U.S. patent application number 10/750570 was filed with the patent office on 2008-08-07 for cyclic ether vitamin d3 compounds, 1alpha(oh)3-epi-vitamin d3 compounds and uses thereof.
This patent application is currently assigned to Women and Infants Hospital. Invention is credited to Satyanarayana G. Reddy.
Application Number | 20080188548 10/750570 |
Document ID | / |
Family ID | 21944848 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080188548 |
Kind Code |
A1 |
Reddy; Satyanarayana G. |
August 7, 2008 |
Cyclic ether vitamin D3 compounds, 1alpha(OH)3-epi-vitamin D3
compounds and uses thereof
Abstract
Novel cyclic ether vitamin D3 compounds having a cyclic ether
side chain are disclosed. These compounds were first identified as
metabolites of 3-epi vitamin D3 produced via a tissue-specific
metabolic pathway which catalyzes the formation of a cyclic ether
structure. Also disclosed are 1.alpha.(OH) 3-epi vitamin D3
compounds, which are produced via the epimerization of a
3-.beta.-hydroxyl group of 1.alpha.(OH) vitamin D3 precursor in
vivo. The vitamin D3 compounds of the present invention can be used
as substitutes for natural and synthetic vitamin D3 compounds.
Inventors: |
Reddy; Satyanarayana G.;
(Barrington, RI) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
Women and Infants Hospital
|
Family ID: |
21944848 |
Appl. No.: |
10/750570 |
Filed: |
December 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10188320 |
Jul 1, 2002 |
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10750570 |
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09617881 |
Jul 17, 2000 |
6479538 |
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10188320 |
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09079942 |
May 15, 1998 |
6100294 |
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09617881 |
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60046690 |
May 16, 1997 |
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Current U.S.
Class: |
514/451 ;
514/729; 549/427; 568/819 |
Current CPC
Class: |
C07C 401/00 20130101;
C07D 307/33 20130101; A61P 17/00 20180101; A61P 19/08 20180101;
A61P 3/02 20180101; A61P 3/14 20180101; A61P 5/18 20180101; C07D
309/06 20130101; C07D 309/04 20130101; A61P 43/00 20180101; A61P
3/00 20180101; A61P 19/10 20180101; C07D 303/14 20130101 |
Class at
Publication: |
514/451 ;
549/427; 514/729; 568/819 |
International
Class: |
A61K 31/351 20060101
A61K031/351; C07D 309/06 20060101 C07D309/06; A61K 31/047 20060101
A61K031/047; C07C 33/14 20060101 C07C033/14; A61P 3/00 20060101
A61P003/00; A61P 19/10 20060101 A61P019/10 |
Claims
1. An isolated cyclic ether vitamin D3 compound having the formula
(I) as follows: ##STR00009## wherein A.sub.1, A.sub.2 and A.sub.3
are a single or a double bond; X, R.sub.1, R.sub.2, R.sub.3,
R.sub.4 and R.sub.5 are selected from the group consisting of a
hydrogen, a halogen, a haloalkyl, a hydroxy, a hydroxy-protecting
group, an alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl group
and a heterocyclic group.
2. An isolated 3-epi form of 1.alpha.-hydroxy-vitamin D3 compounds
having the formula II as follows: ##STR00010## wherein A.sub.1 is a
single, a double, or a triple bond; A.sub.2, A.sub.3 and A4 are
each independently selected from the group consisting of a single
or a double bond; R.sub.2, R.sub.3, R.sub.4, R.sub.7, R.sub.8 and
R.sub.9 are independently selected from the group consisting of a
hydrogen, a deuterium, a deuteroalkyl, a hydroxy, an alkyl, an
alkoxide, an O-acyl, a halogen, a haloalkyl, a hydroxyalkyl, an
amine or a thiol group, and wherein the pairs of R.sub.2 and
R.sub.3, and R.sub.4 and R.sub.7 taken together are an oxygen atom;
and R.sub.5 and R.sub.6 are independently selected from the group
consisting of a hydrogen, a deuterium, a halogen, an alkyl, a
hydroxyalkyl, a haloalkyl, and a deuteroalkyl.
3. The compound of claim 2, which is 1.alpha.(OH) vitamin D3,
1.alpha., 24 dihydroxy 3-epi vitamin D.sub.3, 1.alpha. hydroxy
24-ethyl 3-epi vitamin D.sub.3, 1.alpha. hydroxy 24-methyl 3-epi
vitamin D.sub.3, or 1.alpha., 24-dihydroxy 24-methyl 3-epi vitamin
D.sub.3.
4. A method of treating a disorder characterized by an aberrant
activity of a vitamin D.sub.3-responsive cell, comprising
administering to a subject an effective amount of a vitamin D.sub.3
compound having the formula (I) or (II) of claim 2, such that the
aberrant activity of the vitamin D.sub.3-responsive cell is
reduced.
5. The method of claim 4, wherein the disorder comprises an
aberrant activity of a hyperproliferative skin cell.
6. The method of claim 4, wherein the disorder comprises an
aberrant activity of an endocrine cell.
7. The method of claim 6, wherein the endocrine cell is a
parathyroid cell and the aberrant activity is processing and/or
secretion of parathyroid hormone.
8. The method of claim 7, wherein the disorder is secondary
hyperparathyroidism.
9. The method of claim 8, wherein the disorder comprises an
aberrant activity of a bone cell.
10. The method of claim 9, wherein the disorder is selected from
the group consisting of osteoporosis, osteodystrophy, senile
osteoporosis, osteomalacia, rickets, osteitis fibrosa cystica,
renal osteodystrophy, secondary hyperparathyrodism, cirrhosis, and
chronic renal disease.
11. The method of claim 4, wherein the subject is a mammal.
12. The method of claim 11, wherein the mammal is a human.
13. A method of ameliorating a deregulation of calcium and
phosphate metabolism, comprising administering to a subject a
therapeutically effective amount of a 3-epi vitamin D.sub.3
compound of claim 2, so as to ameliorate the deregulation of the
calcium and phosphate metabolism.
14. The method of claim 13, wherein the deregulation of the calcium
and phosphate metabolism leads to osteoporosis.
15. A pharmaceutical composition comprising, a therapeutically
effective amount of a vitamin D.sub.3 compound of claim 2, and a
pharmaceutically acceptable carrier.
16. The composition of claim 15, which is suitable for topical or
oral administration.
17. A packaged compound, comprising a vitamin D.sub.3 compound of
claim 2, packaged with instructions for use of the compound for
treating a disorder characterized by an aberrant activity of a
vitamin D.sub.3-responsive cell.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Application No. 60/046,690 filed on May 16, 1997, the
contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The importance of the vitamin D in the biological systems of
higher animals has been recognized since its discovery by Mellanby
in 1920 (Mellanby, E. (1921) Spec. Rep. Ser. Med. Res. Council (GB)
SRS 61:4). It was in the interval of 1920-1930 that vitamin D
officially became classified as a "vitamin" that was essential for
the normal development of the skeleton and maintenance of calcium
and phosphorous homeostasis.
[0003] Studies involving the metabolism of vitamin D.sub.3
(cholecalciferol) were initiated with the discovery and chemical
characterization of the plasma metabolite, 25-hydroxyvitamin
D.sub.3 [25(OH).sub.2D.sub.3] (Blunt, J. W. et al. (1968)
Biochemistry 6:3317-3322) and the hormonally active form, 1.alpha.,
25(OH).sub.2D.sub.3 (Myrtle, J. F. et al. (1970) J. Biol. Chem.
245:1190-1196; Norman, A. W. et al. (1971) Science 173:51-54;
Lawson, D. E. M. et al (1971) Nature 230:228-230; Holick, M. F.
(1971) Proc. Natl. Acad. Sci. USA 68:803-804). The formulation of
the concept of a vitamin D endocrine system was dependent both upon
appreciation of the key role of the kidney in producing 1.alpha.,
25(OH).sub.2D.sub.3 in a carefully regulated fashion (Fraser D. R.
and Kodicek, E (1970) Nature 288:764-766; Wong, R. G. et al. (1972)
J. Clin. Invest. 51:1287-1291), and the discovery of a nuclear
receptor for 1.alpha.,25(OH).sub.2D.sub.3 (VD.sub.3R) in the
intestine (Haussler, M. R. et al. (1969) Exp. Cell Res. 58:234-242;
Tsai, H. C. and Norman, A. W. (1972) J. Biol. Chem. 248:5967-5975).
The operation of the vitamin D endocrine system depends on the
following: first, on the presence of cytochrome P450 enzymes in the
liver (Bergman, T. and Postlind, H. (1991) Biochem. J. 276:427-432;
Ohyama, Y and Okuda, K. (1991) J. Biol. Chem. 266:8690-8695) and
kidney (Henry, H. L. and Norman, A. W. (1974) J. Biol. Chem.
249:7529-7535; Gray, R. W. and Ghazarian, J. G. (1989) Biochem. J.
259:561-568), and in a variety of other tissues to effect the
conversion of vitamin D.sub.3 into biologically active metabolites
such as 1.alpha.,25(OH).sub.2D.sub.3 and 24R,25(OH).sub.2D.sub.3;
second, on the existence of the plasma vitamin D binding protein
(DBP) to effect the selective transport and delivery of these
hydrophobic molecules to the various tissue components of the
vitamin D endocrine system (Van Baelen, H. et al. (1988) Ann NY
Acad. Sci. 538:60-68; Cooke, N. E. and Haddad, J. G. (1989) Endocr.
Rev. 10:294-307; Bikle, D. D. et al. (1986) J. Clin. Endocrinol.
Metab. 63:954-959); and third, upon the existence of
stereoselective receptors in a wide variety of target tissues that
interact with the agonist 1.alpha.,25(OH).sub.2D.sub.3 to generate
the requisite specific biological responses for this secosteroid
hormone (Pike, J. W. (1991) Annu. Rev. Nutr. 11: 189-216). To date,
there is evidence that nuclear receptors for
1.alpha.,25(OH).sub.2D.sub.3 (VD.sub.3R) exist in more than 30
tissues and cancer cell lines (Reichel, H. and Norman, A. W. (1989)
Annu. Rev. Med. 40:71-78).
[0004] Vitamin D.sub.3 and its hormonally active forms are
well-known regulators of calcium and phosphorous homeostasis. These
compounds are known to stimulate, at least one of, intestinal
absorption of calcium and phosphate, mobilization of bone mineral,
and retention of calcium in the kidneys. Furthermore, the discovery
of the presence of specific vitamin D receptors in more than 30
tissues has led to the identification of vitamin D.sub.3 as a
pluripotent regulator outside its classical role in calcium/bone
homeostasis. A paracrine role for 1.alpha.,25(OH).sub.2D.sub.3 has
been suggested by the combined presence of enzymes capable of
oxidizing vitamin D.sub.3 into its active forms, e.g.,
25-OHD-1.alpha.-hydroxylase, and specific receptors in several
tissues such as bone, keratinocytes, placenta, and immune cells.
Moreover, vitamin D.sub.3 hormone and active metabolites have been
found to be capable of regulating cell proliferation and
differentiation of both normal and malignant cells (Reichel, H. et
al. (1989) Ann Rev. Med. 40: 71-78).
[0005] Given the pluripotent activities of vitamin D.sub.3 and its
metabolites, much attention has focused on the development of
synthetic analogs of these compounds. However, clinical
applications of vitamin D.sub.3 and its structural analogs have
been limited by the undesired side effects elicited by these
compounds after administration to a subject, such as the
deregulation of calcium and phosphorous homeostasis in vivo that
results in hypercalcemia.
SUMMARY OF THE INVENTION
[0006] The present invention is based, at least in part, on the
discovery of vitamin D3 compounds having a cyclic ether side chain,
referred to hereinafter as "cyclic ether vitamin D3 compounds", and
which are represented by the formula I. This invention also
describes 3-epi forms of 1.alpha.-hydroxy-vitamin D3 compounds,
which are represented by the formula II. The cyclic ether and
1.alpha.-hydroxy-vitamin D3 compounds of formulas I and II,
respectively, referred to hereinafter as "vitamin D3 compounds of
formulas I and II" can be produced in vivo via a pathway which
catalyzes the epimerization 3-.beta.-hydroxy-vitamin D3 in certain
tissues, e.g., keratinocytes, bone cells. The vitamin D3 compounds
of the present invention can be used as substitutes for natural and
synthetic forms of vitamin D3.
[0007] Accordingly, the present invention pertains to cyclic ether
vitamin. D3 compounds having the formula (I) as follows:
##STR00001##
wherein A.sub.1, A.sub.2 and A.sub.3 represent a single or a double
bond; X, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 can, e.g.,
be chosen individually from the group of: a hydrogen, a halogen, a
haloalkyl, a hydroxy, a hydroxy-protecting group, an alkyl, e.g., a
lower alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl group and a
heterocyclic group. The orientation of the X group can be in either
an .alpha.- or a .beta.-configuration.
[0008] In a preferred embodiment, the cyclic ether vitamin D3
compound is in its 3-epi configuration, wherein the orientation of
the X group on the A-ring is in an .alpha.-configuration.
[0009] The present invention also pertains to 3-epi forms of
1.alpha.-hydroxy-vitamin D3 compounds having the formula II as
follows:
##STR00002##
wherein A.sub.1 represents a single, a double, e.g., a
trans-double, a cis-double, or a triple bond; A.sub.2, A.sub.3 and
A.sub.4 represent a single or a double bond; R.sub.2, R.sub.3,
R.sub.4, R.sub.7, R.sub.8 and R.sub.9 can, e.g., be chosen
individually from the group of: a hydrogen, a deuterium, a
deuteroalkyl, a hydroxy, an alkyl, e.g., a lower alkyl, e.g., a
C.sub.1-C.sub.4 alkyl, an alkoxide, an O-acyl, a halogen, e.g., a
fluoride, a haloalkyl (e.g., a fluoroalkyl, --CF.sub.3), a
hydroxyalkyl, e.g., a hydroxyalkyl wherein the alkyl group is a
C.sub.4-C.sub.10 alkyl, an amine or a thiol group, and wherein the
pairs of R.sub.2 and R.sub.3, or R.sub.4 and R.sub.7 taken together
can be an oxygen atom, e.g., as in a carbonyl moiety
##STR00003##
and R.sub.5 and R.sub.6 can, e.g., each be chosen individually from
the group of: a hydrogen, a deuterium, a halogen, e.g., a fluoride,
an alkyl, e.g., a lower alkyl, e.g., a C.sub.1-C.sub.4 alkyl, a
hydroxyalkyl, a haloalkyl, e.g., a fluoroalkyl, and a deuteroalkyl.
The amine or thiol group of R.sub.2, R.sub.3, R.sub.4, R.sub.7,
R.sub.8 and R.sub.9 can be substituted to form, e.g., a primary or
a secondary amine, or a primary or secondary thiol, wherein the
substituents can be an alkyl or an aryl group, e.g., a substituent
having 2- to 10-carbon atoms.
[0010] In another aspect, the present invention further pertains to
a pharmaceutical composition comprising, a therapeutically
effective amount of a vitamin D3 compound having the formulas I or
II, and a pharmaceutically acceptable carrier.
[0011] In yet another aspect, this invention provides a method of
modulating a biological activity of a vitamin D3-responsive cell.
This method comprising contacting the cell with an effective amount
of an isolated vitamin D3 compound of formulas I and II such that
modulation of the activity of the cell occurs.
[0012] Another aspect of the invention provides a method of
treating in a subject, a disorder characterized by aberrant growth
or activity of a cell, comprising administering to the subject an
effective amount of a pharmaceutical composition of a vitamin D3
compound of formulas I and II such that the growth or activity of
the cell is reduced.
[0013] In a preferred embodiment, the vitamin D3 compound of
formulas I and II used in the treatment has improved biological
properties compared to vitamin D3, such as enhanced stability
and/or reduced toxicity.
[0014] In one aspect, a method for inhibiting the proliferation
and/or an inducing the differentiation of a hyperproliferative skin
cell is provided, wherein the hyperproliferative skin cell can be
an epidermal cell or an epithelial cell. Accordingly, therapeutic
methods for treating hyperproliferative skin disorders, e.g.,
psoriasis, are provided.
