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.

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).

Effectiveness of 1alpha-hydroxyvitamin D2 in inhibiting tumor growth in a murine transgenic pigmented ocular tumor model.

OBJECTIVE: To study the effectiveness of the vitamin D analogue 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)) in inhibiting ocular tumor growth in transgenic "Tyr-Tag" mice that developed pigmented ocular tumors produced with the simian virus 40 T and t antigens under the control of the mouse tyrosinase gene. These animals develop pigmented intraocular tumors primarily from the retinal pigment epithelium that closely resemble the histologic features and growth pattern of human choroidal melanoma. METHODS: A total of 73 Tyr-Tag transgenic mice between 6 and 7 weeks old were randomly assigned by sex and litter to 3 treatment groups to receive 0.05 microg/d, 0.1 microg/d, or 0.2 microg/d of 1alpha-OH-D(2); a control group received vehicle (coconut oil). The drug was administered by oral gavage 5 times a week for 5 weeks. The animals were then euthanized and their eyes were enucleated and processed histologically. Three serial sections from each eye were examined microscopically and the mean tumor area measured using Optimus software version 6.5 (Media Cybernetics LP, Silver Spring, Md). Toxic adverse effects were assessed on the basis of mortality, weight loss, and serum calcium levels. RESULTS: The mean tumor size in the 0.1- microg/d and 0.2- microg/d dose groups was smaller than in the controls (P<.001). No significant difference was seen between the 0.05- microg/d dose group and the control group (P =.64). Survival for the 0.1- microg/d and 0.2- microg/d dose groups was lower than for the controls (95% in the controls vs 85.7% and 73.7%, respectively; P<.01). CONCLUSION: In the Tyr-Tag transgenic mouse, 1alpha-OH-D(2) inhibits pigmented ocular tumor growth at moderate drug levels with relatively low mortality. Clinical Relevance Vitamin D analogues merit further preclinical study in the treatment of ocular melanoma.

Effectiveness of vitamin D analogues in treating large tumors and during prolonged use in murine retinoblastoma models.

OBJECTIVE: To investigate the effectiveness of the vitamin D analogues 1,25-(OH)(2)-16-ene-23-yne vitamin D(3) (16,23-D(3)) and 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)) in inhibiting retinoblastoma growth in large tumors in a xenograft model and with prolonged use in a transgenic model. METHODS: For the large-tumor study, the xenograft athymic mouse/human retinoblastoma cell (Y-79) model was used. Subcutaneous tumors were allowed to grow to an average volume of 1600 mm(3). Systemic treatment with 1 of the vitamin D analogues or with vehicle (control groups) was carried out for 5 weeks. For the long-term study, transgenic beta-luteinizing hormone-large T antigen (LHbeta-Tag) mice were systemically treated with 1 of the 2 compounds or vehicle (control groups) for up to 15 weeks. Tumor size and signs of toxicity were assessed. RESULTS: In the large-tumor study, tumor volume ratios for the 1alpha-OH-D(2) and 16,23-D(3) groups were significantly lower than those for controls (P<.002). No significant differences in tumor volume were seen between the 1alpha-OH-D(2) and 16,23-D(3) groups (P =.15). In the long-term study, the 1alpha-OH-D(2) group showed significantly smaller tumor size compared with its control (P<.001). No significant difference was seen between the 16,23-D(3) group and its control. Some toxic effects related to hypercalcemia were seen in both studies. CONCLUSIONS: In athymic mice in the large-tumor study, both 1alpha-OH-D(2) and 16,23-D(3) were effective in inhibiting tumor growth compared with controls. In the long-term study, 1alpha-OH-D(2) inhibited tumor growth but 16,23-D(3) did not. Effective doses of both compounds caused hypercalcemia and a significant increase in mortality. Clinical Relevance Use of 1alpha-OH-D(2) inhibited tumor growth in large tumors and with long-term treatment compared with controls. Because of hypercalcemia-related toxic effects seen in the present experiments, in clinical trials, serum calcium levels should be carefully monitored. This analogue may require use with drugs that lower serum calcium levels or use of relatively lower doses or skipped doses. The ideal alternative solution would be to identify vitamin D analogues that retain the antineoplastic action without the calcemic activity.

Toxicity and dose-response studies of 1-alpha hydroxyvitamin D2 in LH-beta-tag transgenic mice.

