Differential effects of oral doxercalciferol (Hectorol) or paricalcitol (Zemplar) in the Cyp27b1-null mouse model of uremia.

BACKGROUND/AIMS: Kidney disease patients experience declining calcitriol levels and develop secondary hyperparathyroidism (SHPT). Animal models of uremia based on 5/6 nephrectomy (NTX) do not consistently reproduce this calcitriol deficiency. We developed an animal model, the NTX Cyp27b1-null mouse, which completely lacks endogenous calcitriol, and examined the suitability of this model for evaluation of treatment with vitamin D analogs in uremia. METHODS: NTX was performed at 2 months of age. One week post-NTX, animals were treated for 4 weeks with vehicle; doxercalciferol at 30, 100 or 300 pg/g body weight (b.w.); or paricalcitol at 100, 300 or 1,000 pg/g b.w. by gavage 3 times per week. RESULTS: Serum blood urea nitrogen and creatinine were elevated. Vehicle-treated NTX null mice had hypocalcemia and SHPT. Doxercalciferol at 100 or 300 pg/g b.w. normalized serum calcium and parathyroid hormone (PTH) levels. Paricalcitol at 300 or 1,000 pg/g normalized serum calcium, but PTH levels remained elevated. Osteomalacia was corrected by 100 pg/g b.w. of doxercalciferol or 1,000 pg/g b.w. of paricalcitol. The highest dose of doxercalciferol, but not of paricalcitol, significantly reduced osteitis fibrosa. CONCLUSION: Our results reveal the differential efficacy of doxercalciferol and paricalcitol in this novel animal model incorporating both calcitriol deficiency and renal insufficiency.

Hepatic activation and inactivation of clinically-relevant vitamin D analogs and prodrugs.

Like most pharmaceutical agents, vitamin D analogs are subject to hepatic metabolism by a variety of cytochrome P450 (CYP)-based systems. Metabolism can involve activation as well as inactivation of the vitamin D analog and one of the more successful families includes the 1alpha-hydroxyvitamin D prodrugs (1alpha-OH-D2, 1alpha-OH-D3, 1alpha-OH-D4, 1alpha-OH-D5), that all require a step of activation. Some of these prodrugs are in use or clinical trial because they have a therapeutic advantage over calcitriol. However, the nature of the activation of these molecules is poorly understood, particularly with regard to the CYP isoform involved. Various transfected CYPs and hepatic cell lines combined with tandem LC-MS analysis were used to investigate the metabolism of a spectrum of vitamin D analogs, including 1alpha-OH-Ds and the topical analog, calcipotriol. In the case of the 1alpha-OH-Ds, evidence was found of multiple sites of side-chain hydroxylation consistent with the generation of more than one active form. The potential involvement of CYP27A and other putative 25-hydroxylases in 1alpha-OH-D activation was also shown, as well as the potential for CYP24 activation and inactivation. In the case of calcipotriol, the respective roles of non-vitamin D-related CYPs and CYP24 in the catabolism of this anti-psoriatic drug were dissected out using cell lines with or without CYP24 expression, allowing us to demonstrate the potential contribution of CYP24 to "vitamin D resistance". The implications of hepatic metabolism in the context of other facets thought to play a role in the mechanism of action of anticancer and antiproliferative vitamin D analogs are discussed.

1,24(S)-dihydroxyvitamin D2, an endogenous vitamin D2 metabolite, inhibits growth of breast cancer cells and tumors.

BACKGROUND: 1,24-Dihydroxyvitamin D2 (1,24(OH)2D2) is a naturally occurring metabolite of vitamin D2 with low calcemic activity and potent antiproliferative activity. We evaluated the activity of 1,24(OH)2D2 in breast cancer models. MATERIALS AND METHODS: The antiproliferative activity of 1,24(OH)2D2 was quantitated against human and murine breast cancer cell lines. The antitumor activity of 1,24(OH)2D2 was quantitated using MCF-7 xenografts in nude mice. RESULTS: 1,24(OH)2D2 inhibited growth of vitamin D receptor (VDR)-positive, but not VDR-negative, breast cancer cells. 1,24(OH)2D2 (10 microg/kg or 50 microg/kg) reduced MCF-7 xenograft growth by 50% after five weeks. Tumor morphology in treated animals was consistent with replacement of epithelial cells by stromal tissue. Mice treated with 1,24(OH)2D2 showed no loss of body weight, hypercalcemia or kidney calcification. CONCLUSION: 1,24(OH)2D2 inhibits growth of breast cancer cells via VDR-dependent mechanisms; its complete lack of toxicity and significant antitumor activity supports further development for chemotherapeutic applications.

