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.     

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.     

Variations of CYP3A activity induced by antiretroviral treatment in HIV-1 infected patients

Objective To measure the in vivo variations of CYP3A activity induced by anti-HIV drugs in human immunodeficiency virus (HIV)1-positive patients.Methods A low oral dose of midazolam (MID) (0.075 mg) was given to the patients and the 30-min total 1-OH midazolam (1-OHMID)/MID ratio was determined. Patients were phenotyped either before the introduction of antiretroviral treatments (control group, 90 patients) or after a variable period of antiretroviral treatment (56 patients). Twenty-one subjects underwent multiple phenotyping tests (before and during the course of the treatment).Results The median MID ratio was 3.51 in the control group (range 0.20–14.6). It was 5-fold higher in the group with efavirenz (28 patients; median, range: 16.0, 3.81–367; P<0.0001), 13-fold lower with nelfinavir (18 patients; 0.27, 0.06–36.3; P<0.0001), 17-fold lower with efavirenz+ritonavir (three patients; 0.21, 0.05–0.47; P=0.006), 50-fold lower with ritonavir (four patients; 0.07, 0.06–0.17; P=0.0007), and 7-fold lower with nevirapine+(ritonavir or nelfinavir or grapefruit juice) (three patients; 0.48, 0.03–1.83; P=0.03). CYP3A activity was lower in the efavirenz+ritonavir group (P=0.01) and in the ritonavir group (P=0.04) than in the nelfinavir group, although already strongly inhibited in the latter.Conclusion The low-dose MID phenotyping test was successfully used to measure the in vivo variations of CYP3A activity induced by antiretroviral drugs. Efavirenz strongly induces CYP3A activity, while ritonavir almost completely inhibits it. Nelfinavir strongly decreases CYP3A activity, but to a lesser extent than ritonavir. The inhibition of CYP3A by ritonavir or nelfinavir offsets the inductive effects of efavirenz or nevirapine administered concomitantly. Finally, no induction of CYP3A activity was noticeable after long-term administration of ritonavir at low dosages (200 mg/day b.i.d.) or of nelfinavir at standard dosages (2,500 mg/day b.i.d.).This work was supported in part by the Swiss National Research Foundation (project 3345-062092.99 and project 3200-065427.01).     

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.     

Artemether-lumefantrine co-administration with antiretrovirals: population pharmacokinetics and dosing implications

AIM

Drug–drug interactions between antimalarial and antiretroviral drugs may influence antimalarial treatment outcomes. The aim of this study was to investigate the potential drug–drug interactions between the antimalarial drugs, lumefantrine, artemether and their respective metabolites desbutyl-lumefantrine and dihydroartemisinin, and the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir.

METHOD

Data from two clinical studies, investigating the influence of the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir on the pharmacokinetics of the antimalarial drugs lumefantrine, artemether and their respective metabolites, in HIV infected patients were pooled and analyzed using a non-linear mixed effects modelling approach.

RESULTS

Efavirenz and nevirapine significantly decreased the terminal exposure to lumefantrine (decrease of 69.9% and 25.2%, respectively) while lopinavir/ritonavir substantially increased the exposure (increase of 439%). All antiretroviral drugs decreased the total exposure to dihydroartemisinin (decrease of 71.7%, 41.3% and 59.7% for efavirenz, nevirapine and ritonavir/lopinavir, respectively). Simulations suggest that a substantially increased artemether-lumefantrine dose is required to achieve equivalent exposures when co-administered with efavirenz (250% increase) and nevirapine (75% increase). When co-administered with lopinavir/ritonavir it is unclear if the increased lumefantrine exposure compensates adequately for the reduced dihydroartemisinin exposure and thus whether dose adjustment is required.

CONCLUSION

There are substantial drug interactions between artemether-lumefantrine and efavirenz, nevirapine and ritonavir/lopinavir. Given the readily saturable absorption of lumefantrine, the dose adjustments predicted to be necessary will need to be evaluated prospectively in malaria-HIV co-infected patients.     

<Emphasis Type="Italic">CYP2B6</Emphasis> genotype is a strong predictor of systemic exposure to efavirenz in HIV-infected Zimbabweans

Objective  

Efavirenz, an antiretroviral medicine, is extensively metabolized by cytochrome P450 2B6 (CYP2B6), UDP-glucuronosyltransferase 2B7 (UGT2B7), and CYP2A6. In this study, we investigated the association of single nucleotide polymorphisms (SNPs) in these genes with plasma efavirenz levels in Zimbabwean human immunodeficiency virus (HIV)-positive patients treated with efavirenz.     

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.

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.     

Plasma Levels of 25‐Hydroxyvitamin D3 and In Vivo Markers of Cytochrome P450 3A Activity in Swedes and Koreans: Effects of a Genetic Polymorphism and Oral Contraceptives

In vitro studies have shown that vitamin D may induce several cytochrome P450 (CYP) enzymes in general and CYP3A4 in particular. The primary aim of this study was to investigate the relationship between plasma levels of 25‐hydroxyvitamin D₃ and suggested in vivo markers of CYP3A activity in healthy volunteers from Sweden and Korea. Plasma concentrations of 25‐hydroxyvitamin D₃ were analysed in samples from three previously performed studies, and the correlation between these levels and suggested in vivo markers of CYP3A activity was investigated by means of nonparametric correlation. In addition, we studied the modulating effects of three vitamin D receptor promoter polymorphisms on the association between 25‐hydroxyvitamin D₃ and CYP3A enzyme activity in Swedish subjects. The plasma levels of 25‐hydroxyvitamin D₃ were not significantly associated with CYP3A phenotypes in any of the three studies, but after accounting for the vitamin D receptor polymorphism rs4516035, there was a significant positive association between 25‐hydroxyvitamin D₃ and CYP3A activity (p = 0.004). Swedes (n = 65) had significantly higher 25‐hydroxyvitamin D₃ levels than Koreans (n = 67), 75 nM compared with 31 nM (p < 0.001). Swedish women taking oral contraceptives (OC) (n = 19) had somewhat higher plasma levels of 25‐hydroxyvitamin D₃ compared with Swedish women not taking oral contraceptives (n = 21), 89 and 72 nM, respectively (p = 0.02). In conclusion, our results suggest that the overall influence on the CYP3A activity by 25‐hydroxyvitamin D₃ is of marginal importance.     

PODOCYTE INJURY IS SUPPRESSED BY 1,25-DIHYDROXYVITAMIN D3 VIA MODULATION OF TRANSFORMING GROWTH FACTOR-β1/BONE MORPHOGENETIC PROTEIN-7 SIGNALLING IN PUROMYCIN AMINONUCLEOSIDE NEPHROPATHY RATS

• 1 Accumulating evidence suggests that vitamin D and its analogues are renoprotective. However, the precise mechanisms and the molecular targets by which active vitamin D exerts its beneficial effects remain obscure. The objective of the present study was to evaluate the effect of active vitamin D on rats with puromycin aminonucleoside (PAN) nephropathy, a model that is characterized by predominant podocyte injury.
• 2 The PAN nephropathy rats were created by a single intravenous injection of 100 mg/kg PAN. Changes in renal pathology and podocyte numbers were observed. Real‐time polymerase chain reaction (PCR) was performed to examine mRNA expression of nephrin, transforming growth factor (TGF)‐β1 and bone morphogenetic protein (BMP)‐7. Protein expression of nephrin, TGF‐β1, BMP‐7 and p‐Smad2/3 and p‐Smad1/5/8 was examined by immunofluorescence, immunohistochemistry and western blotting, respectively. Rats were treated with 1,25(OH)₂D₃ by gastric gavage at a dose of 2.5 µg/kg per day, starting 2 days before PAN injection and continuing throughout the experiment.
• 3 A single injection of PAN induced massive proteinuria and elevated serum creatinine on Day 7, both of which were significantly suppressed by 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃). Immunofluorescence and real‐time PCR of the podocyte‐associated protein nephrin revealed reduced and discontinuous staining and this change was reversed by 1,25(OH)₂D₃. In PAN nephropathy rats, TGF‐β1 and p‐Smad2/3 expression was upregulated, whereas that of BMP‐7 and p‐Smad1/5/8 was downregulated. Treatment with 1,25(OH)₂D₃ significantly restored BMP‐7/Smad signalling while suppressing TGF‐β1/Smad signalling.
• 4 In conclusion, 1,25(OH)₂D₃ can ameliorate podocyte damage and proteinuria induced by PAN. The beneficial effects of 1,25(OH)₂D₃ on podocytes may be attributable, in part, to direct modulation of TGF‐β1/BMP‐7 signalling.

     

CYP3A induction and inhibition by different antiretroviral regimens reflected by changes in plasma 4β-hydroxycholesterol levels

OBJECTIVE AND METHODS: A member of the major human cytochrome P450 superfamily of hemoproteins, CYP3A4/5, converts cholesterol into 4beta-hydroxycholesterol. We studied plasma 4beta-hydroxycholesterol levels prior to and 4 weeks after initiating antiretroviral therapy that included efavirenz, ritonavir-boosted atazanavir or ritonavir-boosted lopinavir with the aim of exploring the usefulness of plasma 4beta-hydroxycholesterol levels as an endogenous biomarker of CYP3A activity. Efavirenz is an inducer of CYP3A, whereas the ritonavir-boosted regimens are net inhibitors of CYP3A. RESULTS: In patients treated with efavirenz, the median plasma 4beta-hydroxycholesterol level increased by 46 ng/mL (p = 0.004; n = 11). In contrast, patients given ritonavir-boosted atazanavir showed a median decrease in plasma 4beta-hydroxycholesterol of -9.4 ng/mL (p = 0.0003; n = 22), and those given ritonavir-boosted lopinavir showed a median change from baseline of -5.8 ng/mL (p = 0.38; n = 19). There were significant between-group differences in the effects of antiretroviral treatment on plasma 4beta-hydroxycholesterol levels (p < 0.0001). CONCLUSION: Changes in plasma 4beta-hydroxycholesterol following the initiation of efavirenz- or atazanavir/ritonavir-based antiretroviral therapy reflected the respective net increase and decrease of CYP3A activity of these regimens. The plasma 4beta-hydroxycholesterol level did not indicate a net CYP3A inhibition in the lopinavir/ritonavir arm, possibly because of concomitant enzyme induction.     

Separate Effects of 1,25‐Dihydroxyvitamin D and Calcium on Renal Calbindin‐D28k and Intestinal Calbindin‐D9k

Abstract: The effects of normal, low and high plasma concentrations of 1,25‐(OH)₂vitamin D (1,25‐(OH)₂D) combined with normal, low and high concentrations of plasma calcium on renal calbindin‐D28k and intestinal calbindin‐D9k were examined in rats. We found that the expression of renal calbindin‐D28k was significantly (P<0.05) increased by high levels of 1,25‐(OH)₂D, but was not affected by a 50% reduction in 1,25‐(OH)₂D. In contrast the intestinal calbindin‐D9k responded significantly (P<0.005) to both high and low 1,25‐(OH)₂D levels. The effect of 1,25‐(OH)₂D on intestinal calbindin‐D9k was modulated by plasma calcium concentrations. Increased plasma calcium levels did not affect the renal calbindin‐D28k concentrations, but suppressed intestinal calbindin‐D9k (P<0.05). The effect of calcium was not mediated by calciotropic hormones. This suggests the existence of a calcium‐sensing mechanism in the proximal duodenum. It is concluded that intestinal calbindin‐D9k is more sensitive than renal calbindin‐D28k to changes in 1,25‐(OH)₂D and that intestinal calbindin‐D9k in contrast to renal calbindin‐D28k is sensitive to changes in plasma calcium concentrations.     

Proliferation and Differentiation of Cultured Human Keratinocytes is Modulated by 1,25(OH)2D3 and Synthetic Vitamin D3 Analogues in a Cell Density-, Calcium- and Serum-Dependent Manner*

Abstract: The natural form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) decreases proliferation and promotes terminal differentiation of cultured human epidermal keratinocytes. The purpose of this study was to find out to what extent the culture conditions determine the sensitivety of keratinocytes to 1,25(OH)₂D₃ and synthetic vitamin D analogues. Human keratinocytes were grown in microplates. Cell proliferation (MTT-assay) and differentiation (quantity of transglutaminase type I) were measured consecutively in the same monolayer. When vitamin D₃ analogues were added to 50–60% confluent keratinocytes grown in serum-free keratinocyte growth medium with 0.09 mM Ca²⁺, stimulation of the proliferation was either minimal or non-existent, while differentiation was unaffected or slightly inhibited. There was no difference in the sensitivity to 1,25(OH)₂D₃ and the vitamin D₃ analogues. When 1,25(OH)₂D₃ was added to less confluent keratinocytes (30%) a marked antiproliferative effect was observed. Addition of 3% charcoal stripped foetal calf serum further enhanced the antiproliferative effect of 1,25(OH)₂D₃, and a difference in the sensitivity of the vitamin D₃ analogues was noted. If, finally, the Ca²⁺ concentration was raised to 0.3 mM, 1,25(OH)₂D₃ and the vitamin D₃ analogues stimulated differentiation. Also, a biphasic effect on proliferation occurred: stimulation at low vitamin D concentrations and inhibition at higher concentrations. Furthermore, keratinocytes became more sensitive to the synthetic vitamin D₃ analogues than to 1,25(OH)₂D₃: KH1060>EB1089>GS1500≥EB1213>calcipotriol>1,25(OH)₂D₃. For all compounds tested differentiation occurred at concentrations 10 to 30 times lower than for proliferation. These results indicate that the sensitivity to vitamin D₃ analogues as well as the direction of the response to vitamin D₃ analogues is dependent on the keratinocyte density, the availability of serum and Ca²⁺ concentrations.     

Rat CYP24A1 acts on 20-hydroxyvitamin D3 producing hydroxylated products with increased biological activity

20-Hydroxyvitamin D₃ (20(OH)D₃), the major product of CYP11A1 action on vitamin D₃, is biologically active and is produced in vivo. As well as potentially having important physiological actions, it is of interest as a therapeutic agent due to its lack of calcemic activity. In the current study we have examined the ability of CYP24A1, the enzyme that inactivates 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), to metabolize 20(OH)D₃. Rat CYP24A1 was expressed in Escherichia coli, purified by Ni-affinity chromatography and assayed with substrates incorporated into phospholipid vesicles which served as a model of the inner mitochondrial membrane. In this system CYP24A1 metabolized 1,25(OH)₂D₃ with a catalytic efficiency 1.4-fold higher than that seen for 25-hydroxyvitamin D₃ (25(OH)D₃). CYP24A1 hydroxylated 20(OH)D₃ to several dihydroxy-derivatives with the major two identified by NMR as 20,24-dihydroxyvitamin D₃ (20,24(OH)₂D₃) and 20,25-dihydroxyvitamin D₃ (20,25(OH)₂D₃). The catalytic efficiency of CYP24A1 for 20(OH)D₃ metabolism was more than 10-fold lower than for either 25(OH)D₃ or 1,25(OH)₂D₃ and no secondary metabolites were produced. The two major products, 20,24(OH)₂D₃ and 20,25(OH)₂D₃, caused significantly greater inhibition of colony formation by SKMEL-188 melanoma cells than either 1,25(OH)₂D₃ or the parent 20(OH)D₃, showing that CYP24A1 plays an activating, rather than an inactivating role on 20(OH)D₃.     

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.