U.S. patent application number 10/441731 was filed with the patent office on 2003-11-06 for method of treating malignancy associated hypercalcemia using active vitamin d analogues.
This patent application is currently assigned to Bone Care International, Inc.. Invention is credited to Bishop, Charles W., Mazess, Richard B..
Application Number | 20030207810 10/441731 |
Document ID | / |
Family ID | 25398783 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207810 |
Kind Code |
A1 |
Bishop, Charles W. ; et
al. |
November 6, 2003 |
Method of treating malignancy associated hypercalcemia using active
vitamin D analogues
Abstract
Methods utilizing active vitamin D analogs for the treatment of
malignancy-associated hypercalcemia. Methods comprise the
application of an effective amount of a hypocalcemic vitamin D
compound to alleviate hypercalcemia, lower serum parathyroid
hormone related protein (PTHrP) levels.
Inventors: |
Bishop, Charles W.;
(Madison, WI) ; Mazess, Richard B.; (Madison,
WI) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH, LLP
ONE SOUTH PINCKNEY STREET
P O BOX 1806
MADISON
WI
53701
|
Assignee: |
Bone Care International,
Inc.
Middleton
WI
|
Family ID: |
25398783 |
Appl. No.: |
10/441731 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10441731 |
May 20, 2003 |
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09891763 |
Jun 26, 2001 |
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6566353 |
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09891763 |
Jun 26, 2001 |
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09596149 |
Feb 23, 1998 |
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6537982 |
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09596149 |
Feb 23, 1998 |
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08781910 |
Dec 30, 1996 |
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5763429 |
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Current U.S.
Class: |
514/167 ;
514/19.3; 514/19.4 |
Current CPC
Class: |
A61K 31/59 20130101;
A61P 3/02 20180101; A61P 35/00 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 038/00 |
Claims
What is claimed is:
1. A method of treating hypercalcemia associated with malignant or
neoplastic cells, comprising treating the cells with an effective
amount of a hypocalcemic vitamin D compound having a hydrocarbon
moiety at the C.sub.24 position.
2. The method of claim 1, wherein the cells are cancers of the
breast, colon, lung, neck and head, pancreas, endometrium, bladder,
cervix, testes, ovaries, squamous cell carcinoma, myeloid and
lymphocytic leukemia, lymphoma, medullary thyroid carcinoma,
melanoma, multiple myeloma, retinoblastoma or sarcomas of the soft
tissues and bone.
3. The method of claim 1, wherein the hypocalcemic vitamin D is a
compound represented by formula (I) 4wherein A.sup.1 and A.sup.2
each are hydrogen or a carbon-carbon bond, thus forming a double
bond between C-22 and C-23; R.sup.1 and R.sup.2 are identical or
different and are hydrogen, hydroxyl, lower alkyl, lower
fluoroalkyl, O-lower alkyl, lower alkenyl, lower fluoroalkenyl,
O-lower alkenyl, O-lower acyl, O-aromatic acyl, lower cycloalkyl
with the proviso that R.sup.1 and R.sup.2 cannot both be an alkenyl
group, or taken together with the carbon to which they are bonded,
form a C.sub.3-C.sub.8 cyclocarbon ring; R.sup.3 is lower alkyl,
lower alkenyl, lower fluoroalkyl, lower fluoroalkenyl, O-lower
alkyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl or lower
cycloalkyl; X.sup.1 is hydrogen or hydroxyl, or, taken with
R.sup.3, constitutes a bond when R.sup.3 is an alkenyl group, and
X.sup.2 is hydrogen or hydroxyl, or, taken with R.sup.1 or R.sup.2,
constitutes a double bond, and X.sup.3 is hydrogen or hydroxyl
provided that at least one of X.sup.1, X.sup.2 and X.sup.3 is
hydroxyl; and Y is a methylene group if the bond to Y is a double
bond or is a methyl group or hydrogen if the bond to Y is a single
bond.
4. The method of claim 1, wherein said hypocalcemic vitamin D is a
1.alpha.-hydroxvitamin D compound is represented by formula (I)
5wherein A.sup.1 and A.sup.2 each are hydrogen or a carbon-carbon
bond, thus forming a double bond between C-22 and C-23; R.sup.1 and
R.sup.2 are identical or different and are hydrogen, hydroxyl,
lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower
fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl,
lower cycloalkyl with the proviso that R.sup.1 and R.sup.2 cannot
both be an alkenyl group, or taken together with the carbon to
which they are bonded, form a C.sub.3-C.sub.8 cyclocarbon ring;
R.sup.3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower
fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl or lower cycloalkyl; X.sup.1 is hydrogen or
hydroxyl, or, taken with R.sup.3, constitutes a bond when R.sup.3
is an alkenyl group, and X.sup.2 is hydrogen or hydroxyl, or, taken
with R.sup.1 or R.sup.2, constitutes a double bond.
5. The method of claim 4, wherein the compound of formula (I) is
1.alpha.,24-dihydroxyvitamin D.sub.2, 1.alpha.,24-dihydroxyvitamin
D.sub.4, 1.alpha.,25-dihydroxyvitamin D.sub.2,
1.alpha.,25-dihydroxyvitam- in D.sub.4, 1.alpha.-hydroxyvitamin
D.sub.2 or 1.alpha.-hydroxyvitamin D.sub.4.
6. A method in accordance with claim 1, wherein a dosing regimen
for the hypocalcemic vitamin D compound is a daily regimen or an
episodic regimen.
7. A method in accordance with claim 6, wherein the espisodic
regimen is a dose once every 2 to 7 days.
8. A method in accordance with claim 6, wherein the hypocalcemic
vitamin D compound is administered daily at a dose of about 10 to
100 .mu.g/day.
9. A method in accordance with claim 6, wherein the hypocalcemic
vitamin D compound is orally, intravenously or regionally delivered
to a cancer site.
10. A method in accordance with claim 9, wherein the hypocalcemic
vitamin D compound is administered orally.
11. A method in accordance with claim 1, wherein the hypocalcemic
vitamin D compound is co-administered with a cytotoxic agent.
12. A method in accordance with claim 11, wherein the cytotoxic
agent is an antimetabolite, and antimicrotubule agent, an alkyating
agent, a platinum agent, an anthracycline, a topoisomase inhibitor,
or an antibiotic.
13. A method in accordance with claim 12, wherein the
antimetabolite is 5-fluoro-uracil, methotrexate or fludarabine.
14. A method in accordance with claim 12, wherein the
antimicrotubule agent is vincristine, vinblastine or a taxane.
15. A method in accordance with claim 14, wherein the taxane is
paclitaxel or docetaxel.
16. A method in accordance with claim 12, wherein the alkylating
agent is cyclophasphamide, melphalan, biochoroethylnitrosurea or
hydroxyurea.
17. A method in accordance with claim 12, wherein the platinum
agent is cisplatin, carboplatin, oxaliplatin, JM-216 or CI-973.
18. A method in accordance with claim 12, wherein the anthracycline
is doxrubicin or daunorubicin.
19. A method in accordance with claim 12, wherein the antibiotic is
mitomycin, idarubicin, adriamycin or daunomycin.
20. A method in accordance with claim 12, wherein the topoisomerase
inhibitior is etoposide or camptothecins.
21. A method in accordance with claim 12 wherein the cytotoxic
agent is estramustine phosphate or prednimustine.
22. A method of treating a human to alleviate hypercalcemia
associated with breast cancer, colon cancer, prostate cancer,
testicular cancer, pancreatic cancer, endometrial cancer, small
cell and non-small cell cancer of the lung (including squamous,
adneocarcinoma and large cell types), squamous cell of the head and
neck, bladder, ovarian and cervical cancers, myeloid and
lymphocyltic leukemia, lymphoma, hepatic tumors, medullary thyroid
carcinoma, multiple myeloma, melanoma, retinoblastoma or sarcomas
of the soft tissue and bone, comprising administering to the human
therapeutic amount of a hypocalcemic vitamin D compound.