Tissue distribution and activity studies of 1,24-dihydroxyvitamin D2, a metabolite of vitamin D2 with low calcemic activity in vivo.

The active vitamin D compound 1alpha,24(S)-dihydroxyvitamin D(2) (1,24(OH)(2)D(2)) is under development as a therapy for disorders including cancer and secondary hyperparathyroidism. 1,24(OH)(2)D(2) is a potent inhibitor of cell proliferation in vitro and, relative to calcitriol (1,25(OH)(2)D(3)), has reduced calcemic activity in vivo. To examine the mechanisms underlying this reduced calcemic activity, we studied the tissue distribution in rats of radiolabeled 1,24(OH)(2)D(2) or 1,25(OH)(2)D(3) over 24h. Serum levels of 1,24(OH)(2)D(2) were lower than those of 1,25(OH)(2)D(3) at all time points; however, tissue levels of radiolabeled compounds followed different patterns. In duodenum and kidney, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) rose to similar levels at early time points; 1,24(OH)(2)D(2) levels then declined more rapidly. In bone marrow, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were present at similar levels at all time points. In liver, 1,24(OH)(2)D(2) levels were two-fold higher than 1,25(OH)(2)D(3) at 1h post-injection, declining to similar levels by 8h. In vitamin D-deficient rats, doses of 1,24(OH)(2)D(2) 30-fold higher than 1,25(OH)(2)D(3) were required to produce equal stimulation of intestinal calcium absorption. In the same deficient animals, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were nearly equipotent at stimulating bone calcium mobilization. In cultured bone cells, 1,24(OH)(2)D(2) and 1,25(OH)(2)D(3) were equipotent at stimulating osteoclast formation and bone resorption. In summary, the reduced calcemic activity of 1,24(OH)(2)D(2) may result from altered pharmacokinetics relative to 1,25(OH)(2)D(3), resulting in relatively rapid decreases in 1,24(OH)(2)D(2) levels and activity in target organs such as intestine. Further studies will be necessary to confirm these findings and to confirm the clinical utility of 1,24(OH)(2)D(2).

Use of vitamin D(4) analogs to investigate differences in hepatic and target cell metabolism of vitamins D(2) and D(3).

In this study, we used molecules with either of the structural differences in the side chains of vitamin D(2) and vitamin D(3) to investigate which feature is responsible for the significant differences in their respective metabolism, pharmacokinetics and toxicity. We used two cell model systems-HepG2 and HPK1A-ras-to study hepatic and target cell metabolism, respectively. Studies with HepG2 revealed that the pattern of 24- and 26-hydroxylation of the side chain reported for 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)) but not for 1alpha-OH-D(3) is also observed in both 1alpha-OH-D(4) and Delta(22)-1alpha-OH-D(3) metabolism. This suggests that the structural feature responsible for targeting the enzyme to the C24 or C26 site could be either the C24 methyl group or the 22-23 double bond. In HPK1A-ras cells, the pattern of metabolism observed for the 24-methylated derivative, 1alpha,25-(OH)(2)D(4), was the same pattern of multiple hydroxylations at C24, C26 and C28 seen for vitamin D(2) compounds without evidence of side chain cleavage observed for vitamin D(3) derivatives, suggesting that the C24 methyl group plays a major role in this difference in target cell metabolism of D(2) and D(3) compounds. Novel vitamin D(4) compounds were tested and found to be active in a variety of in vitro biological assays. We conclude that vitamin D(4) analogs and their metabolites offer valuable insights into vitamin D analog design, metabolic enzymes and maybe useful clinically.