[0015] In certain embodiments, the instant method can be used for
the treatment of, or prophylactic prevention of a disorder
characterized by aberrant cell growth of vitamin D3-responsive
neoplastic cell, e.g., by administering a pharmaceutical
preparation of a vitamin D3 compound having the formula as shown in
I or II in an amount effective to inhibit growth of the neoplastic
cells.
[0016] In another aspect, the subject method can be used to
modulate an immune response, comprising administering to a subject
a pharmaceutical preparation of a vitamin D compound so as to alter
immune function in the subject. In one embodiment, the method can
be used in the treatment of lymphoid cells, e.g., T cells, natural
killer cells, so as to suppress immune reactions, e.g., to decrease
T cell activity, e.g., to decrease production of lymphokines such
as IL-2 and IFN-.gamma., to decrease T cell proliferation. In
preferred embodiments, the method can be used in treating graft
rejection, autoimmunity and inflammation.
[0017] In yet another aspect, the vitamin D3 compound of the
present invention are useful in the treatment of disorder
characterized by a deregulation of calcium and phosphate
metabolism, comprising administering to a subject a pharmaceutical
preparation of a vitamin D3 compounds of formulas I and II so as to
ameliorate the deregulation in calcium and phosphate
metabolism.
[0018] In a preferred embodiment, the disorder is osteoporosis. In
other embodiments, the vitamin D3 compounds of formulas I and II
can be used to treat diseases characterized by other deregulations
in the metabolism of calcium and phosphate.
[0019] In another aspect, a method for inhibiting PTH secretion in
parathyroid cell using the vitamin D3 compound of formulas I and II
is provided. Furthermore, therapeutic methods for treating
secondary hyperparathyroidism are also provided.
[0020] In yet another aspect, the present invention provides a
method of preventing or protecting against neuronal loss by
contacting a vitamin D3-responsive cell, e.g., a neuronal cell,
with a vitamin D3 compound of formulas I and II to prevent or
retard neuron loss.
[0021] In yet another aspect, the present invention provides a
method of modulating the activity of a vascular smooth muscle cell
by contacting a vitamin D3-responsive smooth muscle cell with a
vitamin D3 compound of formulas I and II to activate or,
preferably, inhibit the activity of the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a compilation of the chemical structures of 266
vitamin D3 compounds (Boullion, R. et al. (1995) Endocrinology
Reviews 16(2): 200-257, the contents of which including the figures
depicted therein are incorporated by reference). Each analog is
identified by its chemical name and a one, two, or three-letter
identification code.
[0023] FIG. 2 shows the HPLC profile and UV spectra of the
metabolites produced in human keratinocytes incubated with
1.alpha., 25(OH).sub.2-3-epi vitamin D3.
[0024] FIG. 3 shows the mass spectra of 1.alpha.,
25(OH).sub.2-3-epi vitamin D3 and its cyclic ether metabolite.
[0025] FIG. 4 shows the proposed metabolic pathway for the
formation of the cyclic ether metabolite of 1.alpha.,
25(OH).sub.2-3-epi vitamin D.sub.3.
[0026] FIG. 5A shows the metabolism of 1.alpha.(OH)-vitamin D3 into
its 3 epi form in the rat osteosarcoma cell line (UMR-106).
[0027] FIG. 5B is a schematic of the 3-epimerization of
1.alpha.(OH)D3 into 1.alpha.OH)-3-epi vitamin D.sub.3.
[0028] FIG. 6 shows the mass spectra of 1.alpha.(OH)D.sub.3 and its
3-epi metabolite.
[0029] FIG. 7 shows the HPLC profile and UV spectra of the
metabolites produced in rat osteosarcoma cell lines (UMR-106) which
were incubated with 1.alpha.(OH)D.sub.3 for 24, 48, or 84
hours.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The language "cyclic ether vitamin D3 compound" is intended
to include all vitamin D3 compounds having a cyclic ether side
chain, including 3-epimeric and non-3-epimeric of vitamin D3 as
represented by the general formula I.
[0031] As used herein, the terms "3-epi vitamin D3" or "3-epi D3"
compounds are intended to include vitamin D3 compounds having a
substituent, e.g., a functional group, e.g., a hydroxyl group,
attached to the carbon at position 3 of the A-ring in an
.alpha.-configuration rather than a .beta.-configuration. The
language "3-epi forms of 1.alpha.-hydroxy-vitamin D3 compounds" or
"1.alpha.-hydroxy-3-epi-vitamin D3 compounds" is intended to
include 1.alpha.-hydroxy-vitamin D3 compounds having the hydroxyl
group, attached to the carbon at position 3 of the A-ring in an
.alpha.-configuration rather than a .beta.-configuration, and which
are represented by the general formula II as described in detail
below.
[0032] The cyclic ether and 1.alpha.-hydroxy-vitamin D3 compounds
of formulae I and II, respectively, referred to hereinafter as
"vitamin D3 compounds of formulas I and II" can be produced in vivo
via a pathway which catalyzes the epimerization
3-.beta.-hydroxy-vitamin D3 in certain tissues, e.g., keratinocytes
or bone cells.
[0033] The language "vitamin D3 compounds" or "cholecalciferols"
(also referred to herein as "D3 compounds") is intended to include
compounds which are structurally similar to vitamin D3. Many of
these compounds are art-recognized and comprise a large number of
natural precursors, metabolites, as well as synthetic analogs of
the hormonally active 1.alpha.,25-dihydroxyvitamin D.sub.3
(1.alpha.,25(OH).sub.2D.sub.3). This language is intended to
include vitamin D3, or an analog thereof, at any stage of its
metabolism, as well as mixtures of different metabolic forms of
vitamin D.sub.3 or analogs thereof. Furthermore, the term "vitamin
D3 compound" also includes synthetic analogs of vitamin D3
illustrated in FIG. 1.
[0034] In the formulas presented herein, the various substituents
are illustrated as joined to the steroid nucleus by one of these
notations: a dotted line (--) indicating a substituent which is in
the .beta.-orientation (i.e., above the plane of the ring), a
wedged solid line indicating a substituent which is in the
.alpha.-orientation (i.e., below the plane of the molecule), or a
solid line (-) indicating a substituent in the plane of the ring.
It should be understood that the stereochemical convention in the
steroid field is opposite from the general chemical field, wherein
a dotted line indicates a substituent which is in an
.alpha.-orientation (i.e., below the plane of the molecule), and a
wedged solid line indicates a substituent which is in the
.beta.-orientation (i.e., above the plane of the ring). As shown,
the A ring of the hormone 1.alpha.,25(OH).sub.2D.sub.3 contains two
asymetric centers at chiral carbons-1 and -3, each one containing a
hydroxyl group in well-characterized configurations, namely the
1.alpha.- and 3.beta.-hydroxyl groups.
[0035] Accordingly, the present invention pertains to cyclic ether
vitamin D3 compounds having the formula (I) as follows:
##STR00004##
wherein A.sub.1, A.sub.2 and A.sub.3 represent a single or a double
bond; X, R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 can, e.g.,
be chosen individually from the group of: a hydrogen, a halogen, a
haloalkyl, a hydroxy, a hydroxy-protecting group, an alkyl, e.g., a
lower alkyl, an alkenyl, an alkynyl, an alkoxy, an aryl group and a
heterocyclic group. The orientation of the X group can be in either
an .alpha.- or a .beta.-configuration.
[0036] In a preferred embodiment, the cyclic ether vitamin D3
compound is represented by the general formula I, wherein the
orientation of the X group on the A-ring is in an
.alpha.-configuration; A.sub.1 is a single bond; A.sub.2 and
A.sub.3 are each a double bond; --X and R.sub.1 are hydroxyl
groups; R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are a hydrogen.
[0037] The present invention also pertains to 3-epi forms of
1.alpha.-hydroxy-vitamin D3 compounds having the formula II:
##STR00005##
wherein A.sub.1 represents a single, a double, e.g., a
trans-double, a cis-double, or a triple bond; A.sub.2, A.sub.3 and
A4 represent a single or a double bond; R.sub.2, R.sub.3, R.sub.4,
R.sub.7, R.sub.8 and R.sub.9 can, e.g., be chosen individually from
the group of: a hydrogen, a deuterium, a deuteroalkyl, a hydroxy,
an alkyl, e.g., a lower alkyl, e.g., a C.sub.1-C.sub.4 alkyl, an
alkoxide, an O-acyl, a halogen, e.g., a fluoride, a haloalkyl
(e.g., a fluoroalkyl, --CF.sub.3), a hydroxyalkyl, e.g., a
hydroxyalkyl wherein the alkyl group is a C.sub.4-C.sub.10 alkyl,
an amine or a thiol group, and wherein the pairs of R.sub.2 and
R.sub.3, or R.sub.4 and R.sub.7 taken together can be an oxygen
atom, e.g., as in a carbonyl moiety
##STR00006##
and R.sub.5 and R.sub.6 can, e.g., each be chosen individually from
the group of: a hydrogen, a deuterium, a halogen, e.g., a fluoride,
an alkyl, e.g., a lower alkyl, e.g., a C.sub.1-C.sub.4 alkyl, a
hydroxyalkyl, a haloalkyl, e.g., a fluoroalkyl, and a deuteroalkyl.
The amine or thiol group of R.sub.2, R.sub.3, R.sub.4, R.sub.7,
R.sub.8 and R.sub.9 can be substituted to form, e.g., a primary or
a secondary amine, or a primary or a secondary thiol, wherein the
substituents can be an alkyl or an aryl group, e.g., a substituent
having 2- to 10-carbon atoms.
[0038] In a preferred embodiment, A.sub.1, A.sub.2 and A.sub.3 are
each a single bond; A.sub.4 is a double bond; R.sub.2, R.sub.3,
R.sub.5, R.sub.6, R.sub.8 and R.sub.9 are each a hydrogen or an
alkyl, e.g., a methyl; and R.sub.4 and R.sub.7 are each a hydrogen,
a hydroxy or an alkyl, e.g., a lower alkyl, e.g., a methyl or an
ethyl group. The chirality of the positions substituted by R.sub.4
and R.sub.7 can be in either an R-- or an S-configuration.
[0039] Exemplary preferred 1.alpha.-hydroxy vitamin D.sub.3
compounds encompassed by formula II include: 1.alpha. hydroxy 3-epi
vitamin D.sub.3, 1.alpha.,24 dihydroxy 3-epi vitamin D.sub.3 (both
1.alpha., 24R-dihydroxy 3-epi vitamin D.sub.3 and 1.alpha.,
24S-dihydroxy 3-epi vitamin D.sub.3), 1.alpha. hydroxy 24-ethyl
3-epi vitamin D.sub.3, 1.alpha. hydroxy 24-methyl 3-epi vitamin
D.sub.3 and 1.alpha., 24-dihydroxy 24-methyl 3-epi vitamin D.sub.3
having the following chemical formulae:
##STR00007## ##STR00008##
[0040] A representation of 1.alpha.-hydroxy-vitamin D3 prior to
3-epi conversion is also depicted as analog BP in FIG. 1.
[0041] In yet another embodiment, the present invention provides
isolated vitamin D3 compounds of formulae I and II, having at least
one biological activity of vitamin D3, and having improved
biological properties compared to vitamin D3, such as enhanced
stability in vivo and/or reduced toxicity.
[0042] The term "epimer" or "epi" compounds is intended to include
compounds having a chiral carbon that varies in the orientation of
a single bond to a substituent on that carbon compared to the
naturally-occurring (or reference) compound, for example, a carbon
where the orientation of the bond to the substituent is in an
.alpha.-configuration, instead of a .alpha.-configuration. The
3-epimer forms of vitamin D3 compounds having the general formulas
I and II have a substituent, e.g., a hydroxyl group, attached to
the carbon at position 3 of the A-ring in an .alpha.-configuration
rather than a .beta.-configuration, whereas all other substituents
can be in either an .alpha.- or a .beta.-configuration.
[0043] As used herein, the term "substituent" refers to a moiety,
for example a functional group, attached to the carbon position 3
of the A ring of the vitamin D.sub.3 compound that allows the
compound to perform its intended function. Accordingly, the term
"substituent" is intended to include hydrogen, halogen, haloalkyl,
hydroxy, hydroxy-protecting group, alkyl, e.g. lower alkyl,
alkenyl, e.g., lower alkenyl, alkynyl, e.g., lower alkynyl, alkoxy,
aryl group and heterocyclic group.
[0044] The term "chiral" refers to molecules which have the
property of non-superimposability of the mirror image partner,
while the term "achiral" refers to molecules which are
superimposable on their mirror image partner. The term
"stereoisomers" or "isomers" refer to compounds which have
identical chemical constitution, but differ with regard to the
arrangement of the atoms or groups in space. In particular,
"enantiomers" refer to two stereoisomers of a compound which are
non-superimposable mirror images of one another. An equimolar
mixture of two enantiomers is called a "racemic mixture" or a
"racemate". "Diastereomers" refer to stereoisomers with two or more
centers of dissymmetry and whose molecules are not mirror images of
one another. With respect to the nomenclature of a chiral center,
terms "d" and "l" configuration are as defined by the IUPAC
Recommendations. As to the use of the terms, diastereomer,
racemate, epimer and enantiomer will be used in their normal
context to describe the stereochemistry of preparations.
[0045] As used herein, the language "isomeric counterparts of
vitamin D3" or "non-epimeric forms" refers to stereoisomers of the
3-epi vitamin D3 compounds. For example, vitamin D3 compounds which
have the orientation of the 3-hydroxy group in a
.alpha.-configuration.
[0046] The terms "isolated" or "substantially purified" as used
interchangeably herein refer to vitamin D.sub.3 compounds in a
non-naturally occurring state. The compounds can be substantially
free of cellular material or culture medium when naturally
produced, or chemical precursors or other chemicals when chemically
synthesized. In other preferred embodiments, the terms "isolated"
or "substantially purified" also refer to preparations of a chiral
compound which substantially lack one of the enantiomers, i.e.,
enantiomerically enriched or non-racemic preparations of a
molecule. Similarly, isolated epimers or diasteromers refers to
preparations of chiral compounds which are substantially free of
other stereochemicai forms. For instance, isolated or substantially
purified vitamin D.sub.3 compounds includes synthetic or natural
preparations of a vitamin D.sub.3 enriched for the stereoisomers
having a substituent attached to the chiral carbon at position 3 of
the A-ring in an .alpha.-configuration, and thus substantially
lacking other isomers having a configuration. Unless otherwise
specified, such terms refer to vitamin D.sub.3 compositions in
which the ratio of a to .beta. forms is greater that 1:1 by weight.
For instance, an isolated preparation of an .alpha. epimer means a
preparation having greater than 50% by weight of the .alpha.-epimer
relative to the .beta. stereoisomer, more preferably at least 75%
by weight, and even more preferably at least 85% by weight. Of
course the enrichment can be much greater than 85%, providing a
"substantially epimer enriched", which refers to preparations of a
compound which have greater than 90% of the .alpha.-epimer relative
to the .beta. stereoisomer, and even more preferably greater than
95%. The term "substantially free of the .beta., stereoisomer" will
be understood to have similar purity ranges.
[0047] As used herein, the language "alkyl" is art-recognized and
includes to the radical of saturated aliphatic groups, including
straight-chain alkyl groups, branched-chain alkyl groups,
cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups,
and cycloalkyl substituted alkyl groups. In preferred embodiments,
a straight chain or branched chain alkyl has 30 or fewer carbon
atoms in its backbone (e.g., C.sub.1-C.sub.30 for straight chain,
C.sub.3-C.sub.30 for branched chain), and more preferably 20 or
fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms
in their ring structure, and more preferably have 5, 6 or 7 carbons
in the ring structure.
[0048] Unless the number of carbons is otherwise specified, "lower
alkyl" as used herein means an alkyl group, as defined above, but
having from one to ten carbons, more preferably from one to six,
and most preferably from one to four carbon atoms in its backbone
structure, which may be straight or branched-chain, which may be
straight or branched-chain. Examples of lower alkyl groups include
methyl, ethyl, n-propyl, i-propyl, tert.-butyl, hexyl, heptyl,
octyl and so forth. Likewise, "lower alkenyl" and "lower alkynyl"
have similar chain lengths. Preferred alkyl groups include lower
alkyls. Examples of alkylene groups are methylene, ethylene,
propylene and so forth.
