Rationale for active vitamin D and analogs in the treatment of osteoporosis

In 1981, Chugai Pharmaceutical succeeded in marketing alfacalcidol, a prodrug of calcitriol, as a therapeutic agent for renal osteodystrophy. In 1983, Chugai succeeded in extending the application of alfacalcidol to the treatment of osteoporosis as well. Clinicians in Japan have accepted alfacalcidol as a remedy for osteoporosis. However, the use of calcitriol and its analogs for the treatment of osteoporosis is still controversial. Some misunderstandings exist internationally about the efficacy of the active form of vitamin D for the treatment of osteoporosis. It is important to emphasize that patients with osteoporosis have intestinal calcium malabsorption and dysfunction in renal activation of vitamin D. When massive doses of parent vitamin D were administered to OVX rats, bone mass increased, but surprisingly, many porotic area were observed in the cortical bone. On the other hand, administration of alfacalcidol increased physiological bone without porotic observation. It is necessary to give the active form of vitamin D, D-hormone, with an RDA-equivalent supply of calcium. Alfacalcidol forms physiological strong bones that are hardly fractured by regulating calcium and bone metabolism. We proposed a new vitamin D analog, 2beta (3-hydroxypropoxy)calcitriol [ED-71] as a therapeutic drug for osteoporosis, which is more potent than calcitriol. ED-71 is now being investigated in phase 2 clinical studies in Japan. ED-71 will appear as more improved drugs for osteoporosis until 2010.     

Inhibitory effects of active vitamin D preparations on PTH secretion in rats

To study the effects of various vitamin D preparations on PTH secretion, serum calcium and urinary excretion of cAMP were monitored in conscious perfused rats, and the influences of a bolus iv injection of the preparations on these parameters were examined. Three hours after the administration of 0.25 microgram/kg (0.6 nmol/kg) of 1 alpha, 24(R)-dihydroxycholecalciferol [1 alpha, 24(OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized (PTX) rats (50% of initial value; p less than 0.05) with no change in the concentration of serum calcium (total and ionized). In PTX rats supplemented with bovine PTH (1 U/h), the vitamin D preparation showed no significant effects either on the urinary excretion of cAMP or on serum calcium. These effects were rather specific for active vitamin D preparations, i.e. 1 alpha, 25(OH)2D3 (0.25 micrograms/kg) and 1 alpha OHD3 (1.25-6.25 micrograms/kg). However, 24,25(OH)2D3 (up to 25 micrograms/kg) had no significant effect on these parameters. These results suggest that, in rats, active vitamin D preparations specifically inhibit PTH secretion without causing a significant increase in the serum calcium concentration, reflecting a direct feedback mechanism between active vitamin D metabolite and the parathyroid glands.     

Experimental study of the immunosuppressive properties of vitamin D

The experiments on CBA mice and guinea-pigs have shown that oral vitamin D administration suppressed natural killer activity, decreased the number of T lymphocytes in the peripheral blood and prolonged allograft survival.     

Computational analysis of the active sites in binary and ternary complexes of the vitamin D receptor

We have developed a program CCOMP that compares overlapping fragments of two protein complexes and identifies differently oriented amino acids. CCOMP initially performs a sequence alignment of the analyzed receptors, then superimposes the corresponding aligned residues, and finally calculates the root mean square deviation (RMSD) of individual atoms, every amino acid and the entire complex. Thus, amino acids important for functional differences between both complexes can be detected. Application of CCOMP to 1,25-(OH)₂D₃-hVDR (1DB1) [Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 5491] and 1,25-(OH)₂D₃-rVDR-peptide (1RK3) [Biochemistry 43 (2004) 4101] complexes revealed that the peptide (KNHPMLMNLLKDN) mimicking a co-activator sequence significantly changes the side chain conformation of 35 amino acids. Four of these residues (K242, I256, K260, E416) actually contact the peptide, but all of them are essential for biological activity. Only two (L309 and L400) of the 35 differently oriented amino acids contact the ligand. Interestingly, when the peptide is present (1RK3) leucine 400 shifts closer (0.7 Å) to the vitamin D 26-methyl group. Applying the CCOMP and DSSP programs to binary and ternary VDR complexes also resulted in establishing that seven amino acids (I238, S252, I256, L413, L415, E416, V417) exhibit significant differences in solvent accessibility and are capable of interacting with co-activators.     

Effect of active vitamin D3 on the levels of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine-binding protein

Effect of 1 alpha-OH-vitamin D3 (1 alpha-OH-D3) and 1,25-(OH)2-vitamin D3 (1,25-(OH)2-dihydroxycholecalciferol)(1,25-(OH)2-D3) on the levels of NADPH-dependent cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) was studied in rats and cultured dRLh cells. Deprivation of rats from vitamin D decreased the activity of cytosolic NADPH-dependent T3 binding in rat kidney and liver. The decrease was restored by administration of 1 alpha-OH-D3(0.2 micrograms/kg). The activity of cytosolic NADPH-dependent T3 binding was increased in the dRLh cells by addition of 1,25-(OH)2-D3 to the culture medium. The maximal binding capacity (MBC) was increased by 1,25-(OH)2-D3 without changes in the affinity constant. These results suggested that active vitamin D3 plays an important role in the regulation of cellular T3 translocation through increasing the binding capacity of NADPH-dependent cytosolic T3-binding protein.     

An in vitro study of vitamin D2 hydroxylases in the chick.

An in vitro study of the liver 25-hydroxylation of vitamin D2 and the kidney 1- and 24-hydroxylations of 25-hydroxyvitamin D2 was undertaken in order to determine whether the discrimination against vitamin D2 seen in chicks in vivo is the result of a block of one or more of the steps in the activation of the vitamin D2 molecule. Vitamin D2 hydroxylation reactions in the chick are virtually identical with those observed with the vitamin D3 series. It is, therefore, concluded that the chick possesses the required enzymatic machinery to synthesize 1,25-dihydroxyvitamin D2 and that the discrimination must be because of some unknown metabolic reaction of the vitamin D2 compounds, to a defect in the transport of vitamin D2 metabolites, or to target organ discrimination.     

Estimation of the cutoff value of vitamin D: the Dong-gu study

Background

Vitamin D plays an essential role in bone health and growth, but the optimal serum 25-hydroxyvitamin D (25(OH)D) concentration is not known. This study was performed to investigate the optimal 25(OH)D concentration in regard to parathyroid hormone (PTH) concentration in the Korean general population aged 50 years or older.

Findings

The study population consisted of 8,857 subjects (3,545 men and 5,312 women) who participated in the baseline survey of the Dong-gu study, conducted in Korea between 2007 and 2010. Serum 25(OH)D and PTH concentrations were measured by chemiluminescent microparticle immunoassay. The optimal 25(OH)D concentration was estimated by using nonlinear regression model. Our data show that PTH concentration reached a theoretical plateau at 38.2 pg/ml and corresponding 25(OH)D concentration was 21.1 ng/ml in men and PTH concentration at 42.9 pg/ml and 25(OH)D concentration at 13.8 ng/ml in women.

Conclusions

These results indicate that, for Korean general population aged 50 years or older, the optimal 25(OH)D concentration is 21.1 ng/ml in men and 13.8 ng/ml in women.     

Regulation of renal vitamin D hydroxylase activity in vitamin D deficient rats

The regulation of renal mitochondrial 1-hydroxylase activity in chronic vitamin D deficiency was studied in male rats. These rats were born of mothers who had been raised from weaning (21 days) on a vitamin D deficient diet and who had no detectable serum 1,25-dihydroxycholecalciferol (1,25-(OH)2D) at the time their offspring were weaned (28 days). In the pups, renal mitochondrial 1-hydroxylase activity was undetectable before the 3rd week of life even though the animals were severely hypocalcemic from birth. The 1-hydroxylase activity first became detectable at 26 days of age, rapidly reached a maximum at day 34, then decreased to become undetectable again by 65 days. Throughout this time serum calcium concentration was less than 5.0 mg/dL and serum parathyroid hormone (PTH) concentration, measured by a midmolecule radioimmunoassay, was two- to five-fold greater than that found in vitamin D replete rats. 1-Hydroxylase activity could be restored in the +65-day-old animals by administration of a single dose of 2.5 micrograms vitamin D3. Enzyme activity was detected within 24 h, was maximal at 72 h, and returned to undetectable levels by 96 h after administration of the vitamin. Serum 1,25-(OH)2D which was undetectable before administration of the vitamin D3, was 108 and 458 pg/mL at 16 and 40 h, respectively, after the injection. The serum concentration of this metabolite then decreased progressively to 80 pg/mL by 6 days. 24-Hydroxylase activity first became detectable 48 h after vitamin D administration, increased to a maximum at 96 h, and thereafter decreased to become undetectable by 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)