U.S. patent application number 14/076932 was filed with the patent office on 2014-03-06 for methods and compounds for vitamin d therapy.
This patent application is currently assigned to Opko IP Holdings II, Inc.. The applicant listed for this patent is Opko IP Holdings II, Inc.. Invention is credited to Charles W. Bishop, Keith H. Crawford, Eric J. Messner, P. Martin Petkovich.
Application Number | 20140066415 14/076932 |
Document ID | / |
Family ID | 39926297 |
Filed Date | 2014-03-06 |
United States Patent
Application |
20140066415 |
Kind Code |
A1 |
Petkovich; P. Martin ; et
al. |
March 6, 2014 |
Methods and Compounds for Vitamin D Therapy
Abstract
Methods and compositions comprising 1,25-dihydroxyvitamin
D.sub.2 are disclosed. A method for lowering or maintaining lowered
serum parathyroid hormone in human patients including administering
to said patients an effective amount of 1,25-dihydroxyvitamin
D.sub.2 to lower or maintain lowered serum parathyroid hormone
levels is disclosed. Dosage forms and dosing regimens are also
disclosed.
Inventors: |
Petkovich; P. Martin;
(Kingston, CA) ; Bishop; Charles W.; (Miami Beach,
FL) ; Messner; Eric J.; (Lake Forest, IL) ;
Crawford; Keith H.; (Highlands Ranch, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Opko IP Holdings II, Inc. |
Miami |
FL |
US |
|
|
Assignee: |
Opko IP Holdings II, Inc.
Miami
FL
|
Family ID: |
39926297 |
Appl. No.: |
14/076932 |
Filed: |
November 11, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12597224 |
Apr 16, 2010 |
8592401 |
|
|
PCT/US08/61586 |
Apr 25, 2008 |
|
|
|
14076932 |
|
|
|
|
60913848 |
Apr 25, 2007 |
|
|
|
Current U.S.
Class: |
514/168 |
Current CPC
Class: |
A61K 9/0053 20130101;
A61K 47/44 20130101; A61P 3/02 20180101; A61K 31/592 20130101; A61P
5/20 20180101; A61K 47/14 20130101; A61P 5/18 20180101; A61K 9/0019
20130101 |
Class at
Publication: |
514/168 |
International
Class: |
A61K 31/592 20060101
A61K031/592 |
Claims
1. A pharmaceutical composition having serum or plasma intact
parathyroid hormone lowering activity, which includes an effective
amount of 1,25-dihydroxyvitamin D.sub.2 and at least one
pharmaceutically acceptable excipient.
2. The pharmaceutical composition of claim 1, in unit dosage
form.
3. The pharmaceutical composition of claim 1, wherein said at least
one pharmaceutically acceptable excipient provides for controlled
release of 1,25-dihydroxyvitamin D.sub.2.
4. The pharmaceutical composition of claim 3, wherein said at least
one pharmaceutically acceptable excipient provides for
substantially constant release of 1,25-dihydroxyvitamin
D.sub.2.
5. The pharmaceutical composition of claim 3, wherein said at least
one pharmaceutically acceptable excipient provides delayed release
of 1,25-dihydroxyvitamin D.sub.2.
6. The pharmaceutical composition of claim 5, wherein the
formulation is an oral formulation and said at least one
pharmaceutically acceptable excipient provides delayed release of
1,25-dihydroxyvitamin D.sub.2 to the small intestine of the
patient.
7. The pharmaceutical composition of claim 5, wherein the
formulation is an oral formulation and said at least one
pharmaceutically acceptable excipient provides delayed release of
1,25-dihydroxyvitamin D.sub.2 to the ileum of the patient.
8. A pharmaceutical composition having serum or plasma intact
parathyroid hormone lowering activity, comprising an effective
amount of 1,25-dihydroxyvitamin D.sub.2 and at least one
pharmaceutically acceptable excipient in an oral dosage form.
9. The pharmaceutical composition of claim 1, comprising an
intravenous formulation.
10. The pharmaceutical composition of claim 2, wherein the unit
dosage form comprises about 0.1 .mu.g to about 10 .mu.g of
1,25-dihydroxyvitamin D.sub.2.
11. The pharmaceutical composition of claim 10, wherein the unit
dosage form comprises about 1 .mu.g to about 4 .mu.g of
1,25-dihydroxyvitamin D.sub.2.
12. The pharmaceutical composition of claim 10, wherein the unit
dosage form comprises about 2 .mu.g to about 10 .mu.g of
1,25-dihydroxyvitamin D.sub.2.
13. The pharmaceutical composition of claim 12, wherein the unit
dosage form comprises about 3 .mu.g to about 5 .mu.g of
1,25-dihydroxyvitamin D.sub.2.
14. The pharmaceutical composition of claim 1, comprising a solid
or semi-solid, waxy mixture comprising a waxy controlled release
carrier agent, a lipoidic agent, an oily vehicle for the vitamin D
compound, and 1,25-dihydroxyvitamin D.sub.2.
15. The pharmaceutical composition of claim 14, wherein the waxy
controlled release carrier agent comprises a non-digestible
wax.
16. The pharmaceutical composition of claim 14, wherein the
lipoidic agent includes a mixture of a lipophilic emulsifier which
has an HLB value of less than 7 and an absorption enhancer which
has an HLB value from 13 to 18.
17. The pharmaceutical composition of claim 14, wherein the
lipoidic agent comprises a polyglycolized glyceride.
18. The pharmaceutical composition of claim 14, wherein the oily
vehicle comprises a non-digestible oil.
19. A pharmaceutical composition having serum or plasma intact
parathyroid hormone lowering activity, comprising an effective
amount of 1,25-dihydroxyvitamin D.sub.2 and at least one
pharmaceutically acceptable excipient, wherein said at least one
pharmaceutically acceptable excipient is effective to control the
release rate of 1,25-dihydroxyvitamin D.sub.2 from the dosage form
to reduce the maximum serum concentration of 1,25-dihydroxyvitamin
D.sub.2 in a dose interval (Cmax) and/or reduce the maximum change
in serum concentration of 1,25-dihydroxyvitamin D.sub.2 and/or
increase the time for the plasma concentration of
1,25-dihydroxyvitamin D.sub.2 to reach its maximum in a dose
interval following administration (Tmax) and/or decrease a ratio of
the maximum serum concentration of 1,25-dihydroxyvitamin D.sub.2
within 24 hours after administration to the concentration 24 hours
after administration (Cmax.sub.24hr/C.sub.24hr), and/or increase
the elimination half-life (t.sub.1/2), as compared to either or
both of (a) an equivalent amount of 1,25-dihydroxyvitamin D.sub.2
administered by bolus IV injection and (b) the same dosage form
omitting the effective amount of the release-modifying agent.
20. The pharmaceutical composition of claim 1, comprising a solid
or semi-solid, waxy mixture comprising paraffin wax, fatty acid
mono- and di-glycerides, mineral oil, a polyglycolized glyceride,
and 1,25-dihydroxyvitamin D.sub.2.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This is a division of U.S. patent application Ser. No.
12/597,224 filed Apr. 16, 2010, which is the National Phase of
International Application No. PCT/US08/61586 filed Apr. 25, 2008,
which claims the benefit of priority under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Patent Application Ser. No. 60/913,848 filed
Apr. 25, 2007. The disclosure of each priority application is
incorporated herein by reference.
BACKGROUND
[0002] 1. Field of the Disclosure
[0003] The disclosure relates generally to methods and compounds
for Vitamin D therapy. More particularly, the disclosure relates to
compositions comprising 1,25-dihydroxyvitamin D.sub.2 and methods
of administration thereof in the treatment and prevention of
disease.
[0004] 2. Brief Description of Related Technology
[0005] Secondary hyperparathyroidism is a disorder which develops
primarily because of Vitamin D deficiency. It is characterized by
abnormally elevated blood levels of parathyroid hormone (PTH) and,
in the absence of early detection and treatment, it becomes
associated with parathyroid gland hyperplasia and a constellation
of metabolic bone diseases. It is a common complication of chronic
kidney disease (CKD), with rising incidence as CKD progresses.
Secondary hyperparathyroidism can also develop in individuals with
healthy kidneys, due to environmental, cultural or dietary factors
which prevent adequate Vitamin D supply.
[0006] As to secondary hyperparathyroidism and its occurrence in
CKD, there is a progressive loss of cells of the proximal nephrons,
the primary site for the synthesis of the vitamin D hormones
(collectively "1,25-dihydroxyvitamin D") from 25-hydroxyvitamin
D.sub.3 and 25-hydroxyvitamin D.sub.2. In addition, the loss of
functioning nephrons leads to retention of excess phosphorus which
combined reduces the activity of the renal 25-hydroxyvitamin
D-1.alpha.-hydroxylase, the enzyme which catalyzes the reaction to
produce the D hormones. These two events account for the low serum
levels of 1,25-dihydroxyvitamin D commonly found in patients with
moderate to severe CKD when vitamin D supply is adequate.
[0007] Reduced serum levels of 1,25-dihydroxyvitamin D cause
increased, and ultimately excessive, secretion of PTH by direct and
indirect mechanisms. The resulting hyperparathyroidism leads to
markedly increased bone turnover and its sequela of renal
osteodystrophy, which may include a variety of other diseases, such
as, osteitis fibrosa cystica, osteomalacia, osteoporosis,
extraskeletal calcification and related disorders, e.g., bone pain,
periarticular inflammation and Mockerberg's sclerosis. Reduced
serum levels of 1,25-dihydroxyvitamin D can also cause muscle
weakness and growth retardation with skeletal deformities (most
often seen in pediatric patients).
[0008] "Vitamin D" is a term that refers broadly to the organic
substances named Vitamin D.sub.2, Vitamin D.sub.3, Vitamin D.sub.4,
etc., and is sometimes used loosely to refer to their metabolites
and hormonal forms that influence calcium and phosphorus
homeostasis. "Vitamin D deficiency" is a term that broadly refers
to reduced or low blood levels of Vitamin D, as defined immediately
above.
[0009] The most widely recognized forms of Vitamin D are Vitamin
D.sub.2 (ergocalciferol) and Vitamin D.sub.3 (cholecalciferol).
Vitamin D.sub.2 is produced in plants from ergosterol during
sunlight exposure and is present, to a limited extent, in the human
diet. Vitamin D.sub.3 is generated from 7-dehydrocholesterol in
human skin during exposure to sunlight and also is found, to a
greater extent than Vitamin D.sub.2, in the human diet, principally
in dairy products (milk and butter), certain fish and fish oils,
and egg yolk. Vitamin D supplements for human use consist of either
Vitamin D.sub.2 or Vitamin D.sub.3.
[0010] Both Vitamin D.sub.2 and Vitamin D.sub.3 are metabolized
into prohormones by one or more enzymes located in the liver. The
involved enzymes are mitochondrial and microsomal cytochrome P450
(CYP) isoforms, including CYP27A1, CYP2R1, CYP3A4, CYP2J3 and
possibly others. These enzymes metabolize Vitamin D.sub.2 into two
prohormones known as 25-hydroxyvitamin D.sub.2 and
24(S)-hydroxyvitamin D.sub.2, and Vitamin D.sub.3 into a prohormone
known as 25-hydroxyvitamin D.sub.3. The two 25-hydroxylated
prohormones are more prominent in the blood, and can be
collectively referred to as "25-hydroxyvitamin D." Vitamin D.sub.2
and Vitamin D.sub.3 can be metabolized into their respective
prohormones outside of the liver in certain epithelial cells, such
as enterocytes, which contain the same (or similar) enzymes, but
extrahepatic prohormone production probably contributes little to
blood levels of 25-hydroxyvitamin D.
[0011] The rates of hepatic and extrahepatic production of the
Vitamin D prohormones are not tightly regulated, and they vary
mainly with intracellular concentrations of the precursors (Vitamin
D.sub.2 and Vitamin D.sub.3). Higher concentrations of either
precursor increase prohormone production, while lower
concentrations decrease production. Hepatic production of
prohormones is inhibited by high levels of 25-hydroxyvitamin D via
a poorly understood mechanism apparently directed to prevention of
excessive blood prohormone levels.
[0012] The Vitamin D prohormones are further metabolized in the
kidneys into potent hormones by an enzyme known as CYP27B1 (or
25-hydroxyvitamin D.sub.3-1.alpha.-hydroxylase) located in the
proximal kidney tubule. The prohormones 25-hydroxyvitamin D.sub.2
and 24(S)-hydroxyvitamin D.sub.2 are metabolized into hormones
known as 1.alpha.,25-dihydroxyvitamin D.sub.2 and
1.alpha.,24(S)-dihydroxyvitamin D.sub.2. Likewise,
25-hydroxyvitamin D.sub.3 is metabolized into a hormone known as
1.alpha.,25-dihydroxyvitamin D.sub.3 (or calcitriol). These
hormones are released by the kidneys into the blood for systemic
delivery. The two 1.alpha.,25-dihydroxylated hormones, usually far
more prominent in the blood than 1.alpha.,24(S)-dihydroxyvitamin
D.sub.2, can be collectively referred to as "1,25-dihydroxyvitamin
D." Vitamin D prohormones can be metabolized into hormones outside
of the kidneys in keratinocytes, lung epithelial cells,
enterocytes, cells of the immune system (e.g., macrophages) and
certain other cells containing CYP27B1 or similar enzymes, but such
extrarenal hormone production is incapable of sustaining normal
blood levels of 1,25-dihydroxyvitamin D in advanced CKD.
[0013] Blood levels of 1,25-dihydroxyvitamin D are precisely
regulated by a feedback mechanism which involves PTH. The renal
1.alpha.-hydroxylase (or CYP27B1) is stimulated by PTH and
inhibited by 1,25-dihydroxyvitamin D. When blood levels of
1,25-dihydroxyvitamin D fall, the parathyroid glands sense this
change via intracellular Vitamin D receptors (VDR) and secrete PTH.
The secreted PTH stimulates expression of renal CYP27B1 and,
thereby, increases production of Vitamin D hormones. As blood
concentrations of 1,25-dihydroxyvitamin D rise again, the
parathyroid glands attenuate further PTH secretion. As blood PTH
levels fall, renal production of Vitamin D hormones decreases.
Rising blood levels of 1,25-dihydroxyvitamin D also directly
inhibit further Vitamin D hormone production by CYP27B1.
[0014] PTH secretion can be abnormally suppressed in situations
where blood 1,25-dihydroxyvitamin D concentrations become
excessively elevated, as can occur in certain disorders such as
sarcoidosis or as a result of bolus doses of Vitamin D hormone
replacement therapies. Oversuppression of PTH secretion can cause
or exacerbate disturbances in calcium homeostasis. The parathyroid
glands and the renal CYP27B1 are exquisitely sensitive to changes
in blood concentrations of Vitamin D hormones so that serum
1,25-dihydroxyvitamin D is tightly controlled, fluctuating up or
down by less than 20% during any 24-hour period. In contrast to
renal production of Vitamin D hormones, extrarenal production is
not under precise feedback control.
[0015] Blood levels of 1,25-dihydroxyvitamin D and substrate
25-hydroxyvitamin D prohormone, and regulation thereof, can also be
affected by vitamin D hormone analogs, such as
1.alpha.-hydroxyvitamin D.sub.2 and 19-nor-1,25 dihydroxyvitamin
D.sub.2.
