History of vitamin D treatment of renal osteodystrophy.

Vitamin D treatment was tried when renal osteodystrophy was first recognized in the early 20th century, using vitamin D2, D3, or dihydrotachysterol. Large doses of vitamin D2 or D3 (150,000-500,000 IU) were prescribed by monitoring serum calcium, phosphate, and alkaline phosphatase. After the discovery of 1,25-dihydroxycholecalciferol, this compound or 1 alpha-hydroxycholecalciferol was applied to the treatment of renal osteodystrophy. In a preclinical study, especially of 1 alpha-hydroxycholecalciferol, nephritogenoside nephritis was the most responsive condition. These active vitamin D preparations are now widely used in patients with chronic renal failure under hemodialysis. Other active vitamin D compounds, such as hexafluoro-1,25-dihydroxycholecalciferol and 22-oxacalcitriol, are also under investigation.

Molecular cloning and expression of serum calcium-decreasing factor (caldecrin).

We previously reported on the purification of a serum calcium-decreasing factor, referred to as caldecrin, from porcine pancreas, that is thought to be a serine protease (Tomomura, A., Fukushige, T., Noda, T., Noikura, T., and Saheki, T. (1992) FEBS Lett. 301, 277-281). In the present study, we purified caldecrin from rat pancreas and determined its primary structure by cDNA cloning. The predicted caldecrin protein is presumed to be synthesized as a preproenzyme of 268 amino acids with a signal peptide of 16 amino acids and an activation peptide of 13 amino acids, and is, with the exception of a central region, almost identical to the reported rat pancreatic elastase IV sequence. The caldecrin gene is selectively expressed in the pancreas, as judged by Northern blot analysis. After expression in BMT-10 cells, immunoreactive caldecrin was found in the culture supernatant, and it inhibited the parathyroid hormone-stimulated 45Ca release from cultured fetal long bones. Catalytic site mutants were synthesized in a baculovirus system, and recombinant mutants also decreased the serum calcium level of mice. These data implicate caldecrin, a protease closely related to elastase IV, in the regulation of blood calcium levels.

Antitumor effect of 22-oxa-calcitriol, a noncalcemic analogue of calcitriol, in athymic mice implanted with human breast carcinoma and its synergism with tamoxifen.

The antitumor effect of 22-oxa-calcitriol (OCT), a newly developed noncalcemic analogue of calcitriol, was examined in vivo in athymic mice implanted with human breast carcinoma with or without estrogen receptor (ER). In ER-positive MCF-7 tumor, the growth of which was dependent on exogenous estrogen, administration p.o. of OCT as well as the antiestrogen tamoxifen five times a week for 4 weeks suppressed tumor growth in a dose-related fashion. The antitumor effect of 1.0 microgram/kg body weight (BW) OCT (mean +/- SEM of tumor weight in 6 mice: 28 +/- 4% of vehicle-treated group) was comparable to that of 2.0 mg/kg BW tamoxifen (25 +/- 6% of control group). In addition, a synergistic antitumor effect of submaximal doses of OCT and tamoxifen was observed in MCF-7 tumor in vivo as well as in ER-positive breast carcinoma cell lines (MCF-7 and ZR-75-1) in vitro. Administration of OCT p.o. three times a week for 4 weeks also suppressed the growth of ER-negative MX-1 tumor in a dose-dependent manner without raising serum calcium concentrations. The antitumor effect of 1.0 microgram/kg BW OCT (mean +/- SEM of tumor weight in 10 mice: 44 +/- 6% of vehicle-treated group) was greater than that of 500 micrograms/kg BW Adriamycin (71 +/- 6% of control group). These results indicate that OCT suppresses the growth of ER-negative as well as ER-positive breast carcinoma in vivo without causing hypercalcemia and that the antitumor effect of OCT can be enhanced by tamoxifen in an ER-positive tumor. It is suggested that OCT may provide a new strategy, either alone or in combination with other anticancer drugs, for systemic adjuvant therapy of breast carcinoma regardless of ER status.

