Coproporphyrin I Can Serve as an Endogenous Biomarker for OATP1B1 Inhibition: Assessment Using a Glecaprevir/Pibrentasvir Clinical Study.

Organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 are involved in the disposition of a variety of commonly prescribed drugs. The evaluation of OATP1B1/1B3 inhibition potential by investigational drugs is of interest during clinical drug development due to various adverse events associated with increased exposures of their substrates. Regulatory guidance documents on the in vitro assessment of OATP1B1/1B3 inhibition potential are conservative with up to a third of predictions resulting in false positives. This work investigated the utility of OATP1B1/1B3 endogenous biomarkers, coproporphyrin (CP)-I and CP-III, to assess clinical inhibition of OATP1B1/1B3 and potentially eliminate the need for prospective clinical drug-drug interaction (DDI) studies. Correlations between CP-I exposures and various OATP1B1 static DDI predictions were also evaluated. Glecaprevir/pibrentasvir (GLE/PIB) 300/120 mg fixed-dose combination is known to cause clinical inhibition of OATP1B1/1B3. In a clinical study evaluating the relative bioavailability of various formulations of GLE/PIB regimen, CP-I peak plasma concentration (Cmax ) ratio and 0-16-hour area under the concentration-time curve (AUC0-16 ) ratio relative to baseline increased with increasing GLE exposures, whereas there was a modest correlation between GLE exposure and CP-III Cmax ratio but no correlation with CP-III AUC0-16 ratio. This suggests that CP-I is superior to CP-III as an endogenous biomarker for evaluation of OATP1B1 inhibition. There was a significant correlation between CP-I and GLE Cmax (R2  = 0.65; P < 0.001) across individual subjects. Correlation analysis between GLE OATP1B1 R values and CP-I exposures (Cmax ratio and AUC0-16 ratio) suggests that an R value of > 3 can predict a biologically meaningful inhibition of OATP1B1 when the inhibitor clinical pharmacokinetic parameters are available.

Assessment of the effect of erdafitinib on cardiac safety: analysis of ECGs and exposure-QTc in patients with advanced or refractory solid tumors.

PURPOSE: To characterize the effect of erdafitinib on electrocardiogram (ECG) parameters and the relationship between erdafitinib plasma concentrations and QTc interval changes in patients with advanced or refractory solid tumors. METHODS: Triplicate ECGs and continuous 12-lead Holter data were collected in the dose escalation part (Part 1) of the first-in-human study, with doses ranging from 0.5 to 12 mg. Triplicate ECG monitoring continued in Parts 2-4 where 2 dose regimens selected from Part 1 were expanded in prespecified tumor types. Analyses of ECG data included central tendency analyses, identification of categorical outliers and morphological assessment. A concentration-QTc analysis was conducted using a linear mixed-effect model based on extracted time matching Holter data. RESULTS: Central tendency, categorical outlier, and ECG morphologic analyses from 187 patients revealed no clinically significant effect of erdafitinib on heart rate, atrioventricular conduction or cardiac depolarization (PR and QRS), and no effect on cardiac repolarization (QTc). Concentration-QTc analysis from 62 patients indicated that the slopes of relationship between total and free erdafitinib plasma concentrations and QTcI (mean exponent of 0.395) were estimated as - 0.00269 ms/(ng/mL) and - 1.138 ms/(ng/mL), respectively. The predicted change in QTcI at the observed geometric mean of total and free concentration at the highest therapeutic erdafitinib dose (9 mg daily) was < 10 ms at the upper bound of the two-sided 90% confidence interval. CONCLUSIONS: ECG data and the concentration-QTc relationships demonstrate that erdafitinib does not prolong QTc interval and has no effects on cardiac repolarization or other ECG parameters. Clinical trial registration numbers NCT01703481, EudraCT: 2012-000697-34.

Assessment of Drug Interaction Potential Between the Hepatitis C Virus Direct-Acting Antiviral Agents Elbasvir/Grazoprevir and the Nucleotide Analog Reverse-Transcriptase Inhibitor Tenofovir Disoproxil Fumarate.

Treatment of individuals coinfected with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) requires careful consideration of potential drug-drug interactions. We evaluated the pharmacokinetic interaction of the direct-acting antiviral agents elbasvir and grazoprevir coadministered with the nucleotide reverse transcriptase inhibitor tenofovir disoproxil fumarate (TDF). Three open-label, multidose studies in healthy adults were conducted. In the first study (N = 10), participants received TDF 300 mg once daily, elbasvir 50 mg once daily, and elbasvir coadministered with TDF. In the second study (N = 12), participants received TDF 300 mg once daily, grazoprevir 200 mg once daily, and grazoprevir coadministered with TDF. In the third study (N = 14), participants received TDF 300 mg once daily and TDF 300 mg coadministered with coformulated elbasvir/grazoprevir 50 mg/100 mg once daily. Pharmacokinetics and safety were evaluated. Following coadministration, the tenofovir area under the plasma concentration-time curve to 24 hours and maximum plasma concentration geometric mean ratios (90% confidence intervals) for tenofovir and coadministered drug(s) versus tenofovir were 1.3 (1.2, 1.5) and 1.5 (1.3, 1.6), respectively, when coadministered with elbasvir; 1.2 (1.1, 1.3) and 1.1 (1.0, 1.2), respectively, when coadministered with grazoprevir; and 1.3 (1.2, 1.4) and 1.1 (1.0, 1.4), respectively, when coadministered with the elbasvir/grazoprevir coformulation. TDF had minimal effect on elbasvir and grazoprevir pharmacokinetics. Elbasvir and/or grazoprevir coadministered with TDF resulted in no clinically meaningful tenofovir exposure increases and was generally well tolerated, with no deaths, serious adverse events (AEs), discontinuations due to AEs, or laboratory AEs reported. No dose adjustments for elbasvir/grazoprevir or TDF are needed for coadministration in HCV/HIV-coinfected people.

Assessment of drug interaction potential between the HCV direct-acting antiviral agents elbasvir/grazoprevir and the HIV integrase inhibitors raltegravir and dolutegravir.

BACKGROUND: Elbasvir/grazoprevir is a once-daily fixed-dose combination therapy for the treatment of chronic HCV infection, including HCV/HIV coinfection. OBJECTIVES: To evaluate the pharmacokinetic interaction of elbasvir and grazoprevir with raltegravir or dolutegravir. METHODS: Three open-label trials in healthy adult participants were conducted. In the raltegravir trials, participants received a single dose of raltegravir 400 mg, a single dose of elbasvir 50 mg or grazoprevir 200 mg, and raltegravir with either elbasvir or grazoprevir. In the dolutegravir trial, participants received a single dose of dolutegravir 50 mg alone or co-administered with once-daily elbasvir 50 mg and grazoprevir 200 mg. RESULTS: The raltegravir AUC0-∞ geometric mean ratio (GMR) (90% CI) was 1.02 (0.81-1.27) with elbasvir and 1.43 (0.89-2.30) with grazoprevir. Dolutegravir AUC0-∞ GMR (90% CI) was 1.16 (1.00-1.34) with elbasvir and grazoprevir. The elbasvir AUC0-∞ GMR (90% CI) was 0.81 (0.57-1.17) with raltegravir and 0.98 (0.93-1.04) with dolutegravir. The grazoprevir AUC0-24 GMR (90% CI) was 0.89 (0.72-1.09) with raltegravir and 0.81 (0.67-0.97) with dolutegravir. CONCLUSIONS: Elbasvir or grazoprevir co-administered with raltegravir or dolutegravir resulted in no clinically meaningful drug-drug interactions and was generally well tolerated. These results support the assertion that no dose adjustments for elbasvir, grazoprevir, raltegravir or dolutegravir are needed for co-administration in HCV/HIV-coinfected people.

Dietary exposure assessment of cyadox based on tissue depletion of cyadox and its major metabolites in pigs, chickens, and carp.

The tissue kinetics of cyadox, an antibacterial agent used in food animals, and its major metabolites in pigs, chickens, and carp were investigated followed by a complete dietary exposure assessment to evaluate the food safety of cyadox. Cyadox and its major metabolites, bisdeoxycyadox (Cy1), 4-desoxycyadox (Cy2), N-(quinoxaline-2-methyl)-cyanide acetyl hydrazine (Cy4), quinoxaline-2-carboxylic acid (Cy6), and 2-hydromethyl-3-hydroxy-quinoxaline (Cy12), were simultaneously quantitated with a high-performance liquid chromatography-ultraviolet (HPLC-UV) method. Pigs, chickens, and carp were fed with 150 mg/kg cyadox in feed for consecutive 60, 40, and 30 days, respectively. The residue amount of cyadox and its major metabolites in liver, kidney, muscle, and fat (skin) tissues was determined. Cy2 was below the limit of quantitation even at the withdrawal time of 6 hr, cyadox, Cy4, Cy6, and Cy12 could be detected at 6-24 hr with low level less than 50 µg/kg. By contrast, Cy1 persisted for 3 days in the kidney of pigs and chickens, and in the liver of carp. Based on these residue depletion data and previous toxicology results, the global estimated chronic dietary exposure assessment of cyadox for general population was conducted, indicating a zero withdrawal time (WDT) may be appropriate for cyadox in food animals when used in feed for prolonged administration. These results provide analytical techniques and safety standards suitable for residue monitoring of cyadox in food animals.

Physiologically based pharmacokinetic model for quinocetone in pigs and extrapolation to mequindox.

Physiologically based pharmacokinetic (PBPK) models are scientific methods used to predict veterinary drug residues that may occur in food-producing animals, and which have powerful extrapolation ability. Quinocetone (QCT) and mequindox (MEQ) are widely used in China for the prevention of bacterial infections and promoting animal growth, but their abuse causes a potential threat to human health. In this study, a flow-limited PBPK model was developed to simulate simultaneously residue depletion of QCT and its marker residue dideoxyquinocetone (DQCT) in pigs. The model included compartments for blood, liver, kidney, muscle and fat and an extra compartment representing the other tissues. Physiological parameters were obtained from the literature. Plasma protein binding rates, renal clearances and tissue/plasma partition coefficients were determined by in vitro and in vivo experiments. The model was calibrated and validated with several pharmacokinetic and residue-depletion datasets from the literature. Sensitivity analysis and Monte Carlo simulations were incorporated into the PBPK model to estimate individual variation of residual concentrations. The PBPK model for MEQ, the congener compound of QCT, was built through cross-compound extrapolation based on the model for QCT. The QCT model accurately predicted the concentrations of QCT and DQCT in various tissues at most time points, especially the later time points. Correlation coefficients between predicted and measured values for all tissues were greater than 0.9. Monte Carlo simulations showed excellent consistency between estimated concentration distributions and measured data points. The extrapolation model also showed good predictive power. The present models contribute to improve the residue monitoring systems of QCT and MEQ, and provide evidence of the usefulness of PBPK model extrapolation for the same kinds of compounds.

Estimation of residue depletion of cyadox and its marker residue in edible tissues of pigs using physiologically based pharmacokinetic modelling.

Physiologically based pharmacokinetic (PBPK) models are powerful tools to predict tissue distribution and depletion of veterinary drugs in food animals. However, most models only simulate the pharmacokinetics of the parent drug without considering their metabolites. In this study, a PBPK model was developed to simultaneously describe the depletion in pigs of the food animal antimicrobial agent cyadox (CYA), and its marker residue 1,4-bisdesoxycyadox (BDCYA). The CYA and BDCYA sub-models included blood, liver, kidney, gastrointestinal tract, muscle, fat and other organ compartments. Extent of plasma-protein binding, renal clearance and tissue-plasma partition coefficients of BDCYA were measured experimentally. The model was calibrated with the reported pharmacokinetic and residue depletion data from pigs dosed by oral gavage with CYA for five consecutive days, and then extrapolated to exposure in feed for two months. The model was validated with 14 consecutive day feed administration data. This PBPK model accurately simulated CYA and BDCYA in four edible tissues at 24-120 h after both oral exposure and 2-month feed administration. There was only slight overestimation of CYA in muscle and BDCYA in kidney at earlier time points (6-12 h) when dosed in feed. Monte Carlo analysis revealed excellent agreement between the estimated concentration distributions and observed data. The present model could be used for tissue residue monitoring of CYA and BDCYA in food animals, and provides a foundation for developing PBPK models to predict residue depletion of both parent drugs and their metabolites in food animals.

A physiologically based pharmacokinetic model for the prediction of the depletion of methyl-3-quinoxaline-2-carboxylic acid, the marker residue of olaquindox, in the edible tissues of pigs.

To estimate the consumer exposure to olaquindox (OLA) residues in porcine edible tissues, a physiologically based pharmacokinetic (PBPK) model for methyl-3-quinoxaline-2-carboxylic acid (MQCA), the marker residue of OLA, was developed in pigs based on the assumptions of the flow-limited distribution, hepatic metabolism, and renal excretion. The model included separate compartments corresponding to blood, muscle, liver, kidney, adipose, and an extra compartment representing the remaining carcass. Physiological parameters were determined from literatures. Plasma protein binding, partition coefficients, and renal clearance for MQCA were determined in in vitro and in vivo studies. The metabolic conversion of OLA to MQCA was assumed as a simple, one-step process, and an apparent first-order rate constant (k) was employed to describe this metabolic process. The PBPK model was optimized and validated with plasma and tissue data from literatures and our study. Sensitivity analysis and Monte Carlo simulation were also implemented to estimate the influence of model parameters on the goodness of fit. When compared with the observed data, the PBPK model underestimated the MQCA level in all compartments at the early time points, whereas gave excellent predictions of MQCA concentration in porcine edible tissues at later time points. The correlation coefficients between the predicted and observed values were over 0.88. The consistency between the model predictions and the real residues of OLA in pigs proved the good applicability of our model in food safety risk assessment.

Quantitative assessment of exposure-response relationships for the efficacy and tolerability of varenicline for smoking cessation.

Population exposure-response analyses involving approximately 2,000 cigarette smokers provided an integrated understanding of dose, exposure, patient characteristics, and response relating to the efficacy and tolerability of varenicline for smoking cessation. Full models with a linear function of area under the concentration-time curve at steady state AUC(0-24)(ss) and covariate effects on the baseline probability of response were constructed. Logistic regression results consistently showed that the end-of-treatment abstinence rate increased with increasing varenicline exposure, from 38% at 0.5 mg b.i.d. to 56% at 1 mg b.i.d. (vs. 22% for placebo). Baseline smoking status and age were predictive of smoking cessation, whereas race and gender showed little or no influence. Nausea was the most common adverse event, with an incidence that was gender-related and that increased with varenicline exposure; at a dosage of 1 mg b.i.d. the predicted probability of nausea relative to placebo was 24 vs. 7% in male subjects and 40 vs. 14% in female subjects. The incidence of nausea also showed a decreasing trend with time.

Varenicline: a first-line treatment option for smoking cessation.

BACKGROUND: Varenicline acts as a partial agonist/antagonist with affinity and selectivity for alpha(4) beta(2) nicotinic acetylcholine receptors. This activity at the nicotine-receptor level may help patients achieve smoking cessation by reducing cravings/withdrawal symptoms and smoking satisfaction. OBJECTIVE: This article reviews the literature on the pharmacologic properties, therapeutic efficacy, and tolerability of varenicline for smoking cessation. METHODS: Pertinent controlled clinical trials, meta-analyses, meeting abstracts, case reports, and review articles published in English between 1966 and May 2008 were identified through searches of MEDLINE and OVID using the terms varenicline, smoking, tobacco cessation, and CP 526555. RESULTS: Eight clinical trials were identified that compared

Fluorescence-enhanced europium-diethylenetriaminepentaacetic (DTPA)-monoamide complexes for the assessment of renal function.

Real-time, noninvasive assessment of glomerular filtration rate (GFR) is essential not only for monitoring critically ill patients at the bedside, but also for staging and monitoring patients with chronic kidney disease. In our pursuit to develop exogenous luminescent probes for dynamic optical monitoring of GFR, we have prepared and evaluated Eu(3+) complexes of several diethylenetriamine pentaacetate (DTPA)-monoamide ligands bearing molecular "antennae" to enhance metal fluorescence via intramolecular ligand-metal fluorescence resonance energy transfer process. The results show that Eu-DTPA-monoamide complex 18b, which contains a quinoxanlinyl antenna, exhibits large (ca. 2700-fold) Eu(3+) fluorescence enhancement. Indeed, complex 18b exhibits the highest fluorescent enhancement observed thus far in the DTPA-type metal complexes. The renal clearance property was assessed using the corresponding radioactive (111)In complex 18a, and the data suggest that this complex clears via a complex mechanism that includes glomerular filtration.

Pharmacokinetics of a novel N-methyl-D-aspartate receptor antagonist (SM-18400): identification of an N-acetylated metabolite and pre-clinical assessment of N-acetylation polymorphism.

(S)-9-chloro-5-[p-aminomethyl-o-(carboxymethoxy)phenylcarbamoylmethyl]-6,7-dihydro-1 H,5 H-pyrido[1,2,3-de]quinoxaline-2,3-dione hydrochloride trihydrate (SM-18400) was given intravenously to rats and dogs and its pharmacokinetics was investigated. By LC/MS/MS analysis, the major metabolite in the rat serum was identified as N-acetylated SM-18400 (SM-NAc). In rats, AUC ratio of SM-NAc to SM-18400 was approximately 50%. However, 71% of the dose was excreted as unchanged SM-18400 and only 9.8% as SM-NAc in the urine and bile, indicating that the contribution of N-acetylation clearance (CL(NAc)) to the total clearance (CL(tot)) is limited to 10-30% in rats. No SM-NAc or other metabolites were detected in the dog serum, urine or bile. The in vitro intrinsic clearance (CL(int), ml/min/mg cytosolic protein) of N-acetyltransferase (NAT) activities of dog liver cytosol towards SM-18400 and hepatic N-acetylation clearance (CL(NAc), ml/min/kg body weight) estimated by well-stirred model were both only 5% of the respective rat value, well reflecting the relative in vivo CL(NAc)/CL(tot) ratios. CL(int) values for human liver cytosol samples (n = 4) and estimated CL(NAc) were all less than 18% and 7% of the rat, respectively. Based on these results, we concluded that the CL(NAc)/CL(tot) of human would be small enough to avoid major inter-individual variance in SM-18400 pharmacokinetics due to N-acetylation polymorphism. In addition, even a human liver cytosol sample lacking polymorphic NAT2 activity as determined by sulfamethazine (SMZ) N-acetylation analysis, proved capable of acetylating SM-18400, suggesting that NAT2 is not the major enzyme responsible for N-acetylation of SM-18400 in human. This fact would also reduce the risk of N-acetylation polymorphism playing a role in clinical use of this drug.

Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment.

Various methods of exposure assessment, such as questionnaires, sometimes combined with pictures of cooked meat, have been employed in investigations on the relationship between heterocyclic amines (HA) and health effects. However, as the content of heterocyclic amines vary greatly with cooking conditions, it is difficult to obtain an accurate estimate of the exposure. To improve the exposure assessment, the use of biomarkers has been investigated. The metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is well characterised. In humans, the major part of the dose is excreted in urine within 24-48 h following a meal. A few percent is excreted as parent compounds, whereas the major part is metabolites. Urinary level of parent HA reflects only recent exposure. However, the pattern of excreted metabolites might indicate the capacity to activate or detoxify HAs. The excretion of glucuronide conjugates of N-hydroxy-PhIP and N-hydroxy-MeIQx could be a marker for the N-hydroxylation capacity and the dose of the proximate metabolites. Recently, we proposed 5-OH-PhIP as a marker for the ultimate reactive metabolite of PhIP, since it is formed from this compound as a by-product along with the formation of PhIP-DNA adducts. In a search for biomarkers reflecting exposure over some time, blood protein adducts with a longer lifespan have been investigated, and PhIP adducts of serum albumin and haemoglobin from meat-consuming humans were recently reported. Many compounds, like drugs, nicotine and narcotics, bind to melanin in hair and give information on exposure for longer time periods. In mice, PhIP is irreversibly incorporated in a dose-dependent manner into hair, and in humans exposed to an ordinary diet, it was found to vary from <50 to 5000 pg PhIP/g hair. The incorporation is also dependent on the content of eumelanin. The use of PhIP in hair as a biomarker of exposure is promising, but needs validation, using other methods of exposure assessment.

Rational dose selection for a nonnucleoside reverse transcriptase inhibitor through use of population pharmacokinetic modeling and Monte Carlo simulation.

In order to choose a rational dose for GW 420867X, we first set a goal of therapy. We hypothesized that, for optimal antiretroviral activity, the trough free drug concentration should remain above the 90% effective concentration (EC90) of human immunodeficiency virus type 1. We performed population pharmacokinetic analysis on three different doses of GW 420867X (50, 100, and 200 mg). Monte Carlo simulation was performed, assuming a log-normal distribution for 1,000 simulated subjects for each dose, and was repeated three times. The trough concentrations were divided by 76 to account for protein binding and for the difference between EC50 and EC90. We then determined the fraction of the simulated population whose free drug trough concentrations would exceed an EC90 over a broad range of values. The target attainment for all three doses exceeded 95% out to a starting EC50 of 10 nM. For 16 viral isolates, the EC50 range encountered for GW 420867X did not exceed 8 nM, implying that the three doses could not be differentiated by effect in a clinical trial in naive patients. This prediction was shown to be correct in a randomized, double-blind trial with 1 week of monotherapy with GW 420867X.

Assessment of the DNA adduction and pharmacokinetics of PhIP and MeIOx in rodents at doses approximating human exposure using the technique of accelerator mass spectrometry (AMS) and 32P-postlabeling.

Estimating the cancer risk posed by heterocyclic amines depends on measuring how chemical dose influences measurable indicators of cancer progression. This data ideally should encompass the range of actual human exposure, at the low dose end, and laboratory animal studies, at the high dose end. Accelerator mass spectrometry (AMS) has been used to measure the absorption, fate, and DNA adduct dosimetry of the heterocyclic amines PhIP and MeIQx at doses equivalent to human consumption following single-dose administration and chronic daily dosing. AMS is a nuclear physics technique which specifically counts nuclei of cosmogenic isotopes, rather than relying on decay. For tracing 14C, sensitivity is increased 10(6)-fold relative to decay counting. We have found that tissue clearance rates for [2-(14)C]-PhIP are rapid (t1/2 = 1 h) at low dose (41 ng/kg), with most of the radiocarbon distributed to the liver and G.I. tract. MeIQx-DNA adduct levels decrease linearly with dose (5 mg/kg-500 ng/kg) in single dose exposures. Likewise, the biologically available dose of [2-(14)C]-MeIQx decreases linearly with decreasing dose (5 mg/kg-1 ng/kg). On chronic daily dosing, it takes 40 days for adducts to reach steady-state in tissues and adduct levels appear to decrease linearly with decreasing dose, except possibly at very low doses. DNA binding of PhIP involves both sulfation or acetylation of the N-hydroxylated PhIP. Quantitatively, sulfation appears to be an important pathway for PhIp activation in rodent tissue cytosols while acetylation appears quantitatively more important in human tissue cytosols. The greatest activity is in liver and intestinal tissues for both pathways. The specific DNA adducts formed in vivo and in vitro from exposure to PhIP and MeIQx are likely guanine adducts. These data suggest that DNA adduct dosimetry responds linearly with dose but may become sub-linear at very low doses for chronic exposure and that factors other than DNA adduction may be critical to explain these heterocyclic amines' tumorigenicity.

Metabolism of the food-borne mutagen/carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in the rat: assessment of biliary metabolites for genotoxicity.

The absorption and kinetics of excretion of [14C]2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) was studied in male Sprague-Dawley rats. Within 72 hr of an oral dose of [14C]MeIQx (20 mg/kg) 33-56% of the radioactivity was excreted in the urine and 37-75% of the radioactivity in the faeces, which accounted for greater than 99% of the dose. Only low levels of radioactivity remained in the body. Radioactivity, when expressed per gram of tissue, was highest in the liver and kidney with smaller amounts detected in the lung and both the small and large intestines. Between 25 and 50% of a dose of MeIQx was recovered in the bile within 24 hr. Biliary metabolites were excreted over a long period of time with one radioactive fraction rapidly excreted at 2-3 hr and a second fraction excreted at 10-12 hr. The metabolites present in bile were assessed for genotoxicity using Salmonella typhimurium TA98 with or without hepatic S-9 activation and were found to be present as detoxified products. The residual mutagenic activity present in bile was attributed primarily to unmetabolized MeIQx.