Potent Vitamin D3 Research Articles

The Combination of the CDK4/6 Inhibitor, Palbociclib, With the Vitamin D3 Analog, Inecalcitol, Has Potent In Vitro and In Vivo Anticancer Effects in Hormone-Sensitive Breast Cancer, But Has a More Limited Effect in Triple-Negative Breast Cancer.

Active vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH)2D3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER+) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER+ (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER+ BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER+ BC both in vitro and in vivo.

Eldecalcitol causes FGF23 resistance for Pi reabsorption and improves rachitic bone phenotypes in the male Hyp mouse

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.

Eldecalcitol Causes FGF23 Resistance for Pi Reabsorption and Improves Rachitic Bone Phenotypes in the Male Hyp Mouse.

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.

Selective use of multiple vitamin D response elements underlies the 1 alpha,25-dihydroxyvitamin D3-mediated negative regulation of the human CYP27B1 gene.

The human 25-hydroxyvitamin D3 (25(OH)D3) 1α-hydroxylase, which is encoded by the CYP27B1 gene, catalyzes the metabolic activation of the 25(OH)D3 into 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), the most biologically potent vitamin D3 metabolite. The most important regulator of CYP27B1 gene activity is 1α,25(OH)2D3 itself, which down-regulates the gene. The down-regulation of the CYP27B1 gene has been proposed to involve a negative vitamin D response element (nVDRE) that is located ∼500 bp upstream from transcription start site (TSS). In this study, we reveal the existence of two new VDR-binding regions in the distal promoter, 2.6 and 3.2 kb upstream from the TSS, that bind vitamin D receptor–retinoid X receptor complexes. Since the down regulation of the CYP27B1 gene is tissue- and cell-type selective, a comparative study was done for the new 1α,25(OH)2D3-responsive regions in HEK-293 human embryonic kidney and MCF-7 human breast cancer cells that reflect tissues that, respectively, are permissive and non-permissive to the phenomenon of 1α,25(OH)2D3-mediated down-regulation of this gene. We found significant differences in the composition of protein complexes associated with these CYP27B1 promoter regions in the different cell lines, some of which reflect the capability of transcriptional repression of the CYP27B1 gene in these different cells. In addition, chromatin architecture differed with respect to chromatin looping in the two cell lines, as the new distal regions were differentially connected with the proximal promoter. This data explains, in part, why the human CYP27B1 gene is repressed in HEK-293 but not in MCF-7 cells.

Anticancer Medicinal Mushrooms Can Provide Significant Vitamin D2 (Ergocalciferol)

Studies since 1966 have documented antitumor activities by the β-glucan and triterpene components of numerous Basidiomycetes mushrooms. However, these active ingredients may not explain all of the observed pharmacologic eff ects. To date, the potential pharmacologic role of vitamin D₂ (ergosterol) in medicinal mushrooms has not been examined. Vitamin D₂, when transformed by the liver into the potent vitamin D₃, is a seco-steroid that, like thyroid hormone, binds to a specifi c receptor within the superfamily of nuclear receptors for steroid hormones. Th e vitamin D nuclear receptor is found not only on intestine, bone, liver, and kidney tissues but also on lymphocytes, monocytes, and macrophages as well as hematopoietic, skin, muscle, heart, pancreas, adrenal, brain, reproductive, lung, pituitary, thyroid, and cartilage tissues. Vitamin D₃ regulates gene expression for multiple physiological functions, including those for the central nervous system and immune system. Th is study tested the hypothesis that in mushroom species with documented anticancer activity, mushroom ergosterol exposure to UV-B energy from sun drying signifi cantly increases vitamin D₂ content.

