Combination of cholecalciferol derivatives for the treatment of renal bone disease

A method and composition for an improved treatment of human renal osteodystrophy, comprising the administration of 6-40 mcg/d (micrograms per day) of 24,25(OH).sub.2 D.sub.3 in combination with amounts of 1-alpha(OH)D.sub. 3 or 1,25(OH).sub.2 D.sub.3 or DHT.sub.2, which will maintain in the serum of patients the calcium concentration at 10-11 mg/dl (milligrams per deciliter) and the phosphate concentration at 5.5-6.0 mg/dl (milligrams per deciliter) and the numerical multiplication product of calcium x phosphate not more than 60-65.

The invention relates to an improved method and pharmaceutical composition 
for the treatment of human bone diseases. More particularly the invention 
provides a method and composition for the treatment of renal 
osteodystrophy diseases in humans comprising the administration to a 
patient of 6-40 micrograms per day of 24,25 dihydroxycholecalciferol 
(hereafter "24,25(OH).sub.2 D.sub.3 ") in combination with 
1-alpha-hydroxycholecalciferol (hereafter "1-alpha(OH)D.sub.3 ") or 
1,25-dihydroxycholecalciferol (hereafter "1,25(OH).sub.2 D.sub.3 ") or 
dihydrotachysterol (hereafter "DHT.sub.2 ") in an amount per day which 
will maintain the patient's calcium serum concentration at 10-11 mg/dl and 
the phosphate concentration at 5.5-6.0 mg/dl. 
As used herein, "d" means "day" and "dl" means "deciliter". Also "mcg" 
means "micrograms" and "mg" means "milligrams". 
BACKGROUND OF THE INVENTION 
Cholecalciferol (vitamin D.sub.3) has been known to be intimately 
associated with bone metabolism and effectively used for the cure of 
rickets and osteomalacia since the early 1920s. Studies carried out during 
the last decade have shown that vitamin D.sub.3 must be metabolically 
activated before functioning biochemically in its target tissues, 
including the intestine, bone and kidney. The dihydroxylated metabolite of 
vitamin D.sub.3, namely 1,25(OH).sub.2 D.sub.3, was believed to be 
responsible for all the known biological functions of the vitamin. This 
compound is produced from cholecalciferol by C-25 hydroxylation in the 
liver followed by C-1 hydroxylation in the kidney. It was thus 
understandable why treatment with cholecalciferol was found to be 
ineffective in metabolic bone diseases, such as renal osteodystrophy, in 
patients who, owing to chronic kidney failure, are incapable of converting 
the cholecalciferol prohormone into its aforesaid dihydroxylated 
metabolite hormone. 
The use of 1,25(OH).sub.2 D.sub.3 is now established in the treatment of 
renal bone diseases. Its administration increases calcium absorption from 
the gut and consequently, plasma calcium, and suppresses secondary 
hyperparathyroidism and its skeletal consequences. It also ameliorates 
osteomalacia in the presence of secondary hyperparathyroidism. The 
putative roles of 1,25(OH).sub.2 D.sub.3 in the bone are controversial and 
many of its actions can be accounted for by its effect to increase the 
ionized fraction of plasma calcium. This therapeutic effect is also the 
major cause of vitamin D toxicity, namely hypercalcemia. Its use is 
therefore contraindicated and indeed of limited value in patients with 
pre-existing hypercalcemia due to aluminum toxicity or tertiary 
hyperparathyroidism. 
The failure of 1,25(OH).sub.2 D.sub.3 to control secondary 
hyperparathyroidism in many patients on dialysis stimulated the continuing 
search for more effective therapeutic means. Intravenous administration of 
1,25(OH).sub.2 D.sub.3 has been recently reported to be more effective 
than the same drug administered by the oral route in suppressing secondary 
hyperparathyroidism (Slatopolsky et al., J. Clin. Invest 74, 2136, 1984). 
However, it has not solved the problem of secondary hyperparathyroidism in 
dialysis patients. 
1-alpha(OH)D.sub.3 is a synthetic analog of 1,25(OH).sub.2 D.sub.3. It is 
converted into the latter in the liver by 25-hydroxylation. 
1-alpha(OH)D.sub.3 is now also in clinical use for renal osteodystrophy, 
and its therapeutic effect is equivalent to that of 1,25(OH).sub.2 D.sub.3 
apart from small differences in its biological half-life and dose 
response. 
Although the treatment of chronic renal failure (CRF) with either 
1,25(OH).sub.2 D.sub.3 or 1-alpha(OH)D.sub.3 proved to be effective in 
maintaining normal concentrations of calcium and phosphate in the plasma, 
the beneficial results on the mineralization of bone matrix were highly 
incomplete and unsatisfactory. 