[0049] Moreover, the term alkyl as herein is intended to include
both "unsubstituted alkyls" and "substituted alkyls", the latter of
which refers to alkyl moieties having substituents replacing a
hydrogen on one or more carbons of the hydrocarbon backbone. Such
substituents can include, for example, halogen, hydroxyl, carbonyl
(including aldehydes, ketones, carboxylates, and esters), alkoxyl,
ether, phosphoryl, cyano, amino, acylamino, amido, amidino, imino,
sulfhydryl, alkylthio, arylthio, thiolcarbonyl (including
thiolformates, thiolcarboxylic acids, and thiolesters), sulfonyl,
nitro, heterocyclyl, aralkyl, or an aromatic or heteroaromatic
moiety. It will be understood by those skilled in the art that the
moieties substituted on the hydrocarbon chain can themselves be
substituted, if appropriate. For instance, the substituents of a
substituted alkyl may include substituted and unsubstituted forms
of amino, acylaminos, iminos, amidos, phosphoryls (including
phosphonates and phosphinates), sulfonyls (including sulfates,
sulfonatos, sulfamoyls, and sulfonamidos), and silyl groups, as
well as ethers, alkylthios, arylthios, carbonyls (including
ketones, aldehydes, carboxylates, and esters), --CF.sub.3, --CN and
the like. Exemplary substituted alkyls are described below.
Cycloalkyls can be further substituted with alkyls, alkenyls,
alkoxys, alkylthios, arylthios, aminoalkyls, carbonyl-substituted
alkyls, CF.sub.3, cyano (--CN), and the like.
[0050] The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl group (e.g., an aromatic or
heteroarornatic group).
[0051] The terms "alkenyl" and "alkynyl" are art-recognized and
include to unsaturated aliphatic groups analogous in length and
possible substitution to the alkyls described above, but that
contain at least one double or triple bond respectively.
[0052] The terms "alkoxyl" is art-recognized and includes to an
group represented by the formula --O-alkyl. Representative alkoxyl
groups include methoxy, ethoxy, propoxy, tert-butoxy and the like.
Unless otherwise specified, an "alkoxy" group can be replaced with
a group represented by --O-alkenyl, --O-alkynyl, --O-aryl (i.e., an
aryloxy group), or --O-heterocyclyl. An "ether" is two substituted
or unsubstituted hydrocarbons covalently linked by an oxygen.
Accordingly, the substituent of, e.g., an alkyl that renders that
alkyl an ether is or resembles an alkoxyl, such as can be
represented by one of --O-alkyl, --O-alkenyl, --O-alkynyl,
--O-aryl, or --O-heterocyclyl. The term "lower alkoxy" includes a
lower alkyl group attached to the remainder of the molecule by
oxygen.
[0053] Examples of alkoxy groups include methoxy, ethoxy,
isopropoxy, tert.-butoxy and so forth. The term "phenyl alkoxy"
refer to an alkoxy group which is substituted by a phenyl ring.
Examples of phenyl alkoxy groups are benzyloxy, 2-phenylethoxy,
4-phenylbutoxy and so forth. The term "alkanoyloxy group" refers to
the residue of an alkylcarboxylic acid formed by removal of the
hydrogen from the hydroxyl portion of the carboxyl group. Examples
of alkanoyloxy groups include formyloxy, acetoxy, butyryloxy,
hexanolyoxy and so forth. The term "substituted" as applied to
"phenyl" refers to phenyl which is substituted with one or more of
the following groups: alkyl, halogen (i.e., fluorine, chlorine,
bromine or iodine), nitro, cyano, trifluoromethly and so forth. The
"alkanol" or a "hydroxyalkyl" refer to a compound derived by
protonation of the oxygen atom of an alkoxy group. Examples of
alkanols include methanol, ethanol, 2-propanol, 2-methyl-2-propanol
and the like.
[0054] As used herein the term "hydroxy-protecting group" includes
any group commonly used for the protection of hydroxy functions
during subsequent reactions, including, for example, acyl or
alkylsilyl groups such as trimethylsilyl, triethylsilyl,
t-butyldimethylsilyl and analogous alkylated silyl radicals, or
alkoxyalkyl groups such as methoxymethyl, ethoxymethyl,
methoxyethoxymethyl, tetrahydroftiranyl or tetrahydropyranyl. A
"protected-hydroxy" is a hydroxy function derivatized by one of the
above hydroxy-protecting groupings.
[0055] As used herein, the term "halogen" designates --F, --Cl,
--Br or --I; the term "sulfhydryl" or "thiol" means --SH; the term
"hydroxyl" means --OH.
[0056] The term "aryl" is art-recognized and includes 5- and
6-membered single-ring aromatic groups that may include from zero
to four heteroatoms, for example, benzene, pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole,
pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl
groups also include polycyclic fused aromatic groups such as
naphthyl, quinolyl, indolyl, and the like. Those aryl groups having
heteroatoms in the ring structure may also be referred to as "aryl
heterocycles", "heteroaryls" or "heteroaromatics". The aromatic
ring can be substituted at one or more ring positions with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, acylamino,
azido, nitro, sulfhydryl, imino, amido, amidino, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, arylthio,
sulfonyl, sulfonamido, sulfamoyl, ketone, aldehyde, ester, a
heterocyclyl, an aromatic or heteroaromatic moiety, --CF.sub.3,
--CN, or the like. Aryl groups can also be fused or bridged with
alicyclic or heterocyclic rings which are not aromatic so as to
form a polycycle (e.g., tetralin).
[0057] The terms "heterocyclyl" or "heterocyclic group" are
art-recognized and include 3- to 10-membered ring structures, more
preferably 4- to 7-membered rings, which ring structures include
one to four heteroatoms. Heterocyclyl groups include pyrrolidine,
oxolane, thiolane, imidazole, oxazole, piperidine, piperazine,
morpholine, lactones, lactams such as azetidinones and
pyrrolidinones, lactones, sultams, sultones, and the like. The
heterocyclic ring can be substituted at one or more positions with
such substituents as described above, as for example, halogen,
alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino,
acylamino, nitro, sulfhydryl, imino, amido, phosphonate,
phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, arylthio,
sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
[0058] The terms "polycyclyl" or "polycyclic group" are
art-recognized and include two or more cyclic rings (e.g.,
cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or
heterocyclyls) in which two or more carbons are common to two
adjoining rings, e.g., the rings are "fused rings". Rings that are
joined through non-adjacent atoms are termed "bridged" rings. Each
of the rings of the polycycle can be substituted with such
substituents as described above, as for example, halogen, alkyl,
aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, acylamino,
nitro, sulfhydryl, imino, amido, phosphonate, phosphinate,
carbonyl, carboxyl, silyl, ether, alkylthio, arylthio, sulfonyl,
ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, --CF.sub.3, --CN, or the like.
Vitamin D Synthesis
[0059] The vitamin D3 compounds of the present invention can be
prepared using a variety of synthetic methods, as are known in the
art. For example, many of the above-described compounds can be
prepared by chemical synthesis, or alternatively by enzymatic
conversion of a 3.beta.-vitamin D3 precursor, e.g., by perfusing a
3.beta.-vitamin D3 precursor, a vitamin D3 compound having the
orientation of the hydroxy group at position 3 of the A-ring in a
.beta.-configuration, in a tissue-containing an enzyme which
catalyzes the epimerization of the 3-.beta.-hydroxyl group to the
3.alpha. form vitamin D3 compounds, e.g., keratinocytes or bone
cells as described in Examples I, II and IV.
[0060] For example, methods for synthesizing vitamin D3 compounds
of formulas I and II are well known in the art (see e.g., Bouillon,
F et al., Endocrine Reviews 16(2):201-204; Ikekawa N. (1987) Med.
Res. Rev. 7:333-366; DeLuca H. F. and Ostrem V. K. (1988) Prog.
Clin. Biol. Res. 259:41-55; Ikekawa N. and Ishizuka S. (1992) CRC
Press 8:293-316; Calverley M. J. and Jones G. (1992) Academic Press
193-270; Pardo R. and Santelli M. (1985) Bull. Soc. Chim.
Fr:98-114; Bythgoe B. (1980) Chem. Soc. Rev. 449-475; Quinkert G.
(1985) Synform 3:41-122; Quinkert G. (1986) Synform 4:131-256;
Quinkert G. (1987) Synform 5:1-85; Mathieu C. et al. (1994)
Diabetologia 37:552-558; Dai H. and Posner G. H. (1994) Synthesis
1383-1398). Exemplary methods of synthesis include the
photochemical ring opening of a 1-hydroxylated side chain-modified
derivative of 7-dehydrocholesterol which initially produces a
previtamin that is easily thermolyzed to vitamin D3 in a well known
fashion (Barton D. H. R et al. (1973) J. Am. Chem. Soc.
95:2748-2749; Barton D. H. R. (1974) JCS Chem. Comm. 203-204);
phosphine oxide coupling method developed by (Lythgoe et al (1978)
JCS Perkin Trans. 1:590-595) which comprises coupling a phosphine
oxide to a Grundmann's ketone derivative to directly produce a
1.alpha.,25(OH).sub.2D3 skeleton as described in Baggiolini E. G.
et al. (1986) J. Org. Chem. 51:3098-3108; DeSchrijver J. and
DeClercq P. J. (1993) Tetrahed Lett 34:4369-4372; Posner G. H and
Kinter C. M. (1990) J. Org. Chem. 55:3967-3969; semihydrogenation
of dienynes to a previtamin structure that undergoes rearrangement
to the corresponding vitamin D3 analog as described by Harrison R.
G. et al. (1974) JCS Perkin Trans. 1:2654-2657; Castedo L. et al.
(1988) Tetrahed Lett 29:1203-1206; Mascarenas J. S. (1991)
Tetrahedron 47:3485-3498; Barrack S. A. et al. (1988) J. Org. Chem.
53:1790-1796) and Okamura W. H. et al. (1989) J. Org. Chem.
54:4072-4083; the vinylallene approach involving intermediates that
are subsequently arranged using heat or a combination of metal
catalyzed isomerization followed by sensitized photoisomerization
(Okamura W. H. et al. (1989) J. Org. Chem. 54:4072-4083; Van
Alstyne E. M. et al. (1994) J. Am. Chem. Soc. 116:6207-6210); the
method described by Trost et al. B. M. et al. J. Am. Chem. Soc.
114:9836-9845; Nagasawa K. et al. (1991) Tetrahed Lett 32:4937-4940
involves an acyclic A-ring precursor which is intramolecular
cross-coupled to the bromoenyne leading directly to the formation
of 1,25(OH).sub.2D3 skeleton; a tosylated derivative which is
isomerized to the i-steroid that can be modified at carbon-1 and
then subsequently back-isomerized under sovolytic conditions to
form 1.alpha.,25(OH).sub.2D2 or analogs thereof (Sheves M. and
Mazur Y. (1974) J. Am. Chem. Soc. 97:6249-6250; Paaren H. E. et al.
(1980) J. Org. Chem. 45:3253-3258; Kabat M. et al. (1991) Tetrahed
Lett 32:2343-2346; Wilson S. R. et al. (1991) Tetrahed Lett
32:2339-2342); the direct modification of vitamin D derivatives to
1-oxygenated 5,6-trans vitamin D as described in (Andrews D. R. et
al. (1986) J. Org. Chem. 51:1635-1637); the Diels-Alders
cycloadduct method of previtamin D3 can be used to cyclorevert to
1.alpha.,25(OH).sub.2D2 through the intermediary of a previtamin
form via thermal isomerization (Vammaele L. et al. (1985)
Tetrahedron 41:141-144); and, a final method entails the direct
modification of 1.alpha.,25(OH).sub.2D2 or an analog through use of
suitable protecting groups such as transition metal derivatives or
by other chemical transformations (Okarmura W. H. et al. (1992) J.
Cell Biochem. 49:10-18). Additional methods for synthesizing
vitamins D2 compounds are described in, for example, Japanese
Patent Disclosures Nos. 62750/73,26858/76, 2685976, and 71456/77;
U.S. Pat. Nos. 3,639,596; 3,715,374; 3,847,955 and 3,739,001.
[0061] Examples of the compounds of this invention having a
saturated side chain can be prepared according to the general
process illustrated and described in U.S. Pat. No. 4,927,815, the
description of which is incorporated herein by reference. Examples
of the compounds of this invention having an unsaturated side chain
is can be prepared according to the general process illustrated and
described in U.S. Pat. No. 4,847,012, the description of which is
incorporated herein by reference. Examples of the compounds of this
invention wherein R groups together represent a cyclopentano group
can be prepared according to the general process illustrated and
described in U.S. Pat. No. 4,851,401, the description of which
incorporated herein by reference.
[0062] Another synthetic strategy for the preparation of
side-chain-modified analogues of
1.alpha.,25-dihydroxyergocalciferol is disclosed in Kutner et al.,
The Journal of Organic Chemistry, 1988, 53:3450-3457. In addition,
the preparation of 24-homo and 26-homo vitamin D analogs are
disclosed in U.S. Pat. No. 4,717,721, the description of which is
incorporated herein by reference.
[0063] The enantioselective synthesis of chiral molecules is now
state of the art. Through combinations of enantioselective
synthesis and purification techniques, many chiral molecules can be
synthesized as an enantiomerically enriched preparation. For
example, methods have been reported for the enantioselective
synthesis of A-ring diastereomers of 1.alpha.,25(OH).sub.2D3 as
described in Muralidharan et al. (1993) J. Organic Chem. 58(7):
1895-1899 and Norman et al. (1993) J. Biol. Chem. 268(27):
20022-30. Other methods for the enantiomeric synthesis of various
compounds known in the art include, inter alia, epoxides (see,
e.g., Johnson, R. A.; Sharpless, K. B. In Catalytic Asymmetric
Synthesis; Ojima, I., Ed.: VCH: New York, 1993; Chapter 4.1.
Jacobsen, E. N. Ibid. Chapter 4.2), diols (e.g., by the method of
Sharpless, J. Org. Chem. (1992) 57:2768), and alcohols (e.g., by
reduction of ketones, E. J. Corey et al., J. Am. Chem. Soc. (1987)
109:5551). Other reactions useful for generating optically enriched
products include hydrogenation of olefins (e.g., M. Kitamura et
al., J. Org. Chem. (1988) 53:708); Diels-Alder reactions (e.g., K.
Narasaka et al., J. Am. Chem. Soc. (1989) 111:5340); aldol
reactions and alkylation of enolates (see, e.g., D. A. Evans et
al., J. Am. Chem. Soc. (1981) 103:2127; D. A. Evans et al., J. Am.
Chem. Soc. (1982) 104:1737); carbonyl additions (e.g., R. Noyori,
Angew. Chem. Int. Ed. Eng. (1991) 30:49); and ring-opening of
meso-epoxides (e.g., Martinez, L. E.; Leighton J. L., Carsten, D.
H.; Jacobsen, E. N. J. Am. Chem. Soc. (1995) 117:5897-5898). The
use of enymes to produce optically enriched products is also well
known in the art (e.g., M. P. Scheider, ed. "Enzymes as Catalysts
in Organic Synthesis", D. Reidel, Dordrecht (1986).
[0064] Chiral synthesis can result in products of high stereoisomer
purity. However, in some cases, the stereoisomer purity of the
product is not sufficiently high. The skilled artisan will
appreciate that the separation methods described herein can be used
to further enhance the stereoisomer purity of the vitamin D3-epimer
obtained by chiral synthesis.
[0065] Separation of isomers can be accomplished in several ways
known in the art. An exemplary straight phase and reverse phase
HPLC system used to separate natural or synthetic diastereomers of
1.alpha.,25(OH).sub.2D3 is detailed in the appended example and
illustrated in FIG. 2. Further methods for separating a racemic
mixture of two enantiomers include chromatography using a chiral
stationary phase (see, e.g., "Chiral Liquid Chromatography", W. J.
Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also
be separated by classical resolution techniques. For example,
formation of diastereomeric salts and fractional crystallization
can be used to separate enantiomers. For the separation of
enantiomers of carboxylic acids, the diastereomeric salts can be
formed by addition of enantiomerically pure chiral bases such as
brucine, quinine, ephedrine, strychnine, and the like.
Alternatively, diastereomeric esters can be formed with
enantiomerically pure chiral alcohols such as menthol, followed by
separation of the diastereomeric esters and hydrolysis to yield the
free, enantiomerically enriched carboxylic acid. For separation of
the optical isomers of amino compounds, addition of chiral
carboxylic or sulfonic acids, such as camphorsulfonic acid,
tartaric acid, mandelic acid, or lactic acid can result in
formation of the diastereomeric salts.
Pharmaceutical Compositions
[0066] In another aspect, the present invention provides
pharmaceutically acceptable compositions which comprise a
therapeutically-effective amount of one or more of the isolated
vitamin D.sub.3 compounds of formulas I and II, formulated together
with one or more pharmaceutically acceptable carrier(s).
[0067] In a preferred embodiment, these pharmaceutical compositions
are suitable for topical or oral administration to a subject. In
other embodiments, as described in detail below, the pharmaceutical
compositions of the present invention may be specially formulated
for administration in solid or liquid form, including those adapted
for the following: (1) oral administration, for example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets,
boluses, powders, granules, pastes; (2) parenteral administration,
for example, by subcutaneous, intramuscular or intravenous
injection as, for example, a sterile solution or suspension; (3)
topical application, for example, as a cream, ointment or spray
applied to the skin; (4) intravaginally or intrarectally, for
example, as a pessary, cream or foam; or (5) aerosol, for example,
as an aqueous aerosol, liposomal preparation or solid particles
containing the compound.
[0068] In certain embodiments, the subject is a mammal, e.g., a
primate, e.g., a human. As used herein, the language "subject" is
intended to include human and non-human animals. Preferred human
animals include a human patient having a disorder characterized by
the aberrant activity of a vitamin D.sub.3-responsive cell. The
term "non-human animals" of the invention includes all vertebrates,
e.g., mammals and non-mammals, such as non-human primates, sheep,
dog, cow, chickens, amphibians, reptiles, etc.
[0069] The phrase "therapeutically-effective amount" as used herein
means that amount of a vitamin D.sub.3 compound(s) of formulas I
and II, or composition comprising such a compound which is
effective for the compound to produce its intended function, e.g.,
the modulation of activity of a vitamin D.sub.3-response cell. The
effective amount can vary depending on such factors as the type of
cell growth being treated or inhibited, the particular type of
vitamin D.sub.3 compound, the size of the subject, or the severity
of the undesirable cell growth or activity. One of ordinary skill
in the art would be able to study the aforementioned factors and
make the determination regarding the effective amount of the
vitamin D.sub.3 compound of formulas I and II without undue
experimentation.
[0070] In certain embodiments, one or more vitamin D.sub.3
compounds as represented by formulas I and II may be administered
alone, or as part of combinatorial therapy. For example, the
vitamin D.sub.3 compounds can be conjointly administered with one
or more agents such as mitotic inhibitors, alkylating agents,
antimetabolites, nucleic acid, intercalating agents, topoisomerase
inhibitors, agents which promote apoptosis, and/or agents which
modulate immune responses. The effective amount of vitamin D.sub.3
compound used can be modified according to the concentrations of
the other agents used.
[0071] In vitro assay using keratinocytes or parathyroid cells, or
an assay similar thereto (e.g., differing in choice of cells, e.g.,
bone cells, intestinal cells, neoplastic cells) can be used to
determine an "effective amount" of the vitamin D.sub.3 compounds of
formulas I and II, or combinations thereof. The ordinarily skilled
artisan would select an appropriate amount of each individual
compound in the combination for use in the aforementioned in vitro
assay or similar assays. Changes in cell activity or cell
proliferation can be used to determine whether the selected amounts
are "effective amount" for the particular combination of compounds.
The regimen of administration also can affect what constitutes an
effective amount. As described in detail below, vitamin D.sub.3
compounds of formulas I and II can be administered to the subject
prior to, simultaneously with, or after the administration of the
other agent(s). Further, several divided dosages, as well as
staggered dosages, can be administered daily or sequentially, or
the dose can be proportionally increased or decreased as indicated
by the exigencies of the therapeutic situation.
[0072] The phrase "pharmaceutically acceptable" is employed herein
to refer to those vitamin D.sub.3 compounds of formulas I and II,
compositions containing such compounds, and/or dosage forms which
are, within the scope of sound medical judgment, suitable for use
in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0073] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent or encapsulating material, involved in carrying or
transporting the subject chemical from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: (1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and
potato starch; (3) cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)
powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa butter and suppository waxes; (9) oils,
such as peanut oil, cottonseed oil, safflower oil, sesame oil,
olive oil, corn oil and soybean oil; (10) glycols, such as
propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and polyethylene glycol; (12) esters, such as ethyl oleate
and ethyl laurate; (13) agar; (14) buffering agents, such as
magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)
pyrogen-free water; (17) isotonic saline; (18) Ringer's solution;
(19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other
non-toxic compatible substances employed in pharmaceutical
formulations.
[0074] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, sweetening, flavoring and perfuming
agents, preservatives and antioxidants can also be present in the
compositions.
[0075] Examples of pharmaceutically-acceptable antioxidants
include: (1) water soluble antioxidants, such as ascorbic acid,
cysteine hydrochloride, sodium bisulfate, sodium metabisulfite,
sodium sulfite and the like; (2) oil-soluble antioxidants, such as
ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol,
and the like; and (3) metal chelating agents, such as citric acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tataric acid,
phosphoric acid, and the like.
[0076] Compositions containing the vitamin D.sub.3 compounds of the
present invention include those suitable for oral, nasal, topical
(including buccal and sublingual), rectal, vaginal, aerosol and/or
parenteral administration. The compositions may conveniently be
presented in unit dosage form and may be prepared by any methods
well known in the art of pharmacy. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will vary depending upon the host being treated, the
particular mode of administration. The amount of active ingredient
which can be combined with a carrier material to produce a single
dosage form will generally be that amount of the compound which
produces a therapeutic effect. Generally, out of one hundred
percent, this amount will range from about 1 percent to about
ninety-nine percent of active ingredient, preferably from about 5
percent to about 70 percent, most preferably from about 10 percent
to about 30 percent.
[0077] Methods of preparing these compositions include the step of
bringing into association a vitamin D.sub.3 compound(s) of formulas
I and II with the carrier and, optionally, one or more accessory
ingredients. In general, the formulations are prepared by uniformly
and intimately bringing into association a vitamin D.sub.3 compound
with liquid carriers, or finely divided solid carriers, or both,
and then, if necessary, shaping the product.
[0078] Compositions of the invention suitable for oral
administration may be in the form of capsules, cachets, pills,
tablets, lozenges (using a flavored basis, usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a
suspension in an aqueous or non-aqueous liquid, or as an
oil-in-water or water-in-oil liquid emulsion, or as an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and
glycerin, or sucrose and acacia) and/or as mouth washes and the
like, each containing a predetermined amount of a vitamin D.sub.3
compound(s) of formulas I and II as an active ingredient. A
compound may also be administered as a bolus, electuary or
paste.
[0079] In solid dosage forms of the invention for oral
administration (capsules, tablets, pills, dragees, powders,
granules and the like), the active ingredient is mixed with one or
more pharmaceutically-acceptable carriers, such as sodium citrate
or dicalcium phosphate, and/or any of the following: (1) fillers or
extenders, such as starches, lactose, sucrose, glucose, mannitol,
and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,
sucrose and/or acacia; (3) humectants, such as glycerol; (4)
disintegrating agents, such as agar-agar, calcium carbonate, potato
or tapioca starch, alginic acid, certain silicates, and sodium
carbonate; (5) solution retarding agents, such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7)
wetting agents, such as, for example, acetyl alcohol and glycerol
monostearate; (8) absorbents, such as kaolin and bentonite clay;
(9) lubricants, such a talc, calcium stearate, magnesium stearate,
solid polyethylene glycols, sodium lauryl sulfate, and mixtures
thereof; and (10) coloring agents. In the case of capsules, tablets
and pills, the pharmaceutical compositions may also comprise
buffering agents. Solid compositions of a similar type may also be
employed as fillers in soft and hard-filled gelatin capsules using
such excipients as lactose or milk sugars, as well as high
molecular weight polyethylene glycols and the like.
[0080] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared using binder (for example, gelatin or hydroxypropylmethyl
cellulose), lubricant, inert diluent, preservative, disintegrant
(for example, sodium starch glycolate or cross-linked sodium
carboxymethyl cellulose), surface-active or dispersing agent.
Molded tablets may be made by molding in a suitable machine a
mixture of the powdered peptide or peptidomimetic moistened with an
inert liquid diluent.
[0081] The tablets, and other solid dosage forms of the
pharmaceutical compositions of the present invention, such as
dragees, capsules, pills and granules, may optionally be scored or
prepared with coatings and shells, such as enteric coatings and
other coatings well known in the pharmaceutical-formulating art.
They may also be formulated so as to provide slow or controlled
release of the active ingredient therein using, for example,
hydroxypropylmethyl cellulose in varying proportions to provide the
desired release profile, other polymer matrices, liposomes and/or
microspheres. They may be sterilized by, for example, filtration
through a bacteria-retaining filter, or by incorporating
sterilizing agents in the form of sterile solid compositions which
can be dissolved in sterile water, or some other sterile injectable
medium immediately before use. These compositions may also
optionally contain opacifying agents and may be of a composition
that they release the active ingredient(s) only, or preferentially,
in a certain portion of the gastrointestinal tract, optionally, in
a delayed manner. Examples of embedding compositions which can be
used include polymeric substances and waxes. The active ingredient
can also be in micro-encapsulated form, if appropriate, with one or
more of the above-described excipients.
[0082] Liquid dosage forms for oral administration of the vitamin
D.sub.3 compound(s) of the invention include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions,
syrups and elixirs. In addition to the active ingredient, the
liquid dosage forms may contain inert diluents commonly used in the
art, such as, for example, water or other solvents, solubilizing
agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol,
ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, oils (in particular,
cottonseed, groundnut, corn, germ, olive, castor and sesame oils),
glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty
acid esters of sorbitan, and mixtures thereof.
[0083] Besides inert diluents, the oral compositions can also
include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, coloring, perfuming and
preservative agents.
[0084] Suspensions, in addition to the active vitamin D.sub.3
compound(s) may contain suspending agents as, for example,
ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters, microcrystalline cellulose, aluminum
metahydroxide, bentonite, agar-agar and tragacanth, and mixtures
thereof.
[0085] Pharmaceutical compositions of the invention for rectal or
vaginal administration may be presented as a suppository, which may
be prepared by mixing one or more vitamin D.sub.3 compound(s) of
formulas I and II with one or more suitable nonirritating
excipients or carriers comprising, for example, cocoa butter,
polyethylene glycol, a suppository wax or a salicylate, and which
is solid at room temperature, but liquid at body temperature and,
therefore, will melt in the rectum or vaginal cavity and release
the active agent.
[0086] Compositions of the present invention which are suitable for
vaginal administration also include pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing such carriers
as are known in the art to be appropriate.
[0087] Dosage forms for the topical or transdermal administration
of a vitamin D.sub.3 compound(s) of formulas I and II include
powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches and inhalants. The active vitamin D.sub.3
compound may be mixed under sterile conditions with a
pharmaceutically-acceptable carrier, and with any preservatives,
buffers, or propellants which may be required.
[0088] The ointments, pastes, creams and gels may contain, in
addition to vitamin D.sub.3 compound(s) of formulas I and II,
excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch, tragacanth, cellulose derivatives, polyethylene
glycols, silicones, bentonites, silicic acid, talc and zinc oxide,
or mixtures thereof.
[0089] Powders and sprays can contain, in addition to a vitamin D3
compound(s) of formulas I and II, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates and polyamide
powder, or mixtures of these substances. Sprays can additionally
contain customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0090] The vitamin D.sub.3 compound(s) of formulas I and II can be
alternatively administered by aerosol. This is accomplished by
preparing an aqueous aerosol, liposomal preparation or solid
particles containing the compound. A nonaqueous (e.g., fluorocarbon
propellant) suspension could be used. Sonic nebulizers are
preferred because they minimize exposing the agent to shear, which
can result in degradation of the compound.
[0091] Ordinarily, an aqueous aerosol is made by formulating an
aqueous solution or suspension of the agent together with
conventional pharmaceutically acceptable carriers and stabilizers.
The carriers and stabilizers vary with the requirements of the
particular compound, but typically include nonionic surfactants
(Tweens, Pluronics, or polyethylene glycol), innocuous proteins
like serum albumin, sorbitan esters, oleic acid, lecithin, amino
acids such as glycine, buffers, salts, sugars or sugar alcohols.
Aerosols generally are prepared from isotonic solutions.
[0092] Transdermal patches have the added advantage of providing
controlled delivery of a vitamin D.sub.3 compound(s) of formulas I
and II to the body. Such dosage forms can be made by dissolving or
dispersing the agent in the proper medium. Absorption enhancers can
also be used to increase the flux of the peptidomimetic across the
skin. The rate of such flux can be controlled by either providing a
rate controlling membrane or dispersing the peptidomimetic in a
polymer matrix or gel.
[0093] Ophthalmic formulations, eye ointments, powders, solutions
and the like, are also contemplated as being within the scope of
this invention.
[0094] Pharmaceutical compositions of this invention suitable for
parenteral administration comprise one or more vitamin D.sub.3
compound(s) of formulas I and II in combination with one or more
pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous
solutions, dispersions, suspensions or emulsions, or sterile
powders which may be reconstituted into sterile injectable
solutions or dispersions just prior to use, which may contain
antioxidants, buffers, bacteriostats, solutes which render the
formulation isotonic with the blood of the intended recipient or
suspending or thickening agents.
[0095] Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0096] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption such as aluminum monostearate and gelatin.
[0097] In some cases, in order to prolong the effect of a drug, it
is desirable to slow the absorption of the drug from subcutaneous
or intramuscular injection. This may be accomplished by the use of
a liquid suspension of crystalline or amorphous material having
poor water solubility. The rate of absorption of the drug then
depends upon its rate of dissolution which, in turn, may depend
upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally-administered drug form is accomplished
by dissolving or suspending the drug in an oil vehicle.
[0098] Injectable depot forms are made by forming microencapsule
matrices of vitamin D.sub.3 compound(s) of formulas I and II in
biodegradable polymers such as polylactide-polyglycolide. Depending
on the ratio of drug to polymer, and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions
which are compatible with body tissue.
[0099] When the vitamin D.sub.3 compound(s) of the present
invention are administered as pharmaceuticals, to humans and
animals, they can be given per se or as a pharmaceutical
composition containing, for example, 0.1 to 99.5% (more preferably,
0.5 to 90%) of active ingredient in combination with a
pharmaceutically acceptable carrier.
[0100] The term "administration," is intended to include routes of
introducing a subject the 3-epimer vitamin D.sub.3 compound of
formula I to perform their intended function. Examples of routes of
administration which can be used include injection (subcutaneous,
intravenous, parenterally, intraperitoneally, intrathecal, etc.),
oral, inhalation, rectal and transdermal. The pharmaceutical
preparations are of course given by forms suitable for each
administration route. For example, these, preparations are
administered in tablets or capsule form, by injection, inhalation,
eye lotion, ointment, suppository, etc. administration by
injection, infusion or inhalation; topical by lotion or ointment;
and rectal by suppositories. Oral administration is preferred. The
injection can be bolus or can be continuous infusion. Depending on
the route of administration, the vitamin D3 compound of formulas I
and II can be coated with or disposed in a selected material to
protect it from natural conditions which may detrimentally effect
its ability to perform its intended function. The vitamin D.sub.3
compound of formulas I and II can be administered alone, or in
conjunction with either another agent as described above or with a
pharmaceutically acceptable carrier, or both. The vitamin D.sub.3
compound can be administered prior to the administration of the
other agent, simultaneously with the agent, or after the
administration of the agent. Furthermore, the vitamin D.sub.3
compound of formulas I and II can also be administered in a preform
which is converted into its active metabolite, or more active
metabolite in vivo.
[0101] The phrases "parenteral administration" and "administered
parenterally" as used herein means modes of administration other
than enteral and topical administration, usually by injection, and
includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal, intracapsular, intraorbital,
intracardiac, intradermal, intraperitoneal, transtracheal,
subcutaneous, subcuticular, intraarticulare, subcapsular,
subarachnoid, intraspinal and intrasternal injection and
infusion.
[0102] The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein mean the administration of a vitamin
D.sub.3 compound(s) of formulas I and II, such that it enters the
patient's system and, thus, is subject to metabolism and other like
processes, for example, subcutaneous administration.
