Survey of Pharmaceutical Industry's Best Practices around In Vitro Transporter Assessment and Implications for Drug Development: Considerations from the International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Quantitation of Plasma Membrane Drug Transporters in Kidney Tissue and Cell Lines Using a Novel Proteomic Approach Enabled a Prospective Prediction of Metformin Disposition.

The successful prospective incorporation of in vitro transporter kinetics in physiologically based pharmacokinetic (PBPK) models to describe drug disposition remains challenging. Although determination of scaling factors to extrapolate in vitro to in vivo transporter kinetics has been facilitated by quantitative proteomics, no robust assessment comparing membrane recoveries between different cells/tissues has been made. HEK293 cells overexpressing OCT2, MATE1, and MATE2K or human kidney cortex were homogenized and centrifuged to obtain the total membrane fractions, which were subsequently subjected to liquid-liquid extraction followed by centrifugation and precipitation to isolate plasma membrane fractions. Plasma membrane recoveries determined by quantitation of the marker Na+/K+-ATPase in lysate and plasma membrane fractions were ≤20% but within 3-fold across different cells and tissues. A separate study demonstrated that recoveries are comparable between basolateral and apical membranes of renal proximal tubules, as measured by Na+/K+-ATPase and γ-glutamyl transpeptidase 1, respectively. The plasma membrane expression of OCT2, MATE1, and MATE2K was quantified and relative expression factors (REFs) were determined as the ratio between the tissue and cell concentrations. Corrections using plasma membrane recovery had minimal impact on REF values (<2-fold). In vitro transporter kinetics of metformin were extrapolated to in vivo using the corresponding REFs in a PBPK model. The simulated metformin exposures were within 2-fold of clinical exposure. These results demonstrate that transporter REFs based on plasma membrane expression enable a prediction of transporter-mediated drug disposition. Such REFs may be estimated without the correction of plasma membrane recovery when the same procedure is applied between different matrices. SIGNIFICANCE STATEMENT: Transporter REFs based on plasma membrane expression enable in vitro-in vivo extrapolation of transporter kinetics. Plasma membrane recoveries as determined by the quantification of sodium-potassium adenosine triphosphatase were comparable between the in vitro and in vivo systems used in the present study, and therefore had minimal impact on the transporter REF values.

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.

Prodrug Strategies to Improve the Solubility of the HCV NS5A Inhibitor Pibrentasvir (ABT-530).

A research program to discover solubilizing prodrugs of the HCV NS5A inhibitor pibrentasvir (PIB) identified phosphomethyl analog 2 and trimethyl-lock (TML) prodrug 9. The prodrug moiety is attached to a benzimidazole nitrogen atom via an oxymethyl linkage to allow for rapid and complete release of the drug for absorption following phosphate removal by intestinal alkaline phosphatase. These prodrugs have good hydrolytic stability properties and improved solubility compared to PIB, both in aqueous buffer (pH 7) and FESSIF (pH 5). TML prodrug 9 provided superior in vivo performance, delivering high plasma concentrations of PIB in PK studies conducted in mice, dogs, and monkeys. The improved dissolution properties of these phosphate prodrugs provide them the potential to simplify drug dosage forms for PIB-containing HCV therapy.

Discovery of 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs for delivering nucleoside HCV NS5B polymerase inhibitors.

Our HCV research program investigated novel 2'-dihalogenated nucleoside HCV polymerase inhibitors and identified compound 1, a 5'-phosphoramidate prodrug of 2'-deoxy-2'-α-bromo-ß-chloro uridine. Although 1 had a favorable in vitro activity profile in HCV replicons, oral dosing in dog resulted in low levels of the active 5'-triphosphate (TP) in liver. Metabolism studies using human hepatocytes provided a simple assay for screening alternative phosphoramidate prodrug analogs. Compounds that produced high TP concentrations in hepatocytes were tested in dog liver biopsy studies. This method identified 2-aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrug 14, which provided 100-fold higher TP concentrations in dog liver in comparison to 1 (4 and 24 h after 5 mg/kg oral dose).

Synthesis and evaluation of 2'-dihalo ribonucleotide prodrugs with activity against hepatitis C virus.

