Vitamin D3 derivatives with adamantane or lactone ring side chains are cell type-selective vitamin D receptor modulators

The vitamin D receptor (VDR) mediates the biological actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], the active form of vitamin D, which regulates calcium homeostasis, immunity, cellular differentiation, and other physiological processes. We investigated the effects of three 1,25(OH)(2)D(3) derivatives on VDR function. AD47 has an adamantane ring and LAC67a and LAC67b have lactone ring substituents at the side chain position. These vitamin D derivatives bind to VDR but do not stabilize an active cofactor conformation. In a VDR transfection assay, AD47 and LAC67b act as partial agonists and all three compounds inhibit VDR activation by 1,25(OH)(2)D(3). The derivatives enhanced the heterodimerization of VDR with the retinoid X receptor, an effect unrelated to agonist/antagonist activity. AD47 and LAC67b weakly induced recruitment of the SRC-1 cofactor to VDR, and all three derivatives inhibited the recruitment of p160 family cofactors to VDR induced by 1,25(OH)(2)D(3). It is noteworthy that AD47 induced DRIP205 recruitment as effectively as 1,25(OH)(2)D(3), whereas LAC67a and LAC67b were not effective. We examined the expression of endogenous VDR target genes and the nuclear protein levels of VDR and cofactors in several cell lines, including cells derived from intestine, bone, and monocytes, and found that the vitamin D(3) derivatives act as cell type-selective VDR modulators. The data indicate that side chain modification is useful in the development of VDR antagonists and tissue-selective modulators. Further elucidation of the molecular mechanisms of action of selective VDR modulators will be essential for their clinical application.     

Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Targeting the vitamin D receptor: advances in drug discovery

The vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the biological action of 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active form of vitamin D. VDR regulates bone and calcium homeostasis, immunity, cellular differentiation and other physiological processes. Mutations in the VDR gene were identified in hereditary vitamin D-resistant rickets (HVDRR), and VDR-null mice exhibit the HVDRR phenotype, characterised by rickets and hypocalcaemia. In addition to the treatment of rickets, vitamin D analogues are important therapeutics in osteoporosis and psoriasis. Vitamin D analogues are effective drugs in experimental models of immune disorders and malignancies, such as breast cancer, prostate cancer and leukaemia. The development of functionally selective VDR-targeted drugs is leading to an enhanced understanding and novel therapies for these VDR-related diseases.     

Retiferols – synthesis and biological activity of a conceptually novel class of vitamin D analogs

Introduction: The hypothesis that retiferols are a novel class of vitamin D analogs with therapeutic potential has been recently proved. The CD-ring of vitamin D, originated from a steroid precursor, is not necessary for biological activity. The retiferol, disubstituted at C-13, was bound to the ligand-binding domain (LBD) of vitamin D receptor (VDR) just like the vitamin D hormone [1,25-(OH)₂D₃]. This finding opens the way for retiferols as a novel class of vitamin D therapeutics.

Areas covered: This review presents the concept of retiferols and their structure evolution. Medicinal chemistry and therapeutic perspective of retiferols are reviewed showing how these vitamin D analogs became a source of potential therapeutics.

Expert opinion: Docking experiments and molecular modeling have shown that positioning of vitamin D analog at the LBD of VDR is not disturbed by deletion of a large portion of the vitamin D, exactly as hypothesized. Twenty years of structural modifications have shown that removal of the CD-ring fragment and regioselective methylation results in an almost complete loss of the undesired calcemic activity of retiferol while gaining the agonistic activity comparable to that of 1,25-(OH)₂D₃.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Differential regulation of intestinal and hepatic CYP3A by 1α,25-dihydroxyvitamin D3: Effects on in vivo oral absorption and disposition of buspirone in rats

1α,25-Dihydroxyvitamin D₃ (also called 1,25(OH)₂D₃ or calcitriol) is the biologically active form of vitamin D, which functions as a ligand to the vitamin D receptor (VDR). It was previously reported that intestinal cytochrome P450 3A (CYP3A) expression was altered by 1,25(OH)₂D₃-mediated VDR activation. However, to clarify whether the change in CYP3A subfamily expression by VDR activation can affect metabolic function, further evidence is needed to prove the effect of 1,25(OH)₂D₃ treatment on CYP3A-mediated drug metabolism and pharmacokinetics. Here, we report the effects of 1,25(OH)₂D₃ on CYP3A activity and in vivo pharmacokinetics of buspirone in Sprague–Dawley rats. CYP3A mRNA expression and CYP3A-mediated testosterone metabolism were enhanced in the intestine but were unaffected in the livers of rats treated with 1,25(OH)₂D₃. Notably, the oral pharmacokinetic profile of buspirone (CYP3A substrate drug) and 6′-hydroxybuspirone (major active metabolite of buspirone formed via CYP3A-mediated metabolism) was significantly altered, while its intravenous pharmacokinetic profile was not affected by 1,25(OH)₂D₃ treatment. To the best of our knowledge, this study provides the first reported data regarding the effects of 1,25(OH)₂D₃ treatment on the in vivo pharmacokinetics of intravenous and oral buspirone in rats, by the differential modulation of hepatic and intestinal CYP3A activity. Our present results could lead to further studies in clinically significant CYP3A-mediated drug–nutrient interactions with 1,25(OH)₂D₃, including 1,25(OH)₂D₃–buspirone interaction.