[0016] The Vitamin D hormones have essential roles in human health
which are mediated by the intracellular VDR. In particular, the
Vitamin D hormones regulate blood calcium levels by controlling
intestinal absorption of dietary calcium and reabsorption of
calcium by the kidneys. The Vitamin D hormones also participate in
the regulation of cellular differentiation and growth and normal
bone formation and metabolism. Further, Vitamin D hormones are
required for the normal functioning of the musculoskeletal, immune
and renin-angiotensin systems. Numerous other roles for Vitamin D
hormones are being postulated and elucidated, based on the
documented presence of intracellular VDR in nearly every human
tissue. For example, vitamin D has been postulated to play a role
in cellular differentiation and cancer, in regulation of the immune
system (immune enhancing or immune suppressing effects, depending
on the situation), and atherosclerosis. Vitamin D deficiency
increases the risk of many common cancers, multiple sclerosis,
rheumatoid arthritis, hypertension, cardiovascular heart disease,
and type I diabetes.
[0017] The actions of Vitamin D hormones on specific tissues depend
on the degree to which they bind to (or occupy) the intracellular
VDR in those tissues. VDR binding increases as the intracellular
concentrations of the hormones rise, and decreases as the
intracellular concentrations fall. In all cells, intracellular
concentrations of the Vitamin D hormones change in direct
proportion to changes in blood hormone concentrations. In cells
containing CYP27B1 (or similar enzymes), intracellular
concentrations of the Vitamin D hormones also change in direct
proportion to changes in blood and/or intracellular prohormone
concentrations, as discussed above.
[0018] Vitamin D.sub.2, Vitamin D.sub.3 and their prohormonal forms
have affinities for the VDR which are estimated to be at least
100-fold lower than those of the Vitamin D hormones and do not
effectively activate the receptor. As a consequence, physiological
concentrations of these hormone precursors exert little, if any,
biological actions without prior metabolism to Vitamin D hormones.
However, supraphysiological levels of these hormone precursors,
especially the prohormones, in the range of 10 to 1,000 fold higher
than normal, can sufficiently occupy the VDR and exert actions like
the Vitamin D hormones.
[0019] Blood levels of Vitamin D.sub.2 and Vitamin D.sub.3 are
normally present at stable concentrations in human blood, given a
sustained, adequate supply of Vitamin D from sunlight exposure and
an unsupplemented diet. Slight, if any, increases in blood Vitamin
D levels occur after meals since unsupplemented diets have low
Vitamin D content, even those containing foods fortified with
Vitamin D. The Vitamin D content of the human diet is so low that
the National Institutes of Health (NIH) cautions "it can be
difficult to obtain enough Vitamin D from natural food sources"
[NIH, Office of Dietary Supplements, Dietary Supplement Fact Sheet:
Vitamin D (2005)]. Almost all human Vitamin D supply comes from
fortified foods, exposure to sunlight or from dietary supplements,
with the last source becoming increasingly important. Blood Vitamin
D levels rise only gradually, if at all, after sunlight exposure
since cutaneous 7-dehydrocholesterol is modified by UV radiation to
pre-Vitamin D.sub.3 which undergoes thermal conversion in the skin
to Vitamin D.sub.3 over a period of several days before circulating
in the blood.
[0020] Blood Vitamin D hormone concentrations also remain generally
constant through the day in healthy individuals, but can vary
significantly over longer periods of time in response to seasonal
changes in sunlight exposure or sustained alterations in Vitamin D
intake. Marked differences in normal Vitamin D hormone levels are
commonly observed between healthy individuals, with some
individuals having stable concentrations as low as approximately 20
pg/mL and others as high as approximately 70 pg/mL. Due to this
wide normal range, medical professionals have difficulty
interpreting isolated laboratory determinations of serum total
1,25-dihydroxyvitamin D; a value of 25 pg/mL may represent a normal
value for one individual or a relative deficiency in another.
[0021] Transiently low blood levels of 1,25-dihydroxyvitamin D
stimulate the parathyroid glands to secrete PTH for brief periods
ending when normal blood Vitamin D hormone levels are restored. In
contrast, chronically low blood levels of 1,25-dihydroxyvitamin D
continuously stimulate the parathyroid glands to secrete PTH,
resulting in a disorder known as secondary hyperparathyroidism.
Chronically low hormone levels also decrease intestinal calcium
absorption, leading to reduced blood calcium concentrations
(hypocalcemia) which further stimulate PTH secretion. Continuously
stimulated parathyroid glands become increasingly hyperplastic and
eventually develop resistance to regulation by vitamin D hormones.
Without early detection and treatment, secondary
hyperparathyroidism progressively increases in severity, causing
debilitating metabolic bone diseases, including osteoporosis and
renal osteodystrophy.
[0022] Chronically low blood levels of 1,25-dihydroxyvitamin D
develop when there is insufficient renal CYP27B1 to produce the
required supply of Vitamin D hormones, a situation which commonly
arises in CKD. The activity of renal CYP27B1 declines as the
Glomerular Filtration Rate (GFR) falls below approximately 60
ml/min/1.73 m.sup.2 due to the loss of functioning nephrons. In
end-stage renal disease (ESRD), when the kidneys fail completely
and hemodialysis is required for survival, renal CYP27B1 often
becomes altogether absent. Any remaining CYP27B1 is greatly
inhibited by elevated serum phosphorous (hyperphosphatemia) caused
by inadequate renal excretion of dietary phosphorous.
[0023] Chronically low blood levels of 1,25-dihydroxyvitamin D also
develop because of a deficiency of Vitamin D prohormones, since
renal hormone production cannot proceed without the required
precursors. Prohormone production declines markedly when
cholecalciferol and ergocalciferol are in short supply, a condition
often described by terms such as "Vitamin D insufficiency,"
"Vitamin D deficiency," or "hypovitaminosis D." Therefore,
measurement of 25-hydroxyvitamin D levels in blood has become the
accepted method among healthcare professionals to monitor Vitamin D
status. Recent studies have documented that the great majority of
CKD patients have low blood levels of 25-hydroxyvitamin D, and that
the prevalence of Vitamin D insufficiency and deficiency increases
as CKD progresses.
[0024] It follows that individuals most vulnerable to developing
chronically low blood levels of 1,25-dihydroxyvitamin D are those
with CKD. Most CKD patients typically have decreased levels of
renal CYP27B1 and a shortage of 25-hydroxyvitamin D prohormones.
Not surprisingly, most CKD patients develop secondary
hyperparathyroidism. Unfortunately, early detection and treatment
of secondary hyperparathyroidism in CKD is rare, let alone
prevention.
[0025] The National Kidney Foundation (NKF) has recently focused
the medical community's attention on the need for early detection
and treatment of secondary hyperparathyroidism by publishing Kidney
Disease Outcomes Quality Initiative (K/DOQI) Clinical Practice
Guidelines for Bone Metabolism and Disease in Chronic Kidney
Disease [Am. J. Kidney Dis. 42:S1-S202, 2003)]. The K/DOQI
Guidelines identified the primary etiology of secondary
hyperparathyroidism as chronically low blood levels of
1,25-dihydroxyvitamin and recommended regular screening in CKD
Stages 3 through 5 for elevated blood PTH levels relative to
Stage-specific PTH target ranges. CKD Stage 3 was defined as
moderately decreased kidney function (GFR of 30-59 mL/min/1.73
m.sup.2) with an intact PTH (iPTH) target range of 35-70 pg/mL;
Stage 4 was defined as severely decreased kidney function (GFR of
15-29 mL/min/1.73 m.sup.2), with an iPTH target range of 70-110
pg/mL; and Stage 5 was defined as kidney failure (GFR of <15
mL/min/1.73 m.sup.2 or dialysis) with an iPTH target range of
150-300 pg/mL. In the event that screening revealed an iPTH value
to be above the ranges targeted for CKD Stages 3 and 4, the
Guidelines recommended a follow-up evaluation of serum total
25-hydroxyvitamin D to detect possible Vitamin D insufficiency or
deficiency. If 25-hydroxyvitamin D below 30 ng/mL was observed, the
recommended intervention was Vitamin D repletion therapy using
orally administered ergocalciferol. If 25-hydroxyvitamin D above 30
ng/mL was observed, the recommended intervention was Vitamin D
hormone replacement therapy using known oral or intravenous Vitamin
D hormones or analogs. The Guidelines did not recommend the
concurrent application of Vitamin D repletion and Vitamin D hormone
replacement therapies, consistent with warnings mandated by the
Food and Drug Administration in package inserts for Vitamin D
hormone replacement products.
[0026] The NKF K/DOQI Guidelines defined Vitamin D sufficiency as
serum 25-hydroxyvitamin D levels .gtoreq.30 ng/mL. Recommended
Vitamin D repletion therapy for patients with "Vitamin D
insufficiency," defined as serum 25-hydroxyvitamin D of 16-30
ng/mL, was 50,000 IU per month of oral Vitamin D.sub.2 for 6
months, given either in single monthly doses or in divided doses of
approximately 1,600 IU per day. Recommended repletion therapy for
patients with "Vitamin D deficiency" was more aggressive: for
"mild" deficiency, defined as serum 25-hydroxyvitamin D of 5-15
ng/mL, the Guidelines recommended 50,000 IU per week of oral
Vitamin D.sub.2 for 4 weeks, followed by 50,000 IU per month for
another 5 months; for "severe" deficiency, defined as serum
25-hydroxyvitamin D below 5 ng/mL, the Guidelines recommended
50,000 IU/week of oral Vitamin D.sub.2 for 12 weeks, followed by
50,000 IU/month for another 3 months. Doses of 50,000 IU per week
are approximately equivalent to 7,000 IU per day.
[0027] Most concepts of vitamin D metabolism and function have been
developed with the rat and/or chick as experimental models.
Studying vitamin D metabolism is hampered by the paucity of data on
the normal circulating levels of vitamin D metabolites in mammals
under normal conditions. Most recent research has focused on the
analysis of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D as
indicators of vitamin D status or aberrant physiological
states.
[0028] Shortly after the discovery of vitamin D, it seemed apparent
that Vitamins D.sub.2 and D.sub.3 had similar biological activities
in most mammals. More recent research, fostered by the discovery of
sensitive analytical techniques and the availability of high
specific activity .sup.3H-labeled vitamin D species, indicated that
differences in the metabolism of Vitamins D.sub.2 and D.sub.3 in
mammals are perhaps widespread. Most notable were the apparent
discrimination against Vitamin D.sub.2 by pigs [Biochem J.
204:185-189], cows [J Nutr 113:2595-2600], and humans [Gene
Regulation, Structure-Function Analysis and Clinical Application,
Walter de Gruyter. Berlin, pp. 765-766] and the apparent preference
of Vitamin D.sub.2 by rats [Biochem J 204:185-189, J Bone Miner Res
5(Supplement 2):5265].
[0029] Vitamin D and its metabolites are transported in the blood
of vertebrates attached to Vitamin D binding protein (DBP). Baird
et al [Recent Prog Horm Res. 25:611-664] have shown that protein
binding increases the solubility of steroids and that the metabolic
clearance rate of steroids is in part dependent on their binding to
specific plasma proteins.
[0030] Hay and Watson [Comp Biochem Physiol 56B:375-380] studied
the affinities of DBP for 25-hydroxyvitamin D.sub.2 and
25-hydroxyvitamin D.sub.3 in 63 vertebrate species. They found that
many of the studied species discriminated against 25-hydroxyvitamin
D.sub.2 in favor of 25-hydroxyvitamin D.sub.3 [Biochem J
204:185-189]. However, in rats the discrimination is against
Vitamin D.sub.3 in favor of Vitamin D.sub.2. The rat DBP is known
to have equal affinity for 25-hydroxyvitamin D.sub.2 and
25-hydroxyvitamin D.sub.3, but a lower affinity for Vitamin D.sub.2
relative to Vitamin D.sub.3 [Steroids 37:581-592]. Reddy et al.,
[Calci Tissue Int 36:524] suggested that the lower affinity for
Vitamin D.sub.2 resulted in its enhanced availability for liver
25-hydroxylation. Hence, in the presence of DBP, more
25-hydroxyvitamin D.sub.2 was made relative to 25-hydroxyvitamin
D.sub.3 when equal amounts of Vitamin D.sub.2 or Vitamin D.sub.3
substrate were perfused into rat livers. In the experiments
conducted by Reddy et al., if binding protein was eliminated from
the perfusion media, equal amounts of 25-hydroxyvitamin D.sub.2 and
25-hydroxyvitamin D.sub.3 were synthesized. Collectively, these
data suggest that discrimination against the different forms of
Vitamin D could likely result from variations in the affinity of
DBP for the parent compound and/or one or more of their
metabolites. Regardless of the mechanism for discrimination, it
appears that these differences are present to afford the species
the most efficient utilization of the most abundant Vitamin D
metabolites available in their environment.
[0031] Critical questions remain unanswered regarding complete
elucidation of the Vitamin D.sub.2 metabolic pathway, and species
differences between Vitamin D.sub.2 and D.sub.3 metabolism are
still virtually unexplored. The introduction of Vitamin D as a
pharmacological intervention has resulted in a totally different
set of issues regarding their metabolism, tissue kinetics,
mechanism of action, and potential therapeutic uses.
[0032] Vitamin D receptors are present throughout the human body in
a wide variety of cells, and there have been reports that vitamin D
hormone has diverse "non-classical" biologic effects on cellular
proliferation, the immune system and the cardiovascular system,
beyond its "classical" effects on the PTH system. It has also been
reported that 25-hydroxyvitamin D.sub.2 has direct effects on
parathyroid cells in suppressing PTH [Kidney International,
70(4):654-659, August 2006]. There has been one report that Vitamin
D.sub.2 was less than one-third as potent as Vitamin D.sub.3 and
exhibited a shorter duration of action relative to Vitamin D.sub.3;
administration of 50,000 IU of ergocalciferol or cholecalciferol to
healthy male humans produced similar rises in serum concentration
of the administered vitamin, indicating equivalent absorption, but
25-hydroxyvitamin D.sub.3 levels peaked at 14 days whereas
25-hydroxyvitamin D.sub.2 levels fell early and were not different
from baseline at 14 days [J. Clin. Endocrinol. Metab.,
89(11):5387-5391 (2004)].
[0033] Thus, the relative contribution of 25-hydroxyvitamin D
compounds and 1,25-dihydroxyvitamin D compounds to PTH suppression,
the relative potency of 1,25-dihydroxyvitamin D.sub.2 and
1,25-dihydroxyvitamin D.sub.3 in vivo, and the spectrum of
non-classical biological effects of each of these hormones has not
clearly been elucidated. There remains a need for alternative
vitamin D hormone therapies that ideally provide beneficial effects
on PTH levels, immune status and/or cardiovascular health, with
reduced toxicity.
SUMMARY
[0034] In one aspect the disclosure provides a method of increasing
or maintaining blood concentrations of 1,25-dihydroxyvitamin D in a
patient by administering an amount of 1,25-dihydroxyvitamin
D.sub.2. As noted hereinbefore, many conditions can lead to
1,25-dihydroxyvitamin D deficiencies, including living in northern
latitudes. Treatment with 1,25-dihydroxyvitamin D.sub.2 of those
patients in need thereof can provide blood concentrations of
1,25-dihydroxyvitamin D that are increased or maintained within a
patient's normal historical range for 1,25-dihydroxyvitamin D. Such
administration can be accomplished without a substantially
increased risk of hypercalcemia, hyperphosphatemia, or over
suppression of plasma intact parathyroid hormone (PTH), all of
which have been recognized as risks when treatment with a vitamin D
compound is incurred. Moreover, blood levels of
1,25-dihydroxyvitamin D can be maintained in the patient's
historical physiological range between doses, eliminating spike and
trough concentration patterns. In another aspect, the disclosure
provides a method of administering an amount of
1,25-dihydroxyvitamin D.sub.2 such that one or more symptoms of
1,25-dihydroxyvitamin D deficiency are alleviated, for example,
symptoms of deficiency in the non-classical effects of vitamin
D.