Differential effects of 1,25-(OH)2D3 and 22-oxacalcitriol on phosphate and calcium metabolism.

1,25-dihydroxyvitamin D3 has been used with success in the treatment of secondary hyperparathyroidism associated with chronic renal failure. However, frequently 1,25-(OH)2D3 induces hypercalcemia, especially in those patients ingesting large doses of calcium carbonate, precluding the administration of therapeutic doses of 1,25-(OH)2D3. In addition, control of serum phosphorus is a persistent problem in patients maintained on chronic hemodialysis and 1,25-(OH)2D3 treatment can aggravate the hyperphosphatemia. Thus, ideally an analog of 1,25-(OH)2D3 that can suppress PTH with minor effects on calcium (Ca) and phosphate (PO4) metabolism would be an ideal tool to control secondary hyperparathyroidism. We have shown that 22-oxa-1,25-(OH)2D3 (OCT), an analog of 1,25-(OH)2D3 with little calcemic activity, can suppress PTH mRNA in normal rats and in cultured bovine parathyroid cells with equipotency to 1,25-(OH)2D3. To further characterize the differential effects of 1,25-(OH)2D3 and OCT on Ca and PO4 metabolism we performed several experiments in intact and parathyroidectomized (PTX) rats. In metabolic studies in four groups of normal rats 1,25-(OH)2D3 treatment (8 ng/day) significantly increased the intestinal Ca absorption from 15.2 +/- 2.68% to 30.5 +/- 2.85% (P < 0.01), while the same dose of OCT had no effect. A dose of 200 ng/day of OCT increased intestinal Ca absorption similarly to the 8 ng/day dose of 1,25-(OH)2D3, from 10.6 +/- 2.49% to 24.8 +/- 2.35% (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of 22-oxacalcitriol on serum calcitriol.

1,25-Dihydroxyvitamin D3 (1,25D) regulates its own levels in circulation by affecting its rates of synthesis and degradation, 22-Oxacalcitriol (OCT), a vitamin D analog with low calcemic activity, decreases circulating PTH levels, one of the regulators of renal 1 alpha-hydroxylase, and stimulates vitamin D degradation in vitro. The purpose of this study was to examine the effects of OCT administration on serum levels of 1,25D. In normal rats, OCT administration (4-200 ng, ip, daily for 5 days) caused a dose-dependent reduction in serum calcitriol levels. At a dose of 200 ng, OCT reduced serum 1,25D from 34.5 +/- 2.7 to 10.9 +/- 0.7 pg/ml (P less than or equal to 0.01) without significant changes in ionized Ca or phosphorus levels. The contribution of the suppression of PTH by OCT to the reduction of serum 1,25D was examined by administering OCT to parathyroidectomized (PTX) rats. Two hundred nanograms of OCT, ip, daily for 5 days significantly reduced serum calcitriol from 29.7 +/- 7.6 to 9.1 +/- 0.5 pg/ml (P less than or equal to 0.01) in rats fed a normal calcium diet. Because OCT increased total calcium (TCa) in this group from 7.4 +/- 0.1 to 9.5 +/- 0.3 mg/dl, similar doses of OCT were given to PTX rats fed a calcium-deficient diet. OCT decreased 1,25D from 58.9 +/- 8.9 to 10.3 +/- 0.4 pg/ml and increased TCa from 4.8 +/- 0.2 to 7.4 +/- 0.1 mg/dl. Comparison of serum 1,25D for identical TCa levels in PTX rats (normal calcium diet controls vs. calcium-deficient diet, OCT-treated) clearly indicates that OCT per se reduced serum 1,25D. Further support for a direct effect of OCT was provided by studies in PTX rats fed a low phosphorus diet. OCT decreased serum 1,25D from 125.8 +/- 15.6 to 10.9 +/- 0.6 pg/ml without significant changes in TCa. To further characterize the mechanisms involved in this effect, similar studies were performed in six normal dogs. Intravenous administration of 0.75 micrograms OCT every other day for 1 week decreased serum calcitriol from 25.4 +/- 3.2 to 12.2 +/- 1.3 pg/ml (P less than or equal to 0.002). Ionized Ca and phosphorus remained unchanged. Despite the short half-life of OCT in the circulation, 1,25D levels returned to basal concentrations 96 h after the last dose of OCT.(ABSTRACT TRUNCATED AT 400 WORDS)

1 alpha,25-Dihydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3 prolong survival time of mice inoculated with myeloid leukemia cells.

