The modulation of transcriptional expression and inhibition of multidrug resistance associated protein 4 (MRP4) by analgesics and their primary metabolites

During the course of a toxic challenge, changes in gene expression can manifest such as induction of metabolizing enzymes as a compensatory detoxification response. We currently report that a single 400 mg/kg acetaminophen (APAP)dose to C57BL/6J mice led to an increase in multidrug resistance-associated (Mrp) 4 (Abcc4) mRNA 12 h after administration. Alanine aminotransferase, as a marker of liver injury, was also elevated indicating hepatotoxicity had occurred. Therefore, induction of Mrp4 mRNA was likely attributable to APAP-induced liver injury. Mrp4 has been shown to be upregulated during oxidative stress, and it is well-established that APAP overdose causes oxidative stress due to depletion of glutathione. Given the importance of Mrp4 upregulation as an adaptive response during cholestatic and oxidative liver injury, we next investigated the extent by which human MRP4 can be inhibited by the analgesics, APAP, diclofenac (DCF), and their metabolites. Using an in vitro assay with inside out human MRP4 vesicles, we determined that APAP-cysteine inhibited MRP4 mediated transport of leukotriene C4 with an apparent IC50 of 125 μM. APAP-glutathione also attenuated MRP4 activity though it achieved only 28% inhibition at 300 μM. Diclofenac acyl glucuronide (DCF-AG) inhibited MRP4 transport by 34% at 300 μM. The MRP4 in vitro inhibition occurs at APAPcysteine and DCF-AG concentrations seen in vivo after toxic doses of APAP or DCF in mice, hence the findings are important given the role that Mrp4 serves as a compensatory response during oxidative stress following toxic challenge.Fil: Scialis, Renato J.. University of Connecticut; Estados UnidosFil: Ghanem, Carolina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Manautou, José E.. University of Connecticut; Estados Unido

Metabolism and Disposition of a Selective ␣ 7 Nicotinic Acetylcholine Receptor Agonist in Humans

ABSTRACT: The The formation of 2 was found to be mediated by CYP2D6, a polymorphically expressed enzyme absent in 5 to 10% of white people, whereas the generation of 4 was catalyzed by CYP2D6, FADcontaining monooxygenase 1 (FMO1), and FMO3. It is of interest that, although no overall gender-related differences in excretory routes, mass recoveries, pharmacokinetics, or metabolite profiles of 1 were evident, the observation of one of eight subjects (13%) showing disparate (relative to all other volunteers) systemic exposures to 1, and urinary and plasma quantitative profiles nearly devoid of 2 with the highest levels of 1, seem consistent with both the identification of CYP2D6 as the only major recombinant cytochrome P450 transforming 1 to 2 and the demographics of white CYP2D6 poor metabolizers. Data also reported herein suggest that 4 is generated predominantly by renal FMO1 in humans

The Modulation of Non-steroidal Anti-inflammatory Toxicity as a Function of the Interplay between Uptake and Efflux Transporters

Diclofenac (DCF) is commonly used in the treatment of arthritis and pain. Although mechanisms of injury following DCF administration have been characterized, the roles that transporters have as modulators of exposure and toxicity remain unclear. To that end, in vivo transporter knockout (KO) models were used to assess the toxicokinetics of DCF and its major metabolites. DCF and its reactive acyl glucuronide (DCF-AG) were shown to be substrates for the efflux transporter Bcrp. Biliary excretion of DCF and DCF-AG decreased in Bcrp KOs while only DCF-AG plasma levels increased compared to wild-type (WT) mice. DCF-AG was likewise determined to be a substrate for the efflux transporter Mrp3 evidenced by reduced plasma concentrations in Mrp3 KOs without changes in biliary excretion. Toxic DCF challenge resulted in increased intestinal injury in Mrp3 KOs compared to WTs. In vitro assays revealed that human MRP3 can transport DCF-AG with apparent high affinity compared to human MRP2. The active uptake of DCF-AG was shown to be mediated by human OATPs in an in vitro system, specifically OATP2B1 exhibited concentration-dependent kinetics. Mechanistic studies indicated that DCF-AG was able to induce oxidative stress through creation of reactive oxygen species and inhibition of superoxide dismutase. Furthermore, DCF-AG was shown to inhibit both COX-1 and COX-2, which may have contributed to intestinal injury. Lastly it was demonstrated that DCF-AG can inhibit human MRP4 that is upregulated during oxidative stress. In summary, the data presented herein demonstrate the multifactorial pathways by which DCF-AG is transported and contributes to tissue injury

Hepatic Disposition of Gemfibrozil and Its Major Metabolite Gemfibrozil 1‑<i>O</i>‑β-Glucuronide

Gemfibrozil (GEM), which decreases serum triglycerides and low density lipoprotein, perpetrates drug–drug interactions (DDIs) with several drugs. These DDIs are primarily attributed to the inhibition of drug transporters and metabolic enzymes, particularly cytochrome P450 (CYP) 2C8 by the major circulating metabolite gemfibrozil 1-<i>O</i>-β-glucuronide (GG). Here, we characterized the transporter-mediated hepatic disposition of GEM and GG using sandwich-cultured human hepatocytes (SCHH) and transporter-transfect systems. Significant active uptake was noted in SCHH for the metabolite. GG, but not GEM, showed substrate affinity to organic anion transporting polypeptide (OATP) 1B1, 1B3, and 2B1. In SCHH, glucuronidation was characterized affinity constants (<i>K</i>_m) of 7.9 and 61.4 μM, and biliary excretion of GG was observed. Furthermore, GG showed active basolateral efflux from preloaded SCHH and ATP-dependent uptake into membrane vesicles overexpressing multidrug resistance-associated protein (MRP) 2, MRP3, and MRP4. A mathematical model was developed to estimate hepatic uptake and efflux kinetics of GEM and GG based on SCHH studies. Collectively, the hepatic transporters play a key role in the disposition and thus determine the local concentrations of GEM and more so for GG, which is the predominant inhibitory species against CYP2C8 and OATP1B1

Cell-to-Medium Concentration Ratio Overshoot in the Uptake of Statins by Human Hepatocytes in Suspension, but Not in Monolayer: Kinetic Analysis Suggesting a Partial Loss of Functional OATP1Bs

Suspended human hepatocytes (SHH) have long been used in assessing hepatic drug uptake, while plated human hepatocytes in short-term monolayer culture (PHH) have gained use in recent years. This study aimed to cross-evaluate SHH and PHH in measuring the hepatic uptake mediated by organic anion transporting polypeptide 1Bs (OATP1Bs). We compared the time courses of cell-to-medium (C/M) concentration ratios and initial uptake clearance values of the OATP1B substrates (pitavastatin, rosuvastatin, cerivastatin, pravastatin, dehydropravastatin, and SC-62807) between SHH and PHH. For all compounds except cerivastatin, the C/M ratios in SHH displayed an apparent overshoot (an initial increase followed by a decrease) during the 180-min uptake experiment, but not in PHH. Based on the literature evidence suggesting the possible internalization of OATP1Bs in primary hepatocytes, separate experiments measured the drug uptake after varying lengths of pre-incubation in the drug-free medium. The initial uptake clearances of pitavastatin and rosuvastatin declined in SHH beyond an apparent threshold time of 20-min drug-free pre-incubation, but not in PHH. Kinetic modeling quantitatively captured the decline in the active uptake clearance in SHH, and more than half of the active uptake clearances of pitavastatin and rosuvastatin were prone to loss during the 180-min uptake experiment. These results suggested a partial, time-delayed loss of the functional OATP1Bs in SHH upon prolonged incubation. Our results indicate that PHH is more appropriate for experiments where a prolonged incubation is required, such as estimation of unbound hepatocyte-to-medium concentration ratio (K-p,K-uu) at the steady-state.Y