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.     

Drug‐Mediated Gene Regulation of Vitamin D3 Metabolism in Primary Human Dermal Fibroblasts

Vitamin D metabolism was studied in primary human dermal fibroblasts with focus on drug‐mediated gene regulation related to adverse side effects of antiretroviral drugs used in HIV therapy. The fibroblasts expressed mRNA for cytochrome P450 (CYP) enzymes catalysing bioactivating (CYP2R1, CYP27A1 and CYP27B1) and catabolic reactions (CYP24A1). The cells produced both 25‐hydroxyvitamin D₃ and 1α,25‐dihydroxyvitamin D₃. The results demonstrate that primary dermal fibroblasts have an active vitamin D₃‐metabolizing system. High incidence of low bone mineral density is a concern for HIV‐infected patients treated with antiretroviral drugs. Osteomalacia and severe vitamin D deficiency have been reported. We investigated whether drug‐mediated gene regulation could be a possible mechanism behind these adverse drug effects. Fibroblasts were treated with different drugs used in HIV therapy, and the 1α,25‐dihydroxyvitamin D₃ levels and relative mRNA levels for crucial enzymes were determined. Efavirenz, stavudine and ritonavir significantly down‐regulated the bioactivating CYP2R1 and up‐regulated the catabolic CYP24A1. The drugs reduced bioactivating enzyme activities and cellular levels of 1α,25‐dihydroxyvitamin D₃. The current results indicate that effects on gene expression may lead to disturbed vitamin D metabolism and decreased cellular levels of active vitamin D₃. The data are consistent with the impaired bone health in patients treated with certain antiretroviral drugs.     

Additional role of serum 25‐hydroxyvitamin D3 levels in atherosclerosis in Chinese middle‐aged and elderly men

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Serum 25‐Hydroxyvitamin D and Parathyroid Hormone Levels in Non‐Lactating Women with Post‐Partum Thyroiditis: The Effect of l‐Thyroxine Treatment

Vitamin D deficiency seems to be implicated in the onset and progression of some autoimmune disorders. No previous study has investigated vitamin D homeostasis in post‐partum thyroiditis. We compared 25‐hydroxyvitamin D and parathyroid hormone (PTH) levels between four groups of non‐lactating women who gave birth within 12 months before the beginning of the study: hypothyroid women with post‐partum thyroiditis (group A; n = 14), euthyroid females with post‐partum thyroiditis (group B; n = 14), women with non‐autoimmune hypothyroidism (group C; n = 16) and healthy euthyroid females without thyroid autoimmunity (group D; n = 15). In the second part of the study, groups A and C were treated for 6 months with l ‐thyroxine. Serum levels of 25‐hydroxyvitamin D were lower, while PTH higher in patients with post‐partum thyroiditis than in patients without thyroid autoimmunity. They were also lower (25‐hydroxyvitamin D) or higher (PTH) in group A than in group B, as well as in group C in comparison with group D. l ‐thyroxine treatment increased 25‐hydroxyvitamin D and reduced PTH levels only in hypothyroid women with post‐partum thyroiditis. Baseline levels of 25‐hydroxyvitamin D correlated with thyroid antibody titres, thyroid function and circulating PTH levels, while the effect of l ‐thyroxine on serum levels of this vitamin correlated with the changes in thyroid antibody titres and PTH levels. The results of our study suggest the association of vitamin D status with post‐partum thyroiditis and l ‐thyroxine treatment of this disorder.     

Serum vitamin D levels are inversely related with non‐alcoholic fatty liver disease independent of visceral obesity in Chinese postmenopausal women