Anticancer Medicinal Mushrooms Can Provide Significant Vitamin D2 (Ergocalciferol)

Studies since 1966 have documented antitumor activities by the β-glucan and triterpene components of numerous Basidiomycetes mushrooms. However, these active ingredients may not explain all of the observed pharmacologic effects. To date, the potential pharmacologic role of vitamin D2 (ergosterol) in medicinal mushrooms has not been examined. Vitamin D2, when transformed by the liver into the potent vitamin D3, is a seco-steroid that, like thyroid hormone, binds to a specific receptor within the superfamily of nuclear receptors for steroid hormones. The vitamin D nuclear receptor is found not only on intestine, bone, liver, and kidney tissues but also on lymphocytes, monocytes, and macrophages as well as hematopoietic, skin, muscle, heart, pancreas, adrenal, brain, reproductive, lung, pituitary, thyroid, and cartilage tissues. Vitamin D3 regulates gene expression for multiple physiological functions, including those for the central nervous system and immune system. This study tested the hypothesis that in mushroom species with documented anticancer activity, mushroom ergosterol exposure to UV-B energy from sun drying significantly increases vitamin D2 content.Three specific medicinal mushroom species with strain-specific documented chains of custody were grown under standard conditions indoors. Fruiting bodies were harvested and dried indoors by commercial dryers or outside under the summer sun for 6−8 hours. The products were then subjected to standardized HPLC analysis in conformity with the Official Methods of Analysis of AOAC International (2000) 17th Ed., AOAC International, Gaithersburg, MD, USA, Official Method 982.29 (Modified).Ganoderma lucidum (W.Curt.:Fr.) Lloyd (Reishi), Lentinus edodes (Berk.) Singer (Shiitake), and Grifola frondosa (Dicks.:Fr.) S.F.Gray (Maitake) dried indoors demonstrated D2 content of 6, 134, and 460 IU per 100 grams, respectively. When dried by sunlight outdoors, the D2 content increased to 2760, 21,400, and 31,900 IU per 100 grams, respectively.These data have three important implications. First, the potential vitamin D2 content of antitumor medicinal mushrooms must be considered as a confounding factor in mushroom research. Vitamin D3 stabilizes chromosomal structure and prevents DNA double-strand breaks induced by either endogenous or exogenous factors. Vitamin D3 induces cell cycle arrest, promotes differentiation, and induces apoptosis. Vitamin D3 acts as an antiproliferation agent against many cancers, including breast, prostate, colon, and bladder. In addition, vitamin D3 inhibits both tumor invasion and tumor angiogenesis.Second, sun-exposed mushrooms may be an excellent dietary response for addressing the significant world-wide incidence of vitamin D deficiency. Inadequate vitamin D status not only places people at risk for osteoporosis, but appears to be a significant risk factor for development of cancer. For many cancers, a significant inverse correlation exists between mortality rates and UV-B radiation exposure. For example, the risk of fatal breast cancer in the major urban areas of the United States is inversely proportional to the intensity of local sunlight (r = −0.80, p = 0.0001), and such inverse correlations also exist for prostate, colon, bladder, ovary, non-Hodgkin's lymphoma, esophageal, kidney, lung, pancreatic, rectal, stomach, and corpus uteri cancers.Third, this study challenges the USDA claim that the average edible mushroom contains 76 IU (1.9 μg) of vitamin D per 100 grams and that shiitake mushrooms contain 1550 IU per 100 grams. For retrospective studies of diet and nutrition, use of these standard references may undermine the scientific legitimacy of both the data and its analysis.Vitamin D2 is not as potent as vitamin D3. For many years, vitamin D2 was considered much safer than vitamin D3 because of the additional metabolism required for activation. Thus, although the current RDA for vitamin D is 400 IU per day, prescription doses of ergocalciferol at 50,000 IU twice a week only slowly increase vitamin D levels in deficient adults. However, vitamin D in excessive doses can be toxic and even fatal. This is particularly true in persons with cancer und other diseases that predispose them to hypercalcemia. Further understanding of vitamin D2’s pharmacokinetics and metabolite biologic activities is necessary.