Alterations in gene expression in vitamin D‐deficiency: Down‐regulation of liver Cyp7a1 and renal Oat3 in mice

The vitamin D‐deficient model, established in the C57BL/6 mouse after 8 weeks of feeding vitamin D‐deficient diets in the absence or presence of added calcium, was found associated with elevated levels of plasma parathyroid hormone (PTH) and plasma and liver cholesterol, and a reduction in cholesterol 7α‐hydroxylase (Cyp7a1, rate‐limiting enzyme for cholesterol metabolism) and renal Oat3 mRNA/protein expression levels. However, there was no change in plasma calcium and phosphate levels. Appraisal of the liver revealed an up‐regulation of mRNA expressions of the small heterodimer partner (Shp) and attenuation of Cyp7a1, which contributed to hypercholesterolemia in vitamin D‐deficiency. When vitamin D‐sufficient or D‐deficient mice were further rendered hypercholesterolemic with 3 weeks of feeding the respective, high fat/high cholesterol (HF/HC) diets, treatment with 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃], active vitamin D receptor (VDR) ligand, or vitamin D (cholecalciferol) to HF/HC vitamin D‐deficient mice lowered the cholesterol back to baseline levels. Cholecalciferol treatment partially restored renal Oat3 mRNA/protein expression back to that of vitamin D‐sufficient mice. When the protein expression of protein kinase C (PKC), a known, negative regulator of Oat3, was examined in murine kidney, no difference in PKC expression was observed for any of the diets with/without 1,25(OH)₂D₃/cholecalciferol treatment, inferring that VDR regulation of renal Oat3 did not involve PKC in mice. As expected, plasma calcium levels were not elevated by cholecalciferol treatment of vitamin D‐deficient mice, while 1,25(OH)₂D₃ treatment led to hypercalcemia. In conclusion, vitamin D‐deficiency resulted in down‐regulation of liver Cyp7a1 and renal Oat3, conditions that are alleviated upon replenishment of cholecalciferol.     

The Yin and Yang of vitamin D receptor (VDR) signaling in neoplastic progression: Operational networks and tissue-specific growth control

Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1α,25-(OH)₂ D₃ in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1α,25-(OH)₂ D₃. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1α,25-(OH)₂ D₃/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1α,25-(OH)₂ D₃ genomic and non-genomic actions, Ca²⁺ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.     

Effects of 1α,25‐dihydroxyvitamin D3 on transporters and enzymes of the rat intestine and kidney in vivo

1α,25‐Dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural ligand of the vitamin D receptor (VDR), was found to regulate bile acid related transporters and enzymes directly and indirectly in the rat intestine and liver in vivo. The kidney is another VDR‐rich target organ in which VDR regulation on xenobiotic transporters and enzymes is ill‐defined. Hence, changes in protein and mRNA expression of nuclear receptors, transporters and enzymes of the rat intestine and kidney in response to 1,25(OH)₂D₃ treatment (0 to 2.56 nmol/kg/day intraperitoneally in corn oil for 4 days) were studied. In the intestine, protein and not mRNA levels of Mrp2, Mrp3, Mrp4 and PepT1 in the duodenum and proximal jejunum were induced, whereas Oat1 and Oat3 mRNA were decreased in the ileum after 1,25(OH)₂D₃ treatment. In the kidney, VDR, Cyp24, Asbt and Mdr1a mRNA and protein expression increased significantly (2‐ to 20‐fold) in 1,25(OH)₂D₃‐treated rats, and a 28‐fold increase of Cyp3a9 mRNA but not of total Cy3a protein nor Cyp3a1 and Cyp3a2 mRNA was observed, implicating that VDR played a significant, renal‐specific role in Cyp3a9 induction. Additionally, renal mRNA levels of PepT1, Oat1, Oat3, Ostα, and Mrp4, and protein levels of PepT1 and Oat1 were decreased in a dose‐dependent manner, and the ～50% concomitant reduction in FXR, SHP, HNF‐1α and HNF‐4α mRNA expression suggests the possibility of cross‐talk among the nuclear receptors. It is concluded that the effects of 1,25(OH)₂D₃ changes are tissue‐specific, differing between the intestine and kidney which are VDR‐rich organs. Copyright © 2009 John Wiley & Sons, Ltd.     