[0103] These vitamin D.sub.3 compound(s) of formulas I and II may
be administered to a "subject", e.g., mammals, e.g., humans and
other animals. Administration can be carried out by any suitable
route of administration, including orally, nasally, as by, for
example, a spray, rectally, intravaginally, parenterally,
intracisternally and topically, as by powders, ointments or drops,
including buccally and sublingually.
[0104] Regardless of the route of administration selected, the
vitamin D.sub.3 compound(s) of formulas I and II, which may be used
in a suitable hydrated form, and/or the pharmaceutical compositions
of the present invention, are formulated into
pharmaceutically-acceptable dosage forms by conventional methods
known to those of skill in the art.
[0105] Actual dosage levels and time course of administration of
the active ingredients in the pharmaceutical compositions of this
invention may be varied so as to obtain an amount of the active
ingredient which is effective to achieve the desired therapeutic
response for a particular patient, composition, and mode of
administration, without being toxic to the patient. Exemplary dose
range is from 0.1 to 10 .mu.g per day.
Uses of the Vitamin D Compounds of the Invention
[0106] Another aspect of the invention pertains to isolated vitamin
D.sub.3 compounds of formulas I and II having at least one
biological activity of vitamin D.sub.3, and having improved
biological properties when administered into a subject than vitamin
D.sub.3 under the same conditions, as well as, methods of testing
and using these compounds to treat disorders involving an aberrant
activity of a vitamin D3-responsive cell, e.g., neoplastic cells,
hyperproliferative skin cells, parathyroid cells, immune cells and
bone cells, among others.
[0107] The language "biological activities" of vitamin D.sub.3 is
intended to include all activities elicited by vitamin D.sub.3
compounds of formulas I and II in a responsive cell. This term
includes genomic and non-genomic activities elicited by these
compounds (Bouillon, R. et al. (1995) Endocrinology Reviews
16(2):206-207; Norman A. W. et al. (1992) J. Steroid Biochem Mol.
Biol. 41:231-240; Baran D. T. et al. (1991) J. Bone Miner Res.
6:1269-1275; Caffrey J. M. and Farach-Carson M. C. (1989) J. Biol.
Chem. 264:20265-20274; Nemere I. et al. (1984) Endocrinology
115:1476-1483).
[0108] As used herein, the term "vitamin D.sub.3-responsive cell"
includes any cell which is is capable of responding to a vitamin
D.sub.3 compound having the formula I or II, and is associated with
disorders involving an aberrant activity of hyperproliferative skin
cells, parathyroid cells, neoplastic cells, immune cells, and bone
cells. These cells can respond to vitamin D.sub.3 activation by
triggering genomic and/or non-genomic responses that ultimately
result in the modulation of cell proliferation, differentiation
survival, and/or other cellular activities such as hormone
secretion. In a preferred embodiment, the ultimate responses of a
cell are inhibition of cell proliferation and/or induction of
differentiation-specific genes. Exemplary vitamin D.sub.3
responsive cells include immune cells, bone cells, neuronal cells,
endocrine cells, neoplastic cells, epidermal cells, endodermal
cells, smooth muscle cells, among others.
[0109] As used herein, the language "vitamin D.sub.3 agonist"
refers to a compound which potentiates, induces or otherwise
enhances a biological activity of vitamin D.sub.3 in a responsive
cell. In certain embodiments, an agonist may induce a genomic
activity, e.g., activation of transcription by a vitamin D.sub.3
nuclear receptor, or a non-genomic vitamin D.sub.3 activity, e.g.,
potentiation of calcium channel activity. In other embodiments, the
agonist potentiates the sensitivity of the receptor to another
vitamin D.sub.3 compound, e.g., treatment with the agonist lowers
the concentration of vitamin D.sub.3 compound required to induce a
particular biological response. The language "vitamin D.sub.3
antagonist" is intended to include those compounds that oppose any
biological activity of a vitamin D.sub.3 compound.
[0110] The language "non-genomic" vitamin D.sub.3 activities
include cellular (e.g., calcium transport across a tissue) and
subcellular activities (e.g., membrane calcium transport opening of
voltage-gated calcium channels, changes in intracellular second
messengers) elicited by vitamin D.sub.3 compounds in a responsive
cell. Electrophysiological and biochemical techniques for detecting
these activities are known in the art. An example of a particular
well-studied non-genomic activity is the rapid hormonal stimulation
of intestinal calcium mobilization, termed "transcaltachia" (Nemere
I. et al. (1984) Endocrinology 115:1476-1483; Lieberherr M. et al.
(1989) J. Biol. Chem. 264:20403-20406; Wali R. K. et al. (1992)
Endocrinology 131:1125-1133; Wali R. K. et al. (1992) Am. J.
Physiol. 262:G945-G953; Wali R. K. et al. (1990) J. Clin. Invest.
85:1296-1303; Bolt M. J. G. et al. (1993) Biochem. J. 292:271-276).
Detailed descriptions of experimental transcaltachia are provided
in Norman, A. W. (1993) Endocrinology 268(27):20022-20030;
Yoshimoto, Y. and Norman, A. W. (1986) Endocrinology 118:2300-2304.
Changes in calcium activity and second messenger systems are well
known in the art and are extensively-reviewed in Bouillion, R. et
al. (1995) Endocrinology Review 16(2): 200-257; the description of
which is incorporated herein by reference.
[0111] Exemplary systems and assays for testing non-genomic
activity are extensively described in the following references,
liver (Baran D. T. et al. (1989) FEBS Lett 259:205-208; Baran D. T.
et al. (1990) J. Bone Miner Res. 5:517-524; rat osteoblasts, e.g.,
ROS 17/2.8 cells (Baran D. T. et al. (1991) J. Bone Miner Res.
6:1269-1275; Caffrey J. M. (1989) J. Biol. Chem. 264:20265-20274;
Ciyitelli R et al. (1990) Endocrinology 127:2253-2262), muscle
(DeBoland A. R. and Boland R. L. (1993) Biochem. Biophys Acta Mol.
Cell. Res. 1179:93-104; Morelli S. et al. (1993) Biochem J.
289:675-679; Selles J. and Boland R. L. (1991) Mol. Cell.
Endocrinol. 82:229-235), and in parathyroid cells (Bourdeau A. et
al. (1990) Endocrinology 127:2738-2743).
[0112] The language "genomic" activities or effects of vitamin
D.sub.3 is intended to include those activities mediated by the
nuclear/cytosol receptor for 1.alpha.,25(OH).sub.2D.sub.3 (VD3R),
e.g., transcriptional activation of target genes. The term "VD3Rs"
is intended to include members of the type II class of
steroid/thyroid superfamily of receptors (Stunnenberg, H. G. (1993)
Bio Essays 15(5):309-15), which are able to bind transactivate
through the vitamin D response element (VDRE) in the absence of a
ligand (Damm et al. (1989) Nature 339:593-97; Sap et al. Nature
343:177-180). As used herein "VDREs" refer to a DNA sequences
composed of half-sites arranged as direct repeats. It is known in
the art that type II receptors do not bind to their respective
binding site as homodimers but require an auxiliary factor, RXR
(e.g. RXR.alpha., RXR.beta., RXR.gamma.) for high affinity binding
Yu et al. (1991) Cell 67:1251-1266; Bugge et al. (1992) EMBO J.
11:1409-1418; KIliewer et al. (1992) Nature 355:446-449; Leid et
al. (1992) EMBO J. 11: 1419-1435; Zhang et al. (1992) Nature
355:441-446).
[0113] Following binding, the transcriptional activity of a target
gene (i.e., a gene associated with the specific DNA sequence) is
enhanced as a function of the ligand bound to the receptor
heterodimer. Exemplary vitamin D.sub.3-responsive genes include
osteocalcin, osteopontin, calbindins, parathyroid hormone (PTH),
24-hydroxylase, and .alpha..sub.V .beta..sub.3-integrin. Genomic
activities elicited by vitamin D3 compounds can be tested by
detecting the transcriptional upregulation of a vitamin D.sub.3
responsive, gene in a cell containing VD3R.sub.S. For example, the
steady state levels of responsive gene mRNA or protein, e.g.
calbindin gene, osteocalcin gene, can be detected in vivo or in
vitro. Suitable cells that can be used include any vitamin
D3-responsive cell, e.g., keratinocytes, parathyroid cells, MG-63
cell line, among others.
[0114] In accordance with a still further embodiment of the present
invention, convenient screening methods can be established in cell
lines containing VD.sub.3R.sub.s, comprising (i) establishing a
culture of these cells which include a reporter gene construct
having a reporter gene which is expressed in an VD.sub.3R-dependent
fashion; (ii) contacting these cells with vitamin D3 compounds of
formulas I and II; and (iii) monitoring the amount of expression of
the reporter gene. Expression of the reporter gene reflects
transcriptional activity of the VD.sub.3R.sub.s protein. Typically,
the reporter gene construct will include a reporter gene in
operative linkage with one or more transcriptional regulatory
elements responsive to VD.sub.3R.sub.s, e.g., the VD.sub.3R.sub.s
response element (VDRE) known in the art. The amount of
transcription from the reporter gene may be measured using. any
method known to those of skill in the art to be suitable. For
example, specific mRNA expression may be detected using Northern
blots or specific protein product may be identified by a
characteristic stain, immunoassay or an intrinsic activity. In
preferred embodiments, the gene product of the reporter is detected
by an intrinsic activity associated with that product. For
instance, the reporter gene may encode a gene product that, by
enzymatic activity, gives rise to a detection signal based on
color, fluorescence, or luminescence. The amount of expression from
the reporter gene is then compared to the amount of expression in
either the same cell in the absence of the test compound or it may
be compared with the amount of transcription in a substantially
identical cell that lacks the specific receptors. Agonistic vitamin
D3 compounds can then be readily detected by the increased activity
or concentration of these reporter genes relative to untransfected
controls.
[0115] After identifying certain test compounds as potential
agonists or antagonists of vitamin 1).sub.3 compounds, the
practitioner of the subject assay will continue to test the
efficacy and specificity of the selected compounds both in vitro
and in vivo. Whether for subsequent in vivo testing, or for
administration to an animal as an approved drug, agents identified
in the subject assay can be formulated in pharmaceutical
preparations, such as described above, for in vivo administration
to an animal, preferably a human.
[0116] As described herein, the vitamin D3 compounds of the present
invention show improved biological properties than vitamin D3. As
used herein, the language "improved biological properties" refers
to any activity inherent in a vitamin D3 compound of formula I or
II that enhances its effectiveness in vivo. In a preferred
embodiment, this term refers to any qualitative or quantitative
improved therapeutic property of a vitamin D.sub.3 compound, such
as enhanced stability in vivo and/or reduced toxicity, e.g.,
reduced hypercalcemic activity. The improved biological property
may occur in both a tissue-specific and non-specific manner. For
example, certain tissues may be capable of metabolizing vitamin
D.sub.3 into unique metabolites that enhance in a tissue-specific
manner the biological activities of this compound.
[0117] The increased stability of the vitamin D3 compounds of
formulas I and II can be demonstrated in incubation studies,
wherein a significantly higher concentration of the such vitamin D3
after prolonged incubations in vivo or in vitro, or an increase in
the binding to plasma vitamin D binding protein (DBP) compared to
vitamin D3. indicates a compound having enhanced stability (See A.
W. Norman et al. J. Biol. Chem. 268 (27): 20022-20030).
[0118] The language "reduced toxicity" is intended to include a
reduction in any undesired side effect elicited by a vitamin D3
compound of formula I or H when administered in vivo, e.g., a
reduction in the hypercalcemic activity. The language
"hypercalcemia" or "hypercalcemic activity" is intended to have its
accepted clinical meaning, namely, increases in calcium serum
levels that are manifested in a subject by the following side,
effects, depression of central and peripheral nervous system,
muscular weakness, constipation, abdominal pain, lack of appetite
and, depressed relaxation of the heart during diastole. Symptomatic
manifestations of hypercalcemia are triggered by a stimulation of
at least one of the following activities, intestinal calcium
transport, bone calcium metabolism and osteocalcin synthesis
(reviewed in Boullion, R. et al. (1995) Endocrinology Reviews
16(2): 200-257).
[0119] Compounds exhibiting reduced hypercalcemic activity can be
tested in vivo or in vitro using methods known in the art and
reviewed by Boullion, R. et al. (1995) Endocrinology Reviews 16(2):
200-257. For example, the serum calcium levels following
administration of a vitamin D3 compounds of formula I or II can be
tested by routine experimentation (Lemire, J. M. (1994)
Endocrinology 135(6):2818-2821). Briefly, vitamin D3 compounds of
formulas I and II can be administered intramuscularly to vitamin
D.sub.3-deficient subjects, e.g., rodents, e.g. mouse, or avian
species, e.g. chick. At appropriate time intervals, serum calcium
levels and extent of calcium uptake can be used to determine the
level of bone calcium mobilization (BCM) and intestinal calcium
absorption (ICA) induced by the tested vitamin D.sub.3 compound as
described in Norman, A. W. et al. (1993) J. Biol. Chem.
268(27):20022-20029. Compounds which upon addition fail to increase
the concentration of calcium in the blood serum, thus showing
decreased BCM and ICA responses compared to their isomeric
counterparts, are considered to have reduced hypercalcemic
activity. Compounds which have reduced toxicity compared to their
isomeric counterparts are considered to have reduced toxicity.
Additional calcium homeostasis-related assays are described below
in the Calcium and Phosphate Homeostasis section.
Hyperproliferative Conditions
[0120] In another aspect the present invention provides a method of
treating in a subject, a disorder characterized by aberrant
activity of a vitamin D3-responsive cell. The method involves
administering to the subject an effective amount of a
pharmaceutical composition of a vitamin D3 compound of formula I or
II such that the activity of the cell is modulated. As used herein,
the language "modulate" refers to increases or decreases in the
activity of a cell in response to exposure to a compound of the
invention, e.g., the inhibition of proliferation and/or induction
of differentiation of at least a sub-population of cells in an
animal such that a desired end result is achieved, e.g. a
therapeutic result. In preferred embodiments, this phrase is
intended to include hyperactive conditions that result in
pathological disorders.
[0121] In certain embodiments, the cells to be treated are
hyperproliferative cells. As described in greater detail below, the
vitamin D3 compounds of formulas I and II can be used to inhibit
the proliferation of a variety of hyperplastic and neoplastic
tissues. In accordance with the present invention, vitamin D3
compounds of formulas I and II can be used in the treatment of both
pathologic and non-pathologic proliferative conditions
characterized by unwanted growth of vitamin D3-responsive cells,
e.g., hyperproliferative skin cells, immune cells, and tissue
having transformed cells, e.g., such as carcinomas, sarcomas and
leukemias. In other embodiments, the cells to be treated are
aberrant secretory cells, e.g., parathyroid cells, immune
cells.
[0122] As used herein, the terms "hyperproliferative" and
"neoplastic" are used interchangeably, and include those cells
having the capacity for autonomous growth, i.e., an abnormal state
or condition characterized by rapidly proliferating cell growth.
Hyperproliferative and neoplastic disease states may be categorized
as pathologic, i.e., characterizing or constituting a disease
state, or may be categorized as non-pathologic, i.e., a deviation
from normal but not associated with a disease state. The term is
meant to include all types of cancerous growths or oncogenic
processes, metastatic tissues or malignantly transformed cells,
tissues, or organs, irrespective of histopathologic type or stage
of invasiveness. "Pathologic hyperproliferative" cells occur in
disease states characterized by malignant tumor growth. Examples of
non-pathologic hyperproliferative cells include proliferation of
cells associated with wound repair.
[0123] The use of vitamin D3 compounds of formulas I and II in
treating hyperproliferative conditions has been limited because of
their hypercalcemic effects. Thus, vitamin D3 compounds of formula
I and II can provide a less toxic alternative to current methods of
treatment.
[0124] In one embodiment, this invention features a method for
inhibiting the proliferation and/or inducing the differentiation of
a hyperproliferative skin cell, e.g., an epidermal or an epithelial
cell, e.g. a keratinocytes, by contacting the cells with a vitamin
D3 compound of formula I or II. In general, the method includes a
step of contacting a pathological or non-pathological
hyperproliferative cell with an effective amount of such vitamin D3
compound to promote the differentiation of the hyperproliferative
cells The present method can be performed on cells in culture, e.g.
in vitro or ex vivo, or can be performed on cells present in an
animal subject, e.g., as part of an in vivo therapeutic protocol.