Hepatitis C virus (HCV) nucleoside inhibitors have been a key focus of nearly 2 decades of HCV drug research due to a high barrier to drug resistance and pan-genotypic activity profile provided by molecules in this drug class. Our investigations focused on several potent 2'-halogenated uridine-based HCV polymerase inhibitors, resulting in the discovery of novel 2'-deoxy-2'-dihalo-uridine analogs that are potent inhibitors in replicon assays for all genotypes. Further studies to improve in vivo performance of these nucleoside inhibitors identified aminoisobutyric acid ethyl ester (AIBEE) phosphoramidate prodrugs 18a and 18c, which provide high levels of the active triphosphate in dog liver. AIBEE prodrug 18c was compared with sofosbuvir (1) by co-dosing both compounds by oral administration in dog (5 mg/kg each) and measuring liver concentrations of the active triphosphate metabolite at both 4 and 24 h post dosing. In this study, 18c provided liver triphosphate concentrations that were 6-fold higher than sofosbuvir (1) at both biopsy time points, suggesting that 18c could be a highly effective agent for treating HCV infected patients in the clinic.

Translation of In Vitro Transport Inhibition Studies to Clinical Drug-Drug Interactions for Glecaprevir and Pibrentasvir.

Glecaprevir and pibrentasvir are oral direct-acting antiviral agents approved in combination for treatment of chronic hepatitis C viral infection. In vitro studies identified the combination as potentially clinically relevant inhibitors of the efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and the hepatic uptake transporters organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Glecaprevir inhibited P-gp, BCRP, OATP1B1, and OATP1B3 with IC50 values of 0.33, 2.3, 0.017, and 0.064 µM, respectively. Pibrentasvir inhibited P-gp, BCRP, and OATP1B1 with IC50 values of 0.036, 14, and 1.3 µM, respectively. Neither agent inhibited organic cation transporter (OCT) 1, OCT2, organic anion transporter (OAT) 1, OAT3, multidrug and toxin extrusion (MATE) 1, or MATE2K. Open-label phase 1 clinical drug-drug interaction studies were conducted in healthy subjects to evaluate interaction potential of glecaprevir/pibrentasvir and coadministered selective substrates for P-gp (digoxin, dabigatran etexilate, and sofosbuvir), BCRP (rosuvastatin and sofosbuvir), and OATP1B1/3 (pravastatin and rosuvastatin). The pharmacokinetic maximum plasma concentration (C max) and area under the concentration-time curve (AUC) parameters were evaluated for probe substrates alone and in combination with glecaprevir/pibrentasvir. The C max central values increased by 72%, 105%, 123%, 462%, and 66% for digoxin, dabigatran, pravastatin, rosuvastatin, and sofosbuvir, respectively, and the AUC central values increased by 48%, 138%, 130%, 115%, and 125% for digoxin, dabigatran, pravastatin, rosuvastatin, and sofosbuvir, respectively. Exposure of sofosbuvir metabolite GS-331007 (nucleoside analog) was similar with or without glecaprevir/pibrentasvir. The outcomes of the clinical drug-drug interaction studies confirmed clinically relevant inhibition of P-gp, BCRP, and OATP1B1/3, and were used to provide dosing guidance for the concomitant use of glecaprevir/pibrentasvir with relevant transporter substrates.

No Inhibition of MATE1/2K-Mediated Renal Creatinine Secretion Predicted With Ritonavir or Cobicistat.

Cobicistat has been reported to increase serum creatinine clinically without affecting glomerular filtration. This was ascribed to transient inhibition of MATE1-mediated renal creatinine secretion. Interestingly, a structurally similar drug, ritonavir, has not been associated with serum creatinine increases at the pharmacoenhancer dose. The present study was aimed to investigate the translation of in vitro MATE1/2K inhibition to clinical creatinine increase (cobicistat) and lack of it (ritonavir) considering their intracellular concentrations in renal proximal tubules. Uptake studies showed ritonavir and cobicistat are unlikely substrates for OCT2. The steady-state unbound concentration in the cytosol of human renal proximal tubule epithelial cells was comparable with the extracellular unbound concentration, suggesting that the entry of these compounds is predominantly mediated by passive diffusion. Ritonavir and cobicistat are MATE1 and MATE2K inhibitors with IC50 values of 3.1 and 90 µM (ritonavir), and 4.4 and 3.2 µM (cobicistat), respectively. However, the unbound cytosolic concentrations (Cu,cytosol) of ritonavir and cobicistat in human renal proximal tubule epithelial cells, 0.065 and 0.10 µM, respectively, after incubation with the clinical maximum total plasma concentrations at pharmacoenhancer doses does not support inhibition in vivo; Cu,cytosol >30 fold lower than IC50s. These results demonstrate that MATE1/2K inhibition is unlikely the mechanism of the clinical creatinine elevations with cobicistat.