23. A method of claim 22, wherein said hypocalcemic vitamin D is a
1.alpha.-hydroxyvitamin D compound represented by formula (III)
6wherein A.sup.1 and A.sup.2 each are hydrogen or a carbon-carbon
bond, thus forming a double bond between C-22 and C-23; R.sup.1 and
R.sup.2 are identical or different and are hydrogen, hydroxyl,
lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower
fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl,
lower cycloalkyl with the proviso that R.sup.1 and R.sup.2 cannot
both be an alkenyl group, or taken together with the carbon to
which they are bonded, form a C.sub.3-C.sub.8 cyclocarbon ring;
R.sup.3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower
fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl or lower cycloalkyl; X.sup.1 is hydrogen or
hydroxyl, or, taken with R.sup.3, constitutes a bond when R.sup.3
is an alkenyl group, and X.sup.2 is hydrogen or hydroxyl, or, taken
with R.sup.1 or R.sup.2, constitutes a double bond.
24. The method of claim 23, wherein said therapeutic amount is 0.01
.mu.g/kg/day to 2.0 .mu.g/kg/day.
25. The method of claim 23, wherein the compound of formula (I) is
1.alpha.,24-dihydroxyvitamin D.sub.2, 1.alpha.,24-dihydroxyvitamin
D.sub.4, 1.alpha.,25-dihydroxyvitamin D.sub.2,
1(1,25-dihydroxyvitamin D.sub.4, 1.alpha.-hydroxyvitamin D.sub.2 or
1.alpha.-hydroxyvitamin D.sub.4.
26. A method of treating a human to alleviate hypercalcemia
associated with malignant cells, comprising administering to the
patient a hypocalcemic vitamin D compound, and a cytotoxic
agent.
27. A method in accordance with claim 26, wherein the hypocalcemic
vitamin D compound is administered from 0.5 to 7 days prior to
administration of the cytotoxic agent.
28. A method in accordance with claim 26, wherein the hypocalcemic
vitamin D compound is administered 2 to 4 days prior to
administration of the cytotoxic agent.
29. A method of claim 26, wherein said hypocalcemic vitamin D is a
1.alpha.-hydroxyvitamin D compound represented by formula (III)
7wherein A.sup.1 and A.sup.2 each are hydrogen or a carbon-carbon
bond, thus forming a double bond between C-22 and C-23; R.sup.1 and
R.sup.2 are identical or different and are hydrogen, hydroxyl,
lower alkyl, lower fluoroalkyl, O-lower alkyl, lower alkenyl, lower
fluoroalkenyl, O-lower alkenyl, O-lower acyl, O-aromatic acyl,
lower cycloalkyl with the proviso that R.sup.1 and R.sup.2 cannot
both be an alkenyl group, or taken together with the carbon to
which they are bonded, form a C.sub.3-C.sub.8 cyclocarbon ring;
R.sup.3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower
fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl or lower cycloalkyl; X.sup.1 is hydrogen or
hydroxyl, or, taken with R.sup.3, constitutes a bond when R.sup.3
is an alkenyl group, and X.sup.2 is hydrogen or hydroxyl, or, taken
with R.sup.1 or R.sup.2, constitutes a double bond.
30. The method of claim 29, wherein the therapeutic amount is 0.01
.mu.g/kg/day to 2.0 .mu.g/kg/day.
31. The method of claim 29, wherein the compound of formula (I) is
1.alpha.,24-dihydroxyvitamin D.sub.2, 1.alpha.,24-dihydroxyvitamin
D.sub.4, 1.alpha.,25-dihydroxyvitamin D.sub.2,
1.alpha.,25-dihydroxyvitam- in D.sub.4, 1.alpha.-hydroxyvitamin
D.sub.2 or 1.alpha.-hydroxyvitamin D.sub.4.
32. A method in accordance with claim 29, wherein the cytotoxic
agent is an antimetabolite, and antimicrotubule agent, an alkyating
agent, a platinum agent, an anthracycline, a topoisomase inhibitor,
or an antibiotic.
33. A method of lowering serum parathyroid hormone related protein
in a human patient by administering to the human an effective
amount of a hypocalcemic vitamin D compound.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 09/596,149 filed Feb. 23, 1998, which is a
continuation-in-part of U.S. application Ser. No. 08/781,910, filed
Dec. 20, 1996, now U.S. Pat. No. 5,763,429, all of which are
incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not Applicable
BACKGROUND OF THE INVENTION
[0003] This invention relates generally to a method of treating
malignancy-associated hypercalcemia (MAH), and in particular, to
the use of active forms of vitamin D to reduce hypercalcemia
associated with inhibit the hyperproliferative diseases.
[0004] Extensive research during the past two decades has
established important biologic roles for vitamin D apart from its
classic role in bone and mineral metabolism. Specific nuclear
receptors for 1.alpha.,25-dihydroxyvitamin D.sub.3, the hormonally
active form of vitamin D, are present in cells from diverse organs
not involved in calcium homeostasis. For example, specific,
biologically active vitamin D receptors have been demonstrated in
the human prostatic carcinoma cell line, LNCaP, (Miller et al., 52
Cancer Res. (1992) 515-520); Vitamin D receptors have also been
described for many other neoplastic cells, e.g., carcinomas of the
breast and carcinomas of the colon.
[0005] It has been reported that certain vitamin D compounds and
analogues are potent inhibitors of malignant cell proliferation and
are inducers/stimulators of cell differentiation. For example, U.S.
Pat. No. 4,391,802 issued to Suda et al. discloses that
1.alpha.-hydroxyvitamin D compounds, specifically
1.alpha.,25-dihydroxyvitamin D.sub.3 and 1.alpha.-hydroxyvitamin
D.sub.3, possess potent antileukemic activity by virtue of inducing
the differentiation of malignant cells (specifically leukemia
cells) to nonmalignant macrophages (monocytes), and are useful in
the treatment of leukemia. Antiproliferative and differentiating
actions of 1.alpha.,25-dihydroxyvitamin D.sub.3 and other vitamin
D.sub.3 analogues have been reported with respect to cancer cell
lines. More recently, an association between vitamin D receptor
gene polymorphism and cancer risk has been reported, suggesting
that vitamin D receptors may have a role in the development, and
possible treatment, of cancer.
[0006] These previous studies have focused exclusively on vitamin
D.sub.3 compounds. Even though these compounds may indeed be highly
effective in promoting differentiation in malignant cells in
culture, their practical use in differentiation therapy as
anticancer agents is severely limited because of their equally high
potency as agents affecting calcium metabolism. At the levels
required in vivo for effective use as, for example, antileukemic
agents, these same compounds can induce markedly elevated and
potentially dangerous blood calcium levels by virtue of their
inherent calcemic activity. That is, the clinical use of
1.alpha.,25-dihydroxyvitamin D.sub.3 and other vitamin D.sub.3
analogues as anticancer agents is precluded, or severely limited,
by the risk of hypercalcemia.