The therapeutic regimen can be carried out on a human or any other
animal subject.
[0125] The vitamin D3 compounds of the present invention can be
used to treat a hyperproliferative skin disorder. Exemplary
disorders include, but are not limited to, psoriasis, basal cell
carcinoma, keratinization disorders and keratosis. Additional
examples of these disorders include eczema; lupus associated skin
lesions; psoriatic arthritis; rheumatoid arthritis that involves
hyperproliferation and inflammation of epithelial-related cells
lining the joint capsule; dermatitides such as seborrheic
dermatitis and solar dermatitis; keratoses such as seborrheic
keratosis, senile keratosis, actinic keratosis photo-induced
keratosis, and keratosis follicularis; acne vulgaris; keloids and
prophylaxis against keloid formation; nevi; warts including
verruca, condyloma or condyloma acuminatum, and human papilloma
viral (HPV) infections such as venereal warts; leukoplakia; lichen
planus; and keratitis.
[0126] In an illustrative example, vitamin D3 compounds of formulas
I and II can be used to inhibit the hyperproliferation of
keratinocytes in treating diseases such as psoriasis by
administering an effective amount of these compounds to a subject
in need of treatment. The term "psoriasis" is intended to have its
medical meaning, namely, a disease which afflicts primarily the
skin and produces raised, thickened, scaling, nonscarring lesions.
The lesions are usually sharply demarcated erythematous papules
covered with overlapping shiny scales. The scales are typically
silvery or slightly opalescent. Involvement of the nails frequently
occurs resulting in pitting, separation of the nail, thickening and
discoloration. Psoriasis is sometimes associated with arthritis,
and it may be crippling. Hyperproliferation of keratinocytes is a
key feature of psoriatic epidermal hyperplasia along with epidermal
inflammation and reduced differentiation of keratinocytes. Multiple
mechanisms have been invoked to explain the keratinocyte
hyperproliferation that characterizes psoriasis. Disordered
cellular immunity has also been implicated in the pathogenesis of
psoriasis.
[0127] Pharmaceutical compositions of vitamin D3 compounds of
formulas I and II can be delivered or administered topically or by
transdermal patches for treating dermal psoriasis. Alternatively,
oral administration is used. Additionally, the compositions can be
delivered parenterally, especially for treatment of arthritis, such
as psoriatic arthritis, and for direct injection of skin lesions.
Parenteral therapy is typically intra-dermal, intra-articular,
intramuscular or intravenous. A preferred way to practice the
invention is to apply the vitamin D3 compounds of formulas I and
II, in a cream or oil based carrier, directly to the psoriatic
lesions. Typically, the concentration of the vitamin D3 compound in
a cream or oil is 1-2%. Alternatively, an aerosol can be used
topically. These compounds can also be orally administered.
[0128] In general, the route of administration is topical
(including administration to the eye, scalp, and mucous membranes),
oral, or parenteral. Topical administration is preferred in
treatment of skin lesions, including lesions of the scalp, lesions
of the cornea (keratitis), and lesions of mucous membranes where
such direct application is practical. Shampoo formulations are
sometimes advantageous for treating scalp lesions such as
seborrheic dermatitis and psoriasis of the scalp. Mouthwash and
oral paste formulations can be advantageous for mucous membrane
lesions, such as oral lesions and leukoplakia Oral administration
is a preferred alternative for treatment of skin lesions and other
lesions discussed above where direct topical application is not as
practical, and it is a preferred route for other applications.
[0129] Intra-articular injection is a preferred alternative in the
case of treating one or only a few (such as 2-6) joints.
Additionally, the therapeutic compounds are injected directly into
lesions (intra-lesion administration) in appropriate cases.
Intra-dermal administration is an alternative for dermal lesions
such as those of psoriasis.
[0130] The amount of the pharmaceutical composition to be
administered varies depending upon the type of the disease of a
patient, the severity of the disease, the type of the active
vitamin D.sub.3 compound of Formulas I or II, among others. For
example, the vitamin D3 compound of formula I or II can be
administered topically for treating hyperproliferative skin
conditions at a dose in the range of 1 to 1000 .mu.g per gram of
topical formulation.
Neoplasia
[0131] Another embodiment features methods for inhibiting the
proliferation and/or reversing the transformed phenotype of vitamin
D3-responsive hyperproliferative cells by contacting the cells with
a vitamin D3 compound of formula I or II. In general, the method
includes a step of contacting pathological or non-pathological
hyperproliferative cells with an effective amount of a vitamin D3
compound of formula I or II for promoting the differentiation of
the hyperproliferative cells. The present method can be performed
on cells in culture, e.g., in vitro or ex vivo, or can be performed
on cells present in an animal subject, e.g., as part of an in vivo
therapeutic protocol. The therapeutic regimen can be carried out on
a human or other animal subject.
[0132] The terms "antineoplastic agent" and "antiproliferative
agent" are used interchangeably herein and includes agents that
have the functional property of inhibiting the proliferation of a
vitamin D3-responsive cells, e.g., inhibit the development or
progression of a neoplasm having such a characteristic,
particularly a hematopoietic neoplasm.
[0133] As used herein, a "therapeutically effective anti-neoplastic
amount" of a vitamin D3 compound of formula I or II refers to an
amount of an agent which is effective, upon single or multiple dose
administration to the patient, in inhibiting the growth of a
neoplastic vitamin D3-responsive cells, or in prolonging the
survivability of the patient with such neoplastic cells beyond that
expected in the absence of such treatment. As used herein,
"inhibiting the growth" of the neoplasm includes the slowing,
interrupting, arresting or stopping its growth and metastases and
does not necessarily indicate a total elimination of the neoplastic
growth.
[0134] As used herein, "a prophylactically effective
anti-neoplastic amount" of a compound refers to an amount of a
vitamin D3 compound of formula I or II which is effective, upon
single or multiple dose administration to the patient, in
preventing or delaying the occurrence of the onset of a neoplastic
disease state.
[0135] The common medical meaning of the term "neoplasia" refers to
"new cell growth" that results as a loss of responsiveness to
normal growth controls, e.g. to neoplastic cell growth. A
"hyperplasia" refers to cells undergoing an abnormally high rate of
growth. However, as used herein, the terms neoplasia and
hyperplasia can be used interchangeably, as their context will
reveal, referring to generally to cells experiencing abnormal cell
growth rates. Neoplasias and hyperplasias include "tumors," which
may be either benign, premalignant or malignant.
[0136] The vitamin D3 compounds of formulas I and II can be tested
initially in vitro for their inhibitory effects in the
proliferation of neoplastic cells. Examples of cell lines that can
be used are transformed cells, e.g., the human promyeloid leukemia
cell line HL-60, and the human myeloid leukemia U-937 cell line
(Abe E. et al. (1981) Proc. Natl. Acad. Sci. USA 78:4990-4994; Song
L. N. and Cheng T. (1992) Biochem Pharmacol 43:2292-2295; Zhou J.
Y. et al. (1989) Blood 74:82-93; U.S. Pat. Nos. 5,401,733, U.S.
Pat. No. 5,087,619). Alternatively, the antitumoral effects of
vitamin D3 compounds of formulas I and II can be tested in vivo
using various animal models known in the art and summarized in
Bouillon, R et al. (1995) Endocrine Reviews 16(2):233 (Table E),
which is incorporated by reference herein. For example, SL mice are
routinely used in the art to test vitamin D3 compounds of formulas
I and II as models for MI myeloid leukemia (Honma et al. (1983)
Cell Biol. 80:201-204; Kasukabe T. et al. (1987) Cancer Res.
47:567-572); breast cancer studies can be performed in, for
example, nude mice models for human MX1 (ER) (Abe J. et al. (1991)
Endocrinology 129:832-837; other cancers, e.g., colon cancer,
melanoma osteosarcoma, can be characterized in, for example, nude
mice models as describe in (Eisman J. A. et al. (1987) Cancer Res.
47:21-25; Kawaura A. et al. (1990) Cancer Lett 55:149-152; Belleli
A. (1992) Carcinogenesis 13:2293-2298; Tsuchiya H. et al. (1993) J.
Orthopaed Res. 11:122-130).
[0137] The subject method may also be used to inhibit the
proliferation of hyperplastic/neoplastic cells of hematopoietic
origin, e.g., arising from myeloid, lymphoid or erythroid lineages,
or precursor cells thereof. For instance, the present invention
contemplates the treatment of various myeloid disorders including,
but not limited to, acute promyeloid leukemia (APML), acute
myelogenous leukemia (AML) and chronic myelogenous leukemia (CML)
(reviewed in Vaickus, L. (1991) Crit. Rev. in Oncol/Hemotol.
11:267-97). Lymphoid malignancies which may be treated by the
subject method include, but are not limited to acute lymphoblastic
leukemia (ALL) which includes B-lineage ALL and T-lineage ALL,
chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),
hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional forms of malignant lymphomas contemplated by the
treatment method of the present invention include, but are not
limited to non-Hodgkin lymphoma and variants thereof, peripheral T
cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous
T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF)
and Hodgkin's disease.
[0138] The term "leukemia" is intended to have its clinical
meaning, namely, a neoplastic disease in which white corpuscle
maturation is arrested at a primitive stage of cell development.
The disease is characterized by an increased number of leukemic
blast cells in the bone marrow, and by varying degrees of failure
to produce normal hematopoietic cells. The condition may be either
acute or chronic. Leukemias are further typically categorized as
being either lymphocytic i.e., being characterized by cells which
have properties in common with normal lymphocytes, or myelocytic
(or myelogenous), i.e., characterized by cells having some
characteristics of normal granulocytic cells. Acute lymphocytic
leukemia ("ALL") arises in lymphoid tissue, and ordinarily first
manifests its presence in bone marrow. Acute myelocytic leukemia
("AML") arises from bone marrow hematopoietic stem cells or their
progeny. The term acute myelocytic leukemia subsumes several
subtypes of leukemia: myeloblastic leukemia, promyelocytic
leukemia, and myelomonocytic leukemia. In addition, leukemias with
erythroid or megakaryocytic properties are considered myelogenous
leukemias as well.
[0139] As used herein the term "leukemic cancer" refers to all
cancers or neoplasias of the hemopoietic and immune systems (blood
and lymphatic system). The acute and chronic leukemias, together
with the other types of tumors of the blood, bone marrow cells
(myelomas), and lymph tissue (lymphomas), cause about 10% of all
cancer deaths and about 50% of all cancer deaths in children and
adults less than 30 years old. Chronic myelogenous leukemia (CML),
also known as chronic granulocytic leukemia (CGL), is a neoplastic
disorder of the hematopoietic stem cell. The term "leukemia" is art
recognized and refers to a progressive, malignant disease of the
blood-forming organs, marked by distorted proliferation and
development of leukocytes and their precursors in the blood and
bone marrow.
[0140] In certain embodiments, the vitamin D3 compounds of formulas
I and II can be used in combinatorial therapy with conventional
cancer chemotherapeutics. Conventional treatment regimens for
leukemia and for other tumors include radiation, drugs, or a
combination of both. In addition to radiation, the following drugs,
usually in combinations with each other, are often used to treat
acute leukemias: vincristine, prednisone, methotrexate,
mercaptopurine, cyclophosphamide, and cytarabine. In chronic
leukemia, for example, busulfan, melphalan, and chlorambucil can be
used in combination. All of the conventional anti cancer drugs are
highly toxic and tend to make patients quite ill while undergoing
treatment. Vigorous therapy is based on the premise that unless
every leukemic cell is destroyed, the residual cells will multiply
and cause a relapse.
[0141] The subject method can also be useful in treating
malignancies of the various organ systems, such as affecting lung,
breast, lymphoid, gastrointestinal, and genito-urinary tract as
well as adenocarcinomas which include malignancies such as most
colon cancers, renal-cell carcinoma, prostate cancer and/or
testicular tumors, non-small cell carcinoma of the lung, cancer of
the small intestine and cancer of the esophagus.
[0142] The term "carcinoma" is art recognized and refers to
malignancies of epithelial or endocrine tissues including
respiratory system carcinomas, gastrointestinal system carcinomas,
genitourinary system carcinomas, testicular carcinomas, breast
carcinomas, prostatic carcinomas, endocrine system carcinomas, and
melanomas. Exemplary carcinomas include those forming from tissue
of the cervix, lung, prostate, breast, head and neck, colon and
ovary. The term also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures.
[0143] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0144] According to the general paradigm of vitamin D3 involvement
in differentiation of transformed cells, exemplary solid tumors
that can be treated according to the method of the present
invention include vitamin D3-responsive phenotypes of sarcomas and
carcinomas such as, but not limited to: fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma,
papillary-carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma.
[0145] Determination of a therapeutically effective anti-neoplastic
amount or a prophylactically effective anti-neoplastic amount of
the vitamin D3 compound of formula I or II, can be readily made by
the physician or veterinarian (the "attending clinician"), as one
skilled in the art, by the use of known techniques and by observing
results obtained under analogous circumstances. The dosages may be
varied depending upon the requirements of the patient in the
judgment of the attending clinician, the severity of the condition
being treated and the particular compound being employed. In
determining the therapeutically effective antineoplastic amount or
dose, and the prophylactically effective antineoplastic amount or
dose, a number of factors are considered by the attending
clinician, including, but not limited to: the specific
hyperplastic/neoplastic cell involved; pharmacodynamic
characteristics of the particular agent and its mode and route of
administration; the desired time course of treatment; the species
of mammal; its size, age, and general health; the specific disease
involved; the degree of or involvement or the severity of the
disease; the response of the individual patient; the particular
compound administered; the mode of administration; the
bioavailability characteristics of the preparation administered;
the dose regimen selected; the kind of concurrent treatment (i.e.,
the interaction of the vitamin D3 compounds of formulas I and II
with other co-administered therapeutics); and other relevant
circumstances. U.S. Pat. No. 5,427,916, for example, describes
method for predicting the effectiveness of antineoplastic therapy
in individual patients, and illustrates certain methods which can
be used in conjunction with the treatment protocols of the instant
invention.
[0146] Treatment can be initiated with smaller dosages which are
less than the optimum dose of the compound. Thereafter, the dosage
should be increased by small increments until the optimum effect
under the circumstances is reached. For convenience, the total
daily dosage may be divided and administered in portions during the
day if desired. A therapeutically effective antineoplastic amount
and a prophylactically effective anti-neoplastic amount of a
vitamin D3 compound of formula I or II is expected to vary from
about 0.1 milligram per kilogram of body weight per day (mg/kg/day)
to about 100 mg/kg/day.
[0147] Compounds which are determined to be effective for the
prevention or treatment of tumors in animals, e.g., dogs, rodents,
may also be useful in treatment of tumors in humans. Those skilled
in the art of treating tumor in humans will know, based upon the
data obtained in animal studies, the dosage and route of
administration of the compound to humans. In general, the dosage
and route of administration in humans is expected to be similar to
that in animals.
[0148] The identification of those patients who are in need of
prophylactic treatment for hyperplastic/neoplastic disease states
is well within the ability and knowledge of one skilled in the art.
Certain of the methods for identification of patients which are at
risk of developing neoplastic disease states which can be treated
by the subject method are appreciated in the medical arts, such as
family history of the development of a particular disease state and
the presence of risk factors associated with the development of
that disease state in the subject patient. The present application
also describes other prognostic tests which can be used to make, or
to augment a clinical predication about the use of the method of
the present invention. A clinician skilled in the art can readily
identify such candidate patients, by the use of, for example,
clinical tests, physical examination and medical/family
history.
Immunomodulatory Effects
[0149] In another aspect, this invention provides a method for
modulating the activity of an immune cell by contacting the cell
with a vitamin D3 compound of formulas I or II. Vitamin D3
compounds are known in the art for their inhibitory effects on the
antigen-specific immune system. As used herein, the phrase
"inhibition of an immune response" is intended to include decreases
in T cell proliferation and activity, e.g., a decrease in IL.sub.2,
interferon-.gamma., GM-CSF synthesis and secretion (Lemire, J. M.