Synergistic inhibition of prostate cancer cell lines by a 19- nor hexafluoride vitamin D3 analogue and anti-activator protein 1 retinoid

The secosteroid hormones, all- trans- and 9- cis -retinoic acid and vitamin D3, have demonstrated significant capacity to control proliferation in itro of many solid tumour cell lines. Cooperative synergistic effects by these two ligands have been reported, and it is, therefore, possible that greater therapeutic effects could be achieved if these compounds were administered together. The role of retinoid-dependent anti-activator protein 1 (anti-AP-1) effects in controlling cancer cell proliferation appears significant. We have utilized an anti- AP-1 retinoid [2-(4,4-dimethyl-3,4-dihydro-2H-1 benzopyran-6-yl)carbonyl-2-(4-carboxyphenyl)-1,3,-dithiane; SR11238], which does not transactivate through a retinoic acid response element (RARE), and a potent vitamin D3analogue [1α,25(OH)2-16-ene-23-yne-26,27-F6-19-nor -D3, code name LH] together at low, physiologically safer doses against a panel of prostate cancer cell lines that represent progressively more transformed phenotypes. The LNCaP (least transformed) and PC-3 (intermediately transformed) cell lines were synergistically inhibited in their clonal growth by the combination of LH and SR11238, whereas SR11238 alone was essentially inactive. DU-145 cells (most transformed) were completely insensitive to these analogues. LNCaP cells, but neither PC-3 nor DU-145, underwent apoptosis in the presence of LH and SR11238. Transactivation of the human osteocalcin vitamin D response element (VDRE) by LH was not enhanced in the presence of SR11238, although the expression of E-cadherin in these cells was additively up-regulated in the presence of both compounds. These data suggest the anti-AP-1 retinoid and the vitamin D3 analogue may naturally act synergistically to control cell proliferation, a process that is interrupted during transformation, and that this combination may form the basis for treatment of some androgen-independent prostate cancer. © 1999 Cancer Research Campaign

Novel vitamin D3 analog (CB1093) when combined with paclitaxel and cisplatin inhibit growth of MCF-7 human breast cancer cells in vivo.

Vitamin D3 compounds paclitaxel (Taxol) and cisplatin (CDDP, cis-diamminodichloroplatinum) inhibit growth of a variety of malignant cells. We examined the ability of a novel 20-epi-vitamin D3 analog (code name, CB1093), Taxol and CDDP either alone or in combination to inhibit the growth of a human mammary cancer (MCF-7) growing in BNX triple immunodeficient mice. Tumors in control animals demonstrated infiltrating poorly differentiated adenocarcinomas. At the doses chosen, the antitumor effect of Taxol alone was greater than that of either CB1093 or CDDP alone; and additive effects were observed when either CB 1093 + Taxol or CB 1093 + CDDP + Taxol were administered together. The combination of CB 1093 + Taxol + CDDP was most potent, inhibiting tumor weights by nearly 83% compared to control tumors and producing extensive necrosis of the remaining tumor mass. No additive effect occurred by combining either CB1093 + CDDP or Taxol + CDDP compared to Taxol alone. For all cohorts, their complete hematopoietic blood counts, serum electrolyte analyses including serum calciums as well as their liver and renal functions were within the normal range. Extensive histological analyses of the liver, spleen, kidneys, bone marrow, skin and subcutaneous fat pads from these mice showed no abnormalities. In summary, combined therapy with CB1093 (a potent vitamin D3 analog), Taxol and CDDP, which have non-cross reactive toxicities, holds promise in the treatment of patients with breast cancer.

Disruption of the vertical calcium gradient in murine epidermis by a potent vitamin D3 analogue, KH 1060.

The hormonal forms of vitamin D3 (1,25-dihydroxyvitamin D3 and synthetic vitamin D3 analogues) are potent regulators of keratinocyte growth and stimulators of keratinocyte differentiation. Recent experiments in vitro on cultured keratinocytes indicate that Ca2+ may be a second messenger mediating the effects related to the induction of keratinocyte differentiation by the hormonal forms of vitamin D3. In this study we employed the technique of ion capture cytochemistry to investigate the effects of a potent vitamin D3 analogue, KH 1060 (20-epi-22-oxa-24 alpha-homo-26,27-dimethyl-1,25-dihydroxyvitamin D3), on the distribution of bound intracellular and extracellular calcium in murine epidermis in vitro. Topical treatment of the skin with KH 1060 (0.4 mmol/l) resulted in the development of epidermal hyperplasia and hyperkeratosis. We observed that epidermis treated with KH 1060 contained fewer calcium deposits in the upper epidermal strata (both intra- and extracellularly) than the control skin. This phenomenon was rapid and occurred after only a single application of KH 1060. Calcium distribution in the basal cell layer was not affected. We propose that observed reduction in the quantity of calcium deposits was caused by the release of sequestered calcium from the intracellular stores and cellular Ca2+ uptake, leading eventually to the increase in the intra-cellular concentration of ionized calcium. The ability of the active vitamin D3 compounds to release Ca2+ may be important for their activity in psoriasis.

