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Background: Most immunosuppressants have a narrow margin between efficacy and side effects. A major goal in the development of immunomodulatory strategies is the discovery of combinations of drugs exerting synergistic immunomodulatory effects. The active form of vitamin D, 1,25(OH)2D3, is an immunomodulator that interacts with T cells but mainly targets antigen-presenting cells. We have demonstrated synergism between 1,25(OH)2D3 and cyclosporine, rapamycin, and FK506. The aim of this study was to investigate whether this synergism could be observed with other immunosuppressants (mycophenolate mofetil, leflunomide, and the methylxanthine A802715) and whether analogs of 1,25(OH)2D3 share this synergistic capacity in vivo., Methods: In vitro, the median effect analysis was applied to the inhibition of phytohemagglutinin A-induced lymphocyte proliferation. In vivo, synergism between analogs of 1,25(OH)2D3 and cyclosporine or mycophenolate mofetil was evaluated in experimental autoimmune encephalomyelitis., Results: In vitro, all combinations with 1,25(OH)2D3 were synergistic. The strongest synergism was seen with the inhibitors of interleukin 2 secretion, cyclosporine and FK506 (indexes 0.16 and 0.27, respectively). The weakest synergism was observed in combinations using A802715, a second-signal inhibitor (index 0.52), or the nucleotide synthesis inhibitor mycophenolate mofetil (index 0.43). In vivo, analogs of 1,25(OH)2D3 share the in vitro-observed synergism with 1,25(OH)2D3. Moreover, the differences in synergism with different immunomodulators were also present in vivo, where the best synergism was again seen in combination with cyclosporine (up to 100% paralysis protection)., Conclusions: These data confirm that 1,25(OH)2D3 and its analogs are potent dose-reducing drugs for other immunomodulators, making them potentially interesting for clinical use in autoimmunity and transplantation.

The active form of vitamin D, 1 alpha, 25-(OH)2D3, displays immunomodulatory effects in vitro and in vivo at pharmacological levels. We evaluated the dose-effect relationship of 1,25(OH)2D3 and sirolimus (rapamycin, RAP) in vitro, on the inhibition of PHA-stimulated PBMC proliferation, by using the median effect analysis. Pharmacological concentrations of 1,25(OH)2D3 (between 10(-9) and 3 x 10(-6) M) interacted synergistically with RAP (combination index value of 0.01 for 50% suppression of PBMC proliferation). In vivo, the effect of 1,25(OH)2D3 and RAP combinations on the evolution of experimental allergic encephalomyelitis in SJL mice was analyzed. 1,25(OH)2D3, given i.p., in monotherapy, at a dose of 2 micrograms/kg every two days, from day -3 until day +19 after disease induction, or RAP, injected daily at a dose of 0.3 mg/kg for the same period, decreased EAE incidence (paralysis in 70 and 55% of the animals, respectively, versus 98% in the placebo treated group, p < 0.001). The combination treatment using the two drugs in these subtherapeutical doses provided near-total clinical (8% paralysis, p < 0.001 compared to monotherapy with 1,25(OH)2D3 or RAP) and histological protection, comparable to that obtained with RAP in monotherapy at a threefold higher dose (1 mg/kg/d). When the two drugs were given using an alternate day administration schedule (RAP at 0.6 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to 19), near total protection was again obtained (13% paralysis, p < 0.001 versus control). These in vitro and in vivo data support the existence of synergistic interactions between 1,25(OH)2D3 and RAP. Considering the narrow therapeutic windows of both RAP and vitamin D-related compounds in autoimmune disease models, combinations of these drugs could find clinical application in reducing their individual therapeutically efficient doses to non-toxic levels.

The hormone 1 alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) has immune modulatory activities in vitro and in vivo, and can prevent or delay the onset of experimental or spontaneous autoimmune diseases. At therapeutical doses, however, hypercalcemic side effects are found. The present experiments examined the effects of combined treatment with subtherapeutic doses of cyclosporine A (CsA) and 1,25(OH)2D3 on the evolution of experimental autoimmune encephalomyelitis (EAE) in SJL mice. 1,25(OH)2D3 at 5 micrograms/kg body weight (given by i.p. injection every 2 days) prevented the appearance of paralysis in 70% of the treated mice. The treatment with 1,25(OH)2D3 at 2 micrograms/kg/2 days alone had less substantial protective effects (22% disease-free animals versus 5% in the control group). However, when this subtherapeutic dose was associated to treatment with a daily dose of CsA (2 or 5 mg/kg/day), which by itself was subtherapeutic (24 and 50% disease-free animals, respectively), the association of both drugs led to near-total protection (86% disease-free animals when combined with the highest dose of CsA). When an alternate day administration schedule (CsA at 10 mg/kg and 1,25(OH)2D3 at 2 micrograms/kg, each given on alternate days from day -3 to +19 after disease induction) was used, all treated mice were completely protected clinically and histologically. The two drugs also showed additive effects on serum osteocalcin and urinary calcium and desoxypyridinoline excretion, but not on serum calcium concentration. Our experiments demonstrate that 1,25(OH)2D3 might be a potential dose-reducing agent for CsA in immunosuppressive therapy.