Effects of 1,25-dihydroxyvitamin D3 and its 20-epi analogues (MC 1288, MC 1301, KH 1060), on clonal keratinocyte growth: evidence for differentiation of keratinocyte stem cells and analysis of the modulatory effects of cytokines

1. Keratinocytes are functionally divided into stem cells, transit amplifying cells and terminally differentiated cells. In a hyperproliferative skin disease, psoriasis, increased mitotic activity of the stem cells is chiefly responsible for epidermal hyperplasia. The effects of 1,25dihydroxyvitamin D₃ (1,25(OH)₂D₃) and potent vitamin D₃ analogues (MC 1288: 20-epi-1,25(OH)₂D₃, MC 1301: 20-epi-24a-homo-26,27-dimethyl-1,25(OH)₂D₃, KH 1060: 20-epi-22-oxa-24a-homo-26,27-dimethyl-1,25(OH)₂D₃) on the stem cells were investigated.
2. Stem cells were identified retrospectively as those giving rise to large keratinocyte colonies in culture (holoclones). 1,25(OH)₂D₃ (10⁻⁸–10⁻⁶ M) suppressed formation of holoclones by stimulating the progenitor cell differentiation into the phenotype expressing differentiation markers (keratins K1/K10 and involucrin).
3. 20-Epi vitamin D₃ analogues were more potent than 1,25(OH)₂D₃ in inhibiting the clonal keratinocyte growth. This activity correlated with the ability to induce cell differentiation (KH 1060>MC 1301>MC 1288>1,25(OH)₂D₃).
4. Cytokines modulated the effects of 1,25(OH)₂D₃ on clonal growth. One of the following cytokines (epidermal growth factor, transforming growth factor α, interleukin-1α, interleukin-1β, interleukin-6, interleukin-8) was required for 1,25(OH)₂D₃ to suppress clonal growth and induce cell differentiation. In contrast, keratinocyte growth factor and insulin-like growth factor I attenuated the effects of 1,25(OH)₂D₃.
5. In conclusion, 1,25(OH)₂D₃ and 20-epi vitamin D₃ analogues suppress clonal growth by directly inducing the differentiation of progenitor cells. It is conceivable that stimulation of stem cells differentiation is a major mechanism of action of vitamin D₃ compounds in psoriasis. Balance between different types of cytokines in psoriatic epidermis may be an important factor determining the clinical effect of vitamin D-based therapy.

     

The potential for vitamin D receptor activation in cardiovascular research

Vitamin D₃ needs to be activated into 1,25-dihydroxyvitamin D₃ in order to bind to vitamin D receptor (VDR) for functional responses. Studies in the past have focussed on the role of VDR in mineral homeostasis, with VDR activators used mainly to treat hyperparathyroidism secondary to chronic kidney disease. Chronic kidney disease patients encounter a higher risk of cardiovascular disease than the general public and experience an extremely high cardiovascular-related mortality rate. Recent clinical observations show that VDR therapy reduces mortality and morbidity in chronic kidney disease. Preclinical studies demonstrate that VDR is likely to be involved in the cardiovascular pathophysiology.     

Vitamin D receptor as a drug discovery target

1alpha, 25-dihydroxyvitamin D3 [1,25 (OH)(2)D(3)], the active metabolite of vitamin D3, is known for the maintenance of normal skeleton architecture and mineral homeostasis. Apart form these traditional calcemic actions, 1,25 (OH)(3)D(1) and its synthetic analogs are increasingly recognized for their potent anti-proliferative, prodifferentiative and immunomodulatory activities. The calcemic and non-calcemic actions of 1,25 (OH)(2)D(3) and its synthetic analogs are mediated through vitamin D receptor (VDR), which belongs to the superfamily of steroid/thyroid hormone nuclear receptors. Physiological and pharmacological actions of 1,25 (OH)(2)D(3) in various systems, along with the detection of VDR in target cells, have indicated potential applications of VDR ligands in inflammation, dermatological indications, osteoporosis, cancers and autoimmune diseases. VDR ligands have shown therapeutic potential in limited clinical trials as well as in animal models of these diseases. As a result, a VDR ligand, calcipotriol is in clinic for psoriasis and another, OCT, [2-oxa-1,25 (OH)(2)D(3)] is being developed as a topical agent for the same indication. Further, 1alpha,-hydroxyvitamin D3 (alphacalcidol), a prodrug of 1,25 (OH)(2)D(3) is in clinic and a synthetic VDR ligand, ED-71, is under consideration for approval in Japan for the treatment of osteoporosis. Interestingly, VDR ligands have shown not only preventive but also potent therapeutic anabolic activities in animal models of osteoporosis. However, the wide spread use of VDR ligands in above-mentioned indications is hampered by their major side effect, namely hypercalcemia. In view of this associated toxicity, synthetic VDR ligands with reduced calcemic potential have been synthesized with the ultimate aim of improving their therapeutic efficacy. This review presents recent advances in VDR biology, novel VDR ligands and therapeutic applications of VDR ligands.     