[0007] Hyperalcemia is frequently associated with malignancy (MAH),
and is often a major contributor to morbidity and complicates
clinical management of the malignancy. Parathyroid hormone related
protein (PTHrP) is closely related to parathyroid hormone (PTH) and
binds to the same receptor as PTH as well as other receptors. PTHrP
is one of the main causative substances of such hypercalcemia, and
is overproduced by malignant cells. 1,25-dihydroxyvitamin D.sub.3
has been found to repress the transcription of the PTHrP gene in
cells, however, the 1,25-dihydroxyvitamin D.sub.3 compounds
themselves increase serum calcium levels. Therefore a need exists
for compounds with greater specific activity and selectivity of
action, i.e., vitamin D compounds with antiproliferative and
differentiating effects but which have less calcemic activity.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides a method of treating
malignancy-associated hypercalcemia (MAH) such as that associated
with hyperproliferative cell growth and/or abnormal cell
differentiation. The method includes use of active vitamin D
compounds to treat hypercalcemia and reduce serum parathyroid
hormone related protein (PTHrP) levels.
[0009] The foregoing, and other advantages of the present
invention, are realized in one aspect thereof in a method of
treating malignancy-associated hypercalcemia from the
hyperproliferative activity of human neoplastic or hyperplastic
cells, comprising treating the cells with an effective amount of a
hypocalcemic hydroxyvitamin D compound having a hydrocarbon moiety
substituted at the C-24 position on the sidechain of the molecule.
The treating step includes inhibiting proliferation of, and
inducing and enhancing differentiation in such cells.
[0010] A hydroxyvitamin D compound in accordance with the present
invention is an active vitamin D and is suitably represented by the
formula (I) described hereafter. Suitable compounds of formula (I)
are 1.alpha.,24-dihydroxyvitamin D.sub.2,
1.alpha.,24-dihydroxyvitamin D.sub.4, 1.alpha.,25-dihydroxyvitamin
D.sub.4, 1.alpha.,25-dihydroxyvitam- in D.sub.2,
1.alpha.-hydroxyvitamin D.sub.2 and 1.alpha.-hydroxyvitamin
D.sub.4.
[0011] The effective or therapeutic amount of the hypocalcemic
hydroxyvitamin D compounds administrable in accordance with the
present invention to patients in need on a daily basis per kilogram
of body weight ranges from 0.01 .mu.g/kg/day to 2.0
.mu.g/kg/day.
[0012] In another aspect of the invention, lowering serum
parathyroid hormone related protein (PTHrP) levels in patients
suffering from hypercalcemia is accomplished by a method
comprising, administering to these patients an effective amount of
a hypocalcemic vitamin D compound, to lower the serum parathyroid
hormone related protein (PTHrP) level.
[0013] The hypocalcemic vitamin D compounds are also valuable for
the treatment of breast, prostate and colon cancer, as well as
other neoplasms such as pancreatic cancer, endometrial cancer,
testicular cancer, small cell and non-small cell cancer of the lung
(including squamous, adneocarcinoma and large cell types), squamous
cell of the head and neck, bladder, ovarian and cervical cancers,
myeloid and lymphocyltic leukemia, lymphoma, hepatic tumors,
medullary thyroid carcinoma, multiple myeloma, retinoblastoma, and
sarcomas of the soft tissue and bone, i.e. neoplasms that express a
vitamin D receptor.
[0014] In accordance with the present invention, when effective
amounts of the hypocalcemic vitamin D compounds are administered to
patients with MAH, significantly redeuced hypercalcemia is observed
than is observed after the same amount of an activated vitamin
D.sub.3 (e.g., 1.alpha.-OH D.sub.3, 1.alpha.,25-(OH).sub.2 D.sub.3)
is administered in previously known formulations. Thus, the
compound in accordance with the present invention has an improved
therapeutic index relative to active forms of vitamin D.sub.3
analogues.
[0015] Accordingly, another aspect of the invention is a method of
treating malignancy associated hyercalcemia comprising
administering to a subject who is suffering therefrom an effective
amount of active vitamin D compound which has, or attains through
metabolism in vivo, a vitamin D receptor (VDR) binding affinity
substantially equivalent to the binding affinity of
1.alpha.,25-dihydroxyvitamin D.sub.3 and has a hypercalcemia risk
substantially lower that that of 1.alpha.,25-dihydroxyvitamin
D.sub.3, to normalize or reduce serum calcium levels.
[0016] For treatment for malignancy-associated hypercalcemia and
the underlying malignant condition in accordance with the present
invention, the active vitamin D is suitably administered alone as
an active ingredient in a pharmaceutical composition, or is
co-administered with an anticancer agent.
[0017] Further, included within the scope of the present invention
is the co-administration of a hypocalcemic vitamin D compound with
a cytotoxic or anticancer agent. Such agents suitably include
antimetabolites (e.g., 5-fluoro-uracil, methotrexate, fludarabine),
antimicrotubule agents (e.g., vincristine, vinblastine, taxanes
such as paclitaxel, docetaxel), an alkylating agent (e.g.,
cyclophasphamide, melphalan, biochoroethylnitrosurea, hydroxyurea),
platinum agents (e.g. cisplatin, carboplatin, oxaliplatin, JM-216,
CI-973), anthracyclines (e.g., doxrubicin, daunorubicin),
antibiolitics (e.g., mitomycin, idarubicin, adriamycin,
daunomycin), topoisomerase inhibitiors (e.g., etoposide,
camptothecins) or any other antineoplastic agents. (estramustine
phosphate, prednimustine).
[0018] It is anticipated that the active vitamin D compounds used
in combination with various anticancer drugs can give rise to a
significantly enhanced cytotoxic effect on cancerous cells, thus
providing an increased therapeutic effect. Specifically, as a
significantly increased growth-inhibitory effect is obtained with
the above disclosed combinations utilizing lower concentrations of
the anticancer drugs compared to the treatment regimes in which the
drugs are used alone, there is the potential to provide therapy
wherein adverse side effects associated with the anticancer drugs
are considerably reduced than normally observed with the anticancer
drugs used alone in larger doses. Possible dose ranges of these
co-administered anticancer agents are about 0.1 to 20
mg/kg/day.
[0019] Also included within the scope of the present invention is
the co-administration of effective dosages of a hypocalcemic
vitamin D compound in conjunction with administration of hormones
or other agents, e.g., estrogens, which are known to ameliorate
bone diseases or disorders. For example, prostate cancer often
metastasizes to bone, causing bone loss and associated pain. Such
bone agents may include conjugated estrogens or their equivalents,
calcitonin, bisphosphonates, calcium supplements, cobalamin,
pertussis toxin and boron.
[0020] In another aspect, the invention is a pharmaceutical
composition which includes an anticancer agent which is an active
hypocalcemic vitamin D compound; an agent selected from the group
consisting of (i) an anticancer agent, (ii) a bone agent, and
combinations thereof; and a physiologically acceptable carrier.
[0021] Other advantages and a fuller appreciation of specific
adaptations, compositional variations, and physical attributes will
be gained upon an examination of the following detailed description
of preferred embodiments, taken in conjunction with the appended
claims.
BRIEF DESCRIPTION OF THE DRAWING(S)
[0022] Not Applicable
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention provides an effective method for the
treatment of hypercalcemia, i.e. unphysiologically high and
deleterious blood calcium levels, associated with neoplastic and
hyperproliferative diseases. Particularly, the present invention
relates to therapeutic methods for ameliorating or alleviating the
hypercalcemia associated with the hyperproliferative cellular
activity of malignant and neoplastic diseases, as well as inducing,
enhancing or promoting cell differentiation in the diseased cells.
The present invention provides a novel treatment of a patient
suffering from a hyperproliferative disease with an active
hypocalcemic vitamin D compound. Preferably, the active vitamin D
analogue is a hydroxyvitamin D compound and is suitably represented
by formula (I) as described hereinbelow. The active vitamin D
analogue is provided to the patient without itself causing
dose-limiting hypercalcemia and hypercalciuria, and in fact,
reduces the hypercalcemia caused by the malignancy. These
attributes are achieved through specific chemical properties of the
hypocalcemic vitamin D compounds as described.