Mechanistic Physiologically Based Pharmacokinetic Modeling of the Dissolution and Food Effect of a Biopharmaceutics Classification System IV Compound-The Venetoclax Story.

Venetoclax, a selective B-cell lymphoma-2 inhibitor, is a biopharmaceutics classification system class IV compound. The aim of this study was to develop a physiologically based pharmacokinetic (PBPK) model to mechanistically describe absorption and disposition of an amorphous solid dispersion formulation of venetoclax in humans. A mechanistic PBPK model was developed incorporating measured amorphous solubility, dissolution, metabolism, and plasma protein binding. A middle-out approach was used to define permeability. Model predictions of oral venetoclax pharmacokinetics were verified against clinical studies of fed and fasted healthy volunteers, and clinical drug interaction studies with strong CYP3A inhibitor (ketoconazole) and inducer (rifampicin). Model verification demonstrated accurate prediction of the observed food effect following a low-fat diet. Ratios of predicted versus observed Cmax and area under the curve of venetoclax were within 0.8- to 1.25-fold of observed ratios for strong CYP3A inhibitor and inducer interactions, indicating that the venetoclax elimination pathway was correctly specified. The verified venetoclax PBPK model is one of the first examples mechanistically capturing absorption, food effect, and exposure of an amorphous solid dispersion formulated compound. This model allows evaluation of untested drug-drug interactions, especially those primarily occurring in the intestine, and paves the way for future modeling of biopharmaceutics classification system IV compounds.

Mechanisms and Predictions of Drug-Drug Interactions of the Hepatitis C Virus Three Direct-Acting Antiviral Regimen: Paritaprevir/Ritonavir, Ombitasvir, and Dasabuvir.

To assess drug-drug interaction (DDI) potential for the three direct-acting antiviral (3D) regimen of ombitasvir, dasabuvir, and paritaprevir, in vitro studies profiled drug-metabolizing enzyme and transporter interactions. Using mechanistic static and dynamic models, DDI potential was predicted for CYP3A, CYP2C8, UDP-glucuronosyltransferase (UGT) 1A1, organic anion-transporting polypeptide (OATP) 1B1/1B3, breast cancer resistance protein (BCRP), and P-glycoprotein (P-gp). Perpetrator static model DDI predictions for metabolizing enzymes were within 2-fold of the clinical observations, but additional physiologically based pharmacokinetic modeling was necessary to achieve the same for drug transporters. When perpetrator interactions were assessed, ritonavir was responsible for the strong increase in exposure of sensitive CYP3A substrates, whereas paritaprevir (an OATP1B1/1B3 inhibitor) greatly increased the exposure of sensitive OATP1B1/1B3 substrates. The 3D regimen drugs are UGT1A1 inhibitors and are predicted to moderately increase plasma exposure of sensitive UGT1A1 substrates. Paritaprevir, ritonavir, and dasabuvir are BCRP inhibitors. Victim DDI predictions were qualitatively in line with the clinical observations. Plasma exposures of the 3D regimen were reduced by strong CYP3A inducers (paritaprevir and ritonavir; major CYP3A substrates) but were not affected by strong CYP3A4 inhibitors, since ritonavir (a CYP3A inhibitor) is already present in the regimen. Strong CYP2C8 inhibitors increased plasma exposure of dasabuvir (a major CYP2C8 substrate), OATP1B1/1B3 inhibitors increased plasma exposure of paritaprevir (an OATP1B1/1B3 substrate), and P-gp or BCRP inhibitors (all compounds are substrates of P-gp and/or BCRP) increased plasma exposure of the 3D regimen. Overall, the comprehensive mechanistic assessment of compound disposition along with mechanistic and PBPK approaches to predict victim and perpetrator DDI liability may enable better clinical management of nonstudied drug combinations with the 3D regimen.

Validation of a total IC50 method which enables in vitro assessment of transporter inhibition under semi-physiological conditions.

1. Accurate predictions of clinical transporter-mediated drug-drug interactions (DDI) from in vitro data can be challenging when compounds have poor solubility and/or high nonspecific binding. Additionally, current DDI predictions for compounds with high plasma-protein binding assume that the unbound fraction in plasma is 0.01, if the experimental value is less than 0.01 or cannot be determined. This approach may result in an overestimation of DDI risk. To overcome these challenges, it may be beneficial to conduct inhibition studies under physiologically relevant conditions. 2. Here, IC50 values, determined in the presence of 4% bovine serum albumin approximating human plasma albumin concentrations, were successfully used to predict DDI for uptake transporters, OATP1B1/1B3, OCT1/2, OAT1/3 and MATE1/2K. 3. The IC50 values of reference inhibitors with 4% bovine serum albumin, considered total IC50, were comparable to the predicted values based on nominal IC50 values determined under protein-free conditions and unbound fraction in plasma. Calculation of R-total and Cmax/IC50,total values using total plasma exposure and total IC50 values explained the clinical DDI or absence of it for these inhibitors. 4. These results suggest that IC50 determinations in the presence of 4% albumin can be used, in the context of clinical total exposure, to predict DDI involving uptake transporters.

