Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes

doi:10.1016/S0009-2797(99)00089-7

Chemico-Biological Interactions

Volume 121, Issue 1, 1 June 1999, Pages 37-48

https://doi.org/10.1016/S0009-2797(99)00089-7 Get rights and content

Abstract

The aim of this study was to evaluate rifabutin (RBT) and rifampicin (RIF) capabilities in inducing various xenobiotic metabolizing enzymes such as cytochromes P450 (CYPs) and UDP-glucuronosyl-transferases (UGTs) in cultured fresh and cryopreserved human hepatocytes. Enzyme induction was assessed through the use of several diagnostic markers, i.e. testosterone, midazolam (MDZ), diazepam (DZP) and 7-ethoxyresorufin for CYP-dependent enzyme reactions; and AZT for UGT-dependent enzyme reactions. RBT concentrations (0.118, 0.708 μM) were selected according to previously published pharmacokinetic data in patients. The known CYP3A4 inducer in humans, RIF, was used as a positive control. At the concentrations used, no sign of cytotoxicity was evidenced. Both compounds were able to dose-dependently induce the overall metabolism of testosterone (∼2-fold for RBT, ∼4-fold for RIF) and the formation of the 6β-hydroxylated-derivative (up to ∼4-fold over control for RBT and ∼10-fold for RIF), which is CYP3A4 dependent. The other hydroxylated metabolites (16α-OH and 2α-OH) were also enhanced. The metabolism of MDZ, which is specifically metabolized by CYP3A4 in humans, was also investigated following drug's exposure to hepatocytes. DZP one, which is governed by various CYPs, including CYP3A, was also investigated. RBT was shown to increase the biotransformation of both benzodiazepines (∼1.9-fold over control). Moreover, the effects of both drugs on ethoxyresorufin O-deethylase activity (EROD), which is representative of CYP1A1/2 isoforms, were tested. Results showed only a moderate induction of this marker (∼2-fold over control) when compared to the high effect observed after hepatocyte exposure to 3-methylcholantene (∼14-fold over control). Finally, the action of RBT and RIF on UGTs expression was investigated by using AZT as diagnostic substrate: glucuronides formation was not significantly affected by the two rifamycin derivatives. On the whole, exposure of fresh or cryopreserved human hepatocytes to RBT dose-dependently affected the levels of drug metabolizing enzymes in a dose-dependent manner. However, as already demonstrated by in vivo pharmacokinetic studies, its inducing properties towards CYPs, CYP3A in particular, are less pronounced than RIF.

Introduction

Rifabutin (RBT) is a rifamycin derivative, like rifampicin (RIF), registered for the treatment of pulmonary tuberculosis and for the prophylaxis and treatment of mycobacterium avium complex in patients with AIDS. The inducing properties of Rifabutin towards rat CYPs have been well established, with interspecies variabilities being reported [1], [2]. The aim of this study was to evaluate RBT and RIF capabilities in inducing various xenobiotic metabolizing enzymes such as CYPs and UGTs (UDP-glucuronosyl-transferases) in cultured fresh and cryopreserved human hepatocytes. To this aim, several diagnostic markers were chosen, i.e. testosterone, midazolam, diazepam, 7-ethoxyresorufin, for CYP-dependent enzyme reactions; AZT for UGT-dependent enzyme reactions.

Indeed, it is well known that steroid hydroxylations are selectively mediated by multiple CYP isoenzymes. For testosterone, hydroxylations occur at different positions and are mediated by various known CYPs: for instance 6α- and 15β-OH testosterone metabolites are formed by at least three different human isoenzymes, which belong to the CYP3A subfamily [3], whereas 16β-OH metabolite is produced by CYP2B6. Therefore, in order to investigate the inducing properties of the rifamycin derivatives on various CYP-dependent activities, we measured the regio- and stereo-selective hydroxylations of testosterone in fresh or cryopreserved human hepatocyte cultures pre-treated by various concentrations of the compounds.

Midazolam (MDZ) is a member of the benzodiazepine class of drugs with a short apparent elimination half-life (1–3 h) and hypnotic properties. In man, this drug is mainly metabolised by CYP3A4 to give 1-hydroxylated-midazolam derivative (half-life=0.7 h) which undergo further conjugation by UGTs [4], [5], [6], [7]. Diazepam (DZP) is also a member of 1,4 benzodiazepine class of drugs, but with hypnotic, anxiolytic and muscle relaxant properties (apparent elimination half-life=44 h). In man, DZP is predominantly cleared in urine as conjugates of polar metabolites, which are formed principally in liver by UGTs, after CYPs-mediated oxygenations. Phase I bioproducts are nordiazepam (N-dealkylation, half-life=50–120 h) and temazepam (C₃ hydroxylation, half-life=5–11 h). These products can undergo further metabolism, leading to oxazepam (half-life=4–13 h) which is the ultimate oxidized metabolite of DZP in humans. Oxazepam (OX) and temazepam (TEM) formations are mediated by CYP3A4, whereas nordiazepam one is CYP2C19-dependent [8], [9]. As RIF is a well established inducer of the CYP3A4 gene expression in vivo and in vitro [10], [11], MDZ and DZP have therefore been chosen as experimental probes to check RBT and RIF effects.

Furthermore, in order to investigate the capability of these two drugs in inducing CYP1A1/2, their effects on ethoxyresorufin O-deethylase activity (EROD) were measured in fresh and cryopreserved human hepatic cells in primary culture pre-treated by various concentrations of each test compound. Indeed, because CYP1A1/2 represent some major CYPs isoforms involved in the biotransformation of xenobiotics [12] to cytotoxic and/or mutagenic derivatives, its potential induction is considered of toxicological importance.

Finally, the comparative inducing effects of RBT and RIF were investigated by using 3′-azido-3′-deoxythymidine (AZT) as UGT diagnostic substrate. AZT is the main antiretroviral drug used against human immunodeficiency virus. This drug undergoes extensive biotransformation mainly by the liver microsomal UGT2 in humans [13]. In contrast, for rats, mice and guinea pigs or dogs, the rate of biotransformation is lower. These results were confirmed by studies on hepatocytes in primary culture, isolated from rats, dogs, monkeys and humans [14].

Our data confirm that CYP3A is the major enzyme system induced by rifamycin derivatives. They also demonstrate that expression and induction of enzymes which play a major role in drug’s biotrasformation are maintained in human hepatocytes after cryopreservation.

Section snippets

Primary cultures of human hepatocytes

Freshly isolated (FIH) and thawed (TH) hepatocytes were obtained as previously described [4], [15], [16], [17]. The cell viability was determined using the Erythrosin B exclusion test and was at least 80%. Hepatocytes were seeded on rat tail collagen type I-coated 96- or 12-well plastic plates, at a final density of 3×10⁴ cells/well (in 0.1 ml of medium) or 4×10⁵ cells/well (in 1 ml of medium), respectively. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂ and

Testosterone

The overall metabolism of this substrate was 4.7 times higher in thawed hepatocytes (0.621 nmol/min=10⁶ cells) than in freshly isolated ones (0.131 nmol/min=10⁶ cells). This can be due to the inter-individual variability which is now well-established or to a better stability of CYPs expression over the 72 h culture period of cryopreserved cells. Nevertheless, in both cases it was possible to evidence the effect of RBT and RIF. After 3 days of treatment, both compounds were able to induce the

Discussion

The early in vitro identification of drug–drug interactions is of increasing interest, allowing the prediction of potential in vivo pharmacokinetic consequences. As the liver is the main organ involved in xenobiotic biotransformation, the use of hepatocytes in primary culture is increasing in order to investigate drug metabolism in human and other species [23]. This model allows early information about the potential metabolic fate of a compound and about the main enzyme systems involved in its

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¹: The two first authors contributed equally to this work.

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Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes

Abstract

Introduction

Section snippets

Primary cultures of human hepatocytes

Testosterone

Discussion

Arch. Bichem. Biophys.

Biochem. Pharmacol.

Methods in Enzymol.

Pharmacol. Ther.

Cryobiology

J. Chrom.

Toxic. in Vitro

Chem. Biol. Interact.