[0024] In accordance with the present invention, when effective
amounts of the hypocalcemic active vitamin D compounds are
administered to patients with malignant diseases, the hypercalcemia
is reduced, the PTHrP serum level is reduced, and the proliferative
activity of the abnormal cells is inhibited, redeuced, or
stabilized, and cell differentiation is induced, promoted or
enhanced. Thus, the hypocalcemic vitamin D compounds of the present
invention have an improved therapeutic index relative to active
forms of vitamin D.sub.3 analogues.
[0025] It is known that vitamin D.sub.3 must be hydroxylated in the
C-1 and C-25 positions before it is activated, i.e., before it will
produce a biological response. A similar metabolism appears to be
required to activate other forms of vitamin D, e.g., vitamin
D.sub.2 and vitamin D.sub.4. Therefore, as used herein, the term
"activated vitamin D" or "active vitamin D" is intended to refer to
a vitamin D compound or analogue that has been hydroxylated in at
least the C-1, C-24 or C-25 position of the molecule and either the
compound itself or its metabolites in the case of a prodrug, such
as 1.alpha.-hydroxyvitamin D.sub.2, binds the vitamin D receptor
(VDR). For example, "prodrugs" are vitamin D compounds which are
hydroxylated in the C-1. Such compounds undergo further
hydroxylation in vivo and their metabolites bind the VDR.
[0026] The term "hypocalcemic vitamin D compound" is in reference
to active vitamin D analogs which demonstrate hypocalcemic
activity, i.e. have low calcemic activity relative to that of
1.alpha.,25-dihydroxyvitam- in D.sub.3, including 24-hydroxyvitamin
D compounds, 25-hydroxyvitamin compounds and I a-hydroxyvitamin
compounds.
[0027] Also, as used herein, the term "lower" as a modifier for
alkyl, alkenyl acyl, or cycloalkyl is meant to refer to a straight
or branched, saturated or unsaturated hydrocarbon radical having 1
to 4 carbon atoms. Specific examples of such hydrocarbon radicals
are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl,
ethenyl, propenyl, butenyl, isobutenyl, isopropenyl, formyl,
acetyl, propionyl, butyryl or cyclopropyl. The term "aromatic acyl"
is meant to refer to a unsubstituted or substituted benzoyl
group.
[0028] As used herein, the term "hydrocarbon moiety" refers to a
lower alkyl, a lower alkenyl, a lower acyl group or a lower
cycloalkyl, i.e., a straight or branched, saturated or unsaturated
C.sub.1-C.sub.4 hydrocarbon radial.
[0029] The compound in accordance with the present invention is an
active hypocalcemic vitamin D compound. The active vitamin D
provided is such that the compound has a hydrocarbon moiety at the
C-24 position, e.g. a lower alkyl, alkenyl or acyl group as the
C-24 position. Further, the active vitamin D in accordance with the
present invention may have an unsaturated sidechain, e.g., there is
suitably a double bond between C-22 and C-23, between C-25 and C-26
or between C-26 and C-27.
[0030] The hypocalcemic hydroxyvitamin D of the present invention
suitably has the general formula described in formula (I) 1
[0031] wherein A.sup.1 and A.sup.2 each are hydrogen or a
carbon-carbon bond, thus forming a double bond between C-22 and
C-23; R.sup.1 and R.sup.2 are identical or different and are
hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl,
lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl, lower cycloalkyl with the proviso that R.sup.1 and
R.sup.2 cannot both be alkenyl, or taken together with the carbon
to which they are bonded, form a C.sub.3-C.sub.8 cyclocarbon ring;
R.sup.3 is lower alkyl, lower alkenyl, lower fluoroalkyl, lower
fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl or lower cycloalkyl; X.sup.1 is hydrogen or
hydroxyl, X.sup.2 is hydrogen or hydroxyl, or, may be taken with
R.sup.1 or R.sup.2, to constitute a double bond, and X.sup.3 is
hydrogen or hydroxyl provided that at least one of X.sup.1,
X.sup.2, or X.sup.3 is hydroxyl, and Y is a methylene group if the
bond to Y is a double bond or is a methyl group or hydrogen if the
bond to Y is a single bond.
[0032] A 1.alpha.-hydroxyvitamin D compound of formula (I) is
characterized by the general formula (II): 2
[0033] wherein A.sup.1 and A.sup.2 each are hydrogen or a
carbon-carbon bond, thus forming a double bond between C-22 and
C-23; R.sup.1 and R.sup.2 are identical or different and are
hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl,
lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl, lower cycloalkyl with the proviso that R.sup.1 and
R.sup.2 cannot both be an alkenyl, or taken together with the
carbon to which they are bonded, form a C.sub.3-C.sub.8 cyclocarbon
ring; R.sup.3 is lower alkyl, lower alkenyl, lower fluoroalkyl,
lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl or lower cycloalkyl; X.sup.1 is hydrogen or
hydroxyl, X.sup.2 is hydrogen or hydroxyl, or, may be taken with
R.sup.1 or R.sup.2, to constitute a double bond, and Y is a
methylene group if the bond to Y is a double bond or is a methyl
group or hydrogen if the bond to Y is a single bond.
[0034] Specifically, 1.alpha.-hydroxyvitamin D compounds in
accordance with the present invention are characterized by the
general formula (III): 3
[0035] wherein A.sup.1 and A.sup.2 each are hydrogen or a
carbon-carbon bond, thus forming a double bond between C-22 and
C-23; R.sup.1 and R.sup.2 are identical or different and are
hydrogen, hydroxyl, lower alkyl, lower fluoroalkyl, O-lower alkyl,
lower alkenyl, lower fluoroalkenyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl, lower cycloalkyl with the proviso that R.sup.1 and
R.sup.2 cannot both be an alkenyl, or taken together with the
carbon to which they are bonded, form a C.sub.3-C.sub.8 cyclocarbon
ring; R.sup.3 is lower alkyl, lower alkenyl, lower fluoroalkyl,
lower fluoroalkenyl, O-lower alkyl, O-lower alkenyl, O-lower acyl,
O-aromatic acyl or lower cycloalkyl; X.sup.1 is hydrogen or
hydroxyl, and X.sup.2 is hydrogen or hydroxyl, or, may be taken
with R.sup.1 or R.sup.2, to constitute a double bond.
[0036] The hypocalcemic hydroxyvitamin D compounds of the present
invention are those that have effective antiproliferative and cell
differentiation activity (i.e., reversal of malignant
transformation), but have a lower tendency or inability to cause
hypercalcemia and/or hypercalciuria i.e. they are hypocalcemic
compounds that have low calcemic activity relative to that of
1.alpha.,25-dihydroxyvitamin D.sub.3. In other words, the compounds
of the present invention can be administered at dosages that allow
them to act as antiproliferative agents and cell differentiation
agents when exposed to malignant or other hyperproliferative cells
and can reduce hypercalcemia associated with the maligancy. This
selectivity and specificity of action makes the hypocalcemic
vitamin D compounds useful and preferred antihypercalcemic agents
as well as safely inhibiting hyperproliferation and promoting
malignant or hyperplastic cell differentiation. The compounds of
the present invention, thus, overcome the shortcomings of the known
active vitamin D.sub.3 compounds described above, and can be
considered preferred agents for the control and treatment of
malignant diseases such breast, prostate, testicular and colon
cancer, as well as other neoplasms such as pancreatic cancer,
endometrial cancer, small cell and non-small cell cancer of the
lung (including squamous, adneocarcinoma and large cell types),
squamous cell of the head and neck, bladder, ovarian and cervical
cancers, myeloid and lymphocyltic leukemia, lymphoma, hepatic
tumors, medullary thyroid carcinoma, multiple myeloma, melanoma
retinoblastoma, and sarcomas of the soft tissue and bone, i.e.
neoplasms that express vitamin D receptors.
[0037] Suitable hypocalcemic vitamin D compounds in accordance with
the present invention include: 1.alpha.,24-dihydroxyvitamin
D.sub.2, 1.alpha.,24-dihydroxyvitamin D.sub.4,
1.alpha.,25-dihydroxyvitamin D.sub.2, 1.alpha.,25-dihydroxyvitamin
D.sub.4, 1.alpha.-hydroxyvitamin D.sub.2, and
1.alpha.-hydroxyvitamin D.sub.4. Among those compounds of formula
(I) that have a chiral center in the sidechain, such as at C-24, it
is understood that both epimers (e.g., R and S) and the racemic
mixture are within the scope of the present invention.
[0038] Thus, the present invention provides a method of treating
hypercalcemia associated with malignant cells with an effective
amount of a hypocalcemic vitamin D compound. The effective dosage
amount on a daily basis per kilogram of body weight of the patient
ranges from about 0.01 .mu.g/kg/day to about 2.0 .mu.g/kg/day.
[0039] The compounds of formula (I) can be prepared as described,
e.g., in U.S. Pat. No. 5,488,120 issued to Knutson et al., U.S.
Pat. Nos. 4,670,190 and 4,554,106 issued to DeLuca et al., U.S.
Pat. No. 5,486,636 issued to DeLuca et al., and Strugnell et al.,
310 Biochem. J. (1995) pp. 233-241, all of which are incorporated
herein by reference.
[0040] The biopotencies of the compounds of formula (I) have been
studied and compared to that of 1.alpha.,25-dihydroxyvitamin
D.sub.3, the active hormonal form of vitamin D and the standard
against which all vitamin D compounds and analogues are measured.
For example, it has been found that the vitamin D receptor (VDR)
binding affinities of the compounds of formula (I), or their active
metabolites, are substantially equivalent to (i.e., equal to or up
to 3 times weaker than) the affinity of
1.alpha.,25-dihydroxyvitamin D.sub.3. Such receptor binding
affinities are indicative of potent biological activity.
[0041] At the same time, it has been found that compounds of
formula (I) are significantly less toxic than their corresponding
vitamin D.sub.3 analogues. For example, in parent co-pending
application, Ser. No. 08/265,438, the disclosure of which is
incorporated herein by reference, the LD.sub.50 for
1.alpha.-hydroxyvitamin D.sub.4 was found to be 1.0 mg/kg in males
and 3.0 mg/kg in females, i.e., substantially less toxic than
1.alpha.-hydroxyvitamin D.sub.3 (LD.sub.50.about.0.2 mg/kg).
Further, in the parent U.S. Pat. No. 5,403,831, and its grandparent
U.S. Pat. No. 5,104,864, both of which are incorporated herein by
reference, it has been shown that 1.alpha.-hydroxyvitamin D.sub.2
has the same biopotency as 1.alpha.-hydroxyvitamin D.sub.3 and
1.alpha.,25-dihydroxyvi- tamin D.sub.3 but is much less toxic. Even
dosages up to 10 .mu.g/day of 1.alpha.-hydroxyvitamin D.sub.2 in
women with postmenopausal osteoporosis elicited only mild
hypercalciuria (U.Ca>300 mg/24 hrs), and no marked hypercalcemia
(S. Ca>1 1.0 mg/dL) solely due to 1.alpha.-hydroxyvitamin
D.sub.2 was evident. Additionally, the compound did not adversely
affect kidney function, as determined by creatinine clearance and
BUN; nor did it increase urinary excretion of hydroxyproline,
indicating the absence of any stimulatory effect on bone
resorption. Administration of 1.alpha.-hydroxyvitamin D.sub.2 to
healthy adult males in dosages up to 8 .mu.g/day showed no
clinically significant hypercalcemia or other adverse effects.
[0042] The hypocalcemic vitamin D compounds of the present
invention are useful as active compounds in pharmaceutical
compositions having reduced side effects and low toxicity as
compared with the known analogues of active forms of vitamin
D.sub.3.
[0043] The pharmacologically active compounds of this invention can
be processed in accordance with conventional methods of pharmacy to
produce medicinal agents for administration to patients, e.g.,
mammals including humans. For example, the hypocalcemic vitamin D
compounds can be employed in admixtures with conventional
excipients, e.g., pharmaceutically acceptable carrier substances
suitable for enteral (e.g., oral), parenteral or topical
application which do not deleteriously react with the active
compounds.
[0044] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt solutions, alcohols, gum arabic,
vegetable oils (e.g., almond oil, corn oil, cottonseed oil, peanut
oil, olive oil, coconut oil), mineral oil, fish liver oils, oily
esters such as Polysorbate 80, polyethylene glycols, gelatine,
carbohydrates (e.g., lactose, amylose or starch), magnesium
stearate, talc, silicic acid, viscous paraffin, fatty acid
monoglycerides and diglycerides, pentaerythritol fatty acid esters,
hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
[0045] The pharmaceutical preparations can be sterilized and, if
desired, be mixed with auxiliary agents, e.g., lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or
one or more other active compounds, for example, vitamin D.sub.3
and its 1.alpha.-hydroxylated metabolites, conjugated estrogens or
their equivalents, anti-estrogens, calcitonin, biphosphonates,
calcium supplements, cobalamin, pertussis toxin and boron.
[0046] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous solution,
as well as suspensions, emulsions, or implants, including
suppositories. Parenteral administration suitably includes
subcutaneous, intramuscular, or intravenous injection,
nasopharyngeal or mucosal absorption, or transdermal absorption.
Ampoules are convenient unit dosages.
[0047] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, lozenges, powders, or
capsules. A syrup, elixir, or the like can be used if a sweetened
vehicle is desired.
[0048] For topical application, suitable nonsprayable viscous,
semi-solid or solid forms can be employed which include a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water, for example, mineral oil, almond
oil, self-emulsifying beeswax, vegetable oil, white soft paraffin,
and propylene glycol. Suitable formulations include, but are not
limited to, creams, ointments, lotions, solutions, suspensions,
emulsions, powders, liniments, salves, aerosols, transdermal
patches, etc., which are, if desired, sterilized or mixed with
auxiliary agents, e.g., preservatives, stabilizers, demulsifiers,
wetting agents, etc. A cream preparation in accordance with the
present invention suitably includes, for example, mixture of water,
almond oil, mineral oil and self-emulsifying beeswax; an ointment
preparation suitably includes, for example, almond oil and white
soft paraffin; and a lotion preparation suitably includes, for
example, dry propylene glycol.
[0049] Topical preparations of the compound in accordance with the
present invention useful for the treatment of skin disorders may
also include epithelialization-inducing agents such as retinoids
(e.g., vitamin A), chromanols such as vitamin E, .beta.-agonists
such as isoproterenol or cyclic adenosine monophosphate (cAMP),
anti-inflammatory agents such as corticosteroids (e.g.,
hydrocortisone or its acetate, or dexamethasone) and keratoplastic
agents such as coal tar or anthralin. Effective amounts of such
agents are, for example, vitamin A about 0.003 to about 0.3% by
weight of the composition; vitamin E about 0.1 to about 10%;
isoproterenol about 0.1 to about 2%; cAMP about 0.1 to about 1%;
hydrocortisone about 0.25 to about 5%; coal tar about 0.1 to about
20%; and anthralin about 0.05 to about 2%.
[0050] For rectal administration, the compound is formed into a
pharmaceutical composition containing a suppository base such as
cacao oil or other triglycerides. To prolong storage life, the
composition advantageously includes an antioxidant such as ascorbic
acid, butylated hydroxyanisole or hydroquinone.
