A series of nonsecosteroidal vitamin D receptor agonists for osteoporosis therapy

In an extension of our study on gamma hydroxy carboxylic acid analogs, we explored a series of nonsecosteroidal vitamin D receptor (VDR) agonists in which 1,3-diol of 1,25(OH)₂D₃ had been replaced by aryl acetic acid. These analogs showed very potent activity in vitro compared with 1,25(OH)₂D₃. An X-ray analysis of 8d showed that the inserted phenyl ring well mimicked the folded methylene linker of the gamma hydroxy carboxylic acid moiety but the carboxylic acid of 8d interacted with VDR in a different manner from gamma hydroxy carboxylic acids. Through our in vivo screening in an osteoporosis rat model using immature rats, we identified a potent active vitamin D₃ analog, compound 7e. In mature rats of the same model, compound 7e also showed good PK profiling and excellent ability to prevent bone mineral density loss without severe hypercalcemia. Our nonsecosteroidal VDR agonist 7e (CH5036249) could be a possible new drug candidate for treating osteoporosis in human.     

Design,synthesis and biological evaluation of nonsecosteroidal vitamin D3 receptor ligands as anti-tumor agents

1α,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃, also known as calcitriol), the active form of vitamin D₃, is being increasingly recognized for cancer therapy. Our previous work showed that phenyl-pyrrolyl pentane analogs, which mimicked anti-proliferative activities against several cancer cell lines of the natural secosteroidal ligand 1,25-(OH)₂D₃. Here, in order to optimize the structural features and discover more potent derivative, a series of nonsecosteroidal vitamin D₃ receptor (VDR) ligands bearing acetylene bond linker was designed, synthesized and evaluated. Most of them showed moderate to good binding affinities and agonistic activities. Especially, compound 19f displayed the most anti-proliferative activities against MCF-7 and PC-3 cells with the IC₅₀ values of 1.80 and 5.35 μM, respectively, which was comparable to positive control 1,25-(OH)₂D₃. Moreover, compound 19f exhibited reduced toxicity against human normal liver cell line (L02) compared with the parental compound 7. Besides, the preliminary structure–activity relationships (SARs) were also analyzed.     

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.     

Intervention in autoimmunity: the potential of vitamin D receptor agonists

Vitamin D receptor (VDR) agonists are well known for their capacity to control calcium metabolism and to regulate growth and differentiation of many cell types. More recently, it has become clear that VDR agonists possess immunoregulatory properties and, in particular, pronounced pro-tolerogenic activities. VDR agonists can act directly on T cells, but DCs appear to be their primary targets. The capacity of VDR agonists to modulate DC and T cell functions is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor NF-kappaB that is downregulated by VDR agonists in APCs and in T cells. A potentially very important activity of VDR agonists is their capacity to induce in vitro and in vivo tolerogenic DCs able to enhance CD4+CD25+ suppressor T cells that, in turn, inhibit Th1 cell responses. These mechanisms of action can explain some of the immunoregulatory properties of VDR agonists in the treatment of Th1-mediated autoimmune diseases, but may also represent a physiologic element in the VDR-mediated regulation of innate and adaptive immune responses.     

Novel nonsecosteroidal vitamin D3 carboxylic acid analogs for osteoporosis, and SAR analysis

Novel vitamin D(3) analogs with carboxylic acid were explored, focusing on a nonsecosteroidal analog, LG190178, with a bisphenyl skeleton. From X-ray analysis of these analogs with vitamin D receptor (VDR), the carboxyl groups had very unique hydrogen bonding interactions in VDR and mimicked 1α-hydroxy group and/or 3β-hydroxy group of 1α,25-dihydroxyvitamin D(3). A highly potent analog, 6a, with good in vitro activity and pharmacokinetic profiles was identified from an SAR study. Compound 6a showed significant prevention of bone loss in a rat osteoporosis model by oral administration.     

Three-dimensional structure-function relationship of vitamin D and vitamin D receptor model

On the basis of conformational analysis of the vitamin D side chain and studies using conformationally restricted synthetic vitamin D analogs, we have suggested the active space region concept of vitamin D: The vitamin D side-chain region was grouped into four regions (A, G, EA and EG) and the A and EA regions were suggested to be important for vitamin D actions. We extended our theory to known highly potent vitamin D analogs and found a new region F. The analogs which occupy the F region have such modifications as 22-oxa, 22-ene, 16-ene and 18-nor. Altogether, the following relationship between the space region and activity was found: Affinity for vitamin D receptor (VDR), EA > A> F > G > EG; Affinity for vitamin D binding protein (DBP), A > G,EA,EG; Target gene transactivation, EA > F > A > EG > or = G; Cell differentiation, EA > F > A > EG > or = G; Bone calcium mobilization, EA > GA > F > or = EG; Intestinal calcium absorption, EA = A > or = G > EG. We modeled the 3D structure of VDR-LBD (ligand binding domain) using hRARgamma as a template, to develop our structure-function theory into a theory involving VDR. 1alpha,25(OH)(2)D(3) was docked into the ligand binding pocket of the VDR with the side chain heading the wide cavity at the H-11 site, the A-ring toward the narrow beta-turn site, and the beta-face of the CD ring facing H3. Amino acid residues forming hydrogen bonds with the 1alpha- and 25-OH groups were specified: S237 and R274 forming a pincer type hydrogen-bond for the 1alpha-OH and H397 for the 25-OH. Mutants of several amino acid residues that are hydrogen-bond candidates were prepared and their biologic properties were evaluated. All of our mutation results together with known mutation data support our VDR model docked with the natural ligand.     

Structure-activity relationship studies on vitamin D lactam derivatives as vitamin D receptor antagonist

Structure–activity relationship studies on 1α,25-dihydroxyvitamin D₃-26,23-lactams (DLAMs), antagonists of vitamin D, were conducted, focusing on the substituents of the phenyl group. One of the derivatives (23S,25S)-DLAM-1P-3,5(OEt)₂, showed potent antagonistic activity with an IC₅₀ of 90 nM.     

多巴胺D1和D2受体激动剂和拮抗剂对脑缺血/再灌注损伤的影响

实验应用开阔法、组织病理学方法、原位末端标记(in situ terminal deoxynucleotidyl transferase-metliated de-oxy-UTP mick end labeling,TUNEL)法及免疫组织化学等方法,探讨多巴胺D1、D2受体激动剂和拮抗剂对沙土鼠前脑缺血／再灌注损伤海马CA1区神经元凋亡及凋亡相关基因bcl-2、bax表达的影响。结果显示：前脑缺血5min可引起沙土鼠探索活动增加;再灌注3d,海马CA1区约95％的锥体细胞凋亡;再灌注7d,海马CA1区仅残存约2％—7％的存活锥体细胞;前脑缺血5min可抑制bcl-2的表达并诱导bax表达增高;预先应用D2受体激动剂培高利特可减轻缺血后沙土鼠行为学异常、抑制海马CA1区锥体细胞凋亡、提高锥体细胞存活数、显著诱导bcl-2的表达并抑制bax的表达。预先应用SKF38393、SCH23390及螺哌隆对以上结果无明显影响。实验结果提示,培高利特具有确切的脑保护作用,诱导bcl-2并抑制bax的表达可能是其脑保护作用机制之一。     

Effects of vitamin D receptor inactivation on the expression of calbindins and calcium metabolism

Hypocalcemia, rickets, and osteomalacia are major phenotypic abnormalities in vitamin D receptor (VDR)-null mice. In an attempt to understand the abnormal regulation of calcium metabolism in these animals, we examined the expression of calbindins (CaBP) as well as calcium handling in the intestine and kidney of VDR null mice. In adult VDR-null mice, intestinal and renal CaBP-D9k expression was reduced by 50 and 90%, respectively, at both the mRNA and protein levels compared with wild-type littermates, whereas renal CaBP-D28k expression was not significantly changed. Intestinal calcium absorption was measured by the rate of (45)Ca disappearance from the intestine after an oral dose of the isotope. (45)Ca absorption was similar in VDR-null and wild-type mice, but the amount of (45)Ca accumulated in the serum and bone was 3-4 times higher in wild-type mice than in VDR-null mice. Despite the hypocalcemia, the urinary excretion of calcium in VDR-null mice was not different from that in wild-type mice. Moreover, 1 wk of a high-calcium diet treatment that normalized the serum ionized calcium level of VDR-null mice increased the urinary calcium level of these mutant mice to twofold higher than that of wild-type mice on the same diet, suggesting impaired renal calcium conservation in VDR-null mice. These data demonstrate that renal CaBP-D9k, but not CaBP-D28k, is highly regulated by the VDR-mediated action of 1,25-dihydroxyvitamin D(3). Furthermore, the results also suggest that impaired calcium conservation in the kidney may be the most important factor contributing to the development of hypocalcemia in VDR-null mice, and CaBP-D9k may be an important mediator of calcium reabsorption in the kidney.     

Genetics and biology of vitamin D receptor polymorphisms

The vitamin D endocrine system is involved in a wide variety of biological processes including bone metabolism, modulation of the immune response, and regulation of cell proliferation and differentiation. Variations in this endocrine system have, thus, been linked to several common diseases, including osteoarthritis (OA), diabetes, cancer, cardiovascular disease, and tuberculosis. Evidence to support this pleiotropic character of vitamin D has included epidemiological studies on circulating vitamin D hormone levels, but also genetic epidemiological studies. Genetic studies provide excellent opportunities to link molecular insights with epidemiological data and have therefore gained much interest. DNA sequence variations, which occur frequently in the population, are referred to as "polymorphisms" and can have modest and subtle but true biological effects. Their abundance in the human genome as well as their high frequencies in the human population have made them targets to explain variation in risk of common diseases. Recent studies have indicated many polymorphisms to exist in the vitamin D receptor (VDR) gene, but the influence of VDR gene polymorphisms on VDR protein function and signaling is largely unknown. So far, three adjacent restriction fragment length polymorphisms for BsmI, ApaI, and TaqI, respectively, at the 3' end of the VDR gene have been the most frequently studied. Because these polymorphisms are probably nonfunctional, linkage disequilibrium with one or more truly functional polymorphisms elsewhere in the VDR gene is assumed to explain the associations observed. Research is therefore focussed on documenting additional polymorphisms across the VDR gene to verify this hypothesis and on trying to understand the functional consequences of the variations. Substantial progress has been made that will deepen our understanding of variability in the vitamin D endocrine system and might find applications in risk assessment of disease and in predicting response-to-treatment.     

