FIG. 6 shows normal human kelp transfected with 1α-OHase cDNA.
25 (OH) D for proliferative activity of Tinocytes_ThreeA graph showing the effect of concentration is shown. Transfected with the vector containing the above 1α-OHase cDNA
Cultured human keratinocytes at different concentrations (1 nM, 10 nM, 100 nM)
18 hours, 25 (OH) D_ThreeAnd incubated with it. [^ThreeH] Thymidine removal
Was measured as described above. Closed circle: [[ ^Three H] Thymidine incorporation (“control”); open circle: [Transfected cells with vector containing 1α-OHase cDNA] [^ThreeH] Thymidine incorporation ("1α-OHase transfection").

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61P 17/02 13/08 17/06 17/02 17/14 17/06 19/08 17 / 14 25/00 19/08 29/00 101 25/00 35/00 29/00 101 43/00 35/00 C12N 9/12 43/00 15/00 ZNAA // C12N 9/12 A61K 37/48 ( 31) Priority claim number 60 / 122,268 (32) Priority date March 1, 1999 (1999.3.1) (33) Priority claim country United States (US) (31) Priority claim number 60 / 123,669 (32) Priority date March 9, 1999 (1999.3.9) (33) Priority country United States (US) (31) Priority claim number 60 / 123,670 (32) Priority date (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, CY, D) E, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, G, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, Z A, ZW (72) inventor Chen, Thailand Sea. United States Massachusetts 01776, Sudbury, Hawthorne Drive 18 (72) Inventor Witlatch, Lyman W. United States Massachusetts 02118, Boston, Blackwood No. 214 12 (72) Inventor Gong, Cianfu United States Massachusetts 02460, Newton, Bowers Street 26 (72) Inventor Holic, Michael F. United States Massachusetts 01776, Sudbury, Bishop Lane 31 F-term (reference) 4B024 AA12 BA08 BA63 CA04 DA02 EA04 GA11 4B050 CC01 CC03 DD11 LL03 4C084 AA13 AA17 BA44 DC25 MA01 NA14 ZB262 ZC192 4C087 AA01 AA02 BA10 MA01 NA14 Z

Claims (105)

[Claims] 1. A method for inhibiting tumor cells, said method increasing the level of a metabolic precursor of 1,25-dihydroxyvitamin D available to target cells, but exposing to UV radiation. Or reducing the risk of vitamin D toxicity, wherein the target cell has a hydroxylase for synthesizing 1,25-dihydroxyvitamin D from the metabolic precursor. 2. The method according to claim 1, wherein said hydroxylase is 25-hydroxyvitamin D.
The method according to claim 1, which is -1α-hydroxylase. 3. The method of claim 1, wherein said target cells are genetically altered to produce and respond to active vitamin D metabolites. 4. The method of claim 1, wherein said target cells are prostate cells. 5. The method of claim 1, wherein the method comprises the steps of: providing an effective amount of a metabolic precursor of 1,25-dihydroxyvitamin D to a patient having cancerous cells in a target organ. Wherein the precursor is metabolized by the cells of the target organ to 1,25-hydroxyvitamin D or a functional equivalent thereof. 6. The method of claim 5, wherein the metabolic precursor is 25-hydroxyvitamin D or an analog, derivative, salt, or functional equivalent thereof. 7. An effective amount of the precursor is about 25 nmol / L and 250 nmo.
7. The method of claim 6, wherein said amount produces an interorgan level of said metabolic precursor between 1 / L. 8. An analog or derivative of 25-hydroxyvitamin D,
Alkylated, arylated, halogenated, glycosylated, or carbon-1
7. The method of claim 6, which is 25-hydroxyvitamin D further hydroxylated at any carbon position other than. 9. The method of claim 1, wherein the 1,25-dihydroxyvitamin D metabolic precursor is administered as a composition comprising the metabolic precursor or a salt thereof, and a pharmaceutically acceptable carrier. . 10. The method of claim 1, wherein said method promotes differentiation of said target cells.
The method described in. 11. A method of preventing the growth of tumor cells, said method increasing the level of a metabolic precursor of 1,25-dihydroxyvitamin D, or an analog or derivative thereof, available to target cells. Comprises reducing the risk of UV irradiation exposure or vitamin D toxicity, wherein the target cells have a hydroxylase for synthesizing 1,25-dihydroxyvitamin D from the metabolic precursor ,Method. 12. A method for inhibiting the invasiveness of a cancer cell, said method comprising the steps of using a metabolic precursor of 1,25-dihydroxyvitamin D, or an analog or derivative thereof, available to target cells. Increasing the level, but reducing the risk of UV irradiation exposure or vitamin D toxicity, wherein the target cells are capable of synthesizing 1,25-dihydroxyvitamin D from the metabolic precursor. A method having a lase enzyme. 13. A method for inhibiting metastasis of a cancer cell, the method comprising:
Metabolic precursors of 1,25-dihydroxyvitamin D available to target cells,
Or increasing the level of an analog or derivative thereof, but reducing the risk of UV irradiation exposure or vitamin D toxicity, wherein the target cell comprises:
A method comprising a hydroxylase enzyme for synthesizing 1,25-dihydroxyvitamin D from said metabolic precursor. 14. The method of claim 1, wherein the inhibiting is inhibiting proliferation of the tumor cell.
