Effects of Combination of 1,25(OH) 2D 3 and TLR-4 Inhibitor on the Damage to HaCaT Cells Caused by UVB Irradiation.

Objective: Vitamin D and Toll-like receptor-4 (TLR-4) inhibition are involved in the protection of keratinocytes. The effects of combination of 1,25(OH) 2D 3 and TLR-4 inhibitor on the protection of keratinocytes against ultraviolet radiation B (UVB) irradiation remain unclear. This study was undertaken to explore the effects of combination of 1,25(OH) 2D 3 and TAK-242 (TLR-4 inhibitor) on the damage to HaCaT cells caused by UVB irradiation. Methods: In vitro, HaCaT cells were treated with 1,25(OH) 2D 3 or/and TAK-242 prior to UVB irradiation at the intensity of 20 mJ/cm 2, then the production of reactive oxygen species (ROS), cell migration, apoptosis of cells, and the expression of oxidative stress, endoplasmic reticulum stress, and apoptosis related proteins were determined. Results: Compared with the HaCaT cells treated with 1,25(OH) 2D 3 or TAK-242, the cells treated with both 1,25(OH) 2D 3 and TAK-242 showed, 1) significantly lower production of ROS ( P < 0.05); 2) significantly less apoptosis of HaCaT cells ( P < 0.05); 3) significantly lower expression of NF- κB, Caspase-8, Cyto-C, Caspase-3 ( P < 0.05). Conclusion: The combination of 1,25(OH) 2D 3 and TAK-242 could produce a better protection for HaCaT cells via inhibiting the oxidative stress, endoplasmic reticulum stress and apoptosis than 1,25(OH) 2D 3 or TAK-242 alone.

Metabolic activation of tachysterol3 to biologically active hydroxyderivatives that act on VDR, AhR, LXRs, and PPARγ receptors.

CYP11A1 and CYP27A1 hydroxylate tachysterol3 , a photoproduct of previtamin D3 , producing 20S-hydroxytachysterol3 [20S(OH)T3 ] and 25(OH)T3 , respectively. Both metabolites were detected in the human epidermis and serum. Tachysterol3 was also detected in human serum at a concentration of 7.3 ± 2.5 ng/ml. 20S(OH)T3 and 25(OH)T3 inhibited the proliferation of epidermal keratinocytes and dermal fibroblasts and stimulated the expression of differentiation and anti-oxidative genes in keratinocytes in a similar manner to 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ]. They acted on the vitamin D receptor (VDR) as demonstrated by image flow cytometry and the translocation of VDR coupled GFP from the cytoplasm to the nucleus of melanoma cells, as well as by the stimulation of CYP24A1 expression. Functional studies using a human aryl hydrocarbon receptor (AhR) reporter assay system revealed marked activation of AhR by 20S(OH)T3 , a smaller effect by 25(OH)T3 , and a minimal effect for their precursor, tachysterol3 . Tachysterol3 hydroxyderivatives showed high-affinity binding to the ligan-binding domain (LBD) of the liver X receptor (LXR) α and ß, and the peroxisome proliferator-activated receptor γ (PPARγ) in LanthaScreen TR-FRET coactivator assays. Molecular docking using crystal structures of the LBDs of VDR, AhR, LXRs, and PPARγ revealed high docking scores for 20S(OH)T3 and 25(OH)T3 , comparable to their natural ligands. The scores for the non-genomic-binding site of the VDR were very low indicating a lack of interaction with tachysterol3 ligands. Our identification of endogenous production of 20S(OH)T3 and 25(OH)T3 that are biologically active and interact with VDR, AhR, LXRs, and PPARγ, provides a new understanding of the biological function of tachysterol3 .

Synthesis of C2-Alkoxy-Substituted 19-Nor Vitamin D3 Derivatives: Stereoselectivity and Biological Activity.