There has been recent interest in other metabolites of vitamin D.sub.3, 
notably 25(OH)D.sub.3 and 24,25(OH).sub.2 D.sub.3 which may have different 
actions on target tissue than 1,25(OH).sub.2 D.sub.3 and 
1-alpha(OH)D.sub.3. 
Until recently it was still controversial whether 24,25(OH).sub.2 D.sub.3 
plays any physiological role in man and in animals, and whether it 
possesses a defined biological activity. In early observations this 
metabolite was considered to be only a by-product of renal 25(OH)D.sub.3 
metabolism. The uncertainty surrounding the physiological importance of 
24,25(OH).sub.2 D.sub.3 stems partially from observations in animals. 
In animal experiments, the effect of 24,25(OH).sub.2 D.sub.3 on stimulation 
of intestinal calcium absorption was contingent upon prior renal 
hydroxylation to 1,24,25(OH).sub.3 D.sub.3. Thus in nephrectomized animals 
24,25(OH).sub.2 D.sub.3 failed to stimulate intestinal transport. 
Conversely in man where 24,25(OH).sub.2 D.sub.3 has been shown to be 
active in calcium metabolism 1,24,25(OH).sub.3 D.sub.3 was found to exert 
only a minimal effect. (Boyle et al., J. Biol. Chem. 248, 4174, 1973). 
The increased calcium retention of 24,25(OH).sub.2 D.sub.3 is not 
associated with hypercalcemia, suggesting that 24,25(OH).sub.2 D.sub.3 may 
be directly or indirectly promoting calcium uptake in compartments other 
than the extracellular fluid. There is some evidence that skeletal 
retention of calcium is augmented. A number of experimental observations 
in animals suggest that both 1,25(OH).sub.2 D.sub.3 and 24,25(OH).sub.2 
D.sub.3 are necessary for some aspects of skeletal metabolism. These 
include the normal differentiation of cartilage tissue, the healing of 
fractures and the mineralization of rachitic bone and cartilage and the 
prevention of parathyroid gland hyperplasia. 
RENAL OSTEODYSTROPHY 
Renal bone disease is a common complication in patients undergoing chronic 
dialysis. 
Osteitis fibrosa cystica and osteomalacia are the two most common forms of 
uremic osteodystrophy. The typical histological features of osteitis 
fibrosa cystica are osteoclastic bone resorption forming cystic defects, 
endosteal and marrow space fibrosis with aggregates of osteoclasts and 
brown tumors. In addition there are morphological indications of rapid 
bone turnover reflected by increased active osteoid surface, increased 
formation of woven bone and enhanced tetracycline uptake. The 
characteristic roentgenographic features are subperiosteal bone 
resorption, cystic defects, frequently associated with osteosclerosis and 
extraskeletal calcifications. Secondary hyperparathyroidism that is 
responsible for osteitis fibrosa cystica, emerges early in the course of 
chronic renal disease. (Popovtzer et al., Clin. Sci. 38 297, 1970). 
In chronic renal disease, loss of filtering units imposes increasing loads 
of phosphate on the remaining nephrons. The increased burden of phosphate 
may have several secondary consequences. The high phosphate in serum level 
causes a reciprocal fall in serum ionized calcium and the latter triggers 
secretion of parathyroid hormone (PTH). PTH restores both the serum 
phosphate and calcium to normal through its phosphaturic and calcemic 
actions. Subsequent reductions in nephron population will evoke a further 
rise in PTH leading to secondary hyperparathyroidism as an end-result 
(Popovtzer Inter. J. Artif. Org., 3, 1, 1980). 
Reduced 1,25(OH).sub.2 D.sub.3 activity, as demonstrated to be present in 
early renal disease, may cause secondary hyperparathyroidism by several 
mechanisms, (a) reduced intestinal absorption of calcium, (b) diminished 
skeletal calcemic response to PTH, and (c) loss of physiological 
suppression of PTH secretion. 
Patients with renal bone diseases are often divided into two subgroups, an 
active bone disease subgroup and a non-active bone disease subgroup. This 
is done in accordance with the severity of the bone disease, particularly 
with regard to osteoclastic activity and bone forming activity, reflected 
by active osteoid and tetracycline uptake levels. 
Often, in active bone disease, the osteoclast number equals or exceeds 0.3 
mm.sup.-1 and the active bone resorption equals or exceeds 2.0%. Patients 
who do not meet these two criteria are included in the non-active bone 
disease subgroup. 