The method of claim 1. 15. A method for inhibiting the growth of tumor cells in an animal and / or inducing differentiation of said cells, said method comprising the steps of: providing a poly-protein comprising a gene expressing 25-hydroxyvitamin D-1α-hydroxylase. Introducing a nucleotide construct into the cells in need thereof, thereby producing 1,25-dihydroxyvitamin D or an analog thereof, and inhibiting the growth of the cells. 16. The method of claim 15, wherein said construct comprises homologous regulatory sequences. 17. The method of claim 16, wherein the homologous regulatory sequence is: (a) at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 3;
And (b) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a) or to a polynucleotide having a component thereof, and (b) a nucleotide sequence selected from the group consisting of: ,Method. 18. The method of claim 17, wherein said homologous regulatory sequence comprises a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth 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 set forth in SEQ ID NO: 3. 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 set forth 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 set forth in SEQ ID NO: 3. 22. The method of claim 17, wherein said homologous regulatory sequence comprises the nucleotide sequence set forth 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 the step of introducing a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cells. 26. The method of claim 25, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 27. The method of claim 25, wherein said gene expressing said vitamin D receptor and 25-hydroxyvitamin D-1α-.
The method wherein the genes expressing hydroxylase are on separate DNA constructs. 28. The prostate cancer cell, breast cancer cell, skin cancer cell,
16. The method of claim 15, wherein the method is selected from the group consisting of colon cancer cells, lung cancer cells, leukemia cells, and lymphoma cells. 29. A method for detecting a risk of cancer, the method comprising measuring the level of vitamin D, a vitamin D metabolite, or a vitamin D metabolizing enzyme in a target organ; and the measured value. Correlating with a cancer risk level. 30. The vitamin D metabolite is 25-hydroxyvitamin D,
30. The method of claim 29, which is or an analog or derivative thereof. 31. The method of claim 29, wherein said vitamin D metabolite is 1,25-dihydroxyvitamin D. 32. The vitamin D metabolizing enzyme is 25-hydroxyvitamin D
30. The method according to claim 29, which is -1α-hydroxylase. 33. The method of claim 32, wherein 25-hydroxyvitamin D-1α-hydroxylase levels are measured indirectly from their activity. 34. A method for predicting a successful treatment of a cancer, comprising: measuring the level of vitamin D, a vitamin D metabolite, or a vitamin D metabolizing enzyme in a target organ; Correlating the measurement with the success of the treatment. 35. A composition for inhibiting the growth, invasion, or metastasis of a cancerous cell, wherein the composition is a vitamin D metabolite that is a precursor of 1,25-dihydroxyvitamin D metabolite, and a pharmaceutical composition. A composition comprising a chemically acceptable carrier. 36. A method of treating benign prostate hyperplasia in an animal, comprising:
The method comprises introducing a polynucleotide construct comprising a gene that expresses 25-hydroxyvitamin D-1α-hydroxylase into cells exhibiting a benign prostate hyperplasia in need thereof, whereby 1,25-dihydroxyvitamin D or A method wherein said analog is produced and said hyperplasia is treated. 37. The method of claim 36, wherein said construct comprises homologous regulatory sequences. 38. The method of claim 37, wherein said homologous regulatory sequence comprises a nucleotide sequence as set forth 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 a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cells. 42. The method of claim 41, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 43. The method according to claim 41, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on separate DNA constructs. 44. A method of treating a hyperproliferative dermatological disorder in an animal, said method comprising the steps of:
Introducing a polynucleotide construct comprising a gene that expresses -1α-hydroxylase, thereby producing 1,25-dihydroxyvitamin D or an analog thereof, and inhibiting the growth of the cell. . 45. The method of claim 44, wherein said construct comprises homologous regulatory sequences. 46. The method of claim 45, wherein said homologous regulatory sequence comprises the nucleotide sequence set forth 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 the step of introducing a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cells. 50. The method of claim 49, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 51. The method of claim 49, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on separate DNA constructs. 52. The method of claim 44, wherein said hyperproliferative skin disorder is psoriasis. 53. The method of claim 44, wherein said hyperproliferative skin disorder is selected from the group consisting of ichthyosis and actinic keratosis. 54. A method of treating a disorder in calcium and bone metabolism in an animal, the method comprising providing, in a cell of the animal in need thereof, a gene expressing 25-hydroxyvitamin D-1α-hydroxylase. A polynucleotide construct comprising 1,25-dihydroxyvitamin D or an analog thereof, and treating the disorder. 55. The method of claim 54, wherein said construct comprises homologous regulatory sequences. 56. The method of claim 55, wherein said homologous regulatory sequence comprises the nucleotide sequence set forth 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. 59. The method of claim 54, further comprising introducing a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cell. 60. The method of claim 59, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 61. The method according to claim 59, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on separate DNA 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 disorder of calcium and bone metabolism comprising the group consisting of vitamin D-dependent rickets I, X-linked hypophosphatemic rickets, vitamin D-dependent rickets II, and osteoporosis. 55. The method of claim 54, wherein the method is selected from: 65. The method of claim 54, wherein said disorder in calcium and bone metabolism is low serum calcium due to parathyroid dysfunction. 66. A method of treating or preventing hair loss in an animal, the method comprising: adding 25-hydroxyvitamin D-1α to the hair cells in need thereof.
