Send reprint requests to: DRUG METABOLISM AND DIsi'osITIoN COMPARATIVE METABOLIC DISPOSITION OF IVERMECTIN IN FAT TISSUES OF CATTLE, SHEEP, AND RATS

ABSTRACT: In the study of the metabolic disposition of ivermectin in cattle, sheep, and rats, a group of nonpolar metabolites was detected in the fat tissues of these animals

Cytochrome P450 3A4 is the major enzyme involved in the metabolism of the substance P receptor antagonist aprepitant DMD #216RR 2 Running Title: P450 Metabolism of Aprepitant

Abstract 207 words Introduction 522 word

Pharmacokinetics and Interactions of a Novel CCR5 Receptor Antagonist with Ritonavir in Rats and Monkeys: Role of CYP3A and P-glycoprotein Running title: CYP3A and P-gp in MRK-1 interactions with ritonavir

Abstract The mechanisms of pharmacokinetic interactions of a novel anti-HIV-1 CCR5 receptor antagonist (MRK-1) with ritonavir were evaluated in rats and monkeys. MRK-1 was a good substrate for the human MDR1 and mouse Mdr1a transporters and was metabolized by CYP3A isozymes in rat, monkey and human liver microsomes. Both the in vitro MDR1-mediated transport and oxidative metabolism of MRK-1 were inhibited by ritonavir. Although the systemic pharmacokinetics of MRK-1 in rats and monkeys were linear, the oral bioavailability increased with increase in dose from 2-to 10-mg/kg. The systemic plasma AUC of MRK-1 was increased 4-6 fold when a 2-or a 10-mg/kg dose was orally co-administered with 10-mg/kg ritonavir. Further pharmacokinetic studies in rats indicated that P-glycoprotein (P-gp) inhibition by ritonavir increased the intestinal absorption of 2-mg/kg MRK-1 maximally by ~30-40%, and a major component of the interaction likely resulted from its reduced systemic clearance via the inhibition of CYP3A isozymes. Oral co-administration of quinidine (10-and 30-mg/kg) increased both the extent and the first-order rate of absorption of MRK-1 (2-mg/kg) by ~40-50% and ~100-300%, respectively, in rats, thus further substantiating the role of P-gp in modulating the intestinal absorption of MRK-1 in this species. At the 10-mg/kg MRK-1 dose, however, the entire increase in its AUC upon co-administration with ritonavir or quinidine could be attributed to a reduced systemic clearance and no effects on intestinal absorption were apparent. In contrast to rats, the effects of P-gp in determining the intestinal absorption of MRK-1 appeared less significant in rhesus monkeys at either dose. JPET/2002/45096 4 The CCR5 chemokine receptor is expressed on both monocytes and T-lymphocytes and is believed to play a pivotal role in the pathogenesis of the human immunodeficiency virus infection. It has been suggested that the entry of HIV-1 into the host cell is facilitated by the interaction of the viral envelope glycoproteins gp120 and gp41 with the host cell CD4 and then either the chemokine receptor CCR5 or CXCR4 (Deng et al., 1996; Dragic et al., 1996). The macrophage-tropic or R5 variants of HIV-1 utilize CCR5 for entry and are predominant during the early asymptomatic stages of infection, while T cell line-tropic or X4 variants can use CCR5 or CXCR4 and appear later in ~50% of patients during persistent infection concomitant with a catastrophic decline in CD4 + T-cell numbers and the development of clinical AIDS (Connor et al., 1997 ). Human genetic evidence supports CCR5 as a potentially attractive antiviral target. A 32-base-pair deletion in the CCR5 coding region (CCR5-32) generates a non-functional receptor and homozygosity for CCR5-32 confers resistance to HIV-1 infection in populations at high risk for exposure but does not manifest any adverse health effect Over the past several years, multi-drug therapy has shown considerable advantage over the use of a single drug in the management of HIV infection This has been propelled by the need to delay the development of resistance and avoid doselimiting adverse effects with a single agent. Currently, a triple or a quadruple therapy with two nucleoside analogues, plus one or two protease inhibitors, is considered essential for optimal JPET/2002/45096 5 efficacy and to avoid rapid development of viral resistance Although the availability of a CCR5 antagonist may offer another powerful pharmacological intervention for the management of HIV infection, it is almost certain that a combination therapy would be required in order to achieve reasonable reductions in disease progression and to circumvent rapid development of resistance. Ritonavir is one of several