[0035] In yet another aspect, the disclosure provides a method
which has one or more of the following effects: concurrently
lowering or maintaining plasma intact parathyroid hormone levels;
increasing or maintaining serum calcium levels; maintaining serum
phosphorous levels; increasing serum 1,25-dihydroxyvitamin D
levels; and maintaining serum 1,25-dihydroxyvitamin D levels, in a
human patient, by administering to the patient an effective amount
of 1,25-dihydroxyvitamin D.sub.2 according to the disclosure
herein. Many diseases manifest abnormal levels of more than one
hormone and mineral. In CKD, for example, patients may experience
decreases in 1,25-dihydroxyvitamin D, increases in PTH, and
increases in serum phosphorous. Treatment in accordance with the
disclosure can provide concurrent leveling and/or maintaining of
these various hormone and mineral levels.
[0036] The disclosure herein provides a method for treating and/or
preventing hyperparathyroidism secondary to chronic kidney disease
by lowering (or maintaining low) serum parathyroid hormone (PTH)
levels in a human patient suffering from the disease by
administering to the patient an effective amount of
1,25-dihydroxyvitamin D.sub.2 according to the disclosure herein.
The method may ameliorate or prevent the renal osteodystrophy which
can develop in such patients.
[0037] In one aspect, a method for lowering or maintaining lowered
serum parathyroid hormone in human patients includes administering
to said patients a therapeutically effective amount of
1,25-dihydroxyvitamin D.sub.2 according to the disclosure herein to
lower or maintain lowered serum parathyroid hormone levels,
preferably an amount that lowers PTH levels by at least 15%, 20%,
25% or 30%, or alternatively the amount need to reduce serum levels
of PTH to the target range for the CKD Stage (e.g., for Stage 3 is
35-70 pg/mL (equivalent to 3.85-7.7 pmol/L), for Stage 4 is 70-110
pg/mL (equivalent to 7.7-12.1 pmol/L), and for Stage 5 is 150-300
pg/mL (equivalent to 16.5-33.0 pmol/L) (defined in K/DOQI Guideline
No. 1)).
[0038] In another aspect, the method includes administering to a
patient suffering from hyperparathyroidism secondary to chronic
kidney disease (Stage 3, 4 or 5) an effective amount of
1,25-dihydroxyvitamin D.sub.2 according to the disclosure herein to
lower the serum PTH level. For secondary hyperparathyroidism as
well as other therapies, the 1,25-dihydroxyvitamin D.sub.2 is
contemplated to be administered in an amount of 0.1 .mu.g per week
to about 100 .mu.g per week, for example.
[0039] The disclosure herein also provides a pharmaceutical
composition having serum (or plasma) PTH lowering activity, which
includes, in unit dosage form, an effective amount of
1,25-dihydroxyvitamin D.sub.2 and a pharmaceutically acceptable
excipient.
[0040] The treatment method described herein is an alternative to
conventional vitamin D replacement therapy with
1,25-dihydroxyvitamin D.sub.3, 1.alpha.-hydroxyvitamin D.sub.3,
19-nor-1,25-dihydroxyvitamin D.sub.2, and other active vitamin D
analogs.
[0041] In embodiments, the method is characterized by providing an
active vitamin D compound which has safety and patient survival
benefits associated with other vitamin D.sub.2 compounds, but
which, in addition, is able to replace the classical and
non-classical functions of 1,25-dihydroxyvitamin D.sub.3 more
completely than known synthetic alternatives. As such, the method
addresses a long felt need in therapy for secondary
hyperparathyroidism and other conditions associated with vitamin D
insufficiency or deficiency.
[0042] The methods and compositions are contemplated to be
associated with one or more benefits, such as significantly:
increasing the bioavailability of the contained
1,25-dihydroxyvitamin D.sub.2 by promoting absorption directly into
the bloodstream rather than into the lymphatic system via
chylomicrons; increasing the bioavailability of the contained
1,25-dihydroxyvitamin D.sub.2 by reducing catabolism in the
enterocytes of the upper small intestine; decreasing the
undesirable first pass effects of the contained
1,25-dihydroxyvitamin D.sub.2 on the duodenum; avoiding production
of adverse supraphysiologic surges in blood levels of
1,25-dihydroxyvitamin D; preventing reduction of blood
concentrations of 1,25-dihydroxyvitamin D below optimal levels;
restoring blood concentrations of 1,25-dihydroxyvitamin D to
optimal levels; maintaining blood concentrations of
1,25-dihydroxyvitamin D at such optimal levels; decreasing
disruptions in Vitamin D metabolism and related aberrations in PTH,
calcium and phosphorus homeostasis; and decreasing the risk of
serious side effects associated with Vitamin D hormone replacement,
including hypercalciuria, hypercalcemia, hyperphosphatemia, and
Vitamin D toxicity.
[0043] In another aspect, the invention provides a method effective
to restore and maintain blood concentrations of
1,25-dihydroxyvitamin D in human suffering from chronic kidney
disease (Stage 3, 4 or 5) through chronic treatment (e.g., at least
30 days, or 2, 3, 4, 5 or 6 months, or continued therapy through
life), while avoiding progressive loss of 25-hydroxyvitamin D and
1,25-dihydroxyvitamin D.sub.3. In exemplary embodiments, treatment
of a human with chronic kidney disease suffering from
1,25-dihydroxyvitamin D insufficiency or deficiency, is
contemplated.
[0044] The methods described herein are also intended to be used in
the treatment or prevention of conditions in humans including, but
not limited to: bone depletive disorders which respond to
administration of active forms of vitamin D; immunoresponsive
disorders which respond to administration of active forms of
vitamin D; high blood pressure; bacterial infection; and
cardiovascular disease malabsorption disorders, cancers, and
1,25-dihydroxyvitamin D insufficiency and deficiency. Expected
beneficial effects include amelioration of the disorder.
[0045] In summary, various aspects of the disclosure can provide
therapeutic methods for preventing and/or treating conditions
associated with low blood concentrations of 1,25-dihydroxyvitamin
D, elevated concentrations of PTH, elevated concentrations of serum
phosphorous, and low concentrations of serum calcium. The methods
are suitable for lowering elevated blood parathyroid hormone
levels, and/or maintaining lowered blood PTH levels in subjects
while maintaining normalized or targeted levels of serum calcium,
serum phosphorous, and serum 1,25-dihydroxyvitamin D.sub.2. The
methods described herein also include reducing the risk of over
suppression of PTH by administering to a subject in need thereof an
amount of 1,25-dihydroxyvitamin D.sub.2 to lower or maintain PTH
levels while avoiding or preventing low bone turnover rate, i.e.
adynamic bone disease.
[0046] Further aspects and advantages will be apparent to those of
ordinary skill in the art from a review of the following detailed
description. While the methods and compositions are susceptible of
embodiments in various forms, the description hereafter includes
specific embodiments with the understanding that the disclosure is
illustrative, and is not intended to limit the invention to the
specific embodiments described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] For further facilitating the understanding of the present
invention, twelve drawing figures are appended hereto.
[0048] FIG. 1 shows measured iPTH levels in control, calcitriol,
and 1,25-dihydroxyvitamin D.sub.2-treated rats having
adenine-induced kidney failure according to Example 3.
[0049] FIG. 2 shows measured serum calcium levels for
calcitriol-treated animals, and
[0050] FIG. 3 shows measured serum calcium levels for
1,25-dihydroxyvitamin D.sub.2-treated animals, according to Example
3.
[0051] FIG. 4 shows measured serum phosphorous levels for
calcitriol-treated animals, and
[0052] FIG. 5 shows measured serum phosphorous levels for
1,25-dihydroxyvitamin D.sub.2-treated animals, according to Example
3.
[0053] FIG. 6 shows survival data for calcitriol-treated animals,
and
[0054] FIG. 7 shows survival data for 1,25-dihydroxyvitamin
D.sub.2-treated animals, according to Example 3.
[0055] FIG. 8 shows relative renal FN1 induction in control,
calcitriol, and 1,25-dihydroxyvitamin D.sub.2-treated rats having
adenine-induced kidney failure according to Example 3.
[0056] FIG. 9 shows % calcitriol and 1,25-dihydroxyvitamin D.sub.2
remaining after incubation with human intestine microsomes
according to Example 4.
[0057] FIG. 10 and FIG. 11 show measured alkaline phosphatase and
CYP24 activity in C2BBe1 cells, respectively, after incubation with
calcitriol and 1,25-dihydroxyvitamin D.sub.2 according to Example
5.
[0058] FIG. 12 shows the intestinal alkaline phosphatase IAP
activity in Caco-2 cells, after incubation with calcitriol,
1,25-dihydroxyvitamin D.sub.2, and vitamin D analogs, according to
Example 6.
DETAILED DESCRIPTION
[0059] 1,25-dihydroxyvitamin D.sub.2 has several important
potential advantages over current therapies for the treatment of
chronic kidney disease (CKD). 1,25-dihydroxyvitamin D.sub.2 belongs
to the class of D.sub.2 analogs which includes
19-nor-1,25-dihydroxyvitamin D.sub.2.
[0060] However, 19-nor-1,25-dihydroxyvitamin D.sub.2 has an
important A-ring modification which is believed to result in
altered interactions with the vitamin D receptor, and therefore
differential gene expression compared to 1,25-dihydroxyvitamin
D.sub.2 and 1,25-dihydroxyvitamin D.sub.3. Chronic treatment with
19-nor-1,25-dihydroxyvitamin D.sub.2 leads to decreased levels of
1,25-dihydroxyvitamin D.sub.3. Also after a period of treatment
(24-48 hours), with 19-nor-1,25-dihydroxyvitamin D.sub.2, tissues
become differentially sensitized to the activity of the compound.
For example, low calcemic activity of 19-nor-1,25-dihydroxyvitamin
D.sub.2 has been attributed to reduced sensitivity of intestine and
bone due to changes in the metabolic activity of these tissues.
[0061] 19-nor-1,25-dihydroxyvitamin D.sub.2 is approximately
10-fold less potent than 1,25-dihydroxyvitamin D.sub.3 in promoting
bone resorption in vivo. Yet in vitro studies examining markers of
bone metabolism indicate that the effects of
19-nor-1,25-dihydroxyvitamin D.sub.2 and 1,25-dihydroxyvitamin
D.sub.3 in culture on VDR expression, suppression of cell
proliferation, regulation of osteocalcin and alkaline phosphatase
activity are indistinguishable. Conversely, however, in a study
using Caco-2 cells 19-nor-1,25-dihydroxyvitamin D.sub.2 did not
show a significant effect on calcium transport, while
1,25-dihydroxyvitamin D.sub.3 stimulated calcium transport by 934%.
Also, 19-nor-1,25-dihydroxyvitamin D.sub.2 exhibits differential
regulation of the CYP3A9 gene at the transcriptional level.
[0062] Without intending to be bound by any particular theory, it
is believed that at the level of the VDR, there are some
distinctive interactions with 19-nor-1,25-dihydroxyvitamin D.sub.2
that result in the altered ability of VDR to act on the
transcriptional regulation of certain genes. Accordingly, it is
believed that 19-nor-1,25-dihydroxyvitamin D.sub.2 is not able to
fully replace all of the functions of 1,25-dihydroxyvitamin
D.sub.3.
[0063] All major circulating vitamin D metabolites bind to DBP and
or albumin or lipoprotein. Normally, DBP occupancy by vitamin D
metabolites is approximately 2%. While some studies have determined
that metabolites of 25-hydroxyvitamin D.sub.3, such as
24,25-dihydroxyvitamin D.sub.3, 1,24,25-trihydroxyvitamin D.sub.3,
and 1,25-dihydroxyvitamin D.sub.3 have higher affinity for DBP than
the corresponding D.sub.2 metabolites, a more recent study of human
DBP indicates that metabolites of D.sub.2 have equivalent, or only
slightly lower affinity for DBP than those of vitamin D.sub.3
metabolites. Accordingly, there is believed to be no significant
difference between 1,25-dihydroxyvitamin D.sub.2 and
1,25-dihydroxyvitamin D.sub.3 with respect to DBP binding.
[0064] In contrast, 19-nor-1,25-dihydroxyvitamin D.sub.2 has a
lower (3-fold) affinity for DBP compared to 1,25-dihydroxyvitamin
D.sub.3. This latter observation may account for the shortened
half-life of 19-nor-1,25-dihydroxyvitamin D.sub.2 (between 4-6
hours in healthy patients compared to 15 hours in patients with
chronic renal failure and 20 hours in patients with Stage 4 CKD). A
short half-life is believed to be detrimental to patients over the
long term, since the hormone spike may be followed by a period of
"vitamin D deficiency" as induction of CYP24 depletes stores of
normal vitamin D hormone and prohormone. Furthermore, a sustained
release delivery system, as described below, is desirable.
[0065] Since 19-nor-1,25-dihydroxyvitamin D.sub.2,
1,25-dihydroxyvitamin D.sub.3 and 1,25-dihydroxyvitamin D.sub.2 all
induce CYP24, chronic treatment with any of these agents will
result in progressive loss of 25-hydroxyvitamin D.sub.3 and
1,25-dihydroxyvitamin D.sub.3. It is therefore important that over
the long term, the vitamin D hormone used for replacement therapy
can replace all of the classical, as well as the non-classical
functions of Vitamin D.sub.3 hormone. Vitamin D.sub.2 has been used
as a nutritional substitute for vitamin D.sub.3.
1,25-dihydroxyvitamin D.sub.2 is believed to have advantages over
1,25-dihydroxyvitamin D.sub.3 based on safety and patient survival
benefits. Further, 1,25-dihydroxyvitamin D.sub.2 is believed to
have advantages over 19-nor-1,25-dihydroxyvitamin D.sub.2 and other
vitamin D hormone analogs because it is more completely able to
replace the classical and non-classical functions of
1,25-dihydroxyvitamin D.sub.3.
[0066] As described above, differential gene regulation by
19-nor-dihydroxyvitamin D.sub.2 and acquired resistance to this
compound in certain tissues such as intestine and kidney, coupled
with treatment-induced CYP24 depletion of 25-hydroxyvitamin D.sub.3
and 1,25-dihydroxyvitamin D.sub.3 may have a net effect over the
long term of treatment of vitamin D deficiency for certain vitamin
D-dependent functions. For example, the reduced efficacy of
19-nor-dihydroxyvitamin D.sub.2 in stimulating osteoclast function
may be an important consideration over long term therapy, since
regulation of osteoclast function is critical for bone remodeling.
Changes in bone remodeling dynamics could eventually alter the
structural integrity of bone. Accordingly 1,25-dihydroxyvitamin
D.sub.2 is believed to have significant benefits for long term
hormone replacement therapy based on its closer biochemical and
physiological equivalence to 1,25-dihydroxyvitamin D.sub.3, with an
increased comparative patient survival benefit.
[0067] In one embodiment, administration of 1,25-dihydroxyvitamin
D.sub.2 according to the methods and compositions described herein
will show physiological equivalence to 1,25-dihydroxyvitamin
D.sub.3, with an increased safety. In another embodiment, For
example, serum calcium (Ca) levels can be compared by techniques
known in the art to assess safety. As another example, inorganic
phosphate (Pi) levels can be compared by techniques known in the
art to assess safety. Plasma intact parathyroid hormone (iPTH)
levels can be compared by techniques known in the art to assess
efficacy.