DESCRIPTION OF PRIOR ART 
Ornoy et al (Nature, 276, 517, 1978) demonstrated that the combined 
administration of 1-alpha(OH)D.sub.3 with 24,25(OH).sub.2 D.sub.3 produced 
a better cure of mineralization defect than 1-alpha(OH)D.sub.3 alone in 
vitamin D deficient chicks. Galus et al (Calcif. Tissue Int., 31, 209, 
1980) have shown that the administration of 24,25(OH).sub.2 D.sub.3 in 
vitamin D-deficient dogs increases bone formation and mineralization and 
decreased bone resorption, while 1-alpha(OH)D.sub.3 increased both 
mineralization and resorption. Pavlovitch et al (J. Clin. Invest., 68, 
803, 1981) demonstrated an inhibition of increased bone resorption after 
acute bilateral nephrectomy by 24,25(OH).sub.2 D.sub.3 in rats. In 
addition 24,25(OH).sub.2 D.sub.3 blocked partially the stimulating effect 
of 1,25(OH).sub.2 D.sub.3 on bone resorption. Rubinger et al (Proc. 6th 
Workshop Vit. D, Italy, 1985, p. 96) demonstrated that 24,25(OH).sub.2 
D.sub.3 suppressed the hypercalcemic effect of 1,25(OH).sub.2 D.sub.3 in 
rats with chronic renal failure. 
Tests in Human Patients 
Kanis et al. (Brit. Med. J. 1382, 1978) suggested that 24,25(OH).sub.2 
D.sub.3 may be an important regulator of skeletal metabolism in man with 
potential value as a therapeutic agent in patients with chronic renal 
failure and osteoporosis. 
Weisman et al (Brit. Med. J. 281, 712, 1980) indicated generally that 
treatment with both 1,25(OH).sub.2 D.sub.3 and 24,25(OH).sub.2 D.sub.3 may 
be needed to prevent and heal renal osteodystrophy. 
In Japanese Patent Application No. 55 139-320, published on Oct. 31, 1980, 
combinations of 24,25(OH).sub.2 D.sub.3 with either 1,25(OH).sub.2 D.sub.3 
or 1-alpha(OH)D.sub.3 are claimed for the treatment of numerous bone and 
calcium metabolic diseases, amongst them also renal bone dystrophia. 
However, this patent application is very general and vague and fails to 
disclose how to use these combinations in any of the diseases mentioned, 
including renal osteodystrophy. 
Dunstan et al. (Mineral Electrolyte Metab. 11, 358, 1985) concluded that 
"1,25(OH).sub.2 D.sub.3 is a useful agent in the treatment of renal bone 
diseases but no therapeutic value is consecture for 24,25(OH).sub.2 
D.sub.3 ". 
Thus, the above publication teaches away from a combination of 
24,25(OH).sub.2 D.sub.3 with 1,25(OH).sub.2 D.sub.3 and consequently also 
with 1-alpha(OH)D.sub.3 which is biochemically and therapeutically 
equivalent to 1,25(OH).sub.2 D.sub.3. 
SUMMARY OF INVENTION 
The invention provides a novel method and pharmaceutical composition for 
the improved treatment of human renal osteodystrophy patients undergoing 
hemodialysis. 
According to one aspect of the invention, the new method comprises the 
administration to such a patient 6-40 mcg/d of 24,25(OH).sub.2 D.sub.3 in 
combination with daily amounts of 1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 
D.sub.3 or DHT.sub.2 which will suffice to maintain normal calcium levels, 
namely at 10-11 mg/dl, and the phosphate concentration at 5.5-6.0 mg/dl, 
and that the numerical value of the multiplication of the calcium and the 
phosphate concentrations in the serum should be about 60-65. 
The ingredients in the combination may be administered separately, all of 
them orally by means of capsules or, if preferred, 1-alpha(OH)D.sub.3 and 
1,25(OH).sub.2 D.sub.3 parenterally. 
The preferred method of administration is orally by means of capsules 
containing 5 mcg of 24,25(OH).sub.2 D.sub.3 and various amounts of 
1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 D.sub.3 such as 0.25 mcg, 0.5 mcg, 
0.75 mcg and 1.0 mcg per capsule. 
The new method of treatment of chronic renal failure (CRF) patients by 
means of the specific combinations of 24,25(OH).sub.2 D.sub.3 with 
1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 D.sub.3 in the relative amounts of 
each components as stated above offers significant improvements in the 
clinical symptoms, in the various bone histological parameters and blood 
biochemistry of these patients. 