Expression of retinoic acid receptor-beta sensitizes prostate cancer cells to growth inhibition mediated by combinations of retinoids and a 19-nor hexafluoride vitamin D3 analog.

Retinoids and analogs of vitamin D3 may achieve greater in vivo applications if the toxic side effects encountered at pharmacologically active doses could be alleviated. These seco-steroid hormones often act in concert, and therefore, we attempted to dissect these interactions by isolating combinations of receptor-selective retinoids and a potent vitamin D3 analog [1alpha,25(OH)2-16ene-23-yne-26,27,F6-19nor-D3, code name LH] that were potent inhibitors of prostate cancer cell growth at low, physiologically safer doses. Using a panel of prostate cancer cell lines representing progressively more transformed phenotypes, we found that the LNCaP cell line (least transformed) was either additively or synergistically inhibited in its clonal growth by LH and various naturally occurring and receptor-selective retinoids, the most potent combination being with a retinoic acid receptor (RAR)betagamma-selective retinoid (SR11262). The effect was not found with either PC-3 (intermediate transformation) or DU-145 (most transformed). We also undertook RT-PCR to examine the subtypes of RARs present, and we found that PC-3 and DU-145 did not express RARbeta. Stable expression of RARbeta into the RARbeta-negative PC-3 cells resulted in increased sensitivity to SR11262 and LH proportional to the amount of RARbeta expressed. This study indicates that RARbeta may play an important role in synergistically controlling cell proliferation, and expression is lost with increased prostate cancer cell transformation. Simultaneous administration of a potent vitamin D3 analog and receptor-selective retinoids may have therapeutic potential for the treatment of androgen-dependent and -independent prostate cancer.

A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

AbstractHuman C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

Effects of potent vitamin D3 analogs on clonal proliferation of human prostate cancer cell lines.

Management of prostate cancer that has spread outside of the prostate capsule is a difficult problem. Innovative, non-toxic approaches to the disease are required. New, relatively non-toxic vitamin D3 analogs have recently been synthesized. We report that several of these compounds have marked antiproliferative effects on prostate cells. The clonal antiproliferative activity of five novel analogs of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3, (cmpd C)] as well as 1,25(OH)2D3 itself was tested on three human prostate cancer cell lines (PC-3, LNCaP, and DU-145). The analogs were 20-epi-22oxa-24a,26a,27a-tri-homo-1 alpha,25(OH)2D3 (code name: KH 1060); 24a26a27a-tri-homo-22,24-diene-1 alpha,25(OH)2D3 (code name: EB 1089); 1,25(OH)2-16ene-D3 (code name: HM); 1,25(OH)2-16ene-23yne-D3 (code name: V); 1,25(OH)2-20-epi-D3 (code name: MC 1288)]. With the parent compound [1,25(OH)2D3], the effective dose that inhibited 50% clonogenic growth of PC-3 and LNCaP was 10(-8)M and 7 x 10(-9)M, respectively. For these prostate cancer cell lines, KH 1060 was the most potent analog by an order of 25- to 35-fold as compared to cmpd C. The second and third most potent analogs were HM and MC 1288. DU-145 was resistant to all the vitamin D3 analogs. The major side-effect of 1,25(OH)2D3 is the production of hypercalcemia. The relative inhibitory index (RII) was determined by comparing the antiproliferative activity of the analog to its ability to produce hypercalcemia in mice injected intraperitoneally every other day. The KH 1060 had the best RTI: 50- to 70-fold greater than 1,25(OH)2D3 for PC-3 and LNCaP, respectively. A trial of one or more of these innovative compounds should be considered for treatment of minimal residual disease of prostate cancer.