[0068] As described above, 1,25-dihydroxyvitamin D.sub.3 has
diverse "non-classical" biologic effects beyond its "classical"
effects on the PTH system, such as effects on cellular
proliferation, the immune system and the cardiovascular system.
Administration of 1,25-dihydroxyvitamin D.sub.2 according to the
methods and compositions described herein will preferably have one
or more analogous effects.
[0069] In one embodiment, administration of 1,25-dihydroxyvitamin
D.sub.2 as described herein will contribute one or more
non-classical effects on the renin-angiotensin system typically
shown by 1,25-dihydroxyvitamin D.sub.3. For example, in one type of
embodiment administration of 1,25-dihydroxyvitamin D.sub.2 as
described herein will provide negative endocrine regulation of the
renin-angiotensin system.
[0070] In another embodiment, administration of
1,25-dihydroxyvitamin D.sub.2 as described herein will contribute
one or more non-classical effects on bone typically shown by
1,25-dihydroxyvitamin D.sub.3, such as calcium and phosphate
homeostasis. The effect of administration of 1,25-dihydroxyvitamin
D.sub.2 can be compared with respect to direct and indirect effects
on bone. For example, the effects on regulation of calcium flux,
osteocalcin and acid and alkaline phosphatase activity, and
interleukin-6 (IL-6) can be determined. Effects on bone
mineralization can be determined directly in animal models.
[0071] In still another embodiment, administration of
1,25-dihydroxyvitamin D.sub.2 as described herein will contribute
one or more non-classical effects on immunomodulatory activity
typically shown by 1,25-dihydroxyvitamin D.sub.3. Immunoregulatory
properties of 1,25-dihydroxyvitamin D.sub.3 have been demonstrated
in different models of autoimmune diseases. For example,
1,25-dihydroxyvitamin D.sub.3 has been shown to inhibit in vitro
differentiation and maturation of dendritic cells, has been shown
to effect induction of T cell hyporesponsiveness, to effect
stimulation of human peripheral blood lymphocytes (PBL), to
inhibited the growth-promoting lymphokine interleukin-2, and to
inhibit the proliferation of mitogen-activated lymphocytes.
[0072] The person of ordinary skill in the art will be able to
determine methods for detecting effects such as those described
above. In addition, comparison of genes regulated by gene array
microchip analysis is also contemplated.
[0073] As used herein, the term "Vitamin D toxicity" is meant to
refer to the side effects suffered from excessively elevated
Vitamin D blood levels, including one or more of nausea, vomiting,
polyuria, hypercalciuria, hypercalcemia and hyperphosphatemia.
[0074] "Vitamin D insufficiency and deficiency" is generally
defined as having serum 25-hydroxyvitamin D levels below 30 ng/mL
(see National Kidney Foundation guidelines, NKF, Am. J. Kidney Dis.
42:S1-S202 (2003), incorporated herein by reference).
[0075] As used herein the term "hypercalcemia" refers to condition
in a patient wherein the patient has corrected serum levels of
calcium above 10.2 mg/dL. Normal corrected serum levels of calcium
for a human are between about 8.6 to 10.2 mg/dL.
[0076] As used herein the term "hyperphosphatemia" refers to a
condition in a patient having normal kidney function, or Stage 3-4
CKD, wherein the patient has serum phosphorous levels above 4.6
mg/dL. In a patient who has Stage 5 CKD, hyperphosphatemia occurs
when the patient has serum levels above 5.5 mg/dL. Normal values
for serum phosphorous in a human are 2.5-4.5 mg/dL.
[0077] As used herein the term "over suppression of plasma iPTH"
refers to a condition in a patient having normal kidney function,
or Stage 1-3 CKD, wherein the patient has levels of plasma iPTH
below 15 pg/mL. In a patient having Stage 4 CKD, over suppression
of plasma iPTH occurs when the patient has levels of plasma iPTH
below 30 pg/mL. In a patient having Stage 5 CKD, over suppression
of plasma iPTH occurs when the patient has levels of plasma iPTH
below 100 pg/mL.
[0078] As used herein, the term "Vitamin D hormone replacement
therapy" refers to the administration to a patient of an effective
amount of 1,25-dihydroxyvitamin D.sub.2, optionally together with
or other metabolites and analogs of Vitamin D which can
substantially occupy the intracellular VDR. Preferably the
administration of active vitamin D is by 1,25-dihydroxyvitamin
D.sub.2 alone.
[0079] As used herein, the term "substantially constant" with
respect to the serum or blood level of 1,25-dihydroxyvitamin
D.sub.2 preferably means that the release profile of the controlled
release formulation should not include increases in total serum or
blood levels of 1,25-dihydroxyvitamin D.sub.2 of greater than
approximately 75 pg/mL each after administration of a unit dose,
optionally over a period of preferably at least 30 minutes or 4
hours, etc.
[0080] As used herein, the term "controlled release," "sustained
release," and "modified release" are used interchangeably, and
refer to the release of the administered 1,25-dihydroxyvitamin
D.sub.2 in a way that deviates from immediate release. The term
"controlled release" optionally includes delayed release
characteristics. For example, a delayed release type of controlled
release formulation will be characterized by Cmax at a time greater
than Cmax for an immediate release formulation. As another example,
a sustained release type of controlled release formulation will be
characterized by release at such a rate that total serum or blood
levels of 1,25-dihydroxyvitamin D.sub.2 are maintained or elevated
above predosing levels for an extended period of time, e.g. 20 to
40 minutes or 1 to 15 hours or even longer.
[0081] "Supraphysiologic" in reference to intralumenal,
intracellular and blood levels of Vitamin D refers to a total
concentration of 1,25-dihydroxyvitamin D markedly greater than the
generally stable levels observed in a Vitamin D-replete subject,
animal or human patient over the course of any 24-hour period by
laboratory measurement when Vitamin D supplementation has been
withheld for at least 30 days. "Adverse supraphysiologic surge"
refers to a local or serum concentration of 1,25-dihydroxyvitamin D
that elicits adverse effects such as excessive extrarenal hormone
production, leading to local adverse effects on calcium and
phosphorus metabolism, inhibition of hepatic 25-hydroxylation of
vitamin D, increased catabolism of both Vitamin D and
25-hydroxyvitamin D, hypercalciuria, hypercalcemia and/or
hyperphosphatemia, with possible cardiovascular sequelae.
[0082] The term "therapeutically effective amount" depends on the
patient's condition and is an amount effective to achieve a desired
clinical effect, e.g. to maintain a laboratory test value within
the normal range or the recommended range for that patient's
condition, or an amount effective to reduce the occurrence or
severity of a clinical sign or symptom of disease. In some
embodiments, a therapeutically effective amount is an amount
effective on average to achieve at least a 15%, 20%, 25% or 30%
reduction in serum parathyroid hormone levels (iPTH) from baseline
levels without treatment. In yet other embodiments, a
therapeutically effective amount is an amount effective on average
to reach CKD Stage-specific iPTH target ranges, which for Stage 3
is 35-70 pg/mL (equivalent to 3.85-7.7 pmol/L), for Stage 4 is
70-110 pg/mL (equivalent to 7.7-12.1 pmol/L), and for Stage 5 is
150-300 pg/mL (equivalent to 16.5-33.0 pmol/L) (defined in K/DOQI
Guideline No. 1).
[0083] As used herein, the term "hyperparathyroidism" refers to
primary hyperparathyroidism, secondary hyperparathyroidism and
hyperparathyroidism secondary to chronic kidney disease (Stage 3, 4
or 5).
[0084] As used herein, the term "patient's normal historical
physiological range of serum 1,25-dihydroxyvitamin D" refers to the
average blood concentration range of 1,25-dihydroxyvitamin D of a
patient based on at least two annual or biannual readings of serum
1,25-dihydroxyvitamin D levels taken while the kidneys are
healthy.
[0085] It also is specifically understood that any numerical value
recited herein includes all values from the lower value to the
upper value, i.e., all possible combinations of numerical values
between the lowest value and the highest value enumerated are to be
considered to be expressly stated in this application. For example,
if a concentration range or a beneficial effect range is stated as
1% to 50%, it is intended that values such as 2% to 40%, 10% to
30%, or 1% to 3%, etc., are expressly enumerated in this
specification. These are only examples of what is specifically
intended.
[0086] It is noted that the medical community currently views
Vitamin D.sub.3 compounds as biologically indistinguishable from
the corresponding Vitamin D.sub.2 compounds. This is evident from
the indiscriminate inclusion of either Vitamin D.sub.2 or D.sub.3
in vitamin supplements prepared for human use, and from the
interchangeable use of either vitamin in treating bone diseases
caused by vitamin D deficiency. Curiously, medical experts consider
the hormonally active forms of the two vitamins to be equivalent
despite lack of confirmation from a single human study. (It is also
interestingly noted that Vitamin D.sub.4 is described in The Merck
Index (Merck Index, 11th ed. (1989) p. 9932) as having doubtful
biological activity.) As described herein, the
1,25-dihydroxyvitamin D.sub.2 compound is useful as an active
compound in a pharmaceutical composition. The hormone can be
produced by any of the various known methods of isolation or
synthesis. See, for example, U.S. Pat. No. 3,880,894 (Apr. 29,
1975).
[0087] The 1,25-dihydroxyvitamin D.sub.2 hormone can be processed
in accordance with conventional methods of pharmacy to produce
pharmaceutical agents for administration to patients, e.g., in
admixtures with conventional excipients such as pharmaceutically
acceptable organic or inorganic carrier substances suitable for
parenteral (e.g., subcutaneous, intravenous, intramuscular, and
depot injection), and nonparenteral such as enteral (e.g., oral) or
topical application which do not deleteriously react with the
active compound. The hormone can also be administered in
alternative fashions, including nasopharyngeal or mucosal
absorption such as intranasally, intrarectally, and
intravaginally.
[0088] Suitable pharmaceutically acceptable carriers include but
are not limited to water, salt (buffer) solutions, alcohols, gum
arabic, mineral and vegetable oils, benzyl alcohols, polyethylene
glycols, gelatine, carbohydrates such as lactose, amylose or
starch, magnesium stearate, talc, silicic acid, viscous paraffin,
perfume oil, fatty acid monoglycerides and diglycerides,
pentaerythritol fatty acid esters, hydroxy methylcellulose,
polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be
sterilized and if desired mixed with auxiliary agents, e.g.,
fillers, lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
coloring, flavoring and/or aromatic active compounds. If a solid
carrier is used, the dosage form of the 1,25-dihydroxyvitamin
D.sub.2 may be, for example, tablets, capsules, powders,
suppositories, or lozenges. If a liquid carrier is used, soft
gelatin capsules, transdermal patches, aerosol sprays, topical
creams, syrups or liquid suspensions, emulsions and solutions are
contemplated.
[0089] The presence of alcohol in a dosage form can interfere with
the ability of 1,25-dihydroxyvitamin D.sub.2 to bind to DBP.
Accordingly, oral dosage forms free of or substantially free of
alcohols are contemplated.
[0090] For topical application, there are employed as nonsprayable
forms, viscous to semi-solid or solid forms comprising a carrier
compatible with topical application and having a dynamic viscosity
preferably greater than water. Suitable formulations include but
are not limited to solutions, suspensions, emulsions, creams,
ointments, powders, liniments, salves, aerosols, etc., which are,
if desired, sterilized or mixed with auxiliary agents, e.g.,
preservatives, etc.
[0091] For parenteral application, particularly suitable are
injectable, sterile solutions, preferably oily or aqueous
solutions, as well as suspensions, emulsions, and implants,
including suppositories. Ampoules are convenient unit dosages. It
is also possible to freeze-dry the 1,25-dihydroxyvitamin D.sub.2
and store and use the lyophilizates obtained in preparation of
products. For example, lyophilizates can be stored in a vial and
used to reconstitute a solution for injection immediately before
administration.
[0092] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, and capsules. A syrup,
elixir, or the like can be used wherein a sweetened vehicle is
employed. For example, in a soft gelatin formulation the capsule
fill suitably contains 1,25-dihydroxyvitamin D.sub.2 dissolved in a
pharmaceutically acceptable oil, e.g., fractionated coconut oil,
and includes an antioxidant which may be, for example, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT) or vitamin E.
The capsule shell can suitably contain gelatin, glycerin, titanium
dioxide and coloring agent. The fill is typically about 58-59% by
weight of the whole capsule.
[0093] Where appropriate, 1,25-dihydroxyvitamin D.sub.2 can be
combined with one or more other active compounds, for example one
or more agents characterized by the ability to reduce loss of bone
mass, or bone mineral content in patients. Such compounds can
include other vitamin D compounds, conjugated estrogens, sodium
fluorides, bisphosphonates, cobalamin, pertussin toxin, or boron.
The dosage forms may also contain adjuvants, such as preserving or
stabilizing adjuvants.
Particularly preferred are oral and IV dosage forms.
[0094] Controlled release/sustained release compositions for dosage
forms are contemplated. In one embodiment, an amount of
1,25-dihydroxyvitamin D.sub.2 is included in a controlled release
formulation and is orally administered to a human in need of
treatment. For example, delayed release, sustained release, and
delayed-sustained release compositions are contemplated.
[0095] A controlled release formulation of 1,25-dihydroxyvitamin
D.sub.2 will have one or more benefits, such as significantly:
increasing the bioavailability of the contained
1,25-dihydroxyvitamin D.sub.2 by promoting absorption directly into
the bloodstream rather than into the lymphatic system via
chylomicrons; increasing the bioavailability of the contained
1,25-dihydroxyvitamin D.sub.2 by reducing catabolism in the
enterocytes of the upper small intestine; decreasing the
undesirable first pass effects of the contained
1,25-dihydroxyvitamin D.sub.2, for example on the duodenum and/or
jejunum; avoiding production of adverse supraphysiologic surges in
blood levels of 1,25-dihydroxyvitamin D; increasing the
effectiveness of orally administered 1,25-dihydroxyvitamin D.sub.2
in restoring blood concentrations of 1,25-dihydroxyvitamin D to
optimal levels (defined for CKD patients as equal to or greater
than 25 pg/mL); increasing the effectiveness of orally administered
1,25-dihydroxyvitamin D.sub.2 in maintaining blood concentrations
of 1,25-dihydroxyvitamin D at such optimal levels (e.g., for at
least 30 days); decreasing disruptions in Vitamin D metabolism and
related aberrations in PTH, calcium and phosphorus homeostasis;
and, decreasing the risk of serious side effects associated with
Vitamin D hormone replacement, including hypercalciuria,
hypercalcemia, hyperphosphatemia, and Vitamin D toxicity.
[0096] Similarly, an amount of 1,25-dihydroxyvitamin D.sub.2 can be
provided in an isotonic sterile formulation suitable for gradual
intravenous administration. Gradual intravenous administration, can
have one or more benefits, such as significantly: increasing the
bioavailability of the contained 1,25-dihydroxyvitamin D.sub.2 by
promoting absorption directly into the bloodstream rather than into
the lymphatic system via chylomicrons; increasing the
bioavailability of the contained 1,25-dihydroxyvitamin D.sub.2 by
reducing catabolism in the enterocytes of the upper small
intestine; decreasing the undesirable first pass effects of the
contained 1,25-dihydroxyvitamin D.sub.2 on the duodenum and
jejunum; avoiding production of adverse supraphysiologic surges in
blood levels of 1,25-dihydroxyvitamin D; increasing the
effectiveness of IV administered 1,25-dihydroxyvitamin D.sub.2 in
restoring blood concentrations of 1,25-dihydroxyvitamin D to
optimal levels (defined for CKD patients as equal to or greater
than 25 pg/mL); increasing the effectiveness of orally administered
1,25-dihydroxyvitamin D.sub.2 in maintaining blood concentrations
of 1,25-dihydroxyvitamin D at such optimal levels (e.g., for at
least 30 days); decreasing disruptions in Vitamin D metabolism and
related aberrations in PTH, calcium and phosphorus homeostasis;
and, decreasing the risk of serious side effects associated with
Vitamin D hormone replacement, including hypercalciuria,
hypercalcemia, hyperphosphatemia, and Vitamin D toxicity.