(1992) J. Cell Biochemistry 49:26-31, Lemire, J. M. et al. (1994)
Endocrinology 135 (6): 2813-2821; Bouillon, R. et al. (1995)
Endocine Review 16 (2):231-32)
[0150] In one embodiment, the present invention provides a method
for suppressing immune activity in an immune cell by contacting a
pathological or non-pathological immune cell with an effective
amount of a vitamin D3 compound of formulas I or II to thereby
inhibit an immune response relative to the cell in the absence of
the treatment. The present method can be performed on cells in
culture, e.g., in vitro or ex vivo, or can be performed on cells
present in an animal subject, e.g., as part of all in vivo
therapeutic protocol. In vivo treatment can be carried out on a
human or other animal subject.
[0151] The vitamin D3 compound of formula I or II can be tested
initially in vitro for their inhibitory effects on T cell
proliferation and secretory activity, as described in Reichel, H.
et al., (1987) Proc. Natl. Acad. Sci. USA 84:3385-3389; Lemire, J.
M. et al. (1985) J. Immunol 34:2032-2035. Alternatively, the
immunosuppressive effects can be tested in vivo using the various
animal models known in the art and summarized by Bouillon, R. et
al. (1995) Endocine Reviews 16(2) 232 (Tables 6 and 7). For
examples, animal models for autoimmune disorders, e.g., lupus,
thyroiditis, encephalitis, diabetes and nephritis are described in
(Lemire J. M. (1992) J. Cell Biochem. 49:26-31; Koizumi T. et al.
(1985) Int. Arch Allergy Appl. Immunol. 77:396-404; Abe J. et al.
(1990) Calcium Regulation and Bone Metabolism 146-151; Fournier C.
et al. (1990) Clin. Immunol Immunopathol. 54:53-63; Lemire J. M.
and Archer D. C. (1991) J. Clin. Invest 87:1103-1107); Lemire, J.
M. et al., (1994) Endocrinology 135 (6):2818-2821; Inaba M. et al.
(1992) Metabolism 41:631-635; Mathieu C. et al. (1992) Diabetes
41:1491-1495; Mathieu C. et al. (1994) Diabetologia 37:552-558;
Lillevang S. T. et al. (1992) Clin. Exp. Immunol. 88:301-306, among
others). Models for characterizing immunosuppressuve activity
during organ transplantation, e.g., skin graft, cardiac graft,
islet graft, are described in Jordan S. C. et al. (1988) v Herrath
D (eds) Molecular, Cellular and Clinical Endocrinology 346-347;
Veyron P. et al. (1993) Transplant Immunol. 1:72-76; Jordan S. C.
(1988) v Herrath D (eds) Molecular, Cellular and Clinical
Endocrinology 334-3.35; Lemire J. M. et al. (1992) Transplantation
54:762-763; Mathieu C. et al. (1994) Transplant Proc.
26:3128-3129).
[0152] After identifying certain test compounds as effective
suppressors of an immune response in vitro, these compounds can be
used in vivo as part of a therapeutic protocol. Accordingly,
another embodiment provides a method of suppressing an immune
response, comprising administering to a subject a pharmaceutical
preparation of a vitamin D3 compound of formula I or II, so as to
inhibit immune reactions such as graft rejection, autoimmune
disorders and inflammation.
[0153] For example, the subject vitamin D3 compounds of formulas I
and II can be used to inhibit responses in clinical situations
where it is desirable to downmodulate T cell responses. For
example, in graft-versus-host disease, cases of transplantation,
autoimmune diseases (including, for example, diabetes mellitus,
arthritis (including rheumatoid arthritis, juvenile rheumatoid
arthritis, osteoarthritis, psoriatic arthritis), multiple
sclerosis, encephalomyelitis, diabetes, myasthenia gravis, systemic
lupus erythematosis, autoimmune thyroiditis, dermatitis (including
atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's
Syndrome, including keratoconjunctivitis sicca secondary to
Sjogren's Syndrome, alopecia greata, allergic responses due to
arthropod bite reactions, Crohn's disease, aphthous ulcer, iritis,
conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma,
allergic asthma, cutaneous lupus erythematosus, scleroderma,
vaginitis, proctitis, drug eruptions, leprosy reversal reactions,
erythema nodosum leprosum, autoimmune uveitis, allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy,
idiopathic bilateral progressive sensorineural hearing loss,
aplastic anemia, pure red cell anemia, idiopathic tirombocytopenia,
polychondritis, Wegener's granulomatosis, chronic active hepatitis,
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's
disease, Graves ophthalmopathy, sarcoidosis, primary biliary
cirrhosis, uveitis posterior, and interstitial lung fibrosis).
Downmodulation of immune activity will also be desirable in cases
of allergy such as, atopic allergy.
[0154] As described before, determination of a therapeutically
effective immunosuppressive amount can be readily made by the
attending clinician, as one skilled in the art, by the use of known
techniques and by observing results obtained under analogous
circumstances. Compounds which are determined to be effective in
animals, e.g., dogs, rodents, may be extrapolated accordingly to
humans by those skilled in the art. Starting dose/regimen used in
animals can be estimated based on prior studies. For example, doses
of vitamin D3 compounds of formulas I and II to treat autoimmune
disorders in rodents can be initially estimated in the range of 0.1
g/kg/day to 1 g/kg/day, administered orally or by injection.
[0155] Those skilled in the art will know based upon the data
obtained in animal studies, the dosage and route of administration
in humans is expected to be similar to that in animals. Exemplary
dose ranges to be used in humans are from 0.25 to 10 .mu.g/day,
preferably 0.5 to 5 .mu.g/day per adult (U.S. Pat. No.
4,341,774).
Calcium and Phosphate Homeostasis
[0156] The present invention also relates to a method of treating
in a subject a disorder characterized by deregulation of calcium
metabolism. This method comprises contacting a pathological or
non-pathological vitamin D3 responsive cell with an effective
amount of a vitamin D3 compound of formula I or II to thereby
directly or indirectly modulate calcium and phosphate homeostasis.
The term "homeostasis" is art-recognized to mean maintenance of
static, or constant, conditions in an internal environment. As used
herein, the term "calcium and phospate homeostasis" refers to the
careful balance of calcium and phosphate concentrations,
intracellularly and extracellularly, triggered by fluctuations in
the calcium and phosphate concentration in a cell, a tissue, an
organ or a system. Fluctuations in calcium levels that result from
direct or indirect responses to vitamin D3 compounds of formulas I
and II are intended to be included by these terms. Techniques for
detecting calcium fluctuation in vivo or in vitro are known in the
art.
[0157] Exemplary Ca.sup.++ homeostasis related assays include
assays that focus on the intestine where intestinal
.sup.45Ca.sup.2+ absorption is determined either 1) in vivo
(Hibberd K. A. and Norman A. W. (1969) Biochem. Pharmacol.
18:2347-2355; Hurwitz S. et al. (1967) J. Nutr. 91:319-323; Bickle
D. D. et al. (1984) Endocrinology 114:260-267), or 2) in vitro with
everted duodenal sacs (Schachter D. et al. (1961) Am. J. Physiol
200:1263-1271), or 3) on the genomic induction of
calbindin-D.sub.28k in the chick or of calbindin-D.sub.9k in the
rat (Thomasset M. et al. (1981) FEBS Lett. 127:13-16; Brehier A.
and Thomasset M. (1990) Endocrinology 127:580-587). The
bone-oriented assays include: 1) assessment of bone resorption as
determined via the release of Ca.sup.2+ from bone in vivo (in
animals fed a zero Ca.sup.2+ diet) (Hibberd K. A. and Norman A. W.
(1969) Biochem. Pharmacol. 18:2347-2355; Hurwitz S. et al. (1967)
J. Nutr. 91:319-323), or from bone explants in vitro (Bouillon R.
et al. (1992) J. Biol. Chem. 267:3044-3051), 2) measurement of
serum osteocalcin levels [osteocalcin is an osteoblast-specific
protein that after its synthesis is largely incorporated into the
bone matrix, but partially released into the circulation (or tissue
culture medium) and thus represents a good market of bone formation
or turnover] (Bouillon R. et al. (1992) Clin. Chem. 38:2055-2060),
or 3) bone ash content (Norman A. W. and Wong R. G. (1972) J. Nutr.
102:1709-1718). Only one kidney-oriented assay has been employed.
In this assay, urinary Ca.sup.2+ excretion is determined
(Hartenbower D. L. et al. (1977) Walter de Gruyter, Berlin pp
587-589); this assay is dependent upon elevations in the serum
Ca.sup.2+ level and may reflect bone Ca.sup.2+ mobilizing activity
more than renal effects. Finally, there is a "soft tissue
calcification" assay that has been employed to detect the
consequences of 1.alpha.,25(OH).sub.2D3 or analog-induced severe
hypercalcemia. In this assay a rat is administered an
intraperitoneal dose of .sup.45C.sup.2+, followed by seven daily
relative high doses of 1.alpha.,25(OH).sub.2D3 or the analog of
interest; in the event of onset of a severe hypercalcemia, soft
tissue calcification can be assessed by determination of the
.sup.45Ca.sup.2+ level. In all these assays, vitamin D3 compounds
or formulas I and II are administered to vitamin D-sufficient or
deficient animals, as a single dose or chronically (depending upon
the assay protocol), at an appropriate time interval before the end
point of the assay is quantified.
[0158] In certain embodiments, vitamin D3 compounds of formulas I
and II can be used to modulate bone metabolism. The language "bone
metabolism" is intended to include direct or indirect effects in
the formation or degeneration of bone structures, e.g., bone
formation, bone resorption, etc., which may ultimately affect the
concentrations in serum of calcium and phosphate. This term is also
intended to include effects of vitamin D3 compounds in bone cells,
e.g. osteoclasts and osteoblasts, that may in turn result in bone
formation and degeneration. For example, it is known in the art,
that vitamin D3 compounds of formulas I and II exert effects on the
bone forming cells, the osteoblasts through genomic and non-genomic
pathways (Walters M. R. et al. (1982) J. Biol. Chem. 257:7481-7484;
Jurutka P. W. et al. (1993) Biochemistry 32:8184-8192; Mellon W. S,
and DeLuca H. F. (1980) J. Biol. Chem. 255:4081-4086). Similarly,
vitamin D3 compounds of formulas I and II are known in the art to
support different activities of bone resorbing osteoclasts such as
the stimulation of differentiation of monocytes and mononuclear
phagocytes into osteoclasts (Abe E. et al. (1988) J. Bone Miner
Res. 3:635-645; Takahashi N. et al. (1988) Endocrinology
123:1504-1510; Udagawa N. et al. (1990) Proc. Natl. Acad. Sci. USA
87:7260-7264). Accordingly, vitamin D3 compounds of formulas I and
II that modulate the production of bone cells can influence bone
formation and degeneration.
[0159] The present invention provides a method for modulating bone
cell metabolism by contacting a pathological or a non-pathological
bone cell with an effective amount of a vitamin D3 compound of
formula I or II to thereby modulate bone formation and
degeneration. The present method can be performed on cells in
culture, e.g., in vitro or ex vivo, or can be performed in cells
present in an animal subject, e.g., cells in vivo. Exemplary
culture systems that can be used include osteoblast cell lines,
e.g., ROS 17/2.8 cell line, monocytes, bone marrow culture system
(Suda T. et al. (1990) Med. Res. Rev. 7:333-366; Suda T. et al.
(1992) J. Cell Biochem. 49:53-58) among others. Selected compounds
can be further tested in vivo, for example, animal models of
osteopetrosis and in human disease (Shapira F. (1993) Clin. Orthop.
294:34-44).
[0160] In a preferred embodiment, a method for treating
osteoporosis is provided, comprising administering to a subject a
pharmaceutical preparation of a vitamin D3 compound of formula I or
II to thereby ameliorate the condition relative to an untreated
subject. The rationale for utilizing vitamin D3 compounds in the
treatment of osteoporosis is supported by studies indicating a
decrease in serum concentration of 1.alpha.,25(OH).sub.2D3 in
elderly subjects (Lidor C. et. al. (1993) Calcif. Tissue Int.
52:146-148). In vivo studies using vitamin D3 compounds in animal
models and humans are described in Bouillon, et al. (1995)
Endocrine Reviews 16(2):229-231.
[0161] Vitamin D3 compounds of formulas I and II can be tested in
ovariectomized animals, e.g., dogs, rodents, to assess the changes
in bone mass and bone formation rates in both normal and
estrogen-deficient animals. Clinical trials can be conducted in
humans by attending clinicians to determine therapeutically
effective amounts of the vitamin D3 compounds of formulas I and II
in preventing and treating osteoporosis.
[0162] Preferred compounds to be tested include 3-epi forms of
3-epi forms of 1.alpha. (OH)D.sub.3 as shown in Example II, which
shows the production of 1.alpha.(OH)-3-epi-D.sub.3 in the rat
osteosarcoma cell line UMR-106. The 3 epi conversion of
1.alpha.(OH)-D3 presents the possibility of a yet improved of this
compound.
[0163] In other embodiments, therapeutic applications of the
vitamin D3 compounds of formulas I and II include treatment of
other diseases characterized by metabolic calcium and phosphate
deficiencies. Exemplary of such diseases are the following:
osteoporosis, osteodystrophy, osteomalacia, rickets, osteitis
fibrosa cystica, renal osteodystrophy, osteosclerosis,
anti-convulsant treatment, osteopenia, fibrogenesis-imperfecta
ossium, secondary hyperparathyrodism, hypoparathyroidism,
hyperparathyroidism, cirrhosis, obstructive jaundice, drug induced
metabolism, medullary carcinoma, chronic renal disease,
hypophosphatemic VDRR, vitamin D-dependent rickets, sarcoidosis,
glucocorticoid antagonism, malabsorption syndrome, steatorrhea,
tropical sprue, idiopathic hypercalcemia and milk fever.
Hormone Secretion
[0164] In yet another aspect, the present invention provides a
method for modulating hormone secretion of a vitamin D3-responsive
cell, e.g., an endocrine cell. The language "hormone secretion" is
art-recognized and includes both genomic and non-genomic activities
of vitamin D3 compounds of formulas I and II that control the
transcription and processing responsible for secretion of a given
hormone e.g., parathyroid hormone (PTH), calcitonin, insulin,
prolactin (PRL) and TRH in a vitamin D3 responsive cell (Bouillon,
R. et al. (1995) Endocrine Reviews 16(2):235-237). The language
"vitamin D3 responsive cells" as used herein is intended to include
endocrine cells which respond to vitamin D3 compounds of formulas I
and II by altering gene expression and/or post-transcriptional
processing secretion of a hormone. Exemplary endocrine cells
include parathyroid cells, pancreatic cells, pituitary cells, among
others.
[0165] The present method can be performed on cells in culture,
e.g. in vitro or ex vivo, or on cells present in an animal subject,
e.g., in vivo. Vitamin D3 compounds of formulas I and II can be
initially tested in vitro using primary cultures of parathyroid
cells. Other systems that can be used include the testing by
prolactin secretion in rat pituitary tumor cells, e.g., GH4C.sub.1
cell line (Wark J. D. and Tashjian Jr. A. H. (1982) Endocrinology
111:1755-1757; Wark J. D. and Tashjian Jr. A. H. (1983) J. Biol.
Chem. 258:2118-2121; Wark J. D. and Gurtler V. (1986) Biochem. J.
233:513-518) and TRH secretion in GH4C1 cells. Alternatively, the
effects of vitamin D3 compounds of formulas I and II can be
characterized in vivo using animals models as described in Nko M.
et al. (1982) Miner Electrolyte Metab. 5:67-75; Oberg F. et al.
(1993) J. Immunol. 150:3487-3495; Bar-Shavit Z. et al. (1986)
Endocrinology 118:679-686; Testa U. et al. (1993) J. Immunol.
150:2418-2430; Nakamaki T. et al. (1992) Anticancer Res.
12:1331-1337; Weinberg J. B. and Larrick J. W. (1987) Blood
70:994-1002; Chambaut-Guerin A. M. and Thomopoulos P. (1991) Eur.
Cytokine New. 2:355; Yoshida M. et al. (1992) Anticancer Res.
12:1947-1952; Momparler R. L. et al. (1993) Leukemia 7:17-20;
Eisman J. A. (1994) Kanis J A (eds) Bone and Mineral Research
2:45-76; Veyron P. et al. (1993) Transplant Immunol. 1:72-76; Gross
M. et al. (1986) J. Bone Miner Res. 1:457-467; Costa E. M. et al.
(1985) Endocrinology 117:2203-2210; Koga M. et al. (1988) Cancer
Res. 48:2734-2739; Franceschi R. T. et al. (1994) J. Cell Physiol.