Regulation of VDR expression in rat and human intestine and liver – Consequences for CYP3A expression

The vitamin D receptor (VDR) regulates the expression of drug metabolizing enzymes and transporters in intestine and liver, but the regulation of VDR expression in intestine and liver is incompletely understood. We studied the regulation of VDR mRNA expression by ligands for VDR, farnesoid X receptor (FXR), glucocorticoid receptor (GR) and protein kinase C α (PKCα) in rat and human ileum and liver using precision-cut slices. 1,25(OH)₂D₃ induced VDR expression in rat ileum and liver, and human ileum but not in liver. Chenodeoxycholic acid (CDCA), but not lithocholic acid (LCA) and GW4064 induced VDR mRNA expression in rat ileum and liver. The PKCα activator, phorbol-12-myristate-13-acetate (PMA) induced the expression of VDR in the rat liver, and the induction of VDR by 1,25(OH)₂D₃ and CDCA was inhibited by the PKCα inhibitor, bisindolyl maleimide I (Bis I). These results show that the expression of VDR is likely to be regulated by PKC but not by FXR or VDR activation at least in the rat liver. The VDR mediated induction of its target genes CYP3A1 and CYP3A2 by 1,25(OH)₂D₃ or LCA in the rat ileum was strongly reduced in the presence of CDCA despite the higher VDR expression. Thus, CDCA might potentiate the toxicity of LCA by inhibiting its metabolism.     

1α,25-dihydroxyvitamin D3 on intestinal transporter function: studies with the rat everted intestinal sac

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃) treatment (2.56 nmol/kg i.p. daily×4) increased PepT1, Mrp2, Mrp4, Asbt, but not Mdr1/P‐gp in the rat small intestine. In this study, the intestinal everted sac technique, together with various select probes: mannitol (paracellular transport), glycylsarcosine (PepT1), 5(and 6)‐carboxy‐2′,7′‐dichlorofluorescein (CDF) diacetate (precursor of CDF for Mrp2), adefovir dipivoxil (precursor of adefovir for Mrp4) and digoxin (P‐gp) was used to examine the functional changes of these transporters. After establishing identical permeabilities (P_app) of mannitol for the apical‐to‐basolateral (A‐to‐B) and basolateral‐to‐apical (B‐to‐A) directions at 20 min in 1,25(OH)₂D₃‐treated vs. vehicle‐treated duodenal, jejunal and ileal everted sacs, a significant enhancement of net A‐to‐B transport of glycylsarcosine in the duodenum, increased B‐to‐A transport of CDF and A‐to‐B and B‐to‐A transport of adefovir in the jejunum were observed with 1,25(OH)₂D₃ treatment. However, the A‐to‐B and B‐to‐A transport of digoxin in the ileum was unchanged. These changes in transporter function in the rat intestinal everted sac corresponded well to changes in proteins that were observed previously. This study confirms that the rat intestinal PepT1, Mrp2 and Mrp4, but not P‐gp are functionally induced by 1,25(OH)₂D₃ treatment via the vitamin D receptor (VDR). Copyright © 2011 John Wiley & Sons, Ltd.     

1,25-Dihydroxyvitamin D3 regulates genes responsible for detoxification in intestine

1α,25-Dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the biologically active form of vitamin D₃, not only plays a major role in mammalian calcium and phosphorous homeostasis but also exerts pleiotropic effects on cell proliferation, differentiation and the immune system. Further, vitamin D is believed to play a significant role in the prevention of colon, prostate, and breast cancer and in reducing the risk of autoimmune diseases. To gain insight into the mechanism whereby vitamin D can have such diverse actions, we have employed microarray technology. We studied the effect of a single dose of 1,25-(OH)₂D₃ on gene expression in the intestine of vitamin D-deficient rats. Within 6 h, 1,25-(OH)₂D₃ stimulates the expression of several phase I and phase II biotransformation genes. There is also an increased expression of antioxidant genes. These results support the idea that vitamin D is a significant factor in detoxification and protection against environmental toxins.