The active form of vitamin D3 (D3), 1a,25-dihydroxyvitamn D3 (1,25D3), plays a central role in calcium and bone metabolism. Many structure-activity relationship (SAR) studies of D3 have been conducted, with the aim of separating the biological activities of 1,25D3 or reducing its side effects, such as hypercalcemia, and SAR studies have shown that the hypercalcemic activity of C2-substituted derivatives and 19-nor type derivatives is significantly suppressed. In the present paper, we describe the synthesis of 19-nor type 1,25D3 derivatives with alkoxy groups at C2, by means of the Julia-Kocienski type coupling reaction between a C2 symmetrical A ring ketone and a CD ring synthon. The effect of C2 substituents on the stereoselectivity of the coupling reaction was evaluated. The biological activities of the synthesized derivatives were evaluated in an HL-60 cell-based assay. The a-methoxy-substituted C2α-7a was found to show potent cell-differentiating activity, with an ED50 value of 0.38 nM, being 26-fold more potent than 1,25D3.

1α,25-(OH)2 vitamin D3 prevents insulin resistance and regulates coordinated exocytosis and insulin secretion.

Vitamin D3 is associated with improvements in insulin resistance and glycemia. In this study, we investigated the short-term effect of 1α,25(OH)2 Vitamin D3 (1,25-D3) and cholecalciferol (vitamin D3) on the glycemia and insulin sensitivity of control and dexamethasone-induced insulin-resistance rats. 45Ca2+ influx responses to 1,25-D3 and its role in insulin secretion were investigated in isolated pancreatic islets from control rats. In vivo, 5 d treatment with 1,25-D3 (i.p.) prevented insulin resistance in dexamethasone-treated rats. Treatment with 1,25-D3 improved the activities of hepatic enzymes, serum lipids and calcium concentrations in insulin-resistant rats. 25-D3 (o.g.) does not affect insulin resistance. In pancreatic islets, 1,25-D3 increased insulin secretion and stimulated rapid response 45Ca2+ influx. The stimulatory effect of 1,25-D3 on 45Ca2+ influx was decreased by diazoxide, apamine, thapsigargin, dantrolene, 2-APB, nifedipine, TEA, PKA, PKC, and cytoskeleton inhibitor, while it was increased by glibenclamide and N-ethylmaleimide. The stimulatory effect of 1,25-D3 on 45Ca2+ influx involves the activation of L-type VDCC, K+-ATP, K+-Ca2+, and Kv channels, which augment cytosolic calcium. These ionic changes mobilize calcium from stores and downstream activation of PKC, PKA tethering vesicle traffic and fusion at the plasma membrane for insulin secretion. This is the first study highlighting the unprecedented role of 1,25-D3 (short-term effect) in the regulation of glucose homeostasis and on prevention of insulin resistance. Furthermore, this study shows the intracellular ß-cell signal transduction of 1,25-D3 through the modulation of pivotal ionic channels and proteins exhibiting a coordinated exocytosis of vesicles for insulin secretion.

Sun Exposure and Vitamin D.

Vitamin D is generally accepted in its importance on the regulation of calcium homeostasis and bone metabolism. Moreover, further health effects due to vitamin D are under discussion. In its effect, vitamin D is more like a hormone. In the classic view, a vitamin is an essential nutrient, which cannot be synthesized independently in the body. Besides nutrition, vitamin D will be produced in the body itself. The skin contains the provitamin D3 7-dehydrocholesterol, a precursor of vitamin D. Provitamin D3 will be photoconverted to previtamin D3 by UVB radiation that penetrates the skin superficially. In this way, the vitamin D metabolism will be started independent of the nutrition. In everyday life, this photosynthesis will be carried out due to the solar UVB radiation penetrating the uncovered skin. In the same spectral waveband range of UVB radiation, which causes the beneficial health effect of starting the vitamin D metabolism, the UVB radiation causes simultaneously acute and chronic harmful health effects as UV erythema (sunburn), skin aging and skin cancer. There is no vitamin D production in the skin without simultaneous DNA damage in the skin. Against this background, risks and benefits have to be balanced carefully.

Probing the Formation and Conformational Relaxation of Previtamin D3 and Analogues in Solution and in Lipid Bilayers.

The photosynthesis of vitamin D3 in mammalian skin results from UV-B irradiation of provitamin D3 (7-dehydrocholesterol, DHC) at ca. 290 nm. Upon return to the ground state, the hexatriene product, previtamin D3, undergoes a conformational equilibration between helical gZg and more planar tZg and tZt forms. The helical gZg forms provide a pathway for the formation of vitamin D3 via a [1,7]-sigmatropic hydrogen shift. Steady state photolysis and UV transient absorption spectroscopy are combined to explore the conformational relaxation of previtamin D3 formed from DHC in isotropic solution and confined to lipid bilayers chosen to model the biological cell membrane. The results are compared with measurements for two analogues: previtamin D2 formed from ergosterol (provitamin D2) and previtamin D3 acetate formed from DHC acetate. The resulting spectral dynamics are interpreted in the context of simulations of optical excitation energy and oscillator strength as a function of conformation. In solution, the relaxation dynamics and steady state product distributions of the three compounds are nearly identical, favoring tZg forms. When confined to lipid bilayers, the heterogeneity and packing forces alter the conformational distributions and enhance the population of a gZg conformer capable of vitamin D formation.

17,20S(OH)2pD Can Prevent the Development of Skin Fibrosis in the Bleomycin-Induced Scleroderma Mouse Model.

Systemic sclerosis (SSc; scleroderma) is a chronic fibrotic disease involving TGF-ß1. Low serum vitamin D (vit D) correlates with the degree of fibrosis and expression of TGF-ß1. This study was designed to determine whether the noncalcemic vit D analog, 17,20S(OH)2pD, suppresses fibrosis and mediators of the TGF-ß1 pathway in the bleomycin (BLM) model of fibrosis. Fibrosis was induced into the skin of female C57BL/6 mice by repeated injections of BLM (50 µg/100 µL) subcutaneously. Mice received daily oral gavage with either vehicle (propylene glycol) or 17,20S(OH)2pD using 5, 15, or 30 µg/kg for 21 days. The injected skin was biopsied; analyzed histologically; examined for total collagen by Sircol; and examined for mRNA expression of MMP-13, BMP-7, MCP-1, Gli1, and Gli2 by TR-PCR. Spleen was analyzed for lymphocytes using flow cytometry. Serum was analyzed for cytokines using a multiplexed ELISA. Results showed that all three doses of 17,20S(OH)2pD suppressed net total collagen production, dermal thickness, and total collagen content in the BLM fibrosis model. 17,20S(OH)2pD also increased MMP-13 expression, decreased MCP-1 and Gli-2 expression in vivo, and suppressed serum levels of IL-13, TNF-α, IL-6, IL-10, IL-17, and IL-12p70. In summary, 17,20S(OH)2pD modulates the mediators of fibrosis in vivo and suppresses total collagen production and dermal thickness. This antifibrotic property of 17,20S(OH)2pD offers new therapeutic approaches for fibrotic disorders.

Chemical Synthesis of Side-Chain-Hydroxylated Vitamin D3 Derivatives and Their Metabolism by CYP27B1.

1α,25-Dihydroxyvitamin D3 (abbreviated here as 1,25D3 ) is a hormonally active form of vitamin D3 (D3 ), and is produced from D3 by CYP27 A1-mediated hydroxylation at C25, followed by CYP27B1-mediated hydroxylation at C1. Further hydroxylation of 25D3 and 1,25D3 occurs at C23, C24 and C26 to generate corresponding metabolites, except for 1,25R,26D3 . Since the capability of CYP27B1 to hydroxylate C1 of side-chain-hydroxylated metabolites other than 23S,25D3 and 24R,25D3 has not been examined, we have here explored the role of CYP27B1 in the C1 hydroxylation of a series of side-chain-hydroxylated D3 derivatives. We found that CYP27B1 hydroxylates the R diastereomers of 24,25D3 and 25,26D3 more effectively than the S diastereomers, but shows almost no activity towards either diastereomer of 23,25D3 . This is the first report to show that CYP27B1 metabolizes 25,26D3 to the corresponding 1α-hydroxylated derivative, 1,25,26D3 . It will be interesting to examine the physiological relevance of this finding.