-Introducing a polynucleotide construct comprising a gene that expresses hydroxylase,
Thereby producing 1,25-dihydroxyvitamin D or an analog thereof and treating or inhibiting the hair loss. 67. The method of claim 66, wherein said construct comprises homologous regulatory sequences. 68. The method of claim 67, wherein said homologous regulatory sequence comprises the nucleotide sequence set forth 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 a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cells. 72. The method of claim 71, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 73. The method of claim 71, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on separate DNA constructs. 74. The method of claim 66, wherein the hair loss is a result of a condition selected from the group consisting of male pattern baldness, female pattern baldness, and chemotherapy-induced alopecia. Is the way. 75. A method for enhancing wound healing in an animal, comprising introducing a polynucleotide construct comprising a gene expressing 25-hydroxyvitamin D-1α-hydroxylase into wound cells in need thereof. And thereby producing 1,25-dihydroxyvitamin D or an analog thereof, and enhancing the healing of the wound. 76. The method of claim 75, wherein said construct comprises homologous regulatory sequences. 77. The method of claim 76, wherein said homologous regulatory sequence comprises the nucleotide sequence set forth 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 a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cells. 81. The method of claim 80, wherein said gene expressing said vitamin D receptor and said 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 82. The method of claim 80, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on separate DNA constructs. 83. A method of treating an autoimmune disease in an animal, said method comprising, in a cell of said animal in need thereof, a polynucleotide comprising a gene that expresses 25-hydroxyvitamin D-1α-hydroxylase. Introducing the construct, thereby producing 1,25-dihydroxyvitamin D or an analog thereof,
And a method wherein said autoimmunity is treated. 84. The method of claim 83, wherein said construct comprises homologous regulatory sequences. 85. The method of claim 84, wherein said homologous regulatory sequence comprises the nucleotide sequence shown 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 a polynucleotide construct comprising a gene that expresses a vitamin D receptor into said cells. 89. The method of claim 88, wherein said gene expressing said vitamin D receptor and said 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on the same DNA construct. 90. The method of claim 88, wherein the gene expressing the vitamin D receptor and the 25-hydroxyvitamin D-1α-
The method, wherein the genes that express hydroxylase are on separate DNA constructs. 91. The method of claim 83, wherein said autoimmune disease is selected from the group consisting of type I diabetes mellitus, multiple sclerosis, rheumatoid arthritis, and psoriatic arthritis. ,Method. 92. An isolated polynucleotide molecule comprising a 25-hydroxyvitamin D-1α-hydroxylase regulatory sequence or its complement. 93. The polynucleotide molecule of claim 92, wherein said regulatory sequence is: (a) at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 3;
A polynucleotide comprising a polynucleotide selected from the group consisting of: a polynucleotide comprising a nucleotide sequence; and (b) a polynucleotide that hybridizes under stringent conditions to the polynucleotide of (a) or a complement thereof. Nucleotide molecule. 94. The polynucleotide molecule of claim 93, wherein said polynucleotide comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence set forth in SEQ ID NO: 3. 95. The polynucleotide molecule of claim 93, wherein said polynucleotide comprises a nucleotide sequence that is at least 97% identical to the nucleotide sequence set forth in SEQ ID NO: 3. 96. The polynucleotide molecule of claim 93, wherein said polynucleotide comprises a nucleotide sequence that is at least 98% identical to the nucleotide sequence set forth in SEQ ID NO: 3. 97. The polynucleotide molecule of claim 93, wherein said polynucleotide comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence set forth in SEQ ID NO: 3. 98. The polynucleotide molecule of claim 93, wherein said polynucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 3. 99. The polynucleotide molecule of claim 92, further comprising a nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase. 100. The polynucleotide molecule of claim 99, wherein the nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase is at least as large as the sequence set forth in SEQ ID NO: 1. A polynucleotide molecule that is 90% identical. 101. The polynucleotide molecule of claim 99, wherein the nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase is at least as large as the sequence set forth in SEQ ID NO: 1. A polynucleotide molecule that is 95% identical. 102. The polynucleotide molecule of claim 99, wherein the nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase is at least as large as the sequence set forth in SEQ ID NO: 1. A polynucleotide molecule that is 97% identical. 103. The polynucleotide molecule of claim 99, wherein the nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase is at least as large as the sequence set forth in SEQ ID NO: 1. A polynucleotide molecule that is 98% identical. 104. The polynucleotide molecule of claim 99, wherein the nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase is at least as large as the sequence set forth in SEQ ID NO: 1. A polynucleotide molecule that is 99% identical. 105. The polynucleotide molecule of claim 99, wherein the nucleotide sequence encoding 25-hydroxyvitamin D-1α-hydroxylase comprises the sequence set forth in SEQ ID NO: 1. Nucleotide molecule.