The aim of the present study was to investigate the association between serum vitamin D levels and both visceral adipose and with non‐alcoholic fatty liver disease (NAFLD) in Chinese postmenopausal women. Four hundred and fifty‐one postmenopausal women between 45 and 74 years of age (mean (± SD) age 57.3 ± 4.6 years) were enrolled in the study. All subjects participated in the Shanghai Obesity Study between June and August 2011 and underwent abdominal magnetic resonance imaging and an abdominal ultrasonography. Patients with a visceral fat area (VFA) ≥ 80 cm² were classified as abdominally obese. Serum 25‐hydroxyvitamin D₃ (25(OH)D₃) levels were measured with an electrochemiluminescence immunoassay. The prevalence of NAFLD in the study population was 34.8% (n = 157). Women with abdominal obesity had significantly lower serum 25(OH)D₃ levels than those without abdominal obesity (median (interquartile range) 11.23 (8.64–14.12) vs 12.56 (9.41–15.98) ng/mL, respectively; P < 0.01). Regardless of abdominal obesity status, serum 25(OH)D₃ levels in patients with NAFLD were lower than those without non‐NAFLD (11.14 (8.63–13.81) vs 12.92 (9.48–16.37) ng/mL (P < 0.05) for those without abdominal obesity; 10.86 (8.61–13.56) vs 11.55 (8.82–16.38) ng/mL (P < 0.05) for those with abdominal obesity). Partial correlation analyses demonstrated a negative correlation between serum 25(OH)D₃ levels and VFA (P < 0.05). Logistic regression analysis revealed that high serum 25(OH)D₃ levels were a protective factor against NAFLD after adjusting for risk factors such as VFA. In conclusion, independent of visceral obesity, vitamin D is inversely correlated with NAFLD in Chinese postmenopausal women.     

Genotype‐sensitive reversible and time‐dependent CYP2D6 inhibition in human liver microsomes

Cytochrome P450 (CYP) 2D6 metabolizes a wide range of xenobiotics and is characterized by a huge interindividual variability. A recent clinical study highlighted differential magnitude of CYP inhibition as a function of CYP2D6 genotype. The aim of this study was to investigate the effect of CYP2D6 genotype on the inhibition of dextromethorphan O‐demethylation by duloxetine and paroxetine in human liver microsomes (HLMs). The study focused on genotypes defined by the combination of two fully functional alleles (activity score 2, AS 2, n = 6), of one fully functional and one reduced allele (activity score 1.5, AS 1.5, n = 4) and of one fully functional and one non‐functional allele (activity score 1, AS 1, n = 6), which all predict extensive metabolizer phenotype. Kinetic experiments showed that maximal reaction velocity was affected by CYP2D6 genotype, with a decrease in 33% of V_max in AS 1 HLMs compared to AS 2 (P = 0.06). No difference in inhibition parameters K_i, K_I and k_inact was observed neither with the competitive inhibitor duloxetine nor with the time‐dependent inhibitor paroxetine. Among the genotypes tested, we found no difference in absolute CYP2D6 microsomal levels with ELISA immunoquantification. Therefore, our results suggest that genotype‐sensitive magnitude of drug‐drug interactions recently observed in vivo is likely to be due to differential amounts of functional enzymes at the microsomal level rather than to a difference in inhibition potencies across genotypes, which motivates for further quantitative proteomic investigations of functional and variant CYP2D6 alleles.     

Impact of CYP3A5 genotype on tolvaptan pharmacokinetics and their relationships with endogenous markers of CYP3A activity and serum sodium level in heart failure patients

Tolvaptan efficacy for heart failure has a large interindividual variation. This study aimed to evaluate the influence of CYP3A5 and ABCB1 genotypes on tolvaptan pharmacokinetics and their relationships with plasma markers of CYP3A activity and laboratory test values in heart failure patients. Fifty‐eight heart failure patients receiving oral tolvaptan for volume overload were enrolled. Blood samples for determination of pre‐dose plasma concentrations of tolvaptan and its metabolites were collected. CYP3A5 and ABCB1 genotypes, plasma 4β‐hydroxycholesterol/total cholesterol ratio (4β‐OHC/TC) and 25‐hydroxyvitamin D (25‐OHD), and serum laboratory test values were evaluated. The CYP3A5*3/*3 genotype was associated with a higher plasma concentration of tolvaptan but not with its metabolic ratios. The ABCB1 3435C > T, 2677G > T/A and 1236C > T polymorphisms affected neither tolvaptan pharmacokinetics nor its metabolism. Plasma 4β‐OHC/TC and 25‐OHD concentration were not correlated with plasma tolvaptan concentration. In a stratified analysis based on CYP3A5 genotype, plasma 4β‐OHC/TC had a negative correlation with plasma tolvaptan concentration in the patients with the CYP3A5*1 allele, while the plasma concentration of 25‐OHD did not. The CYP3A5*3/*3 genotype was associated with a higher serum sodium level in the patients with volume overload. The plasma concentration of 25‐OHD had a positive correlation with the serum total bilirubin level. In conclusion, CYP3A5*3 but not ABCB1 genotypes elevated tolvaptan plasma exposure in heart failure patients. CYP3A5‐deficient patients treated with tolvaptan had a higher serum sodium level. The CYP3A5 genotype altered the relationship between plasma tolvaptan and 4β‐OHC.     

Age‐related Differences in CYP3A Abundance and Activity in the Liver of the Göttingen Minipig

In view of paediatric drug development, regulatory authorities often request safety studies in juvenile animals, including minipigs. Unfortunately, knowledge on the ontogeny of the biotransformation processes in animal models remains scarce and impedes a correct interpretation of the toxicity findings. CYP3A4 is one of the most important drug‐metabolizing enzymes in human beings and shows important similarities with CYP3A in the minipig. Therefore, the aim of this study was to assess the abundance and activity of CYP3A in liver microsomes from foetal, juvenile (days 1, 3, 7 and 28) and adult male and female Göttingen minipigs. CYP3A abundance was studied by an indirect enzyme‐linked immunosorbent assay (ELISA), whereas CYP3A activity was assessed by a biotransformation assay with Luciferin‐IPA. CYP3A abundance could not be detected until day 3. From day 7 onwards, a gradual increase in expression was noted, leading to the highest abundance in adult animals. CYP3A activity was not detectable in foetuses and 1‐day‐old animals. The CYP3A activity was detectable, but below the LLOQ in day 3 animals and increased gradually with age to reach the highest level in adults. The CYP3cide and ketoconazole inhibition, and testosterone and midazolam reduction of Luciferin‐IPA metabolism in minipig liver microsomes substantiate that Luciferin‐IPA is metabolized by CYP3A in minipigs. A positive correlation was found between CYP3A abundance and biotransformation of Luciferin‐IPA (Pearson r = 0.863; p < 0.0001). In conclusion, both abundance and activity of CYP3A increased gradually in juvenile minipigs, but remained below the levels observed in adult animals.     

Association between Common CYP1A2 Polymorphisms and Theophylline Metabolism in Non‐smoking Healthy Volunteers

This study was designed to investigate the impact of cytochrome P450 (CYP) 1A2 polymorphisms on theophylline metabolism in a non‐smoking healthy male Chinese population. Four polymorphisms CYP1A2*1C (G‐3860A), G‐3113A, CYP1A2*1F (C‐163A) and CYP1A2*1B (C‐5347T) were screened in 238 unrelated male volunteers. Then, a single oral 200‐mg dose of theophylline was administered to 37 volunteers, who were selected from 238 volunteers based on the CYP1A2 genotype. CYP1A2 activities were evaluated by plasma 1,7‐dimethylxanthine/caffeine ratios (17X/137X) after administration of 100‐mg caffeine. The plasma concentrations of theophylline, 17X and 137X were determined by high‐performance liquid chromatography. The activity of CYP1A2 was lower in volunteers with the ‐3113 AA genotype compared with those with the ‐3113 AG genotype (0.35 ± 0.04 versus 0.48 ± 0.07, p = 0.016) or the ‐3113 GG genotype (0.35 ± 0.04 versus 0.58 ± 0.22, p = 0.037). CYP1A2*1F polymorphisms were associated with increased CYP1A2 activity in volunteers with ‐3860G/‐3113G/5347C homozygosity (0.66 ± 0.24 versus 0.46 ± 0.05, p = 0.034). However, theophylline metabolism showed no difference among volunteers carrying different haplotype pairs. CYP1A2 genetic polymorphisms influenced CYP1A2 enzyme activity as measured by caffeine, but CYP1A2 gene polymorphisms appeared to have limited influence on theophylline metabolism in our study.     

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.     

Simultaneous evaluation of human CYP3A4 and ABCB1 induction by reporter assay in LS174T cells,stably expressing their reporter genes

The bioavailability of orally administered therapies are often significantly limited in the human intestine by the metabolic activities of cytochrome P450 3A4 (CYP3A4) and P‐glycoprotein (P‐gp). Predicting whether candidate compounds induce CYP3A4 and P‐gp is a crucial stage in the drug development process, as drug–drug interactions may result in the induction of intestinal CYP3A4 and P‐gp. However, the assay systems needed to evaluate both CYP3A4 and P‐gp induction in the intestine are yet to be established. To address this urgent requirement, LS174T cells were used to create two stable cell lines expressing the CYP3A4 or ATP‐binding cassette subfamily B member 1 (ABCB1, encoding P‐gp) reporter genes. First, these stable cells were tested by treatment with 1α,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃), all‐trans‐retinoic acid (ATRA) and 9‐cis‐retinoic acid (9‐cis RA) that induce CYP3A4 and P‐gp in the intestines. All these compounds significantly increased both CYP3A4 and ABCB1 reporter activities in the stable cell lines. To simultaneously assess the induction of CYP3A4 and ABCB1, both stable cells were co‐cultivated to measure their reporter activities. The mixed cells showed a significant increase in the CYP3A4 and ABCB1 reporter activities following treatment with 1,25(OH)₂D₃, ATRA, and 9‐cis RA. These activity levels were maintained after passaging more than 20 times and following multiple freeze–thaw cycles. These results demonstrate that our established cell lines can be used to evaluate simultaneously CYP3A4 and ABCB1 induction in the intestines, providing a valuable in vitro model for the evaluation of future drug candidates. Copyright © 2014 John Wiley & Sons, Ltd.     

Induction of CYP1A1, 2B, 2E1 and 3A in rat liver by organochlorine pesticide dicofol

The present study has determined the ability of dicofol, an organochlorine pesticide, to induce cytochrome P450 using rats treated with 1, 10, and 25 mg/kg dicofol intraperitoneally for 4 days. Treatments with 10 and 25 mg/kg dicofol produced dose-related increases of cytochrome P450 and cytochrome b₅ contents and NADPH-cytochrome c reductase, 7-ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. The treatments also increased glutathione S-transferase and superoxide dismutase activities in liver cytosol. Dicofol at 1 mg/kg produced a general trend towards increases of the aforementioned enzyme levels. The results of immunoblot analyses showed that 10 and 25 mg/kg dicofol increased protein levels of CYP1A1, CYP2B, CYP2E1, and 3A in liver. RT-PCR data indicated that dicofol induced mRNA expression of liver CYP1A1, CYP2B, and CYP3A. Pretreatments of rats with 10 and 25 mg/kg dicofol decreased phenobarbital-induced sleeping time by 34% and 39%, respectively. Dicofol pretreatment at 25 mg/kg increased CCl₄-induced serum alanine aminotransferase activity by 4.3-fold and aspartate aminotransferase activity by 4.1-fold. The present study demonstrates that dicofol has the ability to induce CYP1A1, CYP2B, CYP2E1, and CYP3A in the liver and increase phenobarbital metabolism and CCl₄ toxicity in rats.     

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.

In silico and in vitro studies of two non‐imidazole multiple targeting agents at histamine H3 receptors and cholinesterase enzymes

Recently, multi‐target directed ligands have been of research interest for multifactorial disorders such as Alzheimer's disease (AD). Since H₃ receptors (H₃Rs) and cholinesterases are involved in pathophysiology of AD, identification of dual‐acting compounds capable of improving cholinergic neurotransmission is of importance in AD pharmacotherapy. In the present study, H₃R antagonistic activity combined with anticholinesterase properties of two previously computationally identified lead compounds, that is, compound 3 (6‐chloro‐N‐methyl‐N‐[3‐(4‐methylpiperazin‐1‐yl)propyl]‐1H‐indole‐2‐carboxamide) and compound 4 (7‐chloro‐N‐[(1‐methylpiperidin‐3‐yl)methyl]‐1,2,3,4‐tetrahydroisoquinoline‐2‐carboxamide), was tested. Moreover, molecular docking and binding free energy calculations were conducted for binding mode and affinity prediction of studied ligands toward cholinesterases. Biological evaluations revealed inhibitory activity of ligands in nanomolar (compound 3 : H₃R EC₅₀ = 0.73 nM; compound 4 : H₃R EC₅₀ = 31 nM) and micromolar values (compound 3 : AChE IC₅₀ = 9.09 µM, BuChE IC₅₀ = 21.10 µM; compound 4 : AChE IC₅₀ = 8.40 µM, BuChE IC₅₀ = 4.93 µM) for H₃R antagonism and cholinesterase inhibition, respectively. Binding free energies yielded good consistency with cholinesterase inhibitory profiles. The results of this study can be used for lead optimization where dual inhibitory activity on H₃R and cholinesterases is needed. Such ligands can exert their biological activity in a synergistic manner resulting in higher potency and efficacy.     

The association of serum FGF23 and non‐alcoholic fatty liver disease is independent of vitamin D in type 2 diabetes patients

Recent studies have shown that circulating fibroblast growth factor (FGF) 23 and vitamin D levels are closely correlated with insulin resistance. The aim of this study was to investigate the relationship among serum FGF 23 levels, serum 25‐hydroxyvitamin D [25(OH)D] levels, and non‐alcoholic fatty liver disease (NAFLD) in Chinese patients with type 2 diabetes mellitus (T2DM). This study enrolled 331 hospitalized T2DM patients (209 patients with NAFLD and 122 patients without NAFLD). Serum FGF23 levels were measured using a sandwich enzyme‐linked immunosorbent assay. Serum 25(OH)D levels were determined by an electrochemiluminescence immunoassay. NAFLD was diagnosed by hepatic ultrasound, and the fatty liver index (FLI) was calculated to quantify hepatic steatosis. Results showed that T2DM patients with NAFLD had significantly higher serum FGF23 levels (44.17 [37.92‐51.30] pg/mL vs 40.21 [34.07‐48.33] pg/mL, P = .002), but lower serum 25(OH)D levels (16.43 [12.70‐21.37] ng/mL vs 19.59 [13.78‐26.26] ng/mL, P = .002) than those without NAFLD. Moreover, the incidence rate of NAFLD increased with increasing serum FGF23 levels and decreased with increasing 25(OH)D levels (both P < .05). Logistic regression analysis showed that both serum FGF23 and 25(OH)D levels were independent factors for NAFLD (both P < .05). Furthermore, a multiple stepwise regression analysis also revealed that both serum FGF23 and 25(OH)D levels were independently correlated with FLI (both P < .01). In conclusion, both high FGF23 and low vitamin D levels showed an independent relationship with NAFLD in Chinese T2DM patients, indicating that FGF23 and vitamin D function via different regulatory pathways in the liver.     

Association of MDR1, CYP3A4*18B, and CYP3A5*3 polymorphisms with cyclosporine pharmacokinetics in Chinese renal transplant recipients

Objective  The objective of this study was to retrospectively evaluate the effects of MDR1, CYP3A4*18B, and CYP3A5*3 genetic polymorphisms on cyclosporine A (CsA) pharmacokinetics in Chinese renal transplant patients during the first month after transplantation. Methods  A total of 103 renal transplant recipients receiving CsA were genotyped for MDR1 (C1236T, G2677T/A, and C3435T), CYP3A4*18B, and CYP3A5*3. The predose and 2-h postdose concentrations of CsA (C₀ and C₂, respectively) were determined by fluorescence polarization immunoassay, and their relationships with corresponding genotypes and haplotypes were investigated. Results  Patients with a CYP3A4*1/*1 genotype were found to have a higher dose-adjusted concentration compared with those with CYP3A4*18B/*18B, as follows: for C₂, 19.3% (P = 0.008) during days 8-15, 35.2% (P = 0.008) during days 16–30, and for C₀, 39.7% (P = 0.012) during days 16–30. The dose-adjusted C₀ was higher in patients with MDR1 1236CC compared with those with 1236TT in the first month postoperation. The dose-adjusted C₀ in patients with the CYP3A5*3/*3 genotype was 25.5% and 30.7% higher than those with the wild-type genotype during days 8–15 (P = 0.011) and days 16–30 (P = 0.015), respectively. Haplotype analysis revealed that the dose-adjusted C₀ was higher in the first month following surgery in carriers of haplotype MDR1 CAC than in noncarriers. Polymorphisms of MDR1 and CYP3A5*3 did not affect dose-adjusted C_2. Conclusion  The data suggests that the CYP3A4*18B genotype affects CsA pharmacokinetics during the first month following surgery in Chinese renal transplant recipients. Patients with CYP3A4*18B alleles may require higher doses of CsA to reach the target levels. Large prospective studies may be needed to further explore the impact of MDR1 and CYP3A5*3 polymorphisms on CsA pharmacokinetics in renal transplant recipients.     

Design,synthesis, and biological evaluation of quinazoline derivatives as dual HDAC1 and HDAC6 inhibitors for the treatment of cancer

Fifty‐eight quinazoline‐based compounds were designed and synthesized based on the structural optimizations from the lead compound 23bb in an attempt to search for more potent dual HDAC1 and HDAC6 inhibitors. Among them, 32c (HDAC1, IC₅₀ = 31.10 ± 0.37 nM; HDAC6, IC₅₀ = 16.15 ± 0.62 nM) and 32d (HDAC1, IC₅₀ = 37.00 ± 0.24 nM; HDAC6, IC₅₀ = 35.00 ± 0.71 nM) were not only identified as potent dual‐acting HDAC1 and HDAC6 inhibitors with over 10‐fold selectivity to the other HDACs, but also displayed activities in tubulin acetylation and histone H₃ acetylation induction. Importantly, both of them displayed strong antiproliferative activities against various tumor cell lines in vitro with IC₅₀ values less than 40 nM, especially for hematologic tumors cells (U266 and RPMI8226, IC₅₀ < 1 nM), which were even better than 23bb and SAHA. Furthermore, 32c showed a significant tumor growth inhibition (antitumor rate = 63.98%, p < 0.05) in the resistant MCF‐7/ADR xenograft model without any obvious body weight changes and abnormal behaviors. Our findings validate that 32c is a potent dual inhibitor of HDAC1/6 that can be an efficacious treatment for breast cancer with Adriamycin resistance.     

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.     

Calcitriol Attenuates Weight‐Related Systemic Inflammation and Ultrastructural Changes in the Liver in a Rodent Model

The role of vitamin D in maintaining calcium homoeostasis and bone mineralization is well‐established. The aim of the current investigation was to evaluate the effect of calcitriol treatment on inflammation, insulin resistance and liver changes induced by increased body‐weight. Four groups of mice (n = 11 each) were maintained on either low‐fat diet (LFD) or high‐fat diet (HFD) with and without 1α, 25‐dihydroxyvitamin D3 (calcitriol) for 16 weeks. Body‐weight of animals was recorded at the start of the study and every 4 weeks thereafter. At the end of the experiment, blood samples were collected for the determination of biochemical parameters, and liver tissues were harvested for the histopathological evaluation. A significant gradual decrease in weight was observed in HFD‐fed mice treated with calcitriol compared with a steady increase in controls (p < 0.01). Furthermore, calcitriol treatment reduced concentrations of various inflammatory markers including TNF‐α, CRP and IL‐6 (p < 0.05). Treated animals also exhibited lower levels of C‐peptide and insulin (539.4 ng/ml versus 718.9 ng/ml and 0.77 ng/ml versus 1.7 ng/ml, respectively; p < 0.05), which are consistent with improved insulin resistance. Liver histology and ultrastructural studies showed a marked accumulation of fat droplets in approximately 60–70% of hepatocytes of mice fed on HFD, while calcitriol administration rendered the whole structure more normal. Overall, our data signify an important effect of calcitriol on inflammation under HFD conditions and a protective effect on the liver structure.