Recent Advances in Psoriasis Therapy: Trends and Future Prospects.

BACKGROUND: Psoriasis is a challenging skin disorder due to its chronicity, high rate of prevalence, disability, comorbidity and disfiguration. It is a multi-system disorder that includes joints and metabolic syndromes. Psoriasis is a condition of pathologic interaction among immune cells, biological signaling molecules and skin cells. Several contributing factors are responsible for the exacerbation and onset of psoriasis, i.e. genetic factors and environmental factors such as medications, infectious diseases and lifestyle. OBJECTIVES: To study the new insights in the treatment of psoriasis and future prospects. METHODS: This review article gives an insight on the current concepts of psoriasis and deals with discussing the initiation and development of the diseases. We described the pathogenetic pathway for psoriasis. The article focuses on the treatment approaches for psoriasis that have arisen from the dissection of the inflammatory psoriatic pathways. RESULTS: We aimed to highlight the novel therapies and drugs used in the treatment of psoriasis, including food and drug administration (FDA) approved drugs and drugs under clinical trials. The treatment can be initiated for mild to the moderate diseased condition employing vitamin D3 analogues, corticosteroids and a combination of products as first-line therapy. CONCLUSION: Psoriasis can be managed by a proper understanding of the immune function. We have also discussed medicinal herbs used for psoriasis based on their ethnopharmacological knowledge and reported work of researchers.

Antifibrogenic Activities of CYP11A1-derived Vitamin D3-hydroxyderivatives Are Dependent on RORγ.

Previous studies showed that noncalcemic 20(OH)D3, a product of CYP11A1 action on vitamin D3, has antifibrotic activity in human dermal fibroblasts and in a bleomycin mouse model of scleroderma. In this study, we tested the role of retinoic acid-related orphan receptor γ (RORγ), which is expressed in skin, in the action of CYP11A1-derived secosteroids using murine fibroblasts isolated from the skin of wild-type (RORγ +/+), knockout (RORγ -/-), and heterozygote (RORγ +/-) mice. CYP11A1-derived 20(OH)D3, 20,23(OH)2D3, 1,20(OH)2D3, and 1,20,23(OH)3D3 inhibited proliferation of RORγ +/+ fibroblasts in a dose-dependent manner with a similar potency to 1,25(OH)2D3. Surprisingly, this effect was reversed in RORγ +/- and RORγ -/- fibroblasts, with the most pronounced stimulatory effect seen in RORγ -/- fibroblasts. All analogs tested inhibited TGF-ß1-induced collagen synthesis in RORγ +/+ fibroblasts and the expression of other fibrosis-related genes. This effect was curtailed or reversed in RORγ -/- fibroblasts. These results show that the antiproliferative and antifibrotic activities of the vitamin D hydroxy derivatives are dependent on a functional RORγ. The dramatic changes in the transcriptomes of fibroblasts of RORγ -/- versus wild-type mice following treatment with 20(OH)D3 or 1,20(OH)2D3 provide a molecular basis to explain, at least in part, the observed phenotypic differences.

Simplified LC-MS Method for Analysis of Sterols in Biological Samples.

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Photoprotective Properties of Vitamin D and Lumisterol Hydroxyderivatives.