HIV-protease inhibitors (ritonavir, indinavir, saquinavir, nelfinavir, amprenavir and lopinavir) approved for the management of HIV infection in the US. Protease inhibitors, especially ritonavir, have potent inhibitory effects on drug metabolizing enzymes such as CYP3A4, 2C9, 2C19 and 2D6 (Eagling et al., 1997; This has become an important therapeutic strategy for the pharmacotherapy of HIV. In addition to their inhibitory effects on CYP enzymes, protease inhibitors including ritonavir are also good substrates for the human multi-drug resistance protein MDR1 or P-gp (Kim et al., 1998; Lee et al., 1998; In order to effectively manage and utilize drug-drug interactions towards a therapeutic benefit during the management of HIV infection in the clinic, a thorough understanding of the potential JPET/2002/45096 6 biochemical mechanisms responsible for these interactions is required. Thus, we undertook a series of pharmacokinetic and interaction studies with a novel investigational CCR5 receptor Finke et al., 2002; Methods Materials Identification of Cytochrome P-450 Isozymes Responsible for MRK-1 Metabolism Further confirmation of the CYP isoform(s) responsible for the in vitro metabolism of [ 3 H]MRK-1 (10-µM) in human liver microsomes was obtained by incubating the compound in the presence of monoclonal antibodies against CYP2C9, CYP2D6 and CYP3A4, and also with the cytochrome P-450 isoform-specific inhibitors including sulfaphenazole (CYP2C9), tranylcypromine (CYP2C19), quinidine (CYP2D6), and ketoconazole and troleandomycin (CYP3A4). Effect of the above monoclonal antibodies on MRK-1 metabolism was also examined in male rhesus monkey liver microsomes. In addition, male rat liver microsomes were incubated with polyclonal antibodies against CYP2C11 and 3A2 to examine the role of these CYPs in MRK-1 metabolism in the rat. Each incubation contained 1-mg/mL microsomal protein and 25-µL/mL of the antibody preparation along with the above described buffer and NADPHregenerating system. The disappearance of MRK-1 from the incubation was determined by LC-MS/MS. Metabolite profiles in these incubations were also examined by radiochromatography. JPET/2002/45096 9 The potential of ritonavir to inhibit the metabolism of MRK-1 was also examined in rat, monkey and human liver microsomes. MRK-1 (10-µM) was incubated, as above, with liver microsomes from the three species in the absence and presence of varying concentrations of ritonavir for 15-min. Preliminary studies indicated that metabolism was linear for the duration of the incubation. At the end of the incubation, the reaction was stopped by the addition of an equal volume of acetonitrile. Samples were spun in a centrifuge and the supernatant was analyzed for MRK-1 concentrations using LC-MS/MS. Mesenteric Intestinal Loop Preparation The absorption of MRK-1 was examined in isolated rat intestinal loop preparations in order to determine whether MRK-1 was a substrate for efflux transporters in the rat intestine and if its absorption could be modulated by P-gp inhibitors. The detailed surgical procedures were similar to those described elsewhere The mesenteric vein draining this intestinal segment was cannulated. All mesenteric venous blood draining from the loop was collected via this mesenteric venous cannula at 10-min intervals. The sampled blood was simultaneously replaced with fresh blood from a donor rat by infusion via the femoral vein at approximately the same rate that blood drained from the mesenteric venous cannula (0.1-0.15 mL/min). Blood samples were collected every 10-min for up to 60-min after the injection of a 0.1 mg dose of MRK-1 (in 0.15 mL PEG400:ethanol:water, 20:20:60 by volume) into the intestinal loop. In order to examine the effect of P-gp modulators on MRK-1 absorption, either a 0.1 mg dose of ritonavir or verapamil was included along with MRK-1 in the dosing solution. Plasma was harvested from the collected mesenteric blood samples by centrifugation and analyzed for MRK-1 concentrations using LC-MS/MS. Pharmacokinetics in Rats and Monkeys All animal procedures were approved by the Merck Research Laboratories Institutional Animal Care and Use Committee. Rats and monkeys were housed in temperature and humiditycontrolled rooms with a 12-hr light/dark cycle. Cannulas were implanted in femoral artery and vein of male Sprague-Dawley rats (250-300 g, n=3 or 4/ group) and animals were allowed to recover from surgery for at least one day before experimentation. Similarly, male adult rhesus monkeys (Macaca mulatta, n=4/group) of 5-7 years of age were surgically prepared by placing catheters either into the saphenous vein via percutaneous venipuncture, or by surgically placing indwelling catheters into the femoral vein and connecting them to a subcutaneous vascular access port. Monkeys were transferred to restraint chairs on the day of experiment for dosing and blood collection. Rats and monkeys JPET/2002/45096 14 were fasted overnight before drug administration, whereas access to water was provided ad libitum. Food was restored after the collection of 4-hr blood samples. Intravenous dosing solutions of MRK-1 were prepared in a PEG400:EtOH:Water (2:2:6, by volume) vehicle. The compound was administered as an i.v. bolus via the femoral vein (or saphenous vein in case of monkeys) at 0.5 and 2-mg/kg doses at a dose volume of 1-(rats) or 0.2-mL/kg (monkeys). Oral dosing solutions of MRK-1 were prepared as a suspension in 0.9% NaCl and the doses were 2-and 10-mg/kg in a dosing volume of 1-(monkeys) or 5-mL/kg (rats). Different groups of rats were used for oral and i.v. administration experiments. However, a randomized two-way cross over design was used for the oral and i.v. administration experiments in monkeys. Effect of Ritonavir Oral Co-administration on the Pharmacokinetics of MRK-1 in Rats and Monkeys Separate groups of rats were surgically prepared as above to examine the effect of ritonavir oral co-administration on the pharmacokinetics of MRK-1. However, the same set of rhesus monkeys that were used in the previous pharmacokinetic experiments (vide supra) were used for these studies. Appropriate doses of ritonavir were administered as the commercially available Norvir solution. The oral doses and dosing volumes of MRK-1 were the same as described above for the pharmacokinetic studies. Animals were administered the ritonavir dose via oral gavage followed immediately by the MRK-1 suspension via the same route. MRK-1 was administered either alone (at 2-and 10-mg/kg doses) or in combination with 10-and 30-mg/kg quinidine. Effect of Oral Ritonavir and Quinidine on the Systemic Pharmacokinetics of MRK-1 in Rats Quinidine formulations were prepared at appropriate concentrations, as described above, in EtOH:PEG400:Water (2:2:6, by volume). Norvir solution was used for ritonavir doses. A 0.5 mg/mL solution of MRK-1 was prepared in the EtOH:PEG400:Water (2:2:6, by volume) vehicle. Rats (n=3/group) were administered either vehicle, ritonavir (10-mg/kg), or quinidine (30-mg/kg) doses via oral gavage 30-min before the administration of a 0.5-mg/kg i.v. bolus dose of MRK-1; the 30-min time-point corresponds to plasma concentrations of ritonavir that are near maximal (C max ) with this dosing regimen (data not shown). In all pharmacokinetic and interaction studies, blood samples (250-µL for rats and 1-mL for monkeys) were collected at predetermined time points up to 24-hr after drug administration. JPET/2002/45096 16 Plasma was obtained by centrifugation of the blood and stored at -20 °C until LC-MS/MS analysis. LC-MS/MS Analysis Plasma samples were extracted by a solid phase extraction procedure that utilized Waters Oasis 96-well extraction plates. Briefly, the 96-well plates were equilibrated, successively, in two steps with 1 mL each of methanol and water. An aliquot of 1M phosphoric acid (0.5-mL) was added to each sample well. Appropriate volumes of calibration curve standard solutions, quality control samples (prepared in control rat or monkey plasma), and plasma samples (0.1 mL) were pipetted into the predetermined sample wells. Control plasma (0.1-mL) was included in each of the calibration curve samples. The internal standard (a close analog of MRK-1, 50 ng) was added to all wells and the contents of each well were thoroughly mixed. The plate was eluted slowly under vacuum until the wells were dry and each sample well was then washed with 0.5-mL of distilled water. The sample wells were eluted with 300-µL of acetonitrile:distilled water mixture (90:10, v/v) into a 96-well collection plate and analyzed using LC-MS/MS. Chromatography was performed on an ABZ+ column (100 mm x 2.1 mm, 5 µm, Supelco) and an HPLC system consisting of Perkin Elmer Series 200 Micro Pumps and autosampler using a gradient mobile phase of acetonitrile, methanol and 1-mM ammonium acetate. The HPLC flow rate was 0.35 mL/min. Detection of the analyte and internal standard was performed using a Sciex API 3000 mass spectrometer in the positive ion mode using the Turbo-Ion Spray source at 400°C. Mass transitions (m/z) monitored were 575 → 444 for MRK-1 and 547 → 282 for the internal standard. Triplicate calibration curves were constructed by plotting peak area ratio of JPET/2002/45096 17 the analyte to internal standard against the analyte concentration. The concentrations of MRK-1 in plasma samples were determined by comparing the analyte to internal standard peak area ratios against the calibration curve. Calibration curves for MRK-1 were constructed at a concentration range of 1-1000 ng/mL and the data were fitted to a power model of the form y = ax b . The variability and bias of the LC-MS/MS assay for MRK-1 at all quality control (QC) levels was <15%. Pharmacokinetic Analyses Pharmacokinetic parameters of MRK-1 were calculated by standard pharmacokinetic approaches (Gibaldi and Perrier, 1982). The AUC up to the last sampling point was calculated by the linear trapezoidal rule. Extrapolation to infinity was performed by the factor C last /λ z , where C last is the plasma concentration at the last sampling time and λ z is the terminal elimination rate constant. For determination of the first-order absorption and elimination rate constants in MRK-1-quindine interaction studies, the plasma concentration-time data were fitted to a 1-compartment model with first-order absorption and elimination. where, F a , Dose oral and AUC oral refer to the fraction of orally administered dose absorbed into the circulation, total administered oral dose and systemic blood AUC of MRK-1 following an oral dose, respectively. Assuming linear systemic pharmacokinetics and constant plasma protein binding, the fraction of the orally administered dose that was absorbed following administration of different doses was calculated using the pharmacokinetic data from the i.v. bolus and oral administration experiments and the above two equations. As described in Results, the assumptions of linear systemic pharmacokinetics and constant plasma protein binding were largely true at the plasma concentrations encountered in our studies. JPET/2002/45096 19 Results MRK-1 is a Substrate for the CYP3A Isozymes in Rat, Monkey and Human Liver Microsomes The relative rates of metabolism in liver microsomes from different species followed the rank order monkey ≥ human > rat. Approximately 90, 100 and 60% of the compound was metabolized at the end of a 60-min incubation period when MRK-1 (10-µM) was incubated with rat, monkey and human liver microsomes (1-mg/mL microsomal protein), respectively. The oxidative metabolism of MRK-1 in human and monkey liver microsomal incubations was completely inhibited when microsomes were pre-incubated with monoclonal antibodies against the CYP3A4 isozyme. In contrast, anti-CYP2C8/9 and anti-CYP2D6 antibodies had no significant inhibitory effect on MRK-1 metabolism in either human or monkey liver microsomes. Similarly, anti-rat CYP3A2 antibody inhibited MRK-1 metabolism in rat liver microsomes by MRK-1 is a Substrate for P-glycoprotein MRK-1 showed a substantially greater B-to-A than A-to-B transport in monolayers of human MDR1 or mouse Mdr1a-transfected cell lines, while the transport in the two directions was roughly equal in the parental LLC-PK1 cells Plasma protein binding and blood-to-plasma partitioning of MRK-1 Rat Intestinal Loop Studies Pharmacokinetics of MRK-1 in Rats and Monkeys Pharmacokinetic parameters of MRK-1 in rats and monkeys after i.v. and oral dosing are presented in The average fraction of administered oral dose of MRK-1 that was absorbed from the intestine into the circulation in rats and monkeys was estimated using the well-stirred model of hepatic elimination as described in Methods. The average hepatic blood flow values for rats and adult monkeys were assumed to be 65-and 45-mL/min/kg, respectively, for the purpose of this calculation (Davies and Morris, 1993). The data presented in Effect of Oral Co-administration of Ritonavir on the Pharmacokinetics of MRK-1 in Rats and Monkeys As described above, MRK-1 is a P-gp substrate and is metabolized mainly by the CYP3A isozymes. In vitro studies described herein suggest that ritonavir is a potent inhibitor of MRK-1 metabolism as well as its P-gp mediated efflux transport. Thus, MRK-1 pharmacokinetics can be influenced by ritonavir via inhibition of both these proteins. The data on the effect of oral coadministration of ritonavir on the pharmacokinetics of MRK-1 are presented in when given in combination with ritonavir; thereafter, the concentrations appeared to decline in parallel to those in experiments without ritonavir. Effect of Oral Co-Administration of Quinidine on the Pharmacokinetics of MRK-1 in Rats A nonlinear increase in the oral bioavailability of MRK-1 in rats and monkeys raises the possibility of involvement of P-gp-mediated efflux in the absorptive processes of MRK-1 at the intestinal mucosal surface such that at higher doses the saturation of this efflux transport may lead to increased absorption and bioavailability. In order to confirm this possibility, studies were conducted to examine the oral pharmacokinetics of MRK-1 when administered in combination with quinidine, a known inhibitor of P-gp. Quinidine was selected because of its relatively low inhibitory potential towards CYP3A isozymes Role of Increased Absorption and Reduced Systemic Elimination of MRK-1 in its Pharmacokinetic Interactions with Ritonavir A profound increase in the plasma AUC of MRK-1 upon oral co-administration with ritonavir in both rats and monkeys raises the question of the relative significance of increased absorption (resulting from inhibition of P-gp at the intestinal mucosal surface) and reduced systemic clearance of MRK-1 (because of inhibition of CYP3A mediated metabolism) in this interaction. We chose to address this issue by resolving the systemic clearance component of this interaction from the overall interaction. This was achieved by examining the effect of oral ritonavir administration on the systemic clearance of MRK-1, as shown in We examined also the possibility of quinidine affecting the systemic clearance of MRK-1 via either inhibition of its metabolism and/or biliary, urinary and intestinal secretion or transport. Similar to ritonavir, oral administration of quinidine at 30-mg/kg was found also to impair the systemic clearance of MRK-1, albeit to a lesser extent (~33%). This corresponds to, on average, a ~2 fold reduction in intrinsic hepatic clearance of MRK-1 based on the well-stirred model of liver Although it is relatively easy to identify the substrates and inhibitors of P-gp using cell lines over expressing this transporter or the isolated intestinal loop preparations, the in vivo significance of these findings is somewhat difficult to ascertain. For example, it is difficult to predict whether the oral absorption of a particular P-gp substrate, as identified from in vitro studies, will be markedly influenced by efflux transport at the intestinal mucosal surface. Similarly, it cannot easily be determined whether a P-gp inhibitor such as ritonavir, which inhibits efflux transport in vitro, would exhibit in vivo drug-drug interactions via this mechanism. The majority of the data in support of the significance of P-gp in determining the disposition and pharmacokinetics of drugs comes from comparative studies in wild type (Mdr1a +/+) and Mdr1a deficient (Mdr1a -/-) mice In the present studies, MRK-1 exhibited profound pharmacokinetic interactions following oral co-administration with ritonavir in rats as well as monkeys. From separate studies on MRK-1 disposition in rats and monkeys, we have determined that the systemic clearance of MRK-1 is The role of P-gp in the absorption of MRK-1 in rats was confirmed further by studies in combination with quinidine when an increased and more rapid absorption of MRK-1 was observed. Interestingly, however, a significant component of the quinidine/MRK-1 interactions also appeared to occur via inhibition of systemic clearance of MRK-1. Although the exact mechanism(s) of this interaction remains to be investigated, it could occur via a combination of inhibition of metabolism, and/or biliary and urinary excretion components of MRK-1 clearance. Quinidine, at 10-and 30-mg/kg doses, appeared to result in similar increases in the extent of MRK-1 absorption (40-50%). However, quinidine increased the rate of MRK-1 absorption in a dose-dependent and in a relatively more profound manner (100-300% increase in the first-order absorption rate constant, In comparison to rats, the nonlinearity in both the bioavailability and estimated oral absorption was somewhat less profound in monkeys and was statistically non-significant. Further, the shape of the plasma concentration vs. time profile of MRK-1 following oral administration suggests a more rapid absorption of the compound in monkeys as compared to rats, i.e., the monkeys appear to exhibit a sharper plasma concentration