Syngeneic SL mice inoculated with murine myeloid leukemia cells (M1) all died of leukemia within 30 days. Treatment three times a week with 12.5-50 pmol per mouse of either 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3], the active form of vitamin D3, or its synthetic analog, 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3], considerably prolonged the survival time of mice inoculated with M1 cells. 1 alpha(OH)D3 was more effective than 1 alpha,25(OH)2D3 in increasing the survival time of the mice. 1 alpha(OH)D3 also increased the survival time of nude mice inoculated with M1 cells. The 1 alpha(OH)[3H]D3 administered intraperitoneally to tumor-bearing mice was converted very rapidly to 1 alpha,25(OH)2-[3H]D3. The chronic administration of 25 pmol of 1 alpha(OH)D3 to tumor-bearing mice for 30 days caused no appreciable hypercalcemia. These results indicate clearly that 1 alpha,25(OH)2D3 is effective not only in inducing differentiation of M1 cells in vitro, as previously reported [Abe, E., Miyaura, C., Sakagami, H., Takeda, M., Konno, K., Yamazaki, T., Yoshiki, S. & Suda, T. (1981) Proc. Natl. Acad. Sci. USA 78, 4990-4994], but also in prolonging the survival time of mice inoculated with M1 cells.

Distribution of ornithine decarboxylase activity induced by 1 alpha,25-dihydroxyvitamin D3 in chick duodenal villus mucosa.

The distribution of enzymes involved in polyamine synthesis and that of the polyamine levels were investigated in the duodenal mucosa of vitamin D-deficient chicks and those supplemented with 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. Duodenal epithelial cells were isolated sequentially from the villus tip to the crypt region using a nonenzymatic method. In vitamin D-deficient chicks, the activities of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase were markedly higher in the crypt cells than in the villus cells. Similar gradients were observed in the distribution of putrescine and spermidine. Six hours after iv administration of 625 ng 1 alpha,25-(OH)2D3, ODC activity and putrescine levels markedly increased both in the villus and crypt region. The rate of the increase in ODC activity and putrescine levels, however, was much higher in the villus than in the crypt region. Neither S-adenosylmethionine decarboxylase activity nor spermidine levels were affected by the treatment with 1 alpha,25-(OH)2D3. To investigate differential uptake of 1 alpha,25-(OH)2D3 in the duodenal mucosa, 0.1 nmol 1 alpha,25-(OH)2[3H]D3 with or without a 200-fold excess of unlabeled 1 alpha,25-(OH)2D3 was injected into rachitic chicks, and epithelial cells were sequentially isolated 2 h later. The radioactivity specifically incorporated in the nuclear fraction was distributed uniformly from the villus tip to crypt cells. The cytoplasmic receptor for 1 alpha,25-(OH)2D3 was similarly distributed in the crypt and villus cells. These results suggest that 1 alpha,25-(OH)2D3 plays a physiological role in cellular activities not only in the villus but also in the crypt region through polyamine biosynthesis.

Chemical synthesis, biological activity, and metabolism of 25-hydroxy-24-oxovitamin D3.