[0051] For treatment of hypercalcemia associated with maligancy,
oral administration of the pharmaceutical compositions of the
present invention is preferred. Generally, the compound of this
invention is dispensed by unit dosage form comprising about 0.5
.mu.g to about 25 .mu.g in a pharmaceutically acceptable carrier
per unit dosage. The dosage of the compound according to this
invention generally is about 10 .mu.g to 200 .mu.g/day.
[0052] For topical treatment of skin disorders, the dosage of the
compound of the present invention in a topical composition
generally is about 0.01 .mu.g to about 50 .mu.g per gram of
composition. For treatment of skin cancers, the dosage of the
hypocalcemic vitamin D compound in a locally applied composition
generally is about 0.01 .mu.g to 100 .mu.g per gram
composition.
[0053] It is noted that dosing of the hypocalcemic compounds in
accordance with the present invention can also be done on an
episodic basis, in which case higher doses can be used generally
about 20 .mu.g to about 200 .mu.g given once every 2 to 7 days. The
dose can be given as a single dose or a divided dose in 2 to 5
subdoses, the subdoses given, e.g., one every hour until the total
dose is taken.
[0054] Those of ordinary skill in the art will readily optimize
effective doses and coadministration regimens as determined by good
medical practice and the clinical condition of the individual
patient. Regardless of the manner of administration, it will be
appreciated that the actual preferred amounts of active compound in
a specific case will vary according to the efficacy of the specific
compound employed, the particular compositions formulated, the mode
of application, and the particular situs and organism being
treated. For example, the specific dose for a particular patient
depends on age, body weight, general state of health, on diet, on
the timing and mode of administration, on the rate of excretion,
and on medicaments used in combination and the severity of the
particular disorder to which the therapy is applied. Dosages for a
given host can be determined using conventional considerations,
e.g., by customary comparison of the differential activities of the
subject compounds and of a known agent, such as by means of an
appropriate conventional pharmacological protocol.
[0055] Further, included within the scope of the present invention
is the co-administration of a hypocalcemic vitamin D compound with
a anticancer agent, e.g., a cytotoxic agent, Such agents suitably
include antimetabolites (e.g., 5-fluoro-uracil, methotrexate,
fludarabine), antimicrotubule agents (e.g., vincristine,
vinblastine, taxanes such as paclitaxel, docetaxel), an alkylating
agent (e.g., cyclophasphamide, melphalan, biochoroethylnitrosurea,
hydroxyurea), platinum agents (e.g. cisplatin, carboplatin,
oxaliplatin, JM-216, CI-973), anthracyclines (e.g., doxrubicin,
daunorubicin), antibiolitics (e.g., mitomycin, idarubicin,
adriamycin, daunomycin), topoisomerase inhibitors (e.g., etoposide,
camptothecins) or any other antineoplastic agents. (estramustine
phosphate, prednimustine). It is anticipated that the hypocalcemic
vitamin D compounds used in combination with various anticancer
drugs can give rise to a significantly enhanced cytotoxic effect on
cancerous cells, thus providing an increased therapeutic effect.
Specifically, as a significantly increased growth-inhibitory effect
is obtained with the above disclosed combinations utilizing lower
concentrations of the anticancer drugs compared to the treatment
regimes in which the drugs are used alone, there is the potential
to provide therapy wherein adverse side effects associated with the
anticancer drugs are considerably reduced than normally observed
with the anticancer drugs used alone in larger doses. Possible dose
ranges of these co-administered anticancer agents are about 0.1 to
20 mg/kg/day.
[0056] The term "co-administration" is meant to refer to any
administration route in which two or more agents are administered
to a patient or subject. For example, the agents may be
administered together, or before or after each other. The agents
may be administered by different routes, e.g., one agent may be
administered intravenously while the second agent is administered
intramuscularly, intravenously or orally. The agents may be
administered simultaneously or sequentially, as long as they are
given in a manner sufficient to allow both agents to achieve
effective concentrations in the body. The agents also may be in an
admixture, as, for example, in a single tablet. In sequential
administration, one agent may directly follow administration of the
other or the agents may be give episodically, i.e., one can be
given at one time and the other at a later time, typically within a
week. An example of a suitable co-administration regimen is where a
hypocalcemic vitamin D compound is administered from 0.5 to 7 days
prior to administration of a cytotoxic agent.
[0057] Also included within the scope of the present invention is
the co-administration of effective dosages of the analogue of
formula (I) in conjunction with administration of hormones or other
agents, e.g., estrogens, which are known to ameliorate bone
diseases or disorders. As noted above, prostate cancer often
metastasizes to bone, causing bone loss and associated pain. Such
bone agents may include conjugated estrogens or their equivalents,
calcitonin, bisphosphonates, calcium supplements, cobalamin,
pertussis toxin and boron. It is contemplated that these bone
agents also have an antihypercalcemic effect and may enhance the
treatment of malignancy-associated hypercalcemia. Possible dose
ranges for these co-administered bone agents are provided in Table
1.
1TABLE 1 Possible Oral Dose Ranges for Various Bone Agents
Co-Administered With 1.alpha.-Hydroxyvitamin D of Formula (I) Dose
Ranges Most Agent Broad Preferred Preferred Conjugated Estrogens or
0.3-5.0 0.4-2.4 0.6-1.2 Equivalent (mg/day) Sodium Fluoride
(mg/day) 5-150 30-75 40-60 Calcitonin (IU/day) 5-800 25-500 50-200
Bisphosphonates (mg/day) 0.5-20 1-15 5-10 Calcium Supplements
250-2500 500-1500 750-1000 (mg/day) Cobalamin (.mu.g/day) 5-200
20-100 30-50 Pertussis Toxin (mg/day) 0.1-2000 10-1500 100-1000
Boron (mg/day) 0.10-3000 1-250 2-100 Antiestrogens, such as
Tamoxifen .TM., are also known bone agents and may be suitably used
in conjunction with the hypocalcemic hydroxyvitamin D compounds of
the present invention.
[0058] The present invention is further explained by the following
examples which should not be construed by way of limiting the scope
of the present invention.
VDR BINDING ANALYSES
EXAMPLE 1
1.alpha., 24-dihydroxyvitamin D.sub.2
[1.alpha.,24-(OH).sub.2D.sub.2]
[0059] The affinity of 1.alpha.,24-(OH).sub.2D.sub.2 for the
mammalian vitamin D receptor (VDR) was assessed using a
commercially available kit of bovine thymus VDR and standard
1,25-(OH).sub.2D.sub.3 solutions from Incstar (Stillwater, Minn.).
The half-maximal binding of chemically synthesized
1.alpha.,24-(OH).sub.2D.sub.2 was approximately 150 pg/ml whereas
that of 1.alpha.,25-(OH).sub.2D.sub.3 was 80 pg/ml. Thus, the
1.alpha.,24-(OH).sub.2D.sub.2 had a very similar affinity for
bovine thymus VDR as did 1.alpha.,25-(OH).sub.2D.sub.3, indicating
that 1.alpha.,24-(OH).sub.2D.sub.2 has potent biological
activity.
EXAMPLE 2
1.alpha.,24-dihydroxy Vitamin D.sub.4
[1.alpha.,24-(OH).sub.2D.sub.4]
[0060] The VDR affinity binding of 1.alpha.,24-(OH).sub.2D.sub.4
was investigated. The 1.alpha.,24-(OH).sub.2D.sub.4 was incubated
with vitamin D receptor and radiolabeled tracer
1.alpha.,25-(OH).sub.2D.sub.3. After incubation, the amount of
radioactivity bound to the receptor was determined and compared
with the amount bound after co-incubation of unlabeled and labeled
1.alpha.,25-(OH).sub.2D.sub.3. It was found that 50 pg/tube of
1.alpha.,24-(OH).sub.2D.sub.4 was equivalent to approximately 20 pg
1.alpha.,25-(OH).sub.2D.sub.3.
