Comparative effects of rifabutin and rifampicin on cytochromes P450 and UDP-glucuronosyl-transferases expression in fresh and cryopreserved human hepatocytes
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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The two first authors contributed equally to this work.