25-Hydroxy-24-oxovitamin D3 (25(OH)24-oxo-D3), a metabolite of 25-hydroxyvitamin D3, has been chemically synthesized. The ultraviolet, mass, infrared, and proton nuclear magnetic resonance spectra of the 25(OH)24-oxo-D3 were identical with those of the natural product isolated from chick kidney incubates. The oxo compound showed biological activity similar to 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) in vitamin D-deficient chicks in enhancing intestinal calcium transport and bone calcium mobilization activities. Although 25(OH)24-oxo-D3 partially restored the impaired eggshell weights of Japanese quails fed a vitamin D-deficient diet, it was much less potent than 25-hydroxyvitamin D3 or 1 alpha, 25-dihydroxyvitamin D3. In addition, there was no effect on the calcification of medullary bone. When 25(OH)24-oxo[3H]D3 was incubated with kidney homogenates from vitamin D-deficient chicks, it was metabolized to [3H]1 alpha, 24,25-trihydroxyvitamin D3 and a metabolite which was eluted in a region between authentic 24R,25(OH)2D3 and 1 alpha, 25-dihydroxyvitamin D3 on high pressure liquid chromatography. In the incubates of kidney homogenates from vitamin D-supplemented chicks, those metabolites were not detected. In vitamin D-supplemented chicks, the recovery of radioactivity in the chloroform phase extracted by the method of Bligh and Dyer was only 50%, while that in vitamin D-deficient chicks was 87%. Moreover, the radioactivity eluted in the 25(OH)24-oxo-D3 fraction from vitamin D-supplemented chicks was only one-fifth of that from vitamin D-deficient birds. The present results indicate that the 24-oxidation of 24,25(OH)2D3 may be a route of inactivation of vitamin D3.

Isolation, identification, and biological activity of 25-hydroxy-24-oxovitamin D3: a new metabolite of vitamin D3 generated by in vitro incubations with kidney homogenates.

A metabolite of 25-hydroxyvitamin D3 has been isolated in pure form from incubation mixtures containing kidney homogenates of chicks given large doses of vitamin D3. The isolation involved methanol--chloroform extraction and six steps of column chromatography. The metabolite was identified as 25-hydroxy-24-oxovitamin D3 by means of ultraviolet absorption spectrometry, mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and specific chemical reactions. Use of a sensitive in situ technique revealed that 25-hydroxy-24-oxovitamin D3 enhances intestinal calcium transport in rats approximately as effectively as 24,25-dihydroxyvitamin D3 does. In contrast, 25-hydroxy-24-oxovitamin D3 appeared to be less active than 24,25-dihydroxyvitamin D3 in chicks 24 h after intravenous injection.

Metabolites of 24,25-dihydroxyvitamin D3 made in the kidney of chicks supplemented with vitamin D3.

Metabolism of 25-hydroxyvitamin D3 (25-OH-D3) was examined in chicks supplemented with vitamin D3. Kidney homogenates metabolized in vitro [3H]-25-OH-D3 to 3 new metabolites (peaks A, C and E) by way of 24,25-dihydroxyvitamin D3. The enzymes responsible for the synthesis of these metabolites appeared to be induced by 1 alpha,25-dihydroxyvitamin D3. Production of these metabolites was increased in parallel with the increase of the supplemented levels of vitamin D3, while recovery of the radioactivity in the chloroform phase was sharply decreased. The production of peak C was considered to be closely related to the transfer of the radioactive metabolites to the water-soluble phase. These results may indicate that 24-hydroxylation is a degradation step in the 25-OH-D3 metabolism.

Vitamin D and vitamin D dependency.

Administration of large doses of vitamin D2 brought about a marked increase of 25-hydroxyvitamin D in both the patients with vitamin D dependency and hypophosphatemic vitamin D-resistant rickets. During the administration of vitamin D2, increment of 1,25-dihydroxyvitamin D was marked in hypophosphatemic vitamin D-resistant rickets, but far smaller in vitamin D dependency. In the latter, however, the 1,25-dihydroxyvitamin D reached the level close to the normal adult values. 1 alpha-hydroxyvitamin D3 was found 50 approximately 100 times as effective as vitamin D2 in 2 patients with vitamin D dependency (optimum maintenance dose: 0.05 micrograms/kg/day). It was concluded that 1 alpha-hydroxylation in the renal tubules is markedly defective in the patients with vitamin D dependency, but that a large dose of vitamin D2 is able to cause a definite increase in serum concentration of 1,25-dihydroxyvitamin D resulting in improvement of the rickets.