[0061] These results show that 1.alpha.,24-(OH).sub.2D.sub.4 binds
slightly less tightly to the vitamin D receptor than does
1.alpha.,25-(OH).sub.2D.sub.3. Such data mean that
1.alpha.,24-(OH).sub.2D.sub.4 has high affinity for the VDR and
significant biological activity, similar to that of
1.alpha.,25-(OH).sub.2D.sub.3. These data are consistent with gene
expression studies done (described below) with
1.alpha.,24-(OH).sub.2D.su- b.4 which demonstrate that
1.alpha.,24-(OH).sub.2D.sub.4 is only slightly less active than is
1.alpha.,25-(OH).sub.2D.sub.3.
[0062] These results are surprising and unexpected in view of the
prior art. They are contrary to the normative wisdom in the vitamin
D art regarding the very low degree of biological activity of
vitamin D.sub.4 compounds.
EXAMPLE 3
1.alpha.,24-dihydroxyvitamin D.sub.2
[1.alpha.,24-(OH).sub.2D.sub.2]
[0063] VDR binding of vitamin D compounds by prostate cells is
demonstrated using the techniques of Skowronski et al., 136
Endocrinology (1995) 20-26, which is incorporated herein by
reference. Prostate-derived cell lines are cultured to near
confluence, washed and harvested by scraping. Cells are washed by
centrifugation, and the cell pellet resuspended in a buffered salt
solution containing protease inhibitors. The cells are disrupted by
sonication while cooling on ice. The supernatant obtained from
centrifuging the disrupted cells at 207,000.times.g for 35 min at
4EC is assayed for binding. 200 TL of soluble extract, (1-2 mg
protein/ml supernatant) is incubated with a 1 nM
.sup.3H-1.alpha.,25-(OH).sub.2D.sub.3 and increasing concentrations
of 1.alpha.,24-(OH).sub.2-D.sub.2 (0.01-100 nM) for 16-20 hr at
4EC. Bound and free hormones are separated with hydroxylapatite
using standard procedures. Specific binding is calculated by
subtracting nonspecific binding obtained in the presence of a
250-fold excess of nonradioactive 1.alpha.,25-(OH).sub.2D.sub.3
from the total binding measured. The results demonstrate that
1.alpha.,24-(OH).sub.2D.sub.2 has strong affinity for prostate VDR,
indicating that 1.alpha.,24-(OH).sub.2D.sub.2 has potent biological
activity in respect of prostate cells.
EXAMPLE 4
1.alpha.,24-dihydroxy vitamin D.sub.4
[1.alpha.,24-(OH).sub.2D.sub.4]
[0064] The procedure of Example 3 is repeated using the active
vitamin D analogue 1.alpha.,24-(OH).sub.2D.sub.4, and the specific
binding is determined. The results demonstrate that
1.alpha.,24-(OH).sub.2D.sub.4 has strong affinity for prostate VDR,
indicating that 1.alpha.,24-(OH).sub.2D.sub.4 has potent biological
activity in respect of prostate cells.
EXAMPLE 5
1.alpha.,25-dihydroxyvitamin D.sub.4
[1.alpha.,25-(OH).sub.2D.sub.4]
[0065] The procedure of Example 3 is repeated using the active
vitamin D analogue 1.alpha.,25-(OH).sub.2D.sub.4, and the specific
binding is determined. The results demonstrate that
1.alpha.,25-(OH).sub.2D.sub.4 has strong affinity for prostate VDR,
indicating that 1.alpha.,25-(OH).sub.2D.sub.4 has potent biological
activity in respect of prostate cells.
GENE EXPRESSION
EXAMPLE 6
1.alpha.,24-dihydroxy vitamin D.sub.4
[1.alpha.,24-(OH).sub.2D.sub.4]
[0066] Using the plasmids p(CT4).sup.4TKGH, a vitamin D receptor
(VDR)-expressing plasmid, and pSG5-hVDR1/3, a plasmid containing a
Growth Hormone (GH) gene, under the control of a vitamin
D-responsive element (VDRE), experiments were conducted to explore
the ability of 1.alpha.,24-(OH).sub.2D.sub.4 to induce vitamin
D-dependent growth hormone acting as a reporter gene compared to
that of 1.alpha.,25-(OH).sub.2D.sub.3. Cells in culture were
transfected with these two plasmids. One plasmid contained the gene
for Growth Hormone (GH) under the control of the vitamin D
responsive element (VDRE) and the other plasmid contained the
structural gene for the vitamin D receptor (VDR). These transfected
cultures were incubated with 1.alpha.,24-(OH).sub.2D.sub.4 or
1.alpha.,25-(OH).sub.2D.sub.3, and the production of growth hormone
was measured. Table 2 below shows the results of this assay:
2TABLE 2 Induction of Growth Hormone by Vitamin D Compounds
Concentration Growth Hormone Compound Used (M) Induction (ng/ml)
1,25-(OH).sub.2D.sub.3 1 .times. 10.sup.-10 39
1,25-(OH).sub.2D.sub.3 5 .times. 10.sup.-10 248
1,24-(OH).sub.2D.sub.4 5 .times. 10.sup.-10 165
1,24-(OH).sub.2D.sub.4 1 .times. 10.sup.-9 628
1,24-(OH).sub.2D.sub.4 5 .times. 10.sup.-9 1098
[0067] These data show that the ability of
1.alpha.,24-(OH).sub.2D.sub.4 to stimulate vitamin D-dependent
growth hormone is nearly equivalent to that of
1.alpha.,25-(OH).sub.2D.sub.3. Such results are truly surprising
and would not have been expected by following the teachings of the
prior art.
EXAMPLE 7
1.alpha.,24(S)-dihydroxyvitamin D.sub.2 and
1.alpha.,24(R)-dihydroxy-vitam- in D.sub.2
[1.alpha.,24(S)-(OH).sub.2D.sub.2 and 1.alpha.,24(R)-(OH).sub.2-
D.sub.2]
[0068] The gene expression study described in Example 6 was
conducted to compare the biological activity in vitro of chemically
synthesized 1.alpha.,24(S)-(OH).sub.2D.sub.2 and
1.alpha.,24(R)-(OH).sub.2D.sub.2, with
1.alpha.,25-(OH).sub.2D.sub.3 and 25-OH-D.sub.3. The vitamin
D-dependent transcriptional activation model system was used in
which plasmids pSG5-hVDR1/3 and p(CT4).sup.4TKGH were
co-transfected into Green monkey kidney, COS-1 cells.
[0069] Transfected cells were incubated with vitamin D metabolites
and growth hormone production was measured. As shown in Table 3,
both 1.alpha.,24(S)-(OH).sub.2D.sub.2 and its epimer,
1.alpha.,24(R)-(OH).sub.- 2D.sub.2, had significantly more activity
in this system than 25-OH-D.sub.3, with
1.alpha.,24(S)-(OH).sub.2D.sub.2 having nearly the same activity as
1.alpha.,25-(OH).sub.2D.sub.3.
3TABLE 3 Vitamin D-Inducible Growth Hormone Production In
Transfected COS-1 Cells Vitamin DClnducible Growth Hormone
Production Net vitamin Total GH DCinducible Molar Production*
GH-production Inducer Concentration (ng/ml) (ng/ml) Ethanol 44 0
25-OH-D.sub.3 1 .times. 10.sup.-7 245 201 1 .times. 10.sup.-6 1100
1056 1 .times. 10.sup.-5 775 731 1.alpha.,25-(OH).sub.2D.sub.3 1
.times. 10.sup.-10 74 30 1 .times. 10.sup.-9 925 881 1 .times.