[0097] The preparation of a controlled release form of
1,25-dihydroxyvitamin D.sub.2 suitable for oral administration can
be carried out in accordance with many different principles of
controlled release, and according to many different formulation
techniques. For example, controlled release via dissolution
control, diffusion control, and ion exchange are contemplated.
Non-limiting examples include membrane encapsulated reservoir
devices, bioerodible polymers, matrix systems, and osmotic systems.
Liposomes can also be used as a controlled release carrier for the
hormone. Temperature and/or pH can be used as triggers for release
(e.g., temperature-dependent solubility of a coating or matrix,
and/or pH-dependent solubility of a coating or matrix).
[0098] As one specific example, 1,25-dihydroxyvitamin D.sub.2 can
be embedded for controlled release in a polymer matrix of a
biological degradable polymer, a water-soluble polymer or a mixture
of both, and optionally suitable surfactants. Embedding can mean in
this context the incorporation of micro-particles in a matrix of
polymers. Controlled release formulations can be obtained through
encapsulation of dispersed micro-particles or emulsified
micro-droplets (e.g., via known dispersion or emulsion coating
technologies.
[0099] In another type of formulation, the controlled release
dosage form includes a matrix which binds the 1,25-dihydroxyvitamin
D.sub.2 and permits a slow, relatively steady, preferably
substantially constant, release of the 1,25-dihydroxyvitamin
D.sub.2 over a period of four to eight hours or more, by simple
diffusion and/or gradual disintegration.
[0100] One type of embodiment includes a composition comprising a
controlled release formulation of 1,25-dihydroxyvitamin D.sub.2 and
a method of administering such a formulation to treat
1,25-dihydroxyvitamin D insufficiency and deficiency at a level of
efficiency heretofore unobtainable; without the undesirable first
pass effects of the Vitamin D compounds on the duodenum; without
adverse supraphysiological surges in intralumenal, intracellular
and blood levels of 1,25-dihydroxyvitamin D and their consequences;
and without serious side effects associated with Vitamin D
supplementation, namely Vitamin D toxicity.
[0101] A preferred controlled release composition will be designed
to maintain concentrations of 1,25-dihydroxyvitamin D.sub.2 at or
above 25 pg/mL, or in a range of about 25 pg/mL to about 65 pg/mL,
and is prepared in such a manner as to effect controlled,
preferably substantially constant, release of the
1,25-dihydroxyvitamin D.sub.2 over an extended period of time. An
optional but preferred method practiced with such a composition
will ensure a substantially constant concentration of
1,25-dihydroxyvitamin D.sub.2 in the body and a more sustained
blood level. By providing a slow and steady release of the
1,25-dihydroxyvitamin D.sub.2 over time, blood, intralumenal and
intracellular Vitamin D concentration spikes, i.e., adverse
supraphysiologic levels, are mitigated or eliminated. A gradual
increase in, and then sustained blood levels of
1,25-dihydroxyvitamin D.sub.2 is expected to provide dual
unexpected benefits of unsurpassed effectiveness in restoring blood
1,25-dihydroxyvitamin D.sub.2 to optimal levels, and unsurpassed
safety relative to heretofore known oral formulations of active
Vitamin D or analogs.
[0102] In one optional aspect, the controlled release oral
formulation will also effectively resist disintegration in gastric
juice, and further optionally will avoid substantial (e.g.,
>50%) release of the contained 1,25-dihydroxyvitamin D.sub.2
until it reaches the small intestine, and more preferably the ileum
of the small intestine of humans.
[0103] Once released into the lumen of the ileum the
1,25-dihydroxyvitamin D.sub.2 is absorbed into the bloodstream. In
such an embodiment, preferably the major portion of
1,25-dihydroxyvitamin D.sub.2 is absorbed at a point beyond the
duodenum and jejunum. These proximal portions of the small
intestine can respond to high intralumenal levels of Vitamin D
compounds and, in the process, can catabolize significant
quantities of the 1,25-dihydroxyvitamin D.sub.2. By delaying
release until the ileum, the pharmaceutical composition can
virtually eliminate first pass effects on the proximal intestine,
and reduce unwanted catabolism. Further, transileal absorption of
1,25-dihydroxyvitamin D.sub.2 can be increased with a formulation
described herein, which can be designed to direct the absorbed
1,25-dihydroxyvitamin D.sub.2 onto the serum vitamin D-binding
protein (DBP) versus into chylomicrons. It is believed that
1,25-dihydroxyvitamin D.sub.2 bound to DBP is more protected from
hepatic catabolism. Significant catabolism of administered
1,25-dihydroxyvitamin D.sub.2 prior to or after its absorption into
the bloodstream significantly lowers its systemic bioavailability.
Elimination of first pass effects reduces the risk of Vitamin D
toxicity.
[0104] Thus, one embodiment of the invention is a method of
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to a
patient such that the maximum serum concentration of
1,25-dihydroxyvitamin D in a dose interval (Cmax) is reduced as
compared to an equivalent amount of 1,25-dihydroxyvitamin D.sub.2
administered by bolus IV injection and/or an equivalent
immediate-release, oral dosage form. Similarly, the invention
provides a controlled-release dosage form having a quantity of
1,25-dihydroxyvitamin D.sub.2 that, when administered to a patient,
results in a Cmax of 1,25-dihydroxyvitamin D less than an
equivalent amount of 1,25-dihydroxyvitamin D.sub.2 administered by
bolus IV injection and/or by an equivalent immediate-release, oral
dosage form. For example, the reduction is preferably by a factor
of at least 50%, 60%, 70%, or 80%.
[0105] Another embodiment of the invention is a method of
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to a
patient such that the maximum change in serum concentration of
1,25-dihydroxyvitamin D.sub.2 in a dose interval is reduced as
compared to an equivalent amount of 1,25-dihydroxyvitamin D.sub.2
administered by bolus IV injection and/or an equivalent
immediate-release, oral dosage form. Similarly, the invention
provides a controlled-release dosage form having a quantity of
1,25-dihydroxyvitamin D.sub.2 that, when administered to a patient,
results in a maximum change in serum concentration of
1,25-dihydroxyvitamin D.sub.2 in a dose interval less than an
equivalent amount of 1,25-dihydroxyvitamin D.sub.2 administered by
bolus IV injection and/or by an equivalent immediate-release, oral
dosage form. For example, the reduction is preferably by a factor
of at least 50%, 60%, 70%, or 80%.
[0106] Still another embodiment of the invention is a method of
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to a
patient such that the ratio of the maximum serum concentration
after administration of 1,25-dihydroxyvitamin D.sub.2 to the
concentration 24 hours after administration
(Cmax.sub.24hr/C.sub.24hr) is reduced as compared to an equivalent
amount of 1,25-dihydroxyvitamin D.sub.2 administered by bolus IV
injection and/or an equivalent immediate-release, oral dosage form.
Similarly, the invention provides a controlled-release dosage form
having a quantity of 1,25-dihydroxyvitamin D.sub.2 that, when
administered to a patient, results in Cmax.sub.24hr/C.sub.24h, of
1,25-dihydroxyvitamin D.sub.2 less than an equivalent amount of
1,25-dihydroxyvitamin D.sub.2 administered by bolus IV injection
and/or by an equivalent immediate-release, oral dosage form. For
example, the reduction is preferably by a factor of at least 50%,
60%, 70%, or 80%.
[0107] Yet another embodiment of the invention is a method of
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to a
patient such that the elimination half-life (t.sub.1/2) of
1,25-dihydroxyvitamin D.sub.2 is increased as compared to an
equivalent amount of 1,25-dihydroxyvitamin D.sub.2 administered by
bolus IV injection and/or an equivalent immediate-release, oral
dosage form. Similarly, the invention provides a controlled-release
dosage form having a quantity of 1,25-dihydroxyvitamin D.sub.2
that, when administered to a patient, results in a t.sub.1/2 of
1,25-dihydroxyvitamin D.sub.2 greater than that of an equivalent
amount of 1,25-dihydroxyvitamin D.sub.2 administered by bolus IV
injection and/or by an equivalent immediate-release, oral dosage
form. For example, the increase is preferably by a factor of at
least 25%, 30%, 40%, 50%, or 60%.
[0108] A further embodiment of the invention is a method of
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to a
patient such that the time for the plasma concentration of
1,25-dihydroxyvitamin D.sub.2 to reach its maximum in a dose
interval following administration (Tmax) is increased as compared
to an equivalent amount of 1,25-dihydroxyvitamin D.sub.2
administered by bolus IV injection and/or an equivalent
immediate-release, oral dosage form. Similarly, the invention
provides a controlled-release dosage form having a quantity of
1,25-dihydroxyvitamin D.sub.2 that, when administered to a patient,
results in a Tmax for 1,25-dihydroxyvitamin D.sub.2 greater than
that of an equivalent amount of 1,25-dihydroxyvitamin D.sub.2
administered by bolus IV injection and/or by an equivalent
immediate-release, oral dosage form. For example, the increase is
preferably by a factor of at least 25%, 30%, 40%, 50%, or 60%.
[0109] In one embodiment of the invention, the controlled release
oral formulation of 1,25-hydroxyvitamin D.sub.2 is prepared
generally according to the following procedure. A sufficient
quantity of 1,25-hydroxyvitamin D.sub.2 is completely dissolved in
a minimal volume of USP-grade absolute ethanol (or other suitable
solvent) and mixed with appropriate amounts and types of
pharmaceutical-grade excipients to form a matrix which is solid or
semi-solid at both room temperature and at the normal temperature
of the human body, or a solvent mass which may be in a semi-solid
or liquid form at room temperature and/or at body temperature. The
matrix or solvent mass is completely, almost entirely,
substantially, or partially resistant to digestion in the stomach
and upper small intestine, and it gradually disintegrates in the
lower small intestine.
[0110] In a suitable formulation, the matrix or solvent mass binds
the 1,25-hydroxyvitamin D.sub.2 and permits a slow, relatively
steady, i.e. substantially constant, release of the
1,25-hydroxyvitamin D.sub.2 over a period of four to eight hours or
more, by simple diffusion and/or gradual disintegration, into the
contents of the lumen of the lower small intestine. This preferred
formulation further optionally has an enteric coating that
partially dissolves in aqueous solutions having a pH of about 7.0
to 8.0, or simply dissolves slowly enough that significant release
of 1,25-hydroxyvitamin D.sub.2 is delayed until after the
formulation passes through the duodenum and jejunum.
[0111] As discussed above, the means for providing the controlled
release of 1,25-hydroxyvitamin D.sub.2 may be selected from any of
the known controlled release delivery systems of an active
ingredient over a course of about four or more hours including the
wax matrix system, and the Eudragit RS/RL system (of Rohm Pharma,
GmbH, Weiterstadt, Germany).
[0112] The wax matrix system provides a lipophilic matrix. The wax
matrix system may utilize, bees wax, white wax, cachalot wax or
similar compositions. The active hormone is dispersed in the wax
binder, which slowly disintegrates in intestinal fluids to
gradually release the active ingredient. The wax binder that is
impregnated with the 1,25-hydroxyvitamin D.sub.2 is loaded into
partially-crosslinked, soft gelatin capsules. The wax matrix system
disperses the active ingredient in a wax binder which softens at
body temperature and slowly disintegrates in intestinal fluids to
gradually release the active ingredient. The system suitably
includes a mixture of waxes, with the optional addition of oils, to
achieve a melting point which is higher than body temperature and
preferably lower than the melting temperature of gelatin
formulations typically used to create the shells of either soft
and/or hard gelatin capsules or other formulations used to create
enteric coatings.
[0113] Specifically, in one suitable embodiment, the waxes selected
for the matrix are melted and thoroughly mixed. The desired
quantity of optional oils is added at this time, followed by
sufficient mixing. The waxy mixture is then gradually cooled to a
temperature just above its melting point. The desired amount of
1,25-hydroxyvitamin D.sub.2, dissolved in ethanol, is uniformly
distributed into the molten matrix, and the matrix is loaded into
soft gelatin capsules. The filled capsules are treated for
appropriate periods of time with a solution containing an aldehyde,
such as acetaldehyde, to partially crosslink the gelatin in the
capsule shell. The gelatin shell becomes increasingly crosslinked,
over a period of several weeks and, thereby, more resistant to
dissolution in the contents of stomach and upper intestine. When
properly constructed, this gelatin shell will gradually dissolve
after oral administration and become sufficiently porous (without
fully disintegrating) by the time it reaches the ileum to allow the
1,25-hydroxyvitamin D.sub.2 to diffuse slowly from the wax matrix
into the contents of the lower small intestine.
[0114] Examples of other lipid matrices that may be of value are
glycerides, fatty acids and alcohols, and fatty acid esters.
[0115] Thus, one type of particularly preferred controlled release
formulation is a solid or semi-solid, waxy pharmaceutical
formulation for controlled release of the vitamin D hormone in the
gastrointestinal tract of a subject which ingests the formulation.
The formulation includes a waxy controlled release carrier agent, a
lipoidic agent, an oily vehicle for the vitamin D compound, and the
vitamin D hormone 1,25-dihydroxyvitamin D.sub.2. The formulation
provides for controlled release of the vitamin D compound
incorporated therein. The formulation is free of or essentially
free of disintegrants.
[0116] The waxy controlled release carrier provides for a
formulation which is solid or semi-solid at room temperature and
solid, semi-solid, or liquid at body temperature, preferably
semi-solid or liquid at body temperature. Examples of carriers
suitable for use include waxes, such as synthetic wax,
microcrystalline wax, paraffin wax, carnauba wax, and beeswax;
polyethoxylated castor oil derivatives, hydrogenated vegetable
oils, glyceryl mono-, di- or tribehenates; long-chain alcohols,
such as stearyl alcohol, cetyl alcohol, and polyethylene glycol;
and mixtures of any of the foregoing. Non-digestible waxy
substances, such as hard paraffin wax, are preferred.
[0117] The waxy carrier preferably is present in an amount greater
than about 5% of the formulation, based on the total weight of the
formulation excluding any additional coatings or shells (wt %). For
example, the waxy carrier can comprise greater than 5 wt % of the
formulation, greater than 10 wt % of the formulation, greater than
15 wt % of the formulation, greater than 20 wt % of the
formulation, and greater than 25 wt % of the formulation. The waxy
carrier is preferably present in an amount less than 50 wt %, less
than 40 wt %, less than 35 wt %, or less than 30 wt. %. Suitable
ranges include 5 wt % to 35 wt %, 15 wt % to 35 wt % and 20 to 30
wt %. Examples include 15 wt %, 16 wt %, 17 wt %, 18 wt %, 19 wt %,
20 wt %, 21 wt %, 22 wt %, 23 wt %, 24 wt %, 25 wt %, 26 wt %, 27
wt %, 28 wt %, 29 wt %, and 30 wt %.