Another aspect of the invention is the use of the novel combination of 
24,25(OH).sub.2 D.sub.3 with 1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 D.sub.3 
in the stated relative proportions for CRF patients simultaneously with 
other drugs which may be used in their treatment. In addition 
dihydrotachysterol (DHT.sub.2), which has been shown to be effective in 
combination with 24,25(OH).sub.2 D.sub.3 in the treatment of renal 
osteodystrophy in adults and children can be used in combination with 
24,25(OH).sub.2 D.sub.3 (van Diemen-Steenvoorde, et al., Clin. Nephrol., 
24, 292, 1985).

DETAILED DESCRIPTION OF INVENTION 
The clinical treatment at present in practice for patients with renal bone 
diseases, including those undergoing hemodialysis, comprises the 
administration of either 1,25(OH).sub.2 D.sub.3 or 1-alpha(OH)D.sub.3 or 
DHT.sub.2 orally or parenterally. 
Although these cholecalciferol analogs proved to be effective in 
maintaining normal concentrations of calcium and phosphate in the plasma 
of the patients, they showed little beneficial results on the bone 
mineralization of the bone matrix. The treatment consequently failed to 
improve clinical bone symptoms and bone histological parameters of the 
patients suffering from osteitis fibrosa cystica resulting from secondary 
hyperparathyroidism and from osteomalacia. Although some Prior Art 
publications indicated that both 1,25(OH).sub.2 D.sub.3 and 
24,25(OH).sub.2 D.sub.3 may be necessary for skeletal metabolism and bone 
formation and mineralization, and other publications suggested that the 
combined treatment of the above two cholecalciferol metabolites in renal 
osteodystrophy may have some therapeutic advantages, there was no 
disclosure in the Prior Art of how to perform or use such a combination 
which will result in any advantageous therapeutic effects. 
By virtue of the present invention there was discovered a specific method 
and pharmaceutical combination of 24,25(OH).sub.2 D.sub.3 with either 
1-alpha(OH)D.sub.3, 1,25(OH).sub.2 D.sub.3 or DHT.sub.2 which will offer 
considerable therapeutic advantages to renal osteodystrophy patients (also 
called chronic renal failure - CRF - patients). 
Studies have been provided where the patients were randomly allocated into 
two groups. One group of patients was treated with 1-alpha(OH)D.sub.3 
alone or 1,25(OH).sub.2 D.sub.3 alone (hereafter the "control group") in 
daily dosages to maintain normal serum concentrations of calcium and 
phosphate. To the second group of patients (hereafter the "treatment 
group") was administered 24,25(OH).sub.2 D.sub.3 in daily dosages ranging 
from 10 mcg to 40 mcg in combination with 1-alpha(OH)D.sub.3 or 
1,25(OH).sub.2 D.sub.3 in daily amounts which would maintain the patients 
calcium (Ca) serum concentrations at 10-11 mg/dl and phosphate (P) serum 
concentration at 5.5-6.0 mg/dl, and the Ca.times.P numerical product 
should not exceed 60-65. 
The daily dosages of 1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 D.sub.3 in both 
the "treatment group" and in the "control group" were adjusted for every 
patient individually, in order to maintain the serum concentrations of 
calcium and phosphate at the normal range levels as stated above. 
The actual daily doses of 1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 D.sub.3 
administered to the patients ranged from 0.25 mcg to 3.0 mcg. 
The controlled studies have surprisingly shown that significant therapeutic 
improvements were achieved in the treatment group in comparison with the 
control group. Improvements were observed in both aspects of the renal 
bone disease, in the bone histological parameters as well as in the relief 
of the bone clinical symptoms. 
Bone Histomorphometry 
Two bone biopsies were obtained from the iliac crest, one at the outset of 
the study and the other upon its completion. The histomorphometry of the 
biopsies showed the following favorable results: 
The trabecular bone volume in the treatment group was considerably lower 
than in the control group. 
The active osteoid surface was significantly reduced in the treatment group 
compared to the control group. This was shown in the second biopsy, 
especially in patients with relatively high active osteoid surface levels 
in the first biopsy. 
In the resorption surface, a statistically significant decrease was 
observed in the treatment group, whereas no change could be detected in 
the control group. 
The improvements in the bone resorption were even more pronounced in the 
active bone disease subgroup, which was defined as having an osteoclast 
number equal to or exceeding 0.3 mm.sup.-1 and active bone resorption 
equal to or exceeding 2.0%. All patients with active bone disease in the 
treatment group showed a significant decrease in bone resorption, whereas 
in the control group there was no change or even an increase in bone 
resorption. 
Clinical symptoms 
With respect to clinical manifestations before and after treatment in the 
active bone disease subgroup, such as bone pains and tenderness, muscle 
pains and pruritus, there was a significant improvement in the clinical 
symptoms in the treatment group. In the control group, the clinical 
symptoms remained unchanged, or even worsened. 
Untoward Side Effects 
Careful observation in all studies have not revealed untoward side effects 
in the treatment group that could be attributed to 24,25(OH).sub.2 D.sub.3 
given orally at a daily dose of either 10 mcg or 40 mcg over the whole 
period of the studies. Thus 24,25(OH).sub.2 D.sub.3 can be considered a 
"very safe drug" when administered at the abovementioned daily dosage. In 
addition, it was found that 24,25(OH).sub.2 D.sub.3 administered in 
combination with 1-alpha-hydroxylated D.sub.3 derivatives increased the 
patient's tolerance of these derivatives and reduced the risk of 
hypercalcemia. 
The best mode of carrying out the invention 
The clinical trials and studies of the present invention indicate that the 
best mode of carrying out the invention is as follows: 
The treatment of patients with renal osteodystrophy, including those 
undergoing chronic hemodialysis, should comprise the administration to 
each patient of 10 mcg per day of 24,25(OH).sub.2 D.sub.3 in combination 
with either 1-alpha(OH)D.sub.3, 1,25(OH).sub.2 D.sub.3 or DHT.sub.2 in 
daily doses which will maintain the patient's calcium concentration at the 
upper normal levels of 10 to 11 mg/dl and the phosphate serum 
concentration at 5.5-6.0 mg/dl. Another criterion for the daily dosage of 
1-alpha(OH)D.sub.3, 1,25(OH).sub.2 D.sub.3 or DHT.sub.2 should be that the 
numerical value of the multiplication "calcium.times.phosphate" should not 
exceed 60-65 during the treatment. 
With the specific combination of 10 mcg per day of 24,25(OH).sub.2 D.sub.3 
and the appropriate amounts of 1-alpha(OH)D.sub.3, 1,25(OH).sub.2 D.sub.3 
or DHT.sub.2, the most favorable therapeutic effects were obtained in the 
treatment of renal osteodystrophy patients considering all the aspects of 
the disease: The blood biochemical and hematological measurements, the 
bone histomorphometry and the clinical manifestations. These favorable 
results are outlined in detail in example 7 (Study I). 
The rationale for the administration of 10 mcg of 24,25(OH).sub.2 D.sub.3 
per day to patients can be based on calculations obtained from the 
physiological and therapeutic experiments conducted in the chick. The 
optimal physiological dose of 24,25(OH).sub.2 D.sub.3 in the chick was 
found to be 0.3 mcg, which is about 3 times that of 1,25(OH).sub.2 D.sub.3 
or 1-alpha(OH)D.sub.3. As the therapeutic daily doses of the latter in the 
human are 20 times the physiological doses, the daily therapeutic dose of 
24,25(OH).sub.2 D.sub.3 in the human should be at least 20.times.0.3 mcg, 
namely 6 mcg. 
The best daily dose of 24,25(OH).sub.2 D.sub.3 in CRF patients was found by 
the present invention to be 10 mcg which is only in slight excess of the 
theoretical dose of 6.0 mcg/d. 
24,25(OH).sub.2 D.sub.3 should best be administered by means of 2 soft 
gelatine capsules per day, containing 5 mcg per capsule, one in the 
morning and one in the evening. 
Even more preferable is the administration by means of soft gelatine 
capsules containing a combination of 5 mcg 24,25(OH).sub.2 D.sub.3 and 
amounts of 0.25 or 1.0 mcg of 1-alpha(OH).sub.2 D.sub.3 or 1,25(OH).sub.2 
D.sub.3 in accordance with the needs of each individual patient. 
In the case where 1-alpha(OH)D.sub.3 or 1,25(OH).sub.2 D.sub.3 is 
administered separately from 24,25(OH).sub.2 D.sub.3 it can be done orally 
by means of soft gelatine capsules or tablets containing 0.25 mcg or 1.0 
mcg of the active ingredient. Alternatively, it can also be administered 
parenterally by means of ampoules containing the desired amounts of the 
active ingredient and stabilized by means of sodium ascorbate and a buffer 
to pH of 6.4 to 7.8 (See U.S. Pat. No. 4,308,264). 
The soft gelatine capsules of all the active ingredients 
(1-alpha(OH)D.sub.3, 1,25(OH).sub.2 D.sub.3 or 24,25(OH).sub.2 D.sub.3) 
may be prepared in arachis oil, propylene glycol, glycerin and other 
suitable solvents together with a stabilizer and a preservative. 
Another embodiment of the invention is the use of the combination of 
24,25(OH).sub.2 D.sub.3 in the range given with dihydrotachysterol 
(DHT.sub.2), which is usually administered orally by means of tablets, 
capsules, or solutions. 
The beneficial effect of the simultaneous administration of 24,25(OH).sub.2 
D.sub.3 and DHT.sub.2 was demonstrated by an uncontrolled study in 
patients with dialysis related osteomalacia. (Hodsman, et al., Am. J. Med. 
74, 407, 1983). A more recent study showed also the benefits of this 
combination in children undergoing chronic dialysis with hyperparathyroid 
bone diseases, resulting in a significant decrease in the number of 
osteoblasts. (van Diemen-Steenvoorde, et al., Clin. Nephrol., 24, 292, 
1985). 
The combination of the invention given simultaneously with DHT.sub.2 is 
expected to give even more favorable results in cases of osteomalacia and 
renal bone disease of secondary hyperparathyroidism. 
Assumed Mode of Action of The Invention 
In vitro studies have demonstrated the effective anabolic action of 
24,25(OH).sub.2 D.sub.3 in the presence of 1,25(OH).sub.2 D.sub.3 and PTH. 
Only combined treatment of 1,25(OH).sub.2 D.sub.3, 24,25(OH).sub.2 D.sub.3 
and PTH will induce a significant increase in bone mineral deposition in 
cultured bones in vitro. Each one of the components operates on a 
different cellular function related to bone formation, and jointly they 
have synergistic effect on bone. 
It is well known from the literature that, while both 1,25(OH).sub.2 
D.sub.3 and 24,25(OH).sub.2 D.sub.3 are similar in stimulating calcium 
absorption at the intestinal mucosal level, they differ in the nature of 
their action on the bone level in renal osteodystrophy patients. 
While 1,25(OH).sub.2 D.sub.3 in physiological doses directly stimulates 
bone resorption, 24,25(OH).sub.2 D.sub.3 exerts an opposite action on the 
bone level. Thus, the advantage of simultaneous administration of both 
metabolites in the proportions of the invention to renal osteodystrophy 
patients is in the ability of 24,25(OH).sub.2 D.sub.3 to offset the 
effects of 1,25(OH).sub.2 D.sub.3 (or its analogs 1-alpha(OH)D.sub.3 and 
DHT.sub.2) with regard to the resorbing action on the bone, but on the 
other hand a synergistic action with respect to gut absorption, bone 
mineralization, and less likely, parathyroid hormone (PTH) suppression. 
The following examples provide an illustration of the invention, but should 
not be construed as to limit the scope of the invention. 
EXAMPLE 1 
Soft Gelatine Capsules of 1-alpha(OH)D.sub.3 
Each 0.25 mcg capsule contains: 
______________________________________ 
1-alpha-HYDROXYCHOLECALCIFEROL 
0.275 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 
98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 2 
Soft Gelatine Capsules of 1.0 mcg 1-alpha(OH)D.sub.3 
Each 1.0 mcg capsule contains: 
______________________________________ 
1-alpha-HYDROXYCHOLECALCIFEROL 
1.100 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 
98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 3 
Soft Gelatine Capsules of 0.25 mcg 1,25(OH).sub.2 D.sub.3 
Each 0.25 mcg capsule contains: 
______________________________________ 
1,25-HYDROXYCHOLECALCIFEROL 
0.275 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 4 
Soft Gelatine Capsules of 1.0 mcg of 1,25(OH).sub.2 D.sub.3 
Each 1.0 mcg capsule contains: 
______________________________________ 
1,25-DIHYDROXYCHOLECALCIFEROL 
1.100 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 5 
Soft Gelatine Capsules of 5.0 mcg of 24,25(OH).sub.2 D.sub.3 
Each 5.0 mcg capsule contains: 
______________________________________ 
24R,25-DIHYDROXYCHOLECALCIFEROL 
5.500 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 6 
Soft Gelatine Capsules of 10.0 mcg of 24,25(OH).sub.2 D.sub.3 
Each 10.0 mcg capsule contains: 
______________________________________ 
24R,25-DIHYDROXYCHOLECALCIFEROL 
11.000 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 7 
Soft Gelatine Capsules containing combination of 0.25 mcg or 1.0 mcg of 
1-alpha(OH)D.sub.3 plus 5 mcg of 24,25(OH).sub.2 D.sub.3 
Each 1.0 mcg capsule contains: 
______________________________________ 
1-alpha-HYDROXYCHOLECALCIFEROL 
1.100 mcg* 
24R,25-DIHYDROXYCHOLECALCIFEROL 
5.500 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 8 
Soft Gelatine Capsules containing combination of 0.25 mcg or 1.0 mcg of 
1,25(OH).sub.2 D.sub.3 plus 5 mcg of 24,25(OH).sub.2 D.sub.3 
Each 1.0 mcg capsule contains: 
______________________________________ 
1,25-DIHYDROXYCHOLECALCIFEROL 
1.100 mcg* 
24R,25-DIHYDROXYCHOLECALCIFEROL 
5.500 mcg* 
CITRIC ACID anhydrous 0.015 mg 
PROPYL GALLATE 0.020 mg 
VITAMIN E (dl-alpha TOCOPHEROL) 
0.020 mg 
ALCOHOL dehydrated 1.145 mg 
PEANUT OIL (ARACHIS OIL) 98.800 mg 
Total fill weight per capsule: 
100.0 mg 
______________________________________ 
*Includes 10 percent excess. 