We have previously described new pathways of vitamin D3 activation by CYP11A1 to produce a variety of metabolites including 20(OH)D3 and 20,23(OH)2D3. These can be further hydroxylated by CYP27B1 to produce their C1α-hydroxyderivatives. CYP11A1 similarly initiates the metabolism of lumisterol (L3) through sequential hydroxylation of the side chain to produce 20(OH)L3, 22(OH)L3, 20,22(OH)2L3 and 24(OH)L3. CYP11A1 also acts on 7-dehydrocholesterol (7DHC) producing 22(OH)7DHC, 20,22(OH)27DHC and 7-dehydropregnenolone (7DHP) which can be converted to the D3 and L3 configurations following exposure to UVB. These CYP11A1-derived compounds are produced in vivo and are biologically active displaying anti-proliferative, anti-inflammatory, anti-cancer and pro-differentiation properties. Since the protective role of the classical form of vitamin D3 (1,25(OH)2D3) against UVB-induced damage is recognized, we recently tested whether novel CYP11A1-derived D3- and L3-hydroxyderivatives protect against UVB-induced damage in epidermal human keratinocytes and melanocytes. We found that along with 1,25(OH)2D3, CYP11A1-derived D3-hydroxyderivatives and L3 and its hydroxyderivatives exert photoprotective effects. These included induction of intracellular free radical scavenging and attenuation and repair of DNA damage. The protection of human keratinocytes against DNA damage included the activation of the NRF2-regulated antioxidant response, p53-phosphorylation and its translocation to the nucleus, and DNA repair induction. These data indicate that novel derivatives of vitamin D3 and lumisterol are promising photoprotective agents. However, detailed mechanisms of action, and the involvement of specific nuclear receptors, other vitamin D binding proteins or mitochondria, remain to be established.

Efficient synthesis of 3-TBDMS-11α,25-dihydroxyvitamin D3 and D2 ethers.

Vitamin D deficiency might cause a wide variety of human disorders. As a prerequisite for appropriate diagnosis and therapy, medicinally relevant vitamin D metabolites have to be assayed most accurately and with high specificity. It has been demonstrated, that vitamin D conjugates, linked via a hydroxyl group at C11, might be promising for the development of highly specific antibodies to be employed in competitive protein binding assays. The connective synthesis of 3-TBDMS-11α,25-dihydroxyvitamin D3 and D2 ethers in 500 mg scale, starting from vitamin D2, is described. For installation of a hydroxyl group at C11 a sequence of Pd(OAc)2 mediated oxidation of an enone, epoxidation and subsequent epoxide ring opening was applied to obtain a suitable CD-ring precursor, that was connected with an A-ring diphenylphosphine oxide by Wittig-Horner reaction. Finally, an appropriate side chain was installed, respectively.

Antibacterial effect of indene on Helicobacter pylori correlates with specific interaction between its compound and dimyristoyl-phosphatidylethanolamine.

Recent studies by our group have suggested that the vitamin D3 decomposition product VDP1 [(1R,3aR,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-inden-4-one] confers the potent bactericidal action to Helicobacter pylori by targeting the membranal dimyristoyl-phosphatidylethanolamine (di-14:0 PE). In this study we synthesized a new VDP1 derivative to advance further investigation as for the correlative relationship between VDP1 structure and anti-H. pylori activity or PE vesicle collapse induction activity. The derivative VD3-7 [(1R,7aR)-4-fluoro-7a-methyl-1-((R)-6-methylheptan-2-yl)octahydro-1H-indene] retained a fluorine atom in place of the oxygen atom of VDP1. The fluorination of the carbonyl portion of VDP1 forfeited the effective anti-H. pylori activity. We, therefore, prepared Coomassie brilliant blue (CBB)-containing unilamellar vesicles consisting of various PE molecular species, and examined the vesicle collapse induction activity of either VDP1 or VD3-7 by detecting the CBB eluted from the PE unilamellar vesicles. VDP1 strongly induced CBB elution from the unilamellar vesicles of rectus-PE retaining the same two fatty acid side-chains shorter than carbon numbers 14, indicating that VDP1 specifically disrupted the vesicular conformation of those PE unilamellar vesicles. Meanwhile, VD3-7 had no influence on the structural stability of any PE unilamellar vesicles. This study obtained additional evidence that VDP1 acts as a bactericidal agent on H. pylori by targeting the membranal di-14:0 PE.

UV light suppression of EAE (a mouse model of multiple sclerosis) is independent of vitamin D and its receptor.

Vitamin D and sunlight have each been reported to protect against the development of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (MS). To date, the contribution of each has been unclear as ultra violet (UV) exposure also causes the generation of vitamin D in the skin. To examine whether the UV based suppression of EAE results, at least, in part from the production of vitamin D, we studied the effect of UV light on EAE in mice unable to produce 7-dehydroxycholesterol (7-DHC), the required precursor of vitamin D. Furthermore, we examined UV suppression of EAE in mice devoid of the vitamin D receptor (VDR). Our results demonstrate that UV light suppression of EAE occurs in the absence of vitamin D production and in the absence of VDR. Future investigations will focus on identifying the pathway responsible for the protective action of UV in EAE and presumably human MS.

Development of novel lithocholic acid derivatives as vitamin D receptor agonists.

Lithocholic acid (2) was identified as the second endogenous ligand of vitamin D receptor (VDR), though its binding affinity to VDR and its vitamin D activity are very weak compared to those of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1). 3-Acylated lithocholic acids were reported to be slightly more potent than lithocholic acid (2) as VDR agonists. Here, aiming to develop more potent lithocholic acid derivatives, we synthesized several derivatives bearing a 3-sulfonate/carbonate or 3-amino/amide substituent, and examined their differentiation-inducing activity toward human promyelocytic leukemia HL-60 cells. Introduction of a nitrogen atom at the 3-position of lithocholic acid (2) decreased the activity, but compound 6 bearing a 3-methylsulfonate group showed more potent activity than lithocholic acid (2) or its acylated derivatives. The binding of 6 to VDR was confirmed by competitive binding assay and X-ray crystallographic analysis of the complex of VDR ligand-binding domain (LBD) with 6.

Protective effects of novel derivatives of vitamin D3 and lumisterol against UVB-induced damage in human keratinocytes involve activation of Nrf2 and p53 defense mechanisms.

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

WY 1048, a 17-methyl 19-nor D-ring analog of vitamin D3, in combination with risedronate restores bone mass in a mouse model of postmenopausal osteoporosis.

Bisphosphonates like risedronate inhibit osteoclast-mediated bone resorption and are therefore used in the prevention and treatment of osteoporosis. Also vitamin D3 and calcium supplementation is commonly used in the prevention or treatment of osteoporosis. Combined therapy of risedronate with 1,25(OH)2D3, the active metabolite of vitamin D3, may be advantageous over the use of either monotherapy, but bears a risk of causing hypercalcemia thereby decreasing the therapeutic window for osteoporosis treatment. In this study, we evaluated the effect on bone mass of the combination of risedronate with the 17-methyl 19-nor five-membered D-ring vitamin D3 analog WY 1048 in a mouse ovariectomy model for postmenopausal osteoporosis. Ovariectomy-induced bone loss was restored by administration of risedronate or a combination of risedronate with 1,25(OH)2D3. However, the combination of WY 1048 with risedronate induced an even higher increase on total body and spine bone mineral density and on trabecular and cortical bone mass. Our data indicate that combination therapy of risedronate with WY 1048 was superior in restoring and improving bone mass over a combination of risedronate with 1,25(OH)2D3 with minimal calcemic side effects.

Synthesis and evaluation of third generation vitamin D3 analogues as inhibitors of Hedgehog signaling.

The Hedgehog (Hh) pathway is a developmental pathway with therapeutic potential as a target for a variety of cancers. In recent years, several vitamin D-based compounds have been identified as potent inhibitors of Hh signaling. These analogues contain aromatic phenol A-ring mimics coupled to the CD-ring side chain of vitamin D3 through modified seco-B regions. To continue structure-activity relationship studies on this class of Hh pathway inhibitors, multiple series of vitamin D-based analogues that contain an amine-based seco-B tether and/or incorporate a hydroxyl moiety on C-25 were designed and synthesized. These compounds were evaluated in multiple cell lines for their anti-Hh activity, and we identify analogues 16, 21, 22 as potent vitamin D-based Hh inhibitors (IC50 values of 110-340 nM). We also performed a series of mechanism of action studies in knockout cell lines to further explore whether these analogues inhibit the Hh pathway through a known Hh pathway component or the vitamin D receptor. While the specific cellular target that mediates these effects remains elusive, our studies suggest multiple cellular targets may mediate the anti-Hh activity of this scaffold.