[0118] The lipoidic agent provides for release of the vitamin D
compound from the formulation in the gastrointestinal tract of the
subject being treated. Without intending to be bound by any
particular theory of operation, it is believed that the lipoidic
agent can serve one or more preferred functions such as creating a
micro-emulsion of the oily vehicle in gastrointestinal fluid;
providing prolonged gastric retention, for example by bioadhesive
properties such that the formulation interacts with the mucous
layer of the stomach; and in enhancing absorption of the vitamin D
compound. However, regardless of the mechanism of action, the
invention is not limited by any particular mode of operation.
[0119] The lipoidic agent components preferably are amphiphiles, in
which the molecule or ion contains both hydrophilic and lipophilic
portions. These components can be defined by a numerical value
based on the Hydrophile/Lipophile Balance system ("HLB system").
The HLB scale is a numerical scale, extending from 0 to
approximately 20, where lower numbers denote more lipophilic and
hydrophobic substances, and higher numbers denote more hydrophilic
and lipophobic substances. The affinity of a compound for water, or
for oily substances, is determined and its HLB value is assigned
experimentally. The HLB of the hydrophobic carrier employed herein
preferably will be in a range of about 13 to about 18.
[0120] A variety of pharmaceutically acceptable lipoidic agents may
be incorporated in the formulation. The quantity of lipoidic agent
present in the formulation is preferably at least 5 wt %, at least
15 wt %, at least 35 wt %, at least 40 wt % or at least 45 wt %.
Suitable ranges include about 5 wt % to about 60 wt %, about 20 wt
% to about 60 wt % and about 40 wt % to about 50 wt %.
[0121] In one embodiment, the lipoidic agent is a lipophilic
emulsifier which has an HLB of less than 7 and comprises a member
selected from the group consisting of mixed fatty acid
monoglycerides; mixed fatty acid diglycerides; mixtures of fatty
acid mono- and diglycerides; lipophilic polyglycerol esters;
glycerol esters including glyceryl monooleate, glyceryl dioleate,
glyceryl monostearate, glyceryl distearate, glyceryl monopalmitate,
and glyceryl dipalmitate; glyceryl-lacto esters of fatty acids;
propylene glycol esters including propylene glycol monopalmitate,
propylene glycol monostearate, and propylene glycol monooleate;
sorbitan esters including sorbitan monostearate, sorbitan
sesquioleate; fatty acids and their soaps including stearic acid,
palmitic acid, and oleic acid; and mixtures thereof glyceryl
monooleate, glyceryl dioleate, glyceryl monostearate, glyceryl
distearate, glyceryl monopalmitate, and glyceryl dipalmitate;
glyceryl-lacto esters of fatty acids; propylene glycol esters
including propylene glycol monopalmitate, propylene glycol
monostearate, and propylene glycol monooleate; sorbitan esters
including sorbitan monostearate, sorbitan sesquioleate; fatty acids
and their soaps including stearic acid, palmitic acid, and oleic
acid; and mixtures thereof.
[0122] A preferred lipoidic agent is selected from glycerides and
derivatives thereof. Preferred glycerides are selected from the
group consisting of medium or long chain glycerides, caprylocaproyl
macrogolglycerides, and mixtures thereof.
[0123] Preferred medium chain glycerides include, but are not
limited to, medium chain monoglycerides, medium chain diglycerides,
caprylic/capric triglyceride, glyceryl monolaurate, glyceryl
monostearate, caprylic/capric glycerides, glycerylmonocaprylate,
glyceryl monodicaprylate, caprylic/capric linoleic triglyceride,
and caprylic/capric/succinic triglyceride.
[0124] Monoglycerides having a low melting point are preferred for
making the formulation, and are easily soluble in the intestines.
Preferred monoglycerides include but are not limited to, glyceryl
monostearate, glyceryl monopalmitate, glyceryl monooleate, glyceryl
monocaprylate, glyceryl monocaprate, glyceryl monolaurate, etc.,
preferably glyceryl monostearate (GMS). GMS is a natural
emulsifying agent. It is oil soluble, but poorly soluble in water.
GMS has an HLB value of 3.8. Another preferred monoglyceride is
glyceryl monooleate (GMO). GMO is also a natural emulsifying agent;
it is oil soluble, but poorly soluble in water, and it has an HLB
value of 3.8.
[0125] In another embodiment, the glyceride is an absorption
enhancer selected from caprylocaproyl macrogolglycerides.
Caprylocaproyl macrogolglycerides which may be employed include,
but are not limited to, polyethylene glycosylated glycerides, also
known as polyglycolized glycerides or PEGylated glycerides.
PEGylated glycerides which may be employed in the composition
include, but are not limited to, mixtures of monoglycerides,
diglycerides, and triglycerides and monoesters and diesters of
polyethylene glycol, polyethylene glycosylated almond glycerides,
polyethylene glycosylated corn glycerides, and polyethylene
glycosylated caprylic/capric triglyceride. The absorption enhancer
preferably has an HLB value from 13 to 18, more preferably from 13
to 15.
[0126] One preferred absorption enhancer is known under the trade
name GELUCIRE, and is commercially available from Gattefosse
Corporation, Paramus, N.J., USA. GELUCIRE is a well known excipient
which is a family of fatty acid esters of glycerol and PEG esters,
also known as polyglycolized glycerides. GELUCIRE is used in
various applications including preparing sustained release
pharmaceutical compositions. GELUCIRE compounds are inert,
semi-solid waxy materials which are amphiphilic and are available
with varying physical characteristics such as melting point, HLB,
and solubilities in various solvents. They are surface active in
nature and disperse or solubilize in aqueous media forming
micelles, microscopic globules or vesicles. They are identified by
their melting point/HLB value. The melting point is expressed in
degrees Celsius. One or a mixture of different grades of GELUCIRE
excipient may be chosen to achieve the desired characteristics of
melting point and/or HLB value. The preferred GELUCIRE composition
is GELUCIRE 44/14, a semisolid waxy material with a melting point
of 44.degree. C. and a HLB of 14.
[0127] Another preferred polyglycolyzed glyceride absorption
enhancer is caprylocaproyl macrogol-8-glyceride (CAS No. 85536-07-8
and 84963-88-2). This is a mixture of mono-, di- and triesters of
glycerol and of PEG 400 with medium-chain fatty acids
(C.sub.8-C.sub.10) which is marketed, for example, by Gattefosse
Corporation, Paramus, N.J., USA under the trade name LABRASOL.
LABRASOL has a HLB value of 14 and has the following composition by
weight: C.sub.8-C.sub.10 monoglycerides approximately 4%;
C.sub.8-C.sub.10 diglycerides approximately 17%; C.sub.8-C.sub.10
triglycerides approximately 6%; C.sub.8-C.sub.10 monoesters of PEG
400 approximately 14%; C.sub.8-C.sub.10 diesters of PEG 400
approximately 36%; free PEG 400 approximately 20%; free glycerol
approximately 3%.
[0128] Preferably, the lipoidic agent includes a mixture of a
lipophilic emulsifier which has an HLB of less than 7 and an
absorption enhancer that preferably has an HLB value from 13 to 18.
The lipophilic emulsifier is preferably present in an amount in a
range of about 20 wt % to about 50 wt %, preferably about 30 wt %
to about 40 wt %, and the absorption enhancer is preferably present
in an amount of about 5 to about 20 wt %, preferably about 8 to
about 15 wt %.
[0129] The low melting points of many of the solid lipoidic
compositions provide a means of incorporating the pharmaceutically
active ingredients in them at temperatures from about 0.degree. C.
to about 50.degree. C. above their respective melting points, and
then filling the melt (solution and/or dispersion) in animal or
vegetable gelatin capsules. The melt solidifies inside the capsules
upon cooling to room temperature.
[0130] The oily component serves as a vehicle, preferably the main
vehicle, for the vitamin D compound. Any
pharmaceutically-acceptable oil can be used. Examples include
animal (e.g., fish), vegetable (e.g., soybean), and mineral oils.
The oil preferably will readily dissolve the vitamin D compound
used. Preferred oily components include non-digestible oils, such
as mineral oils, particularly liquid paraffins, and squalene. The
oil vehicle preferably comprises about 10 wt % to about 50 wt % of
the formulation, more preferably about 15 wt % to about 45 wt %
about 20 wt % to about 40 wt %, or about 15 wt % to about 25 wt %.
In one preferred embodiment, the liquid paraffin can be
characterized by one or more of the following parameters: specific
gravity about 0.88 to 0.89; kinematic viscosity (40.degree. C.)
abut 64 to about 70 cSt; molecular weight 424; % paraffinic
hydrocarbons about 59; and pour point -24.degree. C. The ratio
between the waxy component and the oily component can be optimized
in order to achieve the desired rate of release of the vitamin D
compound. Thus, if a heavier oil component is used, relatively less
of the waxy component can be used, and if a lighter oil component
is used, then relatively more waxy component can be used.
[0131] Another suitable controlled-release oral drug delivery
system is the Eudragit RL/RS system in which the active ingredient
1,25-hydroxyvitamin D.sub.2 is formed into granules having a
dimension of 25/30 mesh. The granules are then uniformly coated
with a thin polymeric lacquer which is water insoluble but slowly
water permeable. The coated granules can be mixed with optional
additives such as antioxidants, stabilizers, binders, lubricants,
processing aids and the like. The mixture may be compacted into a
tablet which, prior to use, is hard and dry and can be further
coated, or it may be poured into a capsule. After the tablet or
capsule is swallowed and comes into contact with the aqueous
intestinal fluids, the thin lacquer begins to swell and slowly
allows permeation by intestinal fluids. As the intestinal fluid
slowly permeates the lacquer coating, the contained
1,25-hydroxyvitamin D.sub.2 is slowly released. By the time the
tablet or capsule has passed through the small intestine, about
four to eight hours or more later, the 1,25-hydroxyvitamin D.sub.2
will have been gradually but completely released. Accordingly, the
ingested tablet will release a stream of 1,25-hydroxyvitamin
D.sub.2 as well as any other optional active ingredient.
[0132] The Eudragit system is comprised of high permeability
lacquers (RL) and low permeability lacquers (RS). RS is a water
insoluble film former based on neutral swellable methacrylic acids
esters with a small proportion of trimethylammonioethyl
methacrylate chlorides, and the molar ratio of the quaternary
ammonium groups to the neural ester group is about 1:40. RL is also
a water insoluble swellable film former based on neutral
methacrylic acid esters with a small portion of
trimethylammonioethyl methacrylate chloride, and the molar ratio of
quateranary ammonium groups to neutral ester groups is about 1:20.
The permeability of the coating and thus the time course of drug
release can be titrated by varying the proportion of RS to RL
coating material. For further details of the Eudragit RL/RS system,
reference is made to technical publications available from Rohm
Tech, Inc. 195 Canal Street, Maiden, Mass., 02146. See also, K.
Lehmann, D. Dreher "Coating of tablets and small particles with
acrylic resins by fluid bed technology", Int. J. Pharm. Tech. &
Prod. Mfr. 2(r), 31-43 (1981), incorporated herein by
reference.
[0133] Other examples of insoluble polymers include polyvinyl
esters, polyvinyl acetals, polyacrylic acid esters, butadiene
styrene copolymers and the like.
[0134] Once the coated granules or other formulations are either
formed into a tablet or put into a capsule, the tablet or capsule
is optionally coated with an enteric-coating material which
dissolves at a pH of 7.0 to 8.0. One such pH-dependent
enteric-coating material is Eudragit L/S which dissolves in
intestinal fluid but not in the gastric juices. Other enteric
coating materials may be used, such as cellulose acetate phthalate
(CAP) which is resistant to dissolution by gastric juices but
readily disintegrates due to the hydrolytic effect of the
intestinal esterases.
[0135] The particular choice of enteric-coating material and/or
controlled release material will delay substantial release of the
1,25-hydroxyvitamin D.sub.2, for example until the formulation
reaches the ileum. The particular choice of controlled release
method and material (e.g., coating, matrix, or other medium) will
preferably provide a substantially constant release of the
1,25-hydroxyvitamin D.sub.2 over a period of 4 to 8 hours or
more.
[0136] In one preferred class of embodiments, the modified release
formulation releases at least 70%, more preferably at least 80% of
the vitamin D compound within the first 24 hours after dosing, for
example about 72%.
[0137] Administration of 1,25-dihydroxyvitamin D.sub.2 as described
herein also allows for the efficient and predictable delivery of a
predetermined dosage of vitamin D hormone to a patient. The
temporal and quantitative availability of the active vitamin
D.sub.2 hormone is not dependent on activation in the liver or
other metabolism. Accordingly, lower dosages, compared to other
vitamin D.sub.2 analogs, are considered possible in order to
achieve equivalent effects, while optionally or preferably avoiding
or reducing side effects, as described above.
[0138] As described herein, oral and intravenous dosage
formulations and routes are preferred. The administration of such
therapies can be on an episodic basis, suitably from daily, 6, 5,
4, 3, 2, or 1 times a week.
[0139] In embodiments, the method is contemplated to include
administering a formulation described herein to raise and
preferably also maintain blood 1,25-dihydroxyvitamin D.sub.2 levels
at 25 pg/mL, 30 pg/mL, or higher, e.g. 25-65 pg/mL for an extended
period, for example at least one month, at least three months, at
least six months, or longer.
[0140] The dosage of the 1,25-dihydroxyvitamin D.sub.2 for oral or
parenteral administration generally is about 0.1 .mu.g per week to
100 .mu.g per week, preferably about 0.7 .mu.g per week to about 70
.mu.g per week, which can be split into daily or other periodic
doses, such as three times per week for administration concomitant
with hemodialysis. In exemplary embodiments, a parenteral dosage
equivalent to about 0.5 .mu.g per day to about 2 .mu.g per day is
contemplated, while an oral dosage equivalent to about 0.1, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, or 10 .mu.g per day is contemplated.
[0141] Generally, the 1,25-dihydroxyvitamin D.sub.2 is dispensed by
unit dosage form comprising about 0.1 .mu.g to about 10 .mu.g in a
pharmaceutically acceptable carrier per unit dosage, for example
about 1 .mu.g to about 4 .mu.g. A sustained-release or delayed,
sustained-release unit dosage form including about 2 .mu.g to about
10 .mu.g, or about 3 .mu.g to about 5 .mu.g is also
contemplated.
[0142] The formulation can be prepared by procedures well known to
one of ordinary skill in the art. Typically, the pharmaceutically
acceptable waxes, lipoidic agents, and oils are melted, if
necessary, to provide a flowable liquid thereby making it easier to
obtain a homogeneous mixture. The Vitamin D compound is added to
the thus liquid carrier, for example dissolved in an alcohol such
as anhydrous ethanol, and the ingredients are mixed to provide a
homogeneous mixture. The mixture can be cooled and stored prior to
later division into unit dosage forms, such as filled gelatin
capsules.
[0143] In one preferred method, a portion of the oil vehicle, solid
wax, and a lipophilic emulsifier are heated to a relatively high
temperature (e.g., 65.degree. C.) and mixed prior to adding an
absorption enhancer, followed by additional mixing until
homogenous, then cooling to an intermediate elevated temperature
(e.g., 50.degree. C. to 55.degree. C.). In a separate vessel, an
antioxidant preservative and the remainder of the oil vehicle are
mixed and heated to an intermediate elevated temperature (e.g.,
50.degree. C.), then combined and mixed with the wax mixture until
a homogenous solution is obtained. Next, a solution of vitamin D
compound in alcohol is combined with the homogenous waxy solution,
mixed until a homogenous solution is obtained, preferably filled
into capsules, and then cooled to room temperature. In another
preferred method, a portion of the oil vehicle, solid wax, and a
lipophilic emulsifier are heated at a temperature of 55.degree. C.
to 60.degree. C. and mixed prior to adding an absorption enhancer,
followed by additional mixing until homogenous. In a separate
vessel, an antioxidant preservative and the remainder of the oil
vehicle are mixed and heated to a temperature of 55.degree. C. to
60.degree. C., then combined and mixed with the wax mixture until a
homogenous solution is obtained. Next, a solution of vitamin D
compound in alcohol is combined with the homogenous waxy solution,
mixed until a homogenous solution is obtained, preferably filled
into capsules, and then cooled to room temperature.