EXAMPLE 9 
Treatment of Chronic Renal Failure (CRF) patients with a combination of 
24,25(OH).sub.2 D.sub.3 and 1-alpha(OH)D.sub.3 (Study I). 
The study included 56 CRF male and female adult patients undergoing chronic 
hemodialysis at several medical centers. The patients were randomly 
allocated into two groups, one group of 31 patients was treated with a 
combination of 24,25(OH).sub.2 D.sub.3 and 1-alpha(OH)D.sub.3 (the 
"treatment group") and the other group of patients was treated with 
1-alpha(OH)D.sub.3 along (the "control group"). 
Patients of both groups were administered 1-alpha(OH)D.sub.3 in capsules of 
0.25 mcg or 1.0 mcg per capsule. The dose was adjusted for every patient 
individually so as to maintain the serum calcium at the upper normal 
level, but not higher than 11 mg/dl, and the phosphate level not higher 
than 6.0 mg/dl or that the numerical multiplication product of 
calcium.times.phosphate did not exceed 65. The actual daily doses of 
1-alpha(OH)D.sub.3 ranged from 0.25 mcg to 3.0 mcg. 
The treatment group was administered in addition to 1-alpha(OH)D.sub.3 also 
24,25(OH).sub.2 D.sub.3 at a daily dose of 10 mcg by means of 2 capsules 
containing 5 mcg per capsule, one in the morning and the other in the 
evening. 
The dose of 10 mcg of 24,25(OH).sub.2 D.sub.3 was constant throughout the 
study which lasted 10-16 months. 
Three major categories of variables were measured and monitored during the 
treatment. These were: (1) blood biochemistry, (2) bone histomorphometry 
(static and dynamic), and (3) clinical follow-up. 
Blood Biochemistry 
As stated before, special attention was given to the blood levels of 
calcium, phosphate and to the multiplication product of 
calcium.times.phosphate. 
The serum calcium concentration was found to be significantly higher in the 
treated group during the period 3-8 months of the study, in comparison 
with the control group though the levels remained within the normal range. 
An increase in the 24,25(OH).sub.2 D.sub.3 serum levels in the treated 
groups was the only significant change between the two groups during the 
whole study. 
Bone histomorphometry 
One bone biopsy was taken from all patients under local anesthesia applied 
to both plates of the iliac crest at the onset of the study, and a second 
biopsy was obtained in a similar fashion from the contralateral side of 
the iliac crest approximately one year later, upon the completion of the 
study. 
The results obtained from the biopsies showed statistically significant 
changes between the control group and the treatment group. These were as 
follows: 
trabecular bone volume. This measurement increased significantly during 
treatment in the control group only. At the end of the study it was much 
lower in the treatment group compared to the control group. 
active osteoid surface. The relatively high active osteoid surface levels 
shown by some patients at the first biopsy were significantly lower at the 
second biopsy only in the treatment group and not in the control group. 
resorption surface. Generally, a statistically significant decrease in the 
resorption surface was shown only in the treatment group. There was no 
significant change in the control group. At the end of the study, the 
resorption surface in the treatment group was significantly lower than 
that in the control group. 
Even more pronounced differences in the histomorphological results were 
shown in those patients in whom the respective parameters exhibited high 
levels at the initial biopsy, namely in patients with a a more severe bone 
disease. 
In view of this observation the patients in the study were subdivided into 
two types of bone disease, an active bone disease subgroup and a 
non-active bone disease subgroup. The division was made according to the 
osteoclast number and the active resorption surface. 
Patients with an osteoclast number equal to or exceeding 2.0% were included 
in the active bone disease subgroup, and those with lower values were 
included in the non-active bone disease subgroup. 
The results of this study show that all CRF patients with active bone 
disease in the treatment group showed a most significant decrease in 
active resorption surface, whereas patients with active bone disease in 
the control group either showed no change or even an increase in this 
parameter. 