10.sup.-8 1475 1441 1.alpha.,24(S)-(OH).sub.2D.sub.2 5 .times.
10.sup.-10 425 381 5 .times. 10.sup.-9 1350 1306 5 .times.
10.sup.-8 1182 1138 1.alpha.,24(R)-(OH).sub.2D.sub.2 1 .times.
10.sup.-9 80 36 1 .times. 10.sup.-8 1100 1056 1 .times. 10.sup.-7
1300 1256 *Averages of duplicate determinations
INHIBITION OF CELL PROLIFERATION
EXAMPLE 8
1.alpha.,24-dihydroxyvitamin D.sub.2
[1.alpha.,24-(OH).sub.2D.sub.2]
[0070] Inhibition of cell proliferation is demonstrated using the
techniques of Skowronski et al., 132 Endocrinology (1993) 1952-1960
and 136 Endocrinology (1995) 20-26, both of which are incorporated
herein by reference. The cell lines, LNCaP and PC-3, which are
derived from human prostate adenocarcinoma, are seeded in six-well
tissue culture plates at a density of about 50,000 cells/plate.
After the cells have attached and stabilized, about 2-3 days, the
medium is replenished with medium containing vehicle or the active
vitamin D analogue 1.alpha.,24-(OH).sub.2D.sub.2, at concentrations
from 10.sup.-11 M to 10.sup.-7 M. Medium containing test analogue
or vehicle is replaced every three days. After 6-7 days, the medium
is removed, the cells are rinsed, precipitated with cold 5%
trichloroacetic acid, and washed with cold ethanol. The cells are
solubilized with 0.2 N sodium hydroxide, and the amount of DNA
determined by standard procedures. The results show that cultures
incubated with 1.alpha.,24-(OH).sub.2D.sub.2 in accordance with the
present invention have significantly fewer cells than the control
cultures.
EXAMPLE 9
1.alpha.,24-dihydroxy Vitamin D.sub.4
[1.alpha.,24-(OH).sub.2D.sub.4]
[0071] The procedure of Example 8 is repeated using the active
vitamin D analogue 1.alpha.,24-(OH).sub.2D.sub.4, and the cell
number is determined. Cultures incubated with
1.alpha.,24-(OH).sub.2D.sub.4 have significantly fewer cells than
the control cultures.
EXAMPLE 10
1.alpha.,25-dihydroxyvitamin D.sub.4
[1.alpha.,25-(OH).sub.2D.sub.4]
[0072] The procedure of Example 8 is repeated using the active
vitamin D analogue 1.alpha.,25-(OH).sub.2D.sub.4, and the cell
number is determined. Cultures incubated with
1.alpha.,25-(OH).sub.2D.sub.4 have significantly fewer cells than
the control cultures.
STIMULATION OF CELL DIFFERENTIATION
EXAMPLE 11
1.alpha.,24-dihydroxyvitamin D.sub.2
[1.alpha.,24-(OH).sub.2D.sub.2]
[0073] Using the techniques of Skowronski et al., 132 Endocrinology
(1993) 1952-1960 and 136 Endocrinology (1995) 20-26, both of which
are incorporated herein by reference, cells of the cell line,
LNCaP, which is derived from a human metastatic prostate
adenocarcinoma and known to express PSA, are seeded in six-well
tissue culture plates at a density of about 50,000 cells/plate.
After the cells have attached and stabilized, about 2-3 days, the
medium is replenished with medium containing vehicle or the active
vitamin D analogue, 1.alpha.,24-(OH).sub.2D.sub.2, at
concentrations from 10.sup.-11 M to 10.sup.-7 M. After 6-7 days,
the medium is removed and stored at -20EC for prostate specific
antigen (PSA) analysis.
[0074] The cells from parallel cultures are rinsed, precipitated,
and the amount of DNA determined by standard procedures. PSA is
measured by standard known methods. Cultures incubated with
1.alpha.,24-(OH).sub.2D.s- ub.2 have significantly more PSA than
control cultures when expressed as mass of PSA/cell.
EXAMPLE 12
1.alpha.,24-dihydroxyvitamin D.sub.4
[1.alpha.,24-(OH).sub.2D.sub.4]
[0075] The procedure of Example 12 is repeated except the active
vitamin D analogue is 1.alpha.,24-(OH).sub.2D.sub.4. The PSA is
measured and cultures incubated with 1.alpha.,24-(OH).sub.2D.sub.4
have significantly more PSA than control cultures when expressed as
mass of PSA/cell.
EXAMPLE 13
1.alpha.,25-dihydroxyvitamin D.sub.4
[1.alpha.,24-(OH).sub.2D.sub.4]
[0076] The procedure of Example 12 is repeated except the active
vitamin D analogue is 1.alpha.,25-(OH).sub.2D.sub.4. The PSA is
measured and cultures incubated with 1.alpha.,25-(OH).sub.2D.sub.4
have significantly more PSA than control cultures when expressed as
mass of PSA/cell.
CLINICAL STUDIES
EXAMPLE 14
General Treatment of MAH
[0077] Patients with malignancy-associated hypercalcemia
participate in an open-label study of a hypocalcemic vitamin D
compound in accordance with the present invention. Patients are
restricted to daily calcium intake of about 400-500 mg. Each
patient is also asked to drink 4-6 cups of fluid more than usual
intake to assure adequate oral hydration.
[0078] Each subject is monitored at regular intervals for: (1)
hypercalcemia, serum PTHrP levels, hyperphosphatemia,
hypercalciuria, hyperphosphaturia and other toxicity; (2) evidence
of changes in the progression of metastatic disease; and (3)
compliance with the prescribed test drug dosage.
[0079] The dosing regimen is typically on a daily dose basis of 10
.mu.g or 20 .mu.g per day to about 100 .mu.g/day for 10 weeks.
Alternatively, a non-daily dosing regimen can be used, e.g., 40
.mu.g given every other day, 100 .mu.g given once a week. The route
of administration can vary from oral to intravenous to regional
delivery (e.g., arterial infusion, via the portal vein). Oral is,
of course, the easiest and most cost effective route. Regional
delivery permits high dosing and generally avoids any production of
hypercalcemia. Although, in the case of the compound of the present
invention, the compound is substantially hypocalcemic.
[0080] After the treatment period, CAT, scans, X-rays and bone
scans used for evaluating the progress of metastatic disease show
stable disease or partial remission in many patients treated at the
lower dosage, and stable disease and partial or complete remission
in many patients treated at the higher dosage. Serum calcium levels
are in the normal range and serum levels of PTHrP are redeuced.
EXAMPLE15
Treatment of MAH Using 1.alpha.,24(s)-dihydroxyvitamin D.sub.2
[1.alpha.,24(S)-(OH).sub.2D.sub.2]
[0081] The procedure of example 14 is carried out using
1.alpha.,24-(OH).sub.2D.sub.2. The results show serum calcium
levels in the normal range and serum levels of PTHrP reduced.
EXAMPLE 16
Treatment of MAH Using 1.alpha.-hydroxyvitamin D.sub.2
[1.alpha.-OH-D.sub.2]
[0082] The procedure of example 14 is carried out using
1.alpha.-OH-D.sub.2. The results show serum calcium in the normal
range and serum PTHrP levels reduced.
[0083] While the present invention has now been described and
exemplified with some specificity, those skilled in the art will
appreciate the various modifications, including variations,
additions, and omissions, that may be made in what has been
described. Accordingly, it is intended that these modifications
also be encompassed by the present invention and that the scope of
the present invention be limited solely by the broadest
interpretation lawfully accorded the appended claims.
* * * * *