[0144] The formulation preferably is placed in capsules prior to
administration to the patient in need of treatment. Such capsules
may be hard or soft, and soft capsules are preferred. The
formulation may be filled into gelatin capsules using standard
capsule filling machinery, such as by melting the formulation and
injection filling it into soft capsule shells.
[0145] The formulation and methods of use and making are
contemplated to include embodiments including any combination of
one or more of the additional optional elements, features, and
steps further described below, unless stated otherwise.
[0146] Thus, in one type of embodiment, the formulation further
includes a preservative, such as an antioxidant. Butylated
hydroxytoluene (BHT) is preferred.
[0147] In another type of embodiment, the vitamin D compound is
administered in combination with one or more other therapeutic
agents.
[0148] As described above, the formulation is preferably filled
into gelatin capsules, but it may also be administered in neat
form, or with one or more external coating layers, such as an
enteric coating. It is also contemplated that the formulation can
be pressed into tablets, and in such cases one or more tablet
pressing excipients may be included.
[0149] In the compositions and methods described herein, preferred
steps, preferred components, preferred compositional ranges
thereof, and preferred combinations of the foregoing, can be
selected from the various specific examples provided herein. For
example, a preferred formulation includes a therapeutically
effective amount of 1,25-hydroxyvitamin D.sub.2, about 2 wt %
(e.g., 2.32 wt %) ethanol, about 10 wt % (e.g., 9.75 wt %) GELUCIRE
44/14, about 27 wt % (e.g., 27.51 wt. %) hard paraffin, about 38 wt
% (e.g., 37.85 wt %) GMS, about 22 wt % (e.g., 22.43 wt %) mineral
oil, and optionally a small amount of preservative (e.g., 0.02 wt %
BHT). A variation on this formulation will include about 20% hard
paraffin and about 29% mineral oil.
[0150] Specifications for still another preferred embodiment of a
base capsule fill formulation embodiment, are shown in Table 1
below.
TABLE-US-00001 TABLE 1 Ingredient % w/w 1,25-hydroxyvitamin D.sub.2
effective amount Dehydrated ethanol 2.5 Hard Paraffin 20 Mineral
Oil 30 GELUCIRE 44/14 10 GMS 38 BHT 0.020
[0151] The dosages described herein are contemplated for any of the
therapeutic methods described herein. It will be appreciated that
the actual preferred amount of hormone in a specific case will vary
according the particular compositions formulated, the mode of
application, and the particular situs being treated. Dosages can be
determined using conventional considerations, e.g., by customary
comparison of the differential activity of the hormone and of a
known agent, e.g. by means of an appropriate conventional
pharmacological protocol.
[0152] The specific doses for each particular patient can depend on
a wide variety of factors, for example, on the age, body weight,
general state of health, sex, on the diet, on the timing and mode
of administration, on the rate of excretion, and on medicaments
used in combination and the severity of the particular disorder to
which the therapy is applied.
[0153] Patients in need of vitamin D supplementation include
healthy subjects and subjects at risk for vitamin D insufficiency
or deficiency, for example, subjects with Stage 1, 2, 3, 4 or 5
chronic kidney disease; infants, children and adults that do not
drink vitamin D fortified milk (e.g. lactose intolerant subjects,
subjects with milk allergy, vegetarians who do not consume milk,
and breast fed infants); subjects with rickets; subjects with dark
skin (e.g., in the U.S., 42% of African American women between 15
and 49 years of age were vitamin D deficient compared to 4% of
white women); the elderly (who have a reduced ability to synthesize
vitamin D and also are more likely to stay indoors);
institutionalized adults (who are likely to stay indoors, including
subjects with Alzheimer's disease or mentally ill); subjects who
cover all exposed skin (such as members of certain religions or
cultures); subjects who always use sunscreen (e.g., the application
of sunscreen with a Sun Protection Factor (SPF) value of 8 reduces
production of vitamin D by 95%, and higher SPF values may further
reduce vitamin D); subjects with fat malabsorption syndromes
(including but not limited to cystic fibrosis, cholestatic liver
disease, other liver disease, gallbladder disease, pancreatic
enzyme deficiency, Crohn's disease, inflammatory bowel disease,
sprue or celiac disease, or surgical removal of part or all of the
stomach and/or intestines); subjects with inflammatory bowel
disease; subjects with Crohn's disease; subjects who have had small
bowel resections; subjects with gum disease; subjects taking
medications that increase the catabolism of vitamin D, including
phenyloin, fosphenyloin, phenobarbital, carbamazepine, and
rifampin; subjects taking medications that reduce absorption of
vitamin D, including cholestyramine, colestipol, orlistat, mineral
oil, and fat substitutes; subjects taking medications that inhibit
activation of vitamin D, including ketoconazole; subjects taking
medications that decrease calcium absorption, including
corticosteroids; subjects with obesity (vitamin D deposited in body
fat stores is less bioavailable); subjects with osteoporosis;
and/or postmenopausal women. According to the Institute of
Medicine's report on the Dietary Reference Intakes for vitamin D,
food consumption data suggest that median intakes of vitamin D for
both younger and older women are below current recommendations;
data suggest that more than 50% of younger and older women are not
consuming recommended amounts of vitamin D. Optionally excluded
from the methods of the invention are therapeutic treatment of
subjects suffering from renal osteodystrophy (including
osteomalacia and osteitis fibrosa cystica).
[0154] In other aspects, the compositions and methods of the
invention are useful for prophylactic or therapeutic treatment of
vitamin D-responsive diseases, i.e., diseases where active vitamin
D prevents onset or progression of disease, or reduces signs or
symptoms of disease. Such vitamin D-responsive diseases include
cancer (e.g., breast, lung, skin, melanoma, colon, colorectal,
rectal, prostate and bone cancer). 1,25-dihydroxyvitamin D.sub.2
has been observed to induce cell differentiation and/or inhibit
cell proliferation in vitro for a number of cells. Vitamin
D-responsive diseases also include autoimmune diseases, for
example, type I diabetes, multiple sclerosis, rheumatoid arthritis,
polymyositis, dermatomyositis, scleroderma, fibrosis, Grave's
disease, Hashimoto's disease, acute or chronic transplant
rejection, acute or chronic graft versus host disease, inflammatory
bowel disease, Crohn's disease, systemic lupus erythematosis,
Sjogren's Syndrome, eczema and psoriasis, dermatitis, including
atopic dermatitis, contact dermatitis, allergic dermatitis and/or
chronic dermatitis. Vitamin D-responsive diseases also include
other inflammatory diseases, for example, asthma, chronic
obstructive pulmonary disease, polycystic kidney disease,
polycystic ovary syndrome, pancreatitis, nephritis, hepatitis,
and/or infection. Vitamin D-responsive diseases have also been
reported to include hypertension and cardiovascular diseases. Thus,
the invention contemplates prophylactic or therapeutic treatment of
subjects at risk of or suffering from cardiovascular diseases, for
example, subjects with atherosclerosis, arteriosclerosis, coronary
artery disease, cerebrovascular disease, peripheral vascular
disease, myocardial infarction, myocardial ischemia, cerebral
ischemia, stroke, congestive heart failure, cardiomyopathy, obesity
or other weight disorders, lipid disorders (e.g. hyperlipidemia,
dyslipidemia including associated diabetic dyslipidemia and mixed
dyslipidemia hypoalphalipoproteinemia, hypertriglyceridemia,
hypercholesterolemia, and low HDL (high density lipoprotein)),
metabolic disorders (e.g. Metabolic Syndrome, Type II diabetes
mellitus, Type I diabetes mellitus, hyperinsulinemia, impaired
glucose tolerance, insulin resistance, diabetic complication
including neuropathy, nephropathy, retinopathy, diabetic foot ulcer
and cataracts), and/or thrombosis.
[0155] Diseases which can benefit from a modulation in the levels
of 1,25-dihydroxyvitamin D.sub.2 or its analogs, include, but are
not limited to: (i) in the parathyroid--hypo-parathyroidism,
Pseudohypoparathyroidism, secondary hyperparathyroidism; (ii) in
the pancreas--diabetes; (iii) in the thyroid--medullary carcinoma;
(iv) in the skin--psoriasis; wound healing; (v) in the
lung--sarcoidosis and tuberculosis; (vi) in the kidney--chronic
kidney disease, hypophosphatemic VDRR, vitamin D dependent rickets;
(vii) in the bone--anticonvulsant treatment, fibrogenisis
imperfecta ossium, osteitis fibrosa cystica, osteomalacia,
osteporosis, osteopenia, osteosclerosis, renal osteodytrophy,
rickets; (viii) in the intestine--glucocorticoid antagonism,
idopathic hypercalcemia, malabsorption syndrome, steatorrhea,
tropical sprue; and (ix) autoimmune disorders.
[0156] In embodiments of the invention, the disease that benefits
from a modulation in the levels of 1,25-dihydroxyvitamin D.sub.2,
or an analog thereof, are selected from cancer, dermatological
disorders (for example psoriasis), parathyroid disorders (for
example hyperparathyroidism and secondary hyperparathyroidism),
bone disorders (for example osteoporosis) and autoimmune
disorders.
[0157] The methods and compositions described herein are
particularly useful for treating abnormally elevated blood levels
of PTH. The invention provides a method for treating or preventing
hyperparathyroidism, such as secondary hyperparathyroidism, by
lowering (or maintaining low) serum parathyroid hormone levels in a
patient suffering from the disease. The method at the same time can
ameliorate bone metabolism abnormalities which can develop in such
patients.
[0158] Secondary hyperparathyroidism is a common complication of
chronic kidney disease and thus a particular patient group
contemplated is one with CKD. Patients at Stage 3, 4 and/or 5 CKD
may be treated according to the present invention. Secondary
hyperparathyroidism can also develop in individuals with healthy
kidneys, due to environmental, cultural or dietary factors which
prevent adequate vitamin D supply.
[0159] The methods described herein also intended to be used in the
treatment or prevention of conditions in humans including, but not
limited to: bone depletive disorders which respond to
administration of active forms of vitamin D; immunoresponsive
disorders which respond to administration of active forms of
vitamin D; high blood pressure; bacterial infection; and
cardiovascular disease.
EXAMPLES
[0160] The following examples are provided for illustration and are
not intended to limit the scope of the invention.
Example 1
1,25-Dihydroxyvitamin D.sub.2 for Treating Subjects with Low Serum
1,25-Dihydroxyvitamin D
[0161] 1,25-dihydroxyvitamin D.sub.2 is used as a treatment for
subjects with low serum 1,25-dihydroxyvitamin D in a study
involving 50 adults, ages 18-85 years.
[0162] The subjects have serum 1,25-dihydroxyvitamin D levels below
20 pg/dL and complete an eight-week baseline period and then 24
weeks of therapy with orally administered 1,25-dihydroxyvitamin
D.sub.2.
[0163] The initial dose of 1,25-dihydroxyvitamin D.sub.2 is 1.0
.mu.g, with increases in steps of 0.5 .mu.g/day permitted after
four weeks. The maximum dosage is limited to 5.0 .mu.g/day of
1,25-dihydroxyvitamin D.sub.2. Subjects are monitored at regular
intervals for plasma iPTH, serum calcium and phosphorus, 24-hour
and fasting urinary calcium, bone-specific serum markers, plasma
total 1,25-dihydroxyvitamin D.sub.2 and routine blood chemistries
and hematologies.
[0164] After the 24 week treatment period the subjects treated with
1,25-dihydroxyvitamin D.sub.2 show average serum phosphorous levels
between about 2.5 and 4.5 mg/dL, average corrected serum calcium
levels between about 8.6 and 10.2 mg/dL, average intact serum
parathyroid hormone levels between about 65 pg/mL and 110 pg/mL,
and average blood concentrations of 1,25-dihydroxyvitamin D between
about 20 pg/mL and 60 pg/mL. Testing of serum 1,25-dihydroxyvitamin
D levels between doses of 1,25-dihydroxyvitamin D.sub.2 shows that
serum 1,25-dihydroxyvitamin D levels in the patients are within the
patients' normal historical physiological range for
1,25-dihydroxyvitamin D. Levels of serum bone-specific markers
alkaline phosphatase, N- and C-telopeptides, and osteocalcin in
patients show average normal levels of these markers.
Example 2
Double-Blind Study in End Stage Renal Disease (ESRD) Patients
Exhibiting Secondary Hyperparathyroidism
[0165] Up to 100 ESRD (End Stage Renal Disease) human patients
undergoing chronic hemodialysis are studied in a multicenter,
double-blind, placebo-controlled study. The selected patients
reside in two major metropolitan areas within the continental U.S.,
have ages between 20 and 75 years, and have a history of secondary
hyperparathyroidism. They have been on hemodialysis for at least
four months, have a normal (or near normal) serum albumin, and have
controlled serum phosphorus (often by using oral calcium phosphate
binders).
[0166] On admission to the study, each patient is assigned at
random to one of two treatment groups. One of these groups receives
two consecutive 12-week courses of therapy with
1,25-dihydroxyvitamin D.sub.2; the other group receives one 12-week
course of therapy with 1,25-dihydroxyvitamin D.sub.2 followed,
without interruption, by one 12-week course of placebo therapy.
Each patient discontinues any 1.alpha.,25-dihydroxyvitamin D.sub.3
therapy for eight weeks prior to initiating 1,25-dihydroxyvitamin
D.sub.2 therapy. Throughout this eight-week washout (or control)
period and the two subsequent 12-week treatment periods, patients
are monitored weekly for serum calcium and phosphorus. Serum intact
PTH is monitored weekly or biweekly, and bone-specific serum
markers, serum vitamin D metabolites, serum albumin, blood
chemistries, hemoglobin and hematocrit are monitored at selected
intervals.
[0167] During the study, patients undergo routine hemodialysis
(three times per week) using a 1.24 mM calcium dialysate and ingest
calcium phosphate binders (such as calcium carbonate or calcium
acetate) in an amount sufficient to keep serum phosphate maintained
in a range of 3.5 to 5.5 mg/dL. Patients who develop persistent
mild hypercalcemia or mild hyperphosphatemia during the treatment
periods reduce their 1,25-dihydroxyvitamin D.sub.2 dosage. Patients
who develop marked hypercalcemia (serum levels of total corrected
calcium exceeds 10.2 mg/dL) or marked hyperphosphatemia (serum
levels of phosphorus exceeds 5.5 mg/dL) immediately suspend
treatment. Such patients are monitored at twice-weekly intervals
until the serum calcium or phosphorus is normalized, and resume
1,25-dihydroxyvitamin D.sub.2 dosing.
[0168] During the eight-week washout period, the mean serum level
of PTH increases progressively and significantly. After initiation
of 1,25-dihydroxyvitamin D.sub.2 dosing, mean serum PTH decreases
significantly to less than 50% of pretreatment levels. Due to this
drop in serum PTH, some patients need to reduce their dosage of
1,25-dihydroxyvitamin D.sub.2 to prevent excessive suppression of
serum PTH. In such patients, exhibiting excessive suppression of
serum PTH, transient mild hypercalcemia is observed, which is
corrected by appropriate reductions in 1,25-dihydroxyvitamin
D.sub.2 dosages.
[0169] At the end of the first 12-week treatment period, mean serum
PTH is in the desired range of 150 pg/mL to 300 pg/mL and serum
levels of calcium and phosphorus are normal or near normal for end
stage renal disease patients. At the end of the second 12-week
treatment period (during which time 1,25-dihydroxyvitamin D.sub.2
treatment is suspended and replaced by placebo therapy in one
group), mean serum PTH values markedly increase, reaching
pretreatment levels in the group receiving placebo therapy. Mean
serum PTH remained controlled in the active group with serum levels
of calcium and phosphorus remained normal or near normal. This
study can demonstrate that 1,25-dihydroxyvitamin D.sub.2 is
effective in reducing serum PTH levels, and is safer than currently
used therapies.
Example 3
Administration of Calcitriol and 1,25-Dihydroxyvitamin D.sub.2 to
Rats
[0170] Sprague Dawley rats were given diet containing 0.75% adenine
demonstrated previously to induce kidney failure [Levi et al., J.
Amer. Soc. Neph., 17; 107-112]. After 4 weeks, normal diet was
given to all animals. A control group receiving normal diet during
the entire course of the study was used as normal control animals.
After the 4 weeks of adenine diet treatment, animals were dosed
i.v. 3.times./week for 2 or 8 weeks with calcitriol or
1,25-dihydroxyvitamin D.sub.2 at 0.01, 0.05, 0.1, 0.25 and 0.5
mg/kg. Serum, plasma kidney and parathyroid gland were collected
after 2 and 8 weeks. iPTH and FGF23 in plasma and serum,
respectively, were measured using a commercial Elisa kit. Serum
calcium was measured using an ORTHO-CLINICAL VITROS 250 chemistry
system or an o-cresolphthalein complexone-based assay. Serum
phosphorus was measured using an ORTHO-CLINICAL VITROS 250
chemistry system or an ammonium molybdate-based assay. Fibronectin
1 was measured by real-time PCR from cDNA isolated from kidney.
[0171] FIG. 1 shows measured iPTH levels in adenine treated animals
after two weeks of dosing with either 1,25-dihydroxyvitamin
D.sub.2, 1,25-dihydroxyvitamin D.sub.3, or vehicle. For reference,
PTH values are also shown for animals in the study fed a normal
diet (without adenine). Both 1,25-dihydroxyvitamin D.sub.2 and
1,25-dihydroxyvitamin D.sub.3 in a dose dependant manner can
suppress serum PTH levels. The levels of suppression for the doses
shown are not significantly different between the two compounds.
These results suggest that these compounds are essentially
equivalent with respect to their efficacy in inhibiting the
expression of PTH.
[0172] FIG. 2 shows measured serum calcium levels for
calcitriol-treated animals, and FIG. 3 shows measured serum calcium
levels for 1,25-dihydroxyvitamin D.sub.2-treated animals.
Calcitriol-treated animals showed a significant elevation of
calcium at doses greater than 0.10 .mu.g/kg, whereas
1,25-dihydroxyvitamin D.sub.2-treated animals showed significance
at doses greater than 0.05 .mu.g/kg. A calcitriol dose of 0.10
.mu.g/kg corresponded to about 13.6% PTH inhibition, whereas a
1,25-dihydroxyvitamin D.sub.2 dose of 0.05 .mu.g/kg corresponded to
about 82.7% PTH inhibition. These findings indicate that
1,25-dihydroxyvitamin D.sub.2 appears to be less likely to cause
calcemia than 1,25-dihydroxyvitamin D.sub.3 at doses that have
equivalent efficacy.
[0173] FIG. 4 shows measured serum phosphorous levels for
calcitriol-treated animals, and FIG. 5 shows measured serum
phosphorous levels for 1,25-dihydroxyvitamin D.sub.2-treated
animals. Calcitriol-treated animals showed a significant elevation
of phosphorous at the two highest doses, whereas
1,25-dihydroxyvitamin D.sub.2-treated animals showed significance
only at the highest dose.
[0174] FIG. 6 shows survival data for calcitriol-treated animals,
and FIG. 7 shows survival data for 1,25-dihydroxyvitamin
D.sub.2-treated animals in a prospective study.
[0175] FIG. 8 shows relative renal FN1 induction after 12 days of
treatment. The extent of suppression of renal FN1 expression
observed at 0.5 mg/kg was significantly greater for
1,25-dihydroxyvitamin D.sub.2-treated animals compared to
calcitriol-treated animals. No difference in suppression between
the two compounds was observed at 0.1 mg/kg.
Example 4
Stability of Compounds in Human Intestine
[0176] Equal parts of 0.1M phosphate buffer (pH 7.4), NADPH, G-6-P,
and G-6-P dehydrogenase were mixed to create a NADPH-generating
system, and the final concentration of NADPH, G-6-P, and G-6-P
dehydrogenase were 0.8 mM, 8 mM and 0.8 U/ml.
[0177] 1,25-dihydroxyvitamin D.sub.2 and calcitriol were added in
to the NADPH-generating system. The final concentrations of the two
vitamin D compounds were each 3.3 mM. A reaction was initiated by
the addition of the human intestine microsomes (22 mg/ml) after a 5
min pre-incubation of the reaction at 37.degree. C. After
incubation for 60 min, the reaction was terminated by addition of
cold 100% acetonitrile. An aliquot of the samples was injected into
HPLC to determine the % remaining of the compound.
[0178] Results are shown in FIG. 9, and demonstrate that
1,25-dihydroxyvitamin D.sub.2 is metabolically unstable in human
intestinal epithelia. Selective instability in intestine is an
advantage in vitamin D therapy and may be a factor in accounting
for reduced toxicity of 1,25-dihydroxyvitamin D.sub.2.
Example 5
Alkaline Phosphatase and CYP24 Activity in C2BBe1 Cells
[0179] C2BBe1 cells were treated with 1 mM, 100 nM, and 10 nM
1,25-dihydroxyvitamin D.sub.2 and calcitriol. Cells were incubated
for 8 hours at 37.degree. C. The cells were then lysed in 1 ml of
TRIzol reagent. RNA was isolated from cell lysates through phase
separation, as per the manufacturer's instructions (INVITROGEN).
After cDNA synthesis, real-time PCR was used to quantify alkaline
phosphatase and CYP24.
[0180] Measured alkaline phosphatase and CYP24 activity are shown
in FIG. 10 and FIG. 11, respectively, and demonstrate that
1,25-dihydroxyvitamin D.sub.2 is not a potent inducer of IAP
activity. Furthermore, the similarity between IAP and CYP24
responses suggests metabolic differences in these cells.
Example 6
Intestinal Alkaline Phosphatase IAP Induction in Caco-2 Cells
[0181] FIG. 12 shows the intestinal alkaline phosphatase IAP
activity in Caco-2 cells (change in mRNA optical density per change
in unit time) following treatment with calcitriol (top line),
1,25-dihydroxyvitamin D.sub.2 (next set of data and line down),
19-nor, 1,25-dihydroxyvitamin D.sub.2 (paricalcitol, next set of
data and line down), and an active vitamin D hormone analog which
is disclosed as Formula IX (Compound 1) in U.S. Pat. No. 6,380,408
(col. 6), which is
(5Z,7E,16Z,23E)-(1S,3R)-25-nor-25-t-butylsulfonyl-9,10-seco-5,7,10(19),16-
,23-cholestapentaene-1,3-diol (bottom set of data and line in the
Figure). The data show that 1,25-dihydroxyvitamin D.sub.2 has a
similar effect on IAP compared to 19-nor, 1,25-dihydroxyvitamin
D.sub.2 and another vitamin D analog.
[0182] The foregoing description is given for clearness of
understanding only, and no unnecessary limitations should be
understood therefrom, as modifications within the scope of the
invention may be apparent to those having ordinary skill in the
art.
[0183] All patents, publications and references cited herein are
hereby fully incorporated by reference. In case of conflict between
the present disclosure and incorporated patents, publications and
references, the present disclosure should control.
[0184] Throughout the specification, where compositions are
described as including components or materials, it is contemplated
that the compositions can also consist essentially of, or consist
of, any combination of the recited components or materials, unless
described otherwise.
[0185] The practice of a method disclosed herein, and individual
steps thereof, can be performed manually and/or with the aid of
mechanical and/or electronic equipment. Although processes have
been described with reference to particular embodiments, a person
of ordinary skill in the art will readily appreciate that other
ways of performing the acts associated with the methods may be
used. For example, the order of various of the steps may be changed
without departing from the scope or spirit of the method, unless
described otherwise. In addition, it will be recognized that some
of the individual steps may be combined, omitted, or further
subdivided into additional steps.
[0186] Embodiments contemplated in view of the foregoing
description include the following numbered paragraphs.
[0187] 1. A method for safely lowering or maintaining lowered serum
intact parathyroid hormone in a human patient, comprising
administering to the patient an effective amount of
1,25-dihydroxyvitamin D.sub.2 to lower or maintain lowered serum
parathyroid hormone levels.
[0188] 2. The method of paragraph 1, further comprising
concurrently [0189] (a) increasing and/or maintaining serum calcium
levels in the patient; [0190] (b) maintaining serum phosphorous
levels in the patient; [0191] (c) increasing serum
1,25-dihydroxyvitamin D levels in the patient; and [0192] (d)
maintaining serum 1,25-dihydroxyvitamin D levels in the patient, by
said administration of 1,25-dihydroxyvitamin D2.
[0193] 3. The method according to paragraph 2, comprising
increasing and/or maintaining serum calcium levels in the patient
in a range of about 8.6 to 10.2 mg/dL by said administration of
1,25-dihydroxyvitamin D.sub.2.
[0194] 4. The method according to paragraph 2, comprising
maintaining phosphorous levels in the patient in a range of 2.5 to
4.5 mg/dL by said administration of 1,25-dihydroxyvitamin
D.sub.2.
[0195] 5. The method according to paragraph 2, comprising
increasing serum 1,25-dihydroxyvitamin D levels in the patient to
the patient's normal historical physiological range by said
administration of 1,25-dihydroxyvitamin D.sub.2.
[0196] 6. The method according to paragraph 2, comprising
maintaining serum 1,25-dihydroxyvitamin D levels in the patient's
normal historical physiological range for at least 30 days by said
administration of 1,25-dihydroxyvitamin D.sub.2.
[0197] 7. The method according to paragraph 1, wherein said
effective amount is sufficient to lower serum intact parathyroid
hormone levels by at least 15%.
[0198] 8. A method of safely increasing or maintaining blood
concentrations of 1,25-dihydroxyvitamin D in a human patient by
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to the
patient.
[0199] 9. A method of alleviating one or more symptoms of
1,25-dihydroxyvitamin D deficiency in a human patient, comprising
administering an amount of 1,25-dihydroxyvitamin D.sub.2 to the
patient such that one or more symptoms of 1,25-dihydroxyvitamin D
deficiency are alleviated.
[0200] 10. The method of paragraph 9, wherein said one or more
symptoms of 1,25-dihydroxyvitamin D deficiency comprise symptoms of
deficiency in the non-classical effects of vitamin D.
[0201] 11. The method of paragraph 10, wherein said administration
of 1,25-dihydroxyvitamin D.sub.2 to the patient provides negative
endocrine regulation of the renin-angiotensin system in the
patient.
[0202] 12. The method according to paragraph 1, wherein the patient
has been diagnosed with chronic kidney disease (CKD).
[0203] 13. The method according to paragraph 12, wherein the
patient has been diagnosed with hyperparathyroidism secondary to
chronic kidney disease (SHPT).
[0204] 14. The method according to paragraph 12, wherein said CKD
is Stage 1 or Stage 2 CKD.
[0205] 15. The method according to paragraph 12, wherein said CKD
is Stage 3, Stage 4, or Stage 5 CKD.
[0206] 16. The method according to paragraph 15, wherein the amount
of 1,25-dihydroxyvitamin D.sub.2 administered is sufficient to
reduce serum levels of PTH to 35-70 pg/mL for a Stage 3 CKD
patient, to 70-110 pg/mL for a Stage 4 CKD patient, and to 150-300
pg/mL for a Stage 5 CKD patient.
[0207] 17. The method according to paragraph 1, wherein the amount
1,25-dihydroxyvitamin D.sub.2 administered is in a range of 0.1
.mu.g per week to about 100 .mu.g per week.
[0208] 18. The method according to paragraph 1, comprising
administering 1,25-dihydroxyvitamin D.sub.2 for at least 30
days.
[0209] 19. The method according to paragraph 18, comprising
administering 1,25-dihydroxyvitamin D.sub.2 for at least 2
months.
[0210] 20. The method according to paragraph 18, comprising
administering 1,25-dihydroxyvitamin D.sub.2 for at least 3
months.
[0211] 21. The method according to paragraph 18, comprising
administering 1,25-dihydroxyvitamin D.sub.2 for at least 4
months.
[0212] 22. The method according to paragraph 18, comprising
administering 1,25-dihydroxyvitamin D.sub.2 for at least 5
months.
[0213] 23. The method according to paragraph 18, comprising
administering 1,25-dihydroxyvitamin D.sub.2 for at least 6
months.
[0214] 24. The method according to paragraph 18, further comprising
avoiding progressive loss of 25-hydroxyvitamin D and
1,25-dihydroxyvitamin D.sub.3 in the patient.
[0215] 25. A pharmaceutical composition having serum (or plasma)
intact parathyroid hormone lowering activity, which includes an
effective amount of 1,25-dihydroxyvitamin D.sub.2 and at least one
pharmaceutically acceptable excipient.
[0216] 26. The composition of paragraph 25, in unit dosage
form.
[0217] 27. The composition of paragraph 25, wherein said at least
one pharmaceutically acceptable excipient provides for controlled
release of 1,25-dihydroxyvitamin D.sub.2.
[0218] 28. The composition of paragraph 27, wherein said at least
one pharmaceutically acceptable excipient provides for
substantially constant release of 1,25-dihydroxyvitamin
D.sub.2.
[0219] 29. The composition of paragraph 27, wherein said at least
one pharmaceutically acceptable excipient provides delayed release
of 1,25-dihydroxyvitamin D.sub.2.
[0220] 30. The composition of paragraph 29, wherein said at least
one pharmaceutically acceptable excipient provides delayed release
of 1,25-dihydroxyvitamin D.sub.2 to the small intestine of the
patient.
[0221] 31. The composition of paragraph 29, wherein said at least
one pharmaceutically acceptable excipient provides delayed release
of 1,25-dihydroxyvitamin D.sub.2 to the ileum of the patient.
[0222] 32. The composition of paragraph 26, comprising an oral
formulation.
[0223] 33. The composition of paragraph 25, comprising an
intravenous formulation.
[0224] 34. The composition of paragraph 26, wherein the unit dosage
form comprises about 0.1 .mu.g to about 10 .mu.g of
1,25-dihydroxyvitamin D.sub.2.
[0225] 35. The composition of paragraph 34, wherein the unit dosage
form comprises about 1 .mu.g to about 4 .mu.g of
1,25-dihydroxyvitamin D.sub.2.
[0226] 36. The composition of paragraph 34, comprising about 2
.mu.g to about 10 .mu.g of 1,25-dihydroxyvitamin D.sub.2.
[0227] 37. The composition of paragraph 36, comprising about 3
.mu.g to about 5 .mu.g of 1,25-dihydroxyvitamin D.sub.2.
* * * * *