PURPOSE: To determine the effectiveness of a vitamin D analog, 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)), in inhibiting retinoblastoma in a transgenic retinoblastoma model (LHbeta-Tag mouse) and to evaluate its toxicity. DESIGN: Experimental study using an animal (LHbeta-Tag transgenic mouse) randomized (controlled) trial. PARTICIPANTS AND CONTROLS: Two hundred seventeen LHbeta-Tag transgene-positive 8- to 10-week-old mice total; 179 drug-treated animals, 38 control animals. METHODS: Mice were fed a vitamin D- and calcium-restricted diet and were randomized to treatment groups receiving control (vehicle), or 0.1, 0.3, 0.5, or 1.0 micro g/day of 1alpha-OH-D(2) via oral gavage 5 times weekly for 5 weeks. Body weight was measured at the start of treatment and twice weekly during treatment. Animals were euthanized on the last day of treatment. The eyes were enucleated, processed histologically, and serially sectioned. Representative sections from the superior, middle, and inferior regions of each globe were examined microscopically and tumor areas were measured using Optimas software. Serum was collected for serum calcium levels. Kidneys were removed for histologic processing and were analyzed microscopically for kidney calcification. MAIN OUTCOME MEASURES: Mean tumor area was measured to determine drug effectiveness. Toxicity was assessed by survival, weight loss over the treatment period, serum calcium, and kidney calcification. RESULTS: The mean tumor size in each 1alpha-OH-D(2) group was smaller than controls (all P values < 0.02): control, 90,248 micro m(2); 0.1 micro g, 31,545 micro m(2); 0.3 micro g, 16,750 micro m(2); 0.5 micro g, 30,245 micro m(2); and 1.0 micro g, 16,049 micro m(2). No dose-dependent response curve was evident. The survival percentage for each group was as follows: control, 97%; 0.1 micro g, 91%; 0.3 micro g, 88%; 0.5 micro g, 70%; and 1.0 micro g, 63%. Mortality was higher in the 0.5- micro g and 1.0- micro g doses (P values < 0.01) compared with other treatment groups and with the control group. Serum calcium levels were significant in all treatment groups compared with controls (all P values < 0.0001). CONCLUSIONS: In the LHbeta-Tag mouse, 1alpha-OH-D(2) inhibits retinoblastoma with no significant increase in mortality in lower doses (0.1-0.3 micro g). 1alpha-OH-D(2) has approval by the Food and Drug Administration as an investigative drug for cancer treatment, and has shown efficacy with low toxicity in adult cancer trials. 1alpha-OH-D(2) meets the criteria for human clinical trials.

Toxicity and dose-response studies of 1alpha-hydroxyvitamin D2 in a retinoblastoma xenograft model.

BACKGROUND: Although calcitriol (1,25-dihydroxycholecalciferol) and vitamin D(2) inhibit retinoblastoma growth in the athymic (nude) mouse xenograft (Y-79 cell line) model of retinoblastoma, they can cause severe toxicity. OBJECTIVE: To examine the toxicity of and dose-dependent response for the inhibition of tumor growth for 1alpha-hydroxyvitamin D(2) (1alpha-OH-D(2)), an analogue with reduced systemic toxicity, in the athymic Y-79 mouse model. METHODS: Mice were randomized into treatment and control groups for 5-week toxicity and dose-response studies. Treatment was via oral gavage 5 times per week. Dose-response studies measured tumor inhibition and drug serum levels. Tumor size and body weight were measured weekly together with various criteria for toxicity. Animals were euthanized at the end of the treatment period. Tumors and kidneys were harvested, and serum was analyzed for calcium and drug levels. RESULTS: Doses of 0.1 to 1.2 microg/d were selected on the basis of toxicity studies for the dose-response trial. Tumor weight and volume in the 0.2-microg and 0.3-microg doses were significantly lower than in controls. Mortality rates and kidney calcification in mice treated with doses of 0.1 to 0.3 microg were lower than those observed in studies of calcitriol and vitamin D(2). CONCLUSION: A vitamin D analogue, 1alpha-OH-D(2), inhibits tumor growth in this xenograft model of retinoblastoma with less toxicity than calcitriol and vitamin D(2).

Toxicity and dose-response studies of 1 alpha-hydroxyvitamin D2 in LH beta-Tag transgenic mice.

PURPOSE: The study objective is to determine the effectiveness of a vitamin D analogue, 1 alpha-hydroxyvitamin D2 (1 alpha-OH-D2), in inhibiting retinoblastoma in a transgenic retinoblastoma model (LH beta-Tag mouse) and to evaluate its toxicity. Previous studies of 1 alpha-OH-D2 in athymic mice with human retinoblastoma xenografts suggested efficacy in tumor suppression and suitability for human treatment. METHODS: LH beta-Tag mice (N = 142), 8 to 10 weeks old, were randomly assigned to treatment groups receiving either control (vehicle) or 0.1, 0.3, 0.5, or 1.0 microgram/day of 1 alpha-OH-D2 via oral gavage five times a week for 5 weeks. Animals were then euthanized. The eyes were enucleated, processed histologically, and serially sectioned. Three sections of each eye were microscopically examined, and mean tumor area was measured using Optimus software. Toxicity was assessed by mortality, weight loss, serum calcium levels, and kidney calcification. RESULTS: The mean tumor size in each 1 alpha-OH-D2 group was smaller than in controls (P values < .02): control, 90,248 microns 2; 0.1 microgram, 31,545 microns 2; 0.3 microgram, 16,750 microns 2; 0.5 microgram, 30,245 microns 2; and 1.0 microgram, 16,049 microns 2. No dose-dependent response curve was evident. Mortality was higher in the groups receiving the 0.5 microgram and 1.0 microgram doses (P values < .01) than in the other treatment groups and the control group. CONCLUSION: In the LH beta-Tag mouse, 1 alpha-OH-D2 inhibits retinoblastoma with no increased mortality at lower doses (0.1 to 0.3 microgram). 1 alpha-OH-D2 has been approved by the Food and Drug Administration as an investigative drug for cancer treatment and has shown efficacy with low levels of toxicity in adult cancer trials. 1 alpha-OH-D2 meets the criteria for human clinical trials.