Effects of vitamin D<sub>3</sub> and cyclosporin A on HgCl<sub>2</sub>-induced autoimmunity in Brown Norway rats

Mercuric chloride (HgCl2) induces a lymphoproliferative disorder and autoimmune glomerulonephritis in Brown Norway (BN) rats. This syndrome is the consequence of T cell-dependent polyclonal B cell activation and autoantibody production. We have previously shown that HgCl2-induced autoimmune perturbations can be prevented in BN rats by the administration of cyclosporin A (CsA). The most potent vitamin D3 metabolite 1,25(OH)2 D3 (Vit D3) shares certain immunomodulatory properties with CsA. We therefore chose to compare the effects of Vit D3 to those of CsA in BN rats treated with HgCl2 in order to establish whether Vit D3 either alone or in combination with CsA can attenuate an autoimmune syndrome in vivo. BN rats were treated with HgCl2 according to a standard protocol. Subgroups of rats were also given CsA alone, Vit D3 or synthetic analogues of Vit D3 alone, or combinations of both agents. Different doses and routes of administration were compared. The following markers of disease activity were evaluated: mortality, peak proteinuria, serum IgE concentrations, and renal immunoglobulin deposition. Disease activity was markedly attenuated in all rats treated with CsA alone. Vit D3 and certain of its synthetic analogues administered alone also tempered the autoimmune process, but to a lesser extent than did CsA. The effect of CsA alone was so potent, that no additive or synergistic effects could be demonstrated when CsA was administered in combination with Vit D3. Despite similar described immunomodulatory effects in vitro, CsA is clearly more effective than Vit D3 in preventing HgCl2 autoimmune disease in BN rats. This suggests that there is a difference in the cellular targets of these two agents in vivo, and/or a difference in the potency with which HgCl2-triggered immune activation is suppressed.

An Improved Synthesis of 24,24-Difluoro-1.ALPHA.,25-dihydroxyvitamin D3 from Readily Available Vitamin D2.

An improved synthesis of a highly potent vitamin D3 analog, 24, 24-difluoro-1α, 25-dihydroxyvitamin D3 (1b)has been accomplished via vitamin D2-SO2 adducts. The introduction of fluorine atoms was performed by treating the α-keto ester (11) with diethylaminosulfur trifluoride. The total yield was 12.5% from inexpensive vitamin D2 in 11 steps. This sequence is sufficiently straightforward to be conducted on a gram scale.

An improved synthesis of 24,24-difluoro-1.ALPHA.,25-dihydroxyvitamin D3 from vitamin D2.

An improved synthesis of a highly potent vitamin D3 analog, 24, 24-difluoro-1α, 25-dihydroxyvitamin D3 has been accomplished. The total yield was 9.3% from inexpensive vitamin D2 in 11 steps.

Interaction between 24R,25-dihydroxycholecalciferol and 1,25-dihydroxycholecalciferol on 45Ca release from bone in vitro.

Interaction among vitamin D3 metabolites on bone receptor sites is not known. Therefore, interaction between the most potent vitamin D3 metabolite, 1,25(OH)2D3, and the most abundant dihydroxymetabolite, 24R,25(OH)2D3, was studied on isolated rat fetal bone by measuring 45Ca release from prelabeled bones. 24R,25(OH)2D3 at concentrations of 10-50 ng/ml caused marked inhibition of the bone-resorbing activity of 1,25(OH)2D3 at concentrations of 10-50 pg/ml. 24S,25(OH)2 (unnatural enantiometer), on the other hand, at a concentration of 100 ng/ml did not inhibit the bone-resorbing effect of 10 pg/ml 1,25(OH)2D3. 24R,25(OH)2D3 at a concentration of 20 ng/ml did not inhibit the 45Ca-releasing effect of a submaximal concentration of PTH (500 ng/ml). Therefore, the inhibitory effect of 24R,25(OH)2D3 on the bone response to 1,25(OH)2D3 appeared to be specific and probably due to a competitive inhibitory effect. In addition, the inhibitory effect of 24R,25(OH)2D3 was weak, since it could be partially overcome by increasing the concentration of 1,25(OH)2D3.