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 .

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.

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.

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.

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.

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.

Stereoselective Palladium-Catalyzed Approach to Vitamin D3 Derivatives in Protic Medium.

We describe an efficient convergent synthesis of vitamin D3 metabolites and analogues. The synthetic strategy relies on a tandem Pd-catalyzed A-ring closure and Suzuki-Miyaura coupling to the CD-side chain component to set directly the vitamin D triene system under protic conditions. This strategy enables rapid access to vitamin D3 and 3-epi-vitamin D3 metabolites and analogues modified at the side chain for biological evaluation and structural and metabolic studies.

Effects of 2-substitution on 14-epi-19-nortachysterol-mediated biological events: based on synthesis and X-ray co-crystallographic analysis with the human vitamin D receptor.

Both 2α- and 2ß-hydroxypropyl substituted 14-epi-1α,25-dihydroxy-19-nortachysterols were synthesized to study the human vitamin D receptor (hVDR) binding affinity, binding configurations, and interactions with amino acid residues in the ligand binding domain of hVDR by X-ray co-crystallographic analysis. In conjunction with our previous results on 14-epi-19-nortachysterol, 2-methylidene-, 2α-methyl-, 2ß-methyl, and 2α-hydroxypropoxy-14-epi-19-nortachysterol, we propose a variety of effects of substitution at the C2 position in the 14-epi-19-nortachysterol skeleton on biological activities.

Skin-derived TSLP systemically expands regulatory T cells.

Regulatory T cells (Tregs) are a subset of CD4+ T cells with suppressive function and are critical for limiting inappropriate activation of T cells. Hence, the expansion of Tregs is an attractive strategy for the treatment of autoimmune diseases. Here, we demonstrate that the skin possesses the remarkable capacity to systemically expand Treg numbers by producing thymic stromal lymphopoietin (TSLP) in response to vitamin D receptor stimulation. An ∼2-fold increase in the proportion and absolute number of Tregs was observed in mice treated topically but not systemically with the Vitamin D3 analog MC903. This expansion of Tregs was dependent on TSLP receptor signaling but not on VDR signaling in hematopoietic cells. However, TSLP receptor expression by Tregs was not required for their proliferation. Rather, skin-derived TSLP promoted Treg expansion through dendritic cells. Importantly, treatment of skin with MC903 significantly lowered the incidence of autoimmune diabetes in non-obese diabetic mice and attenuated disease score in experimental autoimmune encephalomyelitis. Together, these data demonstrate that the skin has the remarkable potential to control systemic immune responses and that Vitamin D-mediated stimulation of skin could serve as a novel strategy to therapeutically modulate the systemic immune system for the treatment of autoimmunity.

Eldecalcitol, an Active Vitamin D3 Derivative, Prevents Trabecular Bone Loss and Bone Fragility in Type I Diabetic Model Rats.

Diabetes mellitus is known to adversely affect the bones and be associated with increased fracture risk. We examined whether eldecalcitol (ELD), an active vitamin D3 derivative, could inhibit the diabetic bone loss in streptozotocin-induced type I diabetic rats. ELD (10, 20, or 40 ng/kg), alfacalcidol (ALF; 25, 50, or 100 ng/kg), or vehicle was administered 5 times per week for 12 weeks from 1 week after diabetes induction. Normal control rats received the vehicle. Bone turnover markers, bone mineral density (BMD), and biomechanical strength of the lumbar spine and femur were measured, and bone histomorphometry was performed. Content of advanced glycation end products (AGEs) in the femoral shaft was also determined. In diabetic rats, serum osteocalcin (OC) concentration was lower and urinary excretion of deoxypyridinoline (DPD) tended to be higher than in normal rats. Areal BMD and maximum load of the lumbar vertebrae and femoral shaft were lower in diabetic rats than in normal rats. All doses of ELD and the highest dose of ALF reduced urinary DPD excretion, but had no effect on serum OC. The 20 and 40 ng/kg doses of ELD prevented decreases in BMD and the highest dose of ELD prevented the reduction in maximum load of the lumbar vertebrae, while ALF did not change these parameters. ELD and ALF did not affect areal BMD or biomechanical strength of the femoral shaft. In diabetic rats, bone volume and trabecular thickness in the trabecular bone of the lumbar vertebrae decreased and trabecular separation increased compared to normal rats. ELD and ALF prevented diabetes-induced deterioration of trabecular microstructure. AGE content in the femoral cortical bone increased in the diabetic rats, and ELD and ALF did not change AGE content compared to the diabetic rats. These results indicated that ELD suppressed bone resorption and prevented trabecular bone loss and deterioration of trabecular microstructure, resulting in prevention of reduction in biomechanical strength in type I diabetic rats.

Synthesis and evaluation of vitamin D3 analogues with C-11 modifications as inhibitors of Hedgehog signaling.

Previous structure-activity relationship studies have provided potent and selective analogues of vitamin D3 as inhibitors of the Hedgehog (Hh) signaling pathway. These analogues contain both modified A- and seco-B ring motifs, and have been evaluated for anticancer therapeutic potential. To continue our studies on this scaffold, a new series of compounds were synthesized to explore additional interactions and spatial constraints. These compounds incorporate functional groups of varying size and hydrophobicity at the C-11 position. While large hydrophobic moieties (9c-e) resulted in significant loss of Hh inhibition, smaller or more flexible moieties (9a, 11) maintain anti-Hh activity. These results call for additional and continued studies to identify the binding pocket to better understand these structure-activity relationships.

The role of tachysterol in vitamin D photosynthesis - a non-adiabatic molecular dynamics study.

To investigate the role of tachysterol in the photophysical/photochemical regulation of vitamin D photosynthesis, we studied its electronic absorption properties and excited state dynamics using time-dependent density functional theory (TDDFT), second-order approximate coupled cluster theory (CC2), and non-adiabatic surface hopping molecular dynamics in the gas phase. In excellent agreement with experiments, the simulated electronic spectrum shows a broad absorption band with a remarkably higher extinction coefficient than the other vitamin D photoisomers provitamin D, lumisterol, and previtamin D. The broad band arises from the spectral overlap of four different ground state rotamers. After photoexcitation, the first excited singlet state (S1) decays with a lifetime of 882 fs. The S1 dynamics is characterized by a strong twisting of the central double bond. In 96% of all trajectories this is followed by unreactive relaxation to the ground state near a conical intersection. The double-bond twisting in the chemically unreactive trajectories induces a strong interconversion between the different rotamers. In 2.3% of the trajectories we observed [1,5]-sigmatropic hydrogen shift forming the partly deconjugated toxisterol D1. 1.4% previtamin D formation is observed via hula-twist double bond isomerization. In both reaction channels, we find a strong dependence between photoreactivity and dihedral angle conformation: hydrogen shift only occurs in cEc and cEt rotamers and double bond isomerization occurs mainly in cEc rotamers. Hence, our study confirms the previously formed hypothesis that cEc rotamers are more prone to previtamin D formation than other isomers. In addition, we also observe the formation of a cyclobutene-toxisterol in the hot ground state in 3 trajectories (0.7%). Due to its large extinction coefficient and mostly unreactive behavior, tachysterol acts mainly as a Sun shield suppressing previtamin D formation. Tachysterol shows stronger toxisterol formation than previtamin D and can thus be seen as the major degradation route of vitamin D. Absorption of low energy ultraviolet light by the cEc rotamer can lead to previtamin D formation. In addition, the cyclobutene-toxisterol, which possibly reacts thermally to previtamin D, is also preferably formed at long wavelengths. These two mechanisms are consistent with the wavelength dependent photochemistry found in experiments. Our study reinforces a recent hypothesis that tachysterol constitutes a source of previtamin D when only low energy ultraviolet light is available, as it is the case in winter or in the morning and evening hours of the day.

Lumisterol to Tachysterol Photoisomerization in EPA Glass at 77 K. A Comparative Study.

We present a comparative study of the photoisomerizations of lumisterol (Lumi), previtamin (Pre), and provitamin D3 (Pro) to tachysterol (Tachy) at 77 K in EPA (5:5:2 ether, isopentane, and ethanol by volume) glass. Fluorescence, fluorescence excitation, and UV spectra, measured in the course of these reactions, were analyzed using singular value decomposition with self-modeling (SVD-SM). This represents an extension of previous work that led to the conclusion that in the EPA glass Pre exists as an s-cis,s-cis-conformer (cZc-Pre) which gives, exclusively, an unstable s-cis,s-cis-conformer of Tachy (cEc-Tachy) and Pro gives mainly the tEc-Tachy, that corresponds to a stable s-trans,s-cis-conformer. ( Redwood , C. ; et al. J. Phys. Chem. Lett. 2013 , 4 , 716 - 721 . ) The surprising result was that the major Pre photoproduct from Pro also has a tZc-Pre conformation instead of the expected cZc-Pre conformation. Accordingly, the Pre to Tachy cis-trans photoisomerization proceeds via a conformer specific one-bond-twist (OBT) process as proposed by Havinga ( Maessen , P. A. ; et al. Angew. Chem. Int. Ed. Engl. 1983 , 22 , 718 - 719 . Maessen , P. A. ; et al. Angew. Chem. Int. Ed. Engl. 1983 , 22 , 994 - 1004 . Maessen , P. A. Ph.D. Thesis, State University at Leiden, Leiden, The Netherlands, 1983. ). The role of the EPA glass in controlling conformer distributions and reaction outcomes is further explored by the extension of the studies to Lumi, whose structure differs substantially from that of its stereoisomer, Pro. Initially, the light-induced conrotatory ring openings of Pro and Lumi are expected to give cZc-Pre conformers that differ in the relative orientation of the double bond dihedral angles that define the chiral axis of the triene moiety: (-)cZ(-)c-Pre and (+)cZ(+)c-Pre, respectively. In the case of Pro, much of the cZc-Pre proceeds to tZc-Pre, the precursor of tEc-Tachy. In contrast, we show that under the same conditions most cZc-Pre formed from Lumi retains the cZc-conformation and isomerizes to cEc-Tachy. cZc-Pre from Lumi was not detected by fluorescence, but UV absorption measurements establish its formation as an essential intermediate to Tachy. Aided by theoretical calculations of conformer UV and CD spectra, we conclude that fluorescent thermodynamic Pre and nonfluorescent Pre from Lumi are both (+)cZ(+)c-Pre conformers. They differ in the orientation of the OH in the A ring, pseudoequatorial in the former and pseudoaxial in the latter. The most likely major photochemical sequences starting from Pre and Lumi are (+)cZ(+)c-Pre-eq-OH → (+)cE(+)c-Tachy-eq-OH and Lumi → (+)cZ(+)c-Pre-ax-OH → (+)cE(+)c-Tachy-eq-OH.

Topical application of a vitamin D3 analogue and corticosteroid to psoriasis plaques decreases skin infiltration of TH17 cells and their ex vivo expansion.

BACKGROUND: Topical combination of a vitamin D3 analogue and corticosteroid is widely used for the treatment of psoriasis, a TH17-mediated disorder, but the underlying mechanism remains unclear. OBJECTIVE: We investigated the effect of this topical applicant, focusing on skin-infiltrating TH17 cells. METHODS: In 10 patients with plaque psoriasis, calcipotriol (Cal), betamethasone dipropionate (Bet), or the calcipotriol and betamethasone dipropionate 2-compound formulation (CB) was applied to 3 different psoriatic plaques with similar severity once a day for 14 days. One nonapplied lesion was used as a control. Four-millimeter biopsy specimens were taken from each site, cut into 2 pieces, and subjected to histologic examination and ex vivo expansion of skin-infiltrating T cells with anti-CD3/CD28 antibodies and IL-2. RESULTS: Clinical, histologic, and IL-17A(+) cell-infiltrate improvement was found in the following order: CB > Cal > Bet > control or CB > Bet > Cal > control. Numbers of ex vivo expanded T cells were decreased by topical application of Bet and CB, and CB exhibited the most suppressive result. Numbers and frequencies of TH17 cells were significantly reduced by CB and Cal, suggesting that Cal has a capacity to preferentially suppress TH17 cells. When the stocked T cells from control samples were stimulated with anti-CD3 antibodies in the presence of Bet, Cal, or both, Cal downmodulated IL-17 and IFN-γ production and tended to upregulate IL-4 and IL-6 without apoptosis, but Bet inhibited production of these cytokines with apoptosis. CONCLUSION: These findings suggest that Cal and Bet have different effects on T cells to normalize psoriatic changes, with decreased TH17 cell expansion in the skin lesions.

A Metabolomic and Lipidomic Serum Signature from Nonhuman Primates Administered with a Promising Radiation Countermeasure, Gamma-Tocotrienol.

The development of radiation countermeasures for acute radiation syndrome (ARS) has been underway for the past six decades, leading to the identification of multiple classes of radiation countermeasures. However, to date, only two growth factors (Neupogen and Neulasta) have been approved by the United States Food and Drug Administration (US FDA) for the mitigation of hematopoietic acute radiation syndrome (H-ARS). No radioprotector for ARS has been approved by the FDA yet. Gamma-tocotrienol (GT3) has been demonstrated to have radioprotective efficacy in murine as well as nonhuman primate (NHP) models. Currently, GT3 is under advanced development as a radioprotector that can be administered prior to radiation exposure. We are studying this agent for its safety profile and efficacy using the NHP model. In this study, we analyzed global metabolomic and lipidomic changes using ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) in serum samples of NHPs administered GT3. Our study, using 12 NHPs, demonstrates that alterations in metabolites manifest only 24 h after GT3 administration. Furthermore, metabolic changes are associated with transient increase in the bioavailability of antioxidants, including lactic acid and cholic acid and anti-inflammatory metabolites 3 deoxyvitamin D3, and docosahexaenoic acid. Taken together, our results show that the administration of GT3 to NHPs causes metabolic shifts that would provide an overall advantage to combat radiation injury. This initial assessment also highlights the utility of metabolomics and lipidomics to determine the underlying physiological mechanisms involved in the radioprotective efficacy of GT3.

The action spectrum for vitamin D3: initial skin reaction and prolonged exposure.

Vitamin D3 photosynthesis in the skin is formulated as a set of reaction equations, including side-reactions to lumisterol, tachysterol and toxisterols, and the accompanying reverse reactions, isomerisation of previtamin D3 to vitamin D3 and photodegradation of vitamin D3. The solution of this set is given for the stationary irradiance spectrum. The effective action spectrum for the instantaneous vitamin D3 production changes shape as a function of exposure, and therefore, no single action spectrum can be used. We assessed the action spectrum for unexposed skin and for skin that has been exposed to 7.5 Standard Erythemal Doses (SED). We constructed two new estimates: (1) the RIVM action spectrum, based on absorption spectra, quantum yields and skin transmission spectra, and (2) the modified QUT action spectrum, which is adjusted for self-absorption and skin transmission. For previously unexposed skin, the modified QUT action spectrum gives a qualitatively similar, but larger estimate than the RIVM action spectrum. We have not been able to solve the lack of quantitative agreement between the vitamin D production estimates from the three action spectrum estimates (RIVM, modified QUT and CIE). All new action spectra have stronger emphasis on the short wavelengths than the CIE action spectrum. We showed that, for wavelengths larger than 300 nm, the bandwidth that was used in the experiment that formed the basis of the CIE action spectrum, gives a red-shift of about 1 nm. Generally, with the formation of previtamin D3, the return reaction to provitamin D3 limits the production of vitamin D3. After some exposure, the new action spectrum has negative values for the longer wavelengths in the UVB. For the RIVM action spectrum, this happens after 7.5 SED, for the modified QUT action spectrum already after 1.25 SED, and after 7.5 SED the net production rate is largely cancelled. Thus prolonged exposure of previously unexposed skin saturates vitamin D3 formation. For maximum vitamin D production after 1.25 SED, sunscreens should block wavelengths larger than 310 nm. Sunscreens that block only UVB could result in reduction in vitamin D production after prolonged exposure, or even a destruction of vitamin D that has just been formed.

Synthesis of 24,24-Difluoro-1,3-cis-25-dihydroxy-19-norvitamin D3 Derivatives and Evaluation of Their Vitamin D Receptor-Binding Affinity.

Two vitamin D3 derivatives, namely 24,24-difluoro-1ß,3ß,25-dihydroxy-19-norvitamin D3 (6a) and 24,24-difluoro-1α,3α,25-dihydroxy-19-norvitamin D3 (6b), were synthesized via a convergent route employing Julia-Kocienski olefination as a key step. Compounds 6a and b bound to vitamin D receptor (VDR) with IC50 values of 64.8 and 57.6 nM, respectively, exhibiting about 400- and 30-fold greater binding affinity than the corresponding non-fluorinated derivatives 5a and b.

The Vitamin D Analog, MART-10, Attenuates Triple Negative Breast Cancer Cells Metastatic Potential.

Regarding breast cancer treatment, triple negative breast cancer (TNBC) is a difficult issue. Most TNBC patients die of cancer metastasis. Thus, to develop a new regimen to attenuate TNBC metastatic potential is urgently needed. MART-10 (19-nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3), the newly-synthesized 1α,25(OH)2D3 analog, has been shown to be much more potent in cancer growth inhibition than 1α,25(OH)2D3 and be active in vivo without inducing obvious side effect. In this study, we demonstrated that both 1α,25(OH)2D3 and MART-10 could effectively repress TNBC cells migration and invasion with MART-10 more effective. MART-10 and 1α,25(OH)2D3 induced cadherin switching (upregulation of E-cadherin and downregulation of N-cadherin) and downregulated P-cadherin expression in MDA-MB-231 cells. The EMT(epithelial mesenchymal transition) process in MDA-MB-231 cells was repressed by MART-10 through inhibiting Zeb1, Zeb2, Slug, and Twist expression. LCN2, one kind of breast cancer metastasis stimulator, was also found for the first time to be repressed by 1α,25(OH)2D3 and MART-10 in breast cancer cells. Matrix metalloproteinase-9 (MMP-9) activity was also downregulated by MART-10. Furthermore, F-actin synthesis in MDA-MB-231 cells was attenuated as exposure to 1α,25(OH)2D3 and MART-10. Based on our result, we conclude that MART-10 could effectively inhibit TNBC cells metastatic potential and deserves further investigation as a new regimen to treat TNBC.

Efficacy and safety of eldecalcitol, a new active vitamin D3 analog, in the bone metabolism of postmenopausal women receiving maintenance hemodialysis.

Eldecalcitol (ELD), a new active vitamin D3 analog developed in Japan, has attracted attention as an effective osteoporotic therapeutic drug. However, because ELD leads to greater calcium absorption than does conventional active vitamin D3, it has yet to be used in patients with renal insufficiency. Therefore, we evaluated the efficacy and safety of ELD treatment in 27 postmenopausal women receiving maintenance dialysis in our institution and underwent ELD treatment (starting at 0.5 µg/day) for 6 months. The mean serum albumin-corrected calcium (Caalb) level was significantly increased following treatment (9.01 ± 0.60 before versus 9.56 ± 0.55 after treatment, mean ± SD). Severe hypercalcemia was prevented through cessation or adjustment of the dosage of calcium-containing phosphate binders or existing active vitamin D. The mean serum phosphorus and intact parathyroid hormone levels were well-controlled throughout. The median levels of bone turnover markers, bone-specific alkaline phosphatase and tartrate-resistant acid phosphatase-5b were significantly decreased. The mean lumbar spine bone mineral density (BMD) was increased, a significant difference being observed in age-matched Z-scores (-0.60 ± 1.6 versus -0.36 ± 1.5, p = 0.018). The average change in lumbar spine BMD after ELD treatment was 3.10%, and in patients with a T-score of <-4.0, it was 5.63%. There was no effect on forearm BMD. Although this study is based on short-term observation in a single institution, our results suggest that ELD could be used to increase bone density in dialysis patients.

Design, synthesis, and evaluation of hybrid vitamin D3 side chain analogues as hedgehog pathway inhibitors.

Vitamin D3 (VD3) is a moderately potent and non-selective inhibitor of the Hedgehog (Hh) signaling cascade. Previous studies have established that the CD-ring region of VD3 serves as the Hh inhibitory pharmacophore. Subsequently, compound 3, an ester linked aromatic A-ring and CD-ring derivative was identified as an improved and selective Hh inhibitor. Herein, we report modifications of the CD-ring side chain that afford enhancement of selectivity for Hh modulation thereby diminishing the detrimental effects of concomitant vitamin D receptor activation. In general, linear or moderately branched alkyl chains of five or six carbons were optimal for potent and selective inhibition of Hh signaling. Moreover, hybrid VD3 side chain derivative 20 demonstrated 4-fold improvement in Hh antagonistic activity over VD3(IC50=1.1-1.6 µM) while gaining greater than a 1000-fold selectivity for Hh signaling over canonical activation of the vitamin D receptor pathway.