123:401-409; Cross H. S. et al. (1993) Naunyn Schmiedebergs Arch.
Pharmacol. 347:105-110; Zhao X. and Feldman D. (1993) Endocrinology
132:1808-1814; Skowronski R. J. et al. (1993) Endocrinology
132:1952-1960; Henry H. L. and Norman A. W. (1975) Biochem.
Biophys. Res. Commun. 62:781-788; Wecksler W. R. et al. (1980)
Arch. Biochem. Biophys. 201:95-103; Brumbaugh P. F. et al. (1975)
Am. J. Physiol. 238:384-388; Oldham S. B. et al. (1979)
Endocrinology 104:248-254; Chertow B. S. et al. (1975) J. Clin
Invest. 56:668-678; Canterbury J. M. et al. (1978) J. Clin. Invest.
61:1375-1383; Quesad J. M. et al. (1992) J. Clin. Endocrinol.
Metab. 75:494-501.
[0166] In certain embodiments, the vitamin D3 compounds of the
present invention can be used to inhibit parathyroid hormone (PTH)
processing, e.g., transcriptional, translational processing, and/or
secretion of a parathyroid cell as part of a therapeutic protocol.
Therapeutic methods using these compounds can be readily applied to
all diseases, involving direct or indirect effects of PTH activity,
e.g., primary or secondary responses. For example, it is known in
the art that PTH induces the formation of 1,25-dihydroxy vitamin D3
in the kidneys, which in turn in increases calcium and phosphate
absorption from the intestine that causes hypercalcemia. Thus
inhibition of PTH processing and/or secretion would indirectly
inhibit all of the responses mediated by PTH in vivo. Accordingly,
therapeutic applications for these vitamin D3 compounds of formulas
I and II include treating diseases such as secondary
hyperparathyroidism of chronic renal failure (Slatopolsky E. et al.
(1990) Kidney Int. 38:S41-S47; Brown A. J. et al. (1989) J. Clin.
Invest. 84:728-732). Determination of therapeutically affective
amounts and dose regimen can be performed by the skilled artisan
using the data described in the art.
Protection Against Neuronal Loss
[0167] In yet another aspect, the present invention provides a
method of protecting against neuronal loss by contacting a vitamin
D3 responsive cell, e.g., a neuronal cell, with a vitamin D3
compound of formula I or II to prevent or retard neuron loss. The
language "protecting against" is intended to include prevention,
retardation, and/or termination of deterioration, impairment, or
death of a neurons. The language "vitamin D3 responsive cells" as
used herein is intended to include neuronal cells which respond to
vitamin D3 compounds of formulas I and II by altering gene
expression and/or intracellular metabolism. Exemplary neuronal
cells include hippocampal cells, dopaminergic cells, cholinergic
cells, among others.
[0168] Neuron loss can be the result of any condition of a neuron
in which its normal function is compromised. Neuron deterioration
can be the result of any condition which compromises neuron
function which is likely to lead to neuron loss. Neuron function
can be compromised by, for example, altered biochemistry,
physiology, or anatomy of a neuron. Deterioration of a neuron may
include membrane, dendritic, or synaptic changes which are
detrimental to normal neuronal functioning. The cause of the neuron
deterioration, impairment, and/or death may be unknown.
Alternatively, it may be the result of age- and/or disease-related
changes which occur in the nervous system of a subject.
[0169] When neuron loss is described herein as "age-related", it is
intended to include neuron loss resulting from known and unknown
bodily changes of a subject which are associated with aging. When
neuron loss is described herein as "disease-related", it is
intended to include neuron loss resulting from known and unknown
bodily changes of a subject which are associated with disease. It
should be understood, however, that these terms are not mutually
exclusive and that, in fact, many conditions that result in the
loss of neurons are both age and disease-related.
[0170] Exemplary age-related diseases associated with neuron loss
and changes in neuronal morphology include, for example,
Alzheimer's Disease, Pick's Disease, Parkinson's Disease, Vascular
Disease, Huntington's Disease, and Age-Associated Memory Impairment
In Alzheimer's Disease patients, neuron loss is most notable in the
hippocampus, frontal, parietal, and anterior temporal cortices,
amygdala, and the olfactory system. The most prominently affected
zones of the hippocampus include the CA1 region, the subiculum, and
the entorhinal cortex. Memory loss is considered the earliest and
most representative cognitive change because the hippocampus is
well known to play a crucial role in memory. Pick's Disease is
characterized by severe neuronal degeneration in the neocortex of
the frontal and anterior temporal lobes which is sometimes
accompanied by death of neurons in the striatum. Parkinson's
Disease can be identified by the loss of neurons in the substantia
nigra and the locus ceruleus.
[0171] Huntington's Disease is characterized by degeneration of the
intrastriatal and cortical cholinergic neurons and GABA-ergic
neurons. Parkinson's and Huntington's Diseases are usually
associated with movement disorders, but often show cognitive
impairment (memory loss) as well.
[0172] Age-Associated Memory Impairment (AAMI) is another
age-associated disorder that is characterized by memory loss in
healthy, elderly individuals in the later decades of life. Crook,
T. et al. (1986) Devel Neuropsych. 2(4):261-276. Presently, the
neural basis for AAMI has not been precisely defined. However,
neuron death with aging has been reported to occur in many species
in brain regions implicated in memory, including cortex,
hippocampus, amygdala, basal ganglia, cholinergic basal forebrain,
locus ceruleus, raphe nuclei, and cerebellum. Crook, T. et al.
(1986) Devel. Neuropsych. 2(4):261-276.
[0173] Vitamin D3 compounds of formulas I and II can protect
against neuron loss by genomic or non-genomic mechanisms. Nuclear
vitamin D3 receptors are well known to exist in the periphery but
have also been found in the brain, particularly in the hippocampus
and neocortex. Non-genomic mechanisms may also prevent or retard
neuron loss by regulating intraneuronal and/or peripheral calcium
and phosphate levels. Furthermore, vitamin D3 compounds of formulas
I and II may protect against neuronal loss by acting indirectly,
e.g., by modulating serum PTH levels. For example, a positive
correlation has been demonstrated between serum PTH levels and
cognitive decline in Alzheimer's Disease.
[0174] The present method can be performed on cells in culture,
e.g. in vitro or ex vivo, or on cells present in an animal subject,
e.g., in vivo. Vitamin D3 compounds of formulas I and II can be
initially tested in vitro using neurons from embryonic rodent pups
(See e.g. U.S. Pat. No. 5,179,109-fetal rat tissue culture), or
other mammalian (See e.g. U.S. Pat. No. 5,089,517-fetal mouse
tissue culture) or non-mammalian animal models. These culture
systems have been used to characterize the protection of
peripheral, as well as, central nervous system neurons in animal or
tissue culture models of ischemia, stroke, trauma, nerve crush,
Alzheimer's Disease, Pick's Disease, and Parkinson's Disease, among
others. Examples of in vitro systems to study the prevention of
destruction of neocortical neurons include using in vitro cultures
of fetal mouse neurons and glial cells previously exposed to
various glutamate agonists, such as kainate, NMDA, and
.alpha.-amino-3-hydroxy-5-methyl-4-isoxazolepronate (AMPA). U.S.
Pat. No. 5,089,517. See also U.S. Pat. No. 5,170,109 (treatment of
rat cortical/hippocampal neuron cultures with glutamate prior to
treatment with neuroprotective compound); U.S. Pat. Nos. 5,163,196
and 5,196,421 (neuroprotective excitatory amino acid receptor
antagonists inhibit glycine, kainate, AMPA receptor binding in
rats).
[0175] Alternatively, the effects of vitamin D3 compounds of
formulas I and II can be characterized in vivo using animals
models. Neuron deterioration in these model systems is often
induced by experimental trauma or intervention (e.g. application of
toxins, nerve crush, interruption of oxygen supply). For example,
in order to demonstrate that certain N-methyl-D-aspartate (NMDA),
an excitatory amino acid neurotransmitter receptor, antagonists
were useful as anticonvulsants and neuroprotectants, the inventors
in U.S. Pat. No. 4,957,909 employed a model wherein Swiss-albino
mice and rat hippocampal neurons were subjected to overstimulation
of excitatory amino acid receptors subsequent to treatment with the
NMDA antagonists. A similar study was performed wherein the utility
of certain NMDA antagonists as agents that prevent
neurodegeneration was demonstrated by treating mice with NMDA
subsequent to treatment with the NMDA antagonists. U.S. Pat. No.
5,168,103.
Smooth Muscle Cells
[0176] In yet another aspect, the present invention provides a
method of modulating the activity of a vascular smooth muscle cell
by contacting a vitamin D3-responsive smooth muscle cell with a
vitamin D3 compounds of formulas I or II to activate or,
preferably, inhibit the activity of the cell. The language
"activity of a smooth muscle cell" is intended to include any
activity of a smooth muscle cell, such as proliferation, migration,
adhesion and/or metabolism.
[0177] In certain embodiments, the vitamin D3 compounds of formulas
I and II can be used to treat diseases and conditions associated
with aberrant activity of a vitamin D3-responsive smooth muscle
cell. For example, the present invention can be used in the
treatment of hyperproliferative vascular diseases, such as
hypertension induced vascular remodeling, vascular restenosis and
atherosclerosis. In other embodiments, the present invention can be
used in treating disorders characterized by aberrant metabolism of
a vitamin D3-responsive smooth muscle cell, e.g., arterial
hypertension.
[0178] The present method can be performed on cells in culture,
e.g. in vitro or ex vivo, or on cells present in an animal subject,
e.g., in vivo. Vitamin D3 compounds of formulas I and II can be
initially tested in vitro as described in Catellot et al. (1982),
J. Biol. Chem. 257(19): 11256.
[0179] This invention is further illustrated by the following
examples which in no way should be construed as being further
limiting. It is understood by the ordinary skilled artisan that
production of a vitamin D3 compound of formula I or II in a cell is
indicative that such compound is biologically active in such cell,
and thus that it can be used in treating conditions arising from
aberrant activity of such cells. For example, production of vitamin
D3 compounds of formulas I and II in keratinocytes is indicative
that such vitamin D3 compounds are biologically active in those
cells and can be used in treating conditions such as psoriasis. The
contents of all cited references (including literature references,
issued patents, published patent applications, and co-pending
patent applications) cited throughout this application are hereby
expressly incorporated by reference.
EXAMPLES
Example 1
Isolation and Identification of a Cyclic Ether Metabolite of
1.alpha.,25-dihydroxy-Vitamin D.sub.3 in Human Keratinocytes
[0180] As described herein, 1.alpha.25(OH).sub.2-3-epi-D.sub.3 is
metabolized into a less polar metabolite than
1.alpha.25(OH).sub.2-3-epi-D.sub.3, peak M1, in human keratinocytes
(FIG. 2). FIG. 2 shows the HPLC profile and UV spectra of the
metabolites produced in human keratinocytes incubated with
1.alpha.25(OH).sub.2-3-epi3D.sub.3 (1 uM) for 24H. On a straight
phase HPLC system, this metabolite (M1) is more polar than 25(OH)
D.sub.3 but less polar than 1.alpha.25(OH).sub.2D.sub.3 and similar
to that of 1.alpha.(OH)D.sub.3. Mass spectrometric analysis reveals
a molecular ion of 414 m/z, which is 2 mass units less than the
starting 1.alpha.25(OH).sub.2D.sub.3, shown in FIG. 3. FIG. 3 shows
the mass spectra of 1.alpha.(OH).sub.2-3-epi-D.sub.3 (M) (upper
panel) and its cyclic ether metabolite (M.sub.1) (lower panel)
isolated from human keratinocytes incubated with
1.alpha.25(OH).sub.2-3-epi-D3(1 uM) for 24 h. The typical fragments
at m/z 134 and 152 m/z indicate an unmodified A-ring and cistriene
structure. A double bond introduced at either C, D rings or the
side chain would be consistent with the molecular weight. However,
this type of unsaturated metabolite still possesses a free
25-hydroxyl group and is expected to have similar retention time as
the starting compound; this is contradicting to what was observed.
Furthermore, the absence of mass fragments at 59 m/z suggests the
absence of a 25-hydroxyl group. The absence of the familiar side
chain cleavage fragments at 251,269 and 287 m/z also suggests a
modified 25-hydroxyl group and a possible structural change at C-20
to retard the cleavage at carbons 17 and 20. A cyclic structure as
shown in FIGS. 3 and 4 is supported by these mass spectrometric and
chromatographic evidences. This proposed structure is consistent
with the loss of m/z 58 (acetone) to form m/z 356 and the
subsequent fragments at 338, 320 and 314.
[0181] It is probable that the 3-epi modification of the A-ring
allows alternate side chain reactions to occur. FIG. 4 summarizes
the proposed metabolic pathway for the formulation of the cyclic
ether metabolite of 1.alpha.25(OH).sub.2-3-epi-D.sub.3. The
formation of a cyclic ether structure could result from a
hydroxylation at either C-17 or C-20 and the subsequent reaction
with the 25-hydroxyl group to form an ether linkage as shown in
FIG. 4. This type of metabolic reactions are known to occur in
hydroxylated fatty acids. Thus, it is probable that some of the
unidentified metabolites can be C-17 or C-20-hydroxylated
metabolites of 1.alpha.25(OH).sub.2-3-epi-D.sub.3.
Example II
Isolation and Identification of a 3-Epi Metabolite of 1.alpha.
hydroxy-Vitamin D.sub.3 in Human Keratinocytes
[0182] FIG. 5A shows the metabolism of 1.alpha.(OH)-D.sub.3 into
its 3 epi form in the osteosarcoma cell line UMR-106. Peak A
represents the 3-epi form of 1.alpha.(OH)D.sub.3. Peak B
corresponds to the substrate, 1.alpha.(OH)D.sub.3. The insert
panels show the UV spectra of the various metabolites as monitored
by photodiode array detector. FIG. 5B shows a schematic
representation of the 3-epimerization of 1.alpha.(OH)D.sub.3 into
1.alpha.(OH)-3-epi vitamin D.sub.3. Similar to 1.alpha.(OH)D.sub.3,
1.alpha.(OH)-3-epi vitamin D.sub.3 can be converted into the
25-hydroxylated form in vivo.
[0183] 1.alpha.(OH)D.sub.3 compounds are currently used in the
treatment of osteoporosis. Thus, 3-epi forms of these compounds may
be used as substitutes for 1.alpha.(OH)D.sub.3 compounds in
treating osteoporosis.
Example III
Confirmation of 3-epi Configuration of 1.alpha.(OH) 3-epi Vitamin
D.sub.3
[0184] To confirm the production of 1.alpha.(OH) 3-epi vitamin
D.sub.3 in bone cells the metabolites of 1.alpha.-3-epi-D.sub.3
produced by the osteosarcoma cell line (UMR-106) were analyzed by
mass spectroscopy. FIG. 6 shows the mass spectra of
1.alpha.(OH)D.sub.3 (upper panel) and its 3-epi metabolite (lower
panel). A comparison of these two mass spectra revealed difference
in peaks observed only in the 3-epi metabolite, for example,
fragments having molecular weights of approximately m/z 57, 217,
312 and 529 (lower panel). The mass spectrum of the 3-epi
metabolite was independently confirmed to be 1.alpha.(OH) 3-epi
vitamin D.sub.3.
Example IV
Enhanced Stability In Vivo of 1.alpha.(OH) 3-epi Vitamin D.sub.3
Compared to Its Isomeric Counterpart
[0185] The stability of 1.alpha.(OH) 3-epi vitamin D.sub.3 in vivo
was characterized by monitoring the changes in the concentration of
1.alpha.(OH) 3-epi vitamin D.sub.3 and its isomeric counterpart at
various time intervals. In particular, FIG. 7 shows the HPLC
profile and UV spectra of the metabolites produced in rat
osteosarcoma cell lines (UMR-106) which were incubated with
1.alpha.(OH)D.sub.3 for 24, 48, or 84 hours. Peak M and S represent
the relative concentrations of 1.alpha.(OH) 3-epi vitamin D.sub.3
and its isomeric counterpart, respectively, at the time intervals
tested. The persistent duration of peak M relative to peak S after
48 and 84 hour-incubations indicates that 3-epi metabolite of
1.alpha.(OH)D.sub.3 are more stable in vivo than its isomeric
counterpart
EQUIVALENTS
[0186] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents of the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *