Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia

Abstract

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Conflict of interest statement

All authors state that they have no conflicts of interest.

Copyright © 2013 American Society for Bone and Mineral Research.

Figures

Figure 1. Expression of CYP3A4, CYP24A1, CYP27A1, CYP27B1, CYP2R1, VDR, and PXR genes in human hepatocytes

Human hepatocytes from three different donors were treated with 10 μM rifampin (RIF), 400 μM phenobarbital (PB), 0.5 μM hyperforin (HF), 50 μM carbamazepine (CBZ), 200 μM levitiracetam (LEV) or vehicle control (CT, 0.1% v/v) for 48 hr, as indicated. Total RNA from each sample was isolated and the expression of CYP3A4, CYP27A1, CYP27B1, CYP24A1, CYP2R1, VDR, and PXR was determined by qRT-PCR assay. Data represent mean ± S.E. (from four replicate determinations of three different liver donors) of the fold-induction in treated cells, compared to those of vehicle treated cells, after normalization to the 18s ribosomal RNA level. *: p < 0.05 for the inductive effect of PXR agonists, compared to corresponding control group.

Figure 2. Formation of three 25OHD3 metabolites and expression of VDR, PXR and related genes in human hepatocytes after treatment of 25OHD3 with or without rifampin pretreatment

After treatment with rifampin (10 μM) for 48 hr, hepatocytes were incubated with various concentrations of 25OHD3 for 4 hr. Media were collected for LC-MS/MS analysis and cell lysates were used for mRNA extraction and RT-PCR. A) Formation of vitamin D metabolites from 25OHD3 metabolism. B) mRNA levels for VDR, PXR and their target genes. Data are represented as mean ± S.E. from three different liver donors. *: p < 0.05 for the effect of rifampin, compared to corresponding control group.

Figure 3. Time-dependent formation of 25OHD3 metabolites and related gene expression after rifampin treatment

After treated with rifampin (10 μM) for 48 hr, hepatocytes were incubated with 25OHD3 (2 μM) for different periods of incubation time, as indicated. After incubation, media were collected for LC-MS/MS analysis and the total mRNA were extracted for RT-PCR analysis. A) mRNA levels for VDR, PXR and their targeted genes. Data are represented as mean ± S.E. from three different liver donors. B) Formation of vitamin D metabolites. *: p < 0.05 for the effect of rifampin, compared to corresponding control group. #: p < 0.05 for the effect of time, compared to corresponding control group (t = 0).

Figure 4. Effects of DHB on the formation of 25OHD3 metabolites in hepatocytes

After treatment with rifampin (10 μM) for 48 hr, hepatocytes were pre-incubated with DHB (20 μM) for 4 hr, followed by incubation with 25OHD3 and DHB mixture under the certain conditions, as indicated: A) incubation with 25OHD3 (2 μM) and DHB for 2 hr; B) incubation with 25OHD3 (2 μM) and DHB for 4 hr; C) 25OHD3 (5 μM) and DHB for 24 hr. Media were collected for LC-MS/MS analysis. Data are represented as mean ± S.E. from three different liver donors.

Figure 5. Formation of 4β,25(OH)2D3 and 4α,25(OH)2D3 conjugates in human hepatocytes

A) Representative chromatograms of 4β,25(OH)2D3 and 4α,25(OH)2D3; B) Time-dependent formation of 4β,25(OH)2D3 and 4α,25(OH)2D3 in hepatocytes after β-glucuronidase treatment. Hepatocytes (n = 3) were pretreated with 10 μM rifampin for 48 hr and then treated with 50 nM 25OHD3 with various incubation times. In parallel, cells were pre-incubated with 20 μM DHB for 4 hr and then co-incubated with 25OHD3. Medium was collected and incubated with β-glucuronidase, extracted and analyzed as described previously. The released amounts of 4β,25(OH)2D3 and 4α,25(OH)2D3 represent the levels of the corresponding conjugates in the culture medium. Both primary oxidation products were undetectable following incubation of 50 nM 25OHD3 without β-glucuronidase treatment of the culture media.

Figure 6. Effect of PXR agonists on expression of VDR, PXR and their target genes, and formation of 24R,25(OH)2D3 in HK2 cells

A) HK2 cells were treated with 10 μM rifampin (RIF), 400 μM phenobarbital (PB), 0.5 μM hyperforin (HF), 50 μM carbamazepine (CBZ), 200 μM levitiracetam (LEV) or vehicle (CT, 0.1% v/v) for 48 hr. 1α,25(OH)2D3 (VD3, 0.5 nM) was used as a positive control for CYP24A1 induction and incubated for 24 hr, as previously described (24). Total RNA was isolated and the expression of CYP3A4, CYP27B1, CYP24A1, VDR, and PXR was determined by qRT-PCR assay. B) Formation of 24R,25(OH)2D3 and 4β,25(OH)2D3. HK2 cells were pretreated with 10 μM rifampin for 48 hr. Cells were washed with PBS twice and then treated with 2 μM 25OHD3 for 4 hr. Media were collected for LC-MS/MS analysis.

Figure 7. Effect of short-term rifampin treatment on the plasma concentrations of vitamin D metabolites

Plasma samples were collected before (pre-RIF) and after (post-RIF) rifampin administration (300 mg, PO, 6 days). Paired comparisons were conducted to show the direct effect of rifampin on the levels of four metabolites in each individual. Statistical analysis was conducted using a paired t-test; a p value less than 0.05 indicates significance.

Figure 8. Correlation between the absolute change in plasma 4β,25(OH)2D3 concentration with absolute change in plasma 1α,25(OH)2D3, 24R,25(OH)2D3, and 25OHD3 levels

The absolute concentration change was calculated as the post-rifampin minus pre-rifampin concentrations. Sample correlation coefficients and corresponding p-values were calculated; the null hypothesis that the population correlation is zero was tested with a t-test, under the assumption that the population was bivariate and normally distributed.

Figure 9. Correlation between 4β,25(OH)2D3 formation and midazolam elimination in both human hepatocytes and healthy volunteers

A). After treated with rifampin (10 μM), phenobarbital (400 μM), hyperforin (0.5 μM), carbamazepine (50 μM), or vehicles (0.1% v/v) for 48 hr, human hepatocytes (from three different liver donors) were incubated with either midazolam (2 μM) for 30 min or 25OHD3 (2 μM) for 4 hr. The rate of 1′-hydroxylation of midazolam was correlated with the concentration of 4β,25(OH)2D3 using linear regression. B). Correlation between the ratio of plasma 4β,25(OH)2D3/25OHD3 and oral midazolam clearance for pre-RIF and post-RIF treatment periods. Open circle: constitutive condition; closed circle: rifampin-induced condition. Oral midazolam clearance was estimated using the Pharsight WinNonlin. Correlation coefficients and corresponding p-values were calculated according to the null hypothesis.

Scheme 1. Proposed mechanism for the tissue-specific induction of CYP3A4 and CYP24A1 gene expression on regulation of 25OHD3 and 1α,25(OH)2D3 catabolism in humans

Both CYP3A4 and CYP24A1 can catalyze 25OHD3 and 1α,25(OH)2D3 hydroxylation. Under the constitutive conditions in the liver, CYP3A4 is the most abundant enzyme, catalyzing 4β-hydroxylation of 25OHD3 and 23R-hydroxylation of 1α,25(OH)2D3. While under the constitutive conditions in the kidney, CYP24A1 is the most abundant enzyme, catalyzing 24R-hydroxylation of 25OHD3 and 1α,25(OH)2D3. In the liver and small intestine, drugs (PXR agonists, e.g. rifampin) induce CYP3A4, but not CYP24A1 gene expression via a PXR pathway. In the kidney, neither CYP3A4 nor CYP24A1 is significantly induced by PXR activation; however CYP24A1 can be induced by vitamin D metabolites [e.g. 1α,25(OH)2D3] via a VDR pathway. Relative induction capability: ++ > +