However, also in the non-active bone disease subgroup 11 out of 12 patients 
who were treated with the combination showed a significant reduction in 
the active resorption surface; in contrast, the control group showed no 
reduction. 
Clinical follow-up. With respect to clinical manifestations of the disease 
of the treated patients, such as bone pains and tenderness, muscle pains, 
pruritus and others, there was generally a marked improvement only in the 
treatment group. This improvement was especially marked in the treatment 
active bone disease subgroup where most patients became asymptomatic. No 
relief of these clinical symptoms was observed in the control group. 
No untoward side effects could be detected in the patients treated with the 
combination which could be attributed to 24,25(OH).sub.2 D.sub.3 
administered orally at a daily dose of 10 mcg. 
This study thus demonstrates that the administration of 24,25(OH).sub.2 
D.sub.3 to renal osteodystrophy patients in daily doses of 10 mcg in 
combination with 1-alpha(OH)D.sub.3 in doses sufficient to maintain the 
levels of calcium and phosphate in the upper normal range, results in 
marked improvements in the bone parameters as well as in clinical 
manifestations, in comparison with the control group which was treated 
with 1-alpha(OH)D.sub.3 alone in similar doses. 
EXAMPLE 10 
Treatment of CRF patients with a combination of 24,25(OH).sub.2 D.sub.3 and 
1-alpha(OH)D.sub.3 (Study II) 
The study included 62 CRF male and female adult patients undergoing chronic 
hemodialysis and lasted for 10-22 months. As in Study I, the CRF patients 
were randomly allocated into two groups: 34 patients were allocated to the 
combination treatment of 24,25(OH).sub.2 D.sub.3 and 1-alpha(OH)D.sub.3. 
In this study the daily dose of 24,25(OH).sub.2 D.sub.3 was 40 mcg and 
that of 1-alpha(OH)D.sub.3 was sufficient to maintain the concentration of 
calcium and phosphate in the serum at the upper normal level in a manner 
as stated in Study I. 
The patients in the control group received 1-alpha(OH)D.sub.3 alone in 
similar doses as in Study I. 
The blood chemistry results were similar to those obtained in Study I. 
The bone histomorphometric measurements performed in 50 patients (25 in 
each group) showed that the combination treatment resulted in the 
following favorable results: 
(1) a decrease in the trabecular bone volume 
(2) a decrease in the resorption surface 
(3) a slight decrease in the active resorption surface 
(4) a slight decrease in osteoclast number and 
(5) a slight decrease in fibrotic surface. 
These improvements in the bone parameters were not as significant as in 
Study I. The improvements in the clinical symptoms were also not as 
prominent in the treatment group in comparison with the control group as 
was observed in Study I. 
EXAMPLE 11 
Combined treatment of CRF patients with 24,25(OH).sub.2 D.sub.3 and 
1,25(OH).sub.2 D.sub.3 (Study III) 
The study included 62 CRF patients with bone diseases, such as osteitis 
fibrosa cystica and osteomalacia. The patients were randomly allocated 
into three groups: 
(1) The control group A which included 25 patients who were treated with 
24,25(OH).sub.2 D.sub.3 alone at doses of 20 mcg/d. 
(2) The control group B which included 24 patients who were treated with 
1,25(OH).sub.2 D.sub.3 alone at varying daily doses ranging from 0.25 mcg 
to 2.0 mcg so as to maintain the concentration of calcium and phosphate at 
the upper normal range as in Study I. 
(3) The treatment group which included 13 patients. These patients were 
administered a combination of 24,25(OH).sub.2 D.sub.3 at 20 mcg/d and 
1,25(OH).sub.2 D.sub.3 at daily doses similar to those administered to 
patients in control group B. 
The study showed the following results: 
The combined treatment induced a less marked increment in serum calcium 
than that observed in the control group treated with 1,25(OH).sub.2 
D.sub.3 alone. 
However, the decrement in alkaline phosphatase activity in the treatment 
group appeared to be less marked than in patients given 1,25(OH).sub.2 
D.sub.3 alone. 
The following conclusions may be drawn from the study: 
(1) The addition of 24,25(OH).sub.2 D.sub.3 with 1,25(OH).sub.2 D.sub.3 for 
the treatment of renal bone disease does not aggravate hypercalcemia, nor 
does it impair the apparent effectiveness of 1,25(OH).sub.2 D.sub.3 in the 
treatment of hyperparathyroid bone disease. 
(2) The combination of 1,25(OH).sub.2 D.sub.3 and 24,25(OH).sub.2 D.sub.3 
in the treatment of hyperparathyroid bone disease is of value in those 
patients who had previously been shown to be refractory to treatment with 
1,25(OH).sub.2 D.sub.3 alone.