The biological activity of 16-epoxy side-chain analogs of 1 alpha,25-dihydroxyvitamin D3, (1 alpha,25(OH)2D3) was evaluated in vitro and in vivo. Compared to 1 alpha,25(0H)2D3, all analogs had lower affinities for the pig duodenal vitamin D receptor and also for the human serum vitamin D binding protein. The in vitro effects on cell proliferation or differentiation of human promyeloid leukemia (induction of superoxide production in HL-60 cells), human osteosarcoma MG-63 cells (osteocalcin secretion), or human breast cancer cells (incorporation of thymidine in MCF-7 cells), was markedly inhibited by several epoxy analogs, compared to 1 alpha,25(OH)2D3, but the rank order of their activity widely varied among different cancer cells. The most potent analogs (24S,25S-24-hydroxy-25,26-epoxy-22-ene-1 alpha-OHD3, 25,26-epoxy-23-yne-1 alpha-OHD3, and 25,26-epoxy-23-yne-20-epi-1 alpha-OHD3 or compounds, 16, 5, and 7, respectively) were equipotent (16 and 5) or 30-fold (compound 7 on MG-63 cells) to 40-fold (compound 7 on MCF-7 cells) more active than 1 alpha,25-(OH)2D3. These analogs were nevertheless poorly antirachitic (< 3%) when tested in vitamin D-deficient chicks (using serum and bone calcium, serum osteocalcin and duodenal calbindin D-28K, as end points). Compound 7 was also 100-fold more active than 1 alpha,25-(OH)2D3 in inhibition of proliferation of human foreskin keratinocytes. Some epoxy analogs of 1 alpha,25-(OH)2D3 thus display interesting dissociations between their receptor affinity and their potency to induce cell differentiation, whereas their effect on cell proliferation/differentiation exceed their calcemic effects more than 100- to 1000-fold.

The hormone 1 alpha, 25 dihydroxyvitamin D3 (1,25(OH)2D3) has potent immunosuppressive effects in vitro. Recent publications also described a protective effect of the hormone in various animal models of immune-mediated diseases. To test its in vivo activity we induced active Heymann nephritis in Lewis rats that were either untreated or treated with 1,25(OH)2D3 or its synthetic 20-epi analogue, KH1060. Treatment with cyclosporine A (CsA) was used as an immunosuppressive control. In this nephrotic model the administration of 1,25(OH)2D3 (0.5 microgram/kg body weight) given on alternate days during the first 13 days after active immunization significantly reduced the proteinuria as measured by weeks 7-9. This reduction was comparable to the reduction observed in rats treated with CsA (20 mg/kg) on alternate days. A second series of experiments with 1,25(OH)2D3 confirmed these findings. The level of autoantibodies was found to be significantly suppressed during the treatment time in the CsA (20 mg/kg) group, whereas the limit of significance (P = 0.06) was reached in the 1,25(OH)2D3 (0.5 microgram/kg) group. The size of the immune deposits also was found to be substantially smaller in the groups that developed less proteinuria. The administration of 1,25(OH)2D3 transiently increased the mean serum calcium concentration with 2.5 mg/dl above the pretreatment values, and the urinary calcium excretion by a factor of 3-5 during the short treatment time. Treatment with the analogue KH1060 did not reduce the proteinuria significantly. Our experiments add evidence to the hypothesis that 1,25(OH)2D3 in pharmacological doses has immunosuppressive potency.

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) generates biological responses via both genomic and nongenomic mechanisms. This article addresses activity differences between the 6-s-trans (extended) and the 6-s-cis (steroid-like) conformation of 1,25-(OH)2D3 to initiate these responses. Because of facile interconversion of the 6-s-trans and 6-s-cis conformers of 1,25-(OH)2D3, kinetically competent amounts of both conformers exist to interact with any potential receptors for 1,25-(OH)2D3. We have chemically synthesized 1,25-(OH)2-9,14,19,19,19-pentadeuterio-pre-D3 (1,25-(OH)2-d5-pre-D3), an analog of the 6-s-cis conformation of 1,25-(OH)2D3. We found that 1,25-(OH)2-d5-pre-D3 and 1,25-(OH)2D3 were equivalently active in two nongenomic systems (transcaltachia as measured in the perfused chick intestine and 45Ca2+ uptake through voltage-gated Ca2+ channels in ROS 17/2.8 cells). 1,25-(OH)2-d5-pre-D3 was significantly less active both in binding in vitro to the plasma vitamin D-binding protein (7%) and to the chick (10%) and pig (4%) intestinal nuclear 1,25-(OH)2D3 receptors generating genomic biological responses in vivo (induction of plasma levels of osteocalcin, < 5%) or in cultured cells (inhibition of HL-60 cell differentiation, < 5%; inhibition of MG-63 proliferation, < 2%; and induction of osteocalcin, < 2%). These results suggest that the genomic and nongenomic responses are mediated by separate receptors. Further, the 6-s-cis form (steroid-like conformation) of the natural hormone, 1,25-(OH)2D3, may be selectively responsible for its nongenomic function(s).