Effects of serotonin1-like receptor agonists on autonomic neurotransmission.

Serotonin1A receptor agonists, 8-hydroxy-2-(di-n-propylamino)tetralin and 10-methyl-11-hydroxyaporphine, inhibited electrical stimulation-induced contraction of the guinea-pig ileum. These agonists also inhibited the pressor and tachycardiac responses to low frequency (0.25 Hz) but not to high frequency (2.0 Hz) electrical stimulation of the sympathetic nervous system in pithed rats. Serotonin1B receptor agonist RU 24969 inhibited pressor and tachycardiac responses to both low and high frequencies of stimulation in pithed rats. In the cat nictitating membrane, serotonin1A receptor agonists did not alter contractions elicited by electrical stimulation (0.1-3.0 Hz). Serotonin not only contracted the cat nictitating membrane but also facilitated contractile responses to low frequency (0.1-1.0 Hz) stimulation. The contractile effect of serotonin in the cat nictitating membrane was blunted by bretylium, methysergide, and ketanserin, but not by metoclopramide. The facilitatory effect of serotonin was antagonized by methysergide, but not by ketanserin, pindolol, propranolol, or metoclopramide. These results suggest that serotonin1A receptors modulate autonomic neurotransmission in the guinea-pig ileum and pithed rats, but not in the cat nictitating membrane. Serotonin contracts the cat nictitating mebrane via serotonin2 subtypes, while facilitating stimulated contractile responses through the serotonin1-like receptors.     

Effects of vitamin D metabolites on axolotl limb regeneration

Vitamin D is essential for normal metabolism of phosphorus and calcium, and differentiation of skeletal elements. 1,25 dihydroxyvitamin-D₃, the biologically active metabolite, acts as an induction/proliferation switch in various cell types and promotes chondrogenesis of chick limb bud mesenchymal cells. The function of vitamin D is mediated through its nuclear receptor, the vitamin D receptor (VDR). The proliferative actions of 1,25(OH)₂-D₃ on limb bud mesenchymal cells are similar to the ones produced by retinoids, such as all- trans retinoic acid (RA) or 9- cis retinoic acid (9- cis ). The retinoids have been shown to be compounds of extreme importance in the field of limb development and regeneration. In order to examine possible roles of vitamin D metabolites on limb regeneration, the effects of 1,25(OH)₂-D₃, 24,25(OH)₂-D₃ and KH1060 (a more potent metabolite) alone or in conjunction with all- trans RA or 9- cis RA on the regenerating axolotl limb. Vitamin D affects limb morphogenesis by generating abnormalities in skeletal elements. Synergism of vitamin D with retinoic acid in affecting pattern formation is suggested by the results.     

Protective effects of activated vitamin D receptor on radiation-induced intestinal injury

Radiation-induced intestinal injury (RIII) is a common complication after radiation therapy in patients with pelvic, abdominal, or retroperitoneal tumours. Recently, in the model of DSS (Dextran Sulfate Sodium Salt) -induced intestinal inflammatory injury, it has been found in the study that transgenic mice expressing hVDR in IEC (Intestinal Epithelial Cell) manifest highly anti-injury properties in colitis, suggesting that activated VDR in the epithelial cells of intestine may inhibit colitis by protecting the mucosal epithelial barrier. In this study, we investigated the effect of the expression and regulation of VDR on the protection of RIII, and the radiosensitivity in vitro experiments, and explored the initial mechanism of VDR in regulating radiosensitivity of IEC. As a result, we found that the expression of VDR in intestinal tissues and cells in mice can be induced by ionizing radiation. VDR agonists are able to prolong the average survival time of mice after radiation and reduce the radiation-induced intestinal injury. For lack of vitamin D, the radiosensitivity of intestinal epithelial cells in mice increased, which can be reduced by VDR activation. Ensuing VDR activation, the radiation-induced intestinal stem cells damage is decreased, and the regeneration and differentiation of intestinal stem cells is promoted as well. Finally, on the basis of sequencing analysis, we validated and found that VDR may target the HIF/PDK1 pathway to mitigate RIII. We concluded that agonism or upregulation of VDR expression attenuates radiation-induced intestinal damage in mice and promotes the repair of epithelial damage in intestinal stem cells.