In vitro OATP1B1 and OATP1B3 inhibition is associated with observations of benign clinical unconjugated hyperbilirubinemia.

1. Transient benign unconjugated hyperbilirubinemia has been observed clinically with several drugs including indinavir, cyclosporine, and rifamycin SV. Genome-wide association studies have shown significant association of OATP1B1 and UGT1A1 with elevations of unconjugated bilirubin, and OATP1B1 inhibition data correlated with clinical unconjugated hyperbilirubinemia for several compounds. 2. In this study, inhibition of OATP1B3 and UGT1A1, in addition to OATP1B1, was explored to determine whether one measure offers value over the other as a potential prospective tool to predict unconjugated hyperbilirubinemia. OATP1B1 and OATP1B3-mediated transport of bilirubin was confirmed and inhibition was determined for atazanavir, rifampicin, indinavir, amprenavir, cyclosporine, rifamycin SV and saquinavir. To investigate the intrinsic inhibition by the drugs, both in vivo Fi (fraction of intrinsic inhibition) and R-value (estimated maximum in vivo inhibition) for OATP1B1, OATP1B3 and UGT1A1 were calculated. 3. The results indicated that in vivo Fi values >0.2 or R-values >1.5 for OATP1B1 or OATP1B3, but not UGT1A1, are associated with previously reported clinical cases of drug-induced unconjugated hyperbilirubinemia. 4. In conclusion, inhibition of OATP1B1 and/or OATP1B3 along with predicted human pharmacokinetic data could be used pre-clinically to predict potential drug-induced benign unconjugated hyperbilirubinemia in the clinic.

Prediction of clinical drug-drug interactions of veliparib (ABT-888) with human renal transporters (OAT1, OAT3, OCT2, MATE1, and MATE2K).

Veliparib (ABT-888) is largely eliminated as parent drug in human urine (70% of the dose). Renal unbound clearance exceeds glomerular filtration rate, suggesting the involvement of transporter-mediated active secretion. Clinically relevant pharmacokinetic interactions in the kidney have been associated with OAT1, OAT3, OCT2, MATE1, and MATE2K. In the present study, interactions of veliparib with these transporters were investigated. Veliparib inhibited OAT1, OAT3, OCT2, MATE1, and MATE2K with IC50 values of 1371, 505, 3913, 69.9, and 69.5 µM, respectively. The clinical unbound maximum plasma concentration of veliparib after single oral dose of 50 mg (0.45 µM) is manyfold lower than IC50 values for OAT1, OAT3, OCT2, MATE1, or MATE2K. These results indicate a low potential for drug-drug interaction (DDI) with OAT1/3, OCT2, or MATE1/2K. Additional studies demonstrated that veliparib is a substrate of OCT2. In Oct1/Oct2 double-knockout mice, the plasma exposure of veliparib was increased by 1.5-fold, and the renal clearance was decreased by 1.8-fold as compared with wild-type mice, demonstrating that organic cation transporters contribute to the renal elimination in vivo. In summary, the in vitro transporter data for veliparib predicts minimal potential for an OAT1/3-, OCT2-, and MATE1/2K-mediated DDI given the clinical exposure after single oral dose of 50 mg.

Localization of P-gp (Abcb1) and Mrp2 (Abcc2) in freshly isolated rat hepatocytes.

Freshly isolated hepatocytes are widely accepted as the "gold standard" for providing reliable data on drug uptake across the sinusoidal (basolateral) membrane. However, the suitability of freshly isolated hepatocytes in suspension to assess efflux by canalicular (apical) proteins or predict biliary excretion in the intact organ is unclear. After collagenase digestion, hepatocytes rapidly lose polarity, but localization of canalicular transport proteins in the first few hours after isolation has not been well characterized. In this study, immunostaining and confocal microscopy have provided, for the first time, a detailed examination of canalicular transport protein localization in freshly isolated rat hepatocytes fixed within 1 h of isolation and in cells cultured for 1 h. Organic anion transporting polypeptide 1a1 (Oatp1a1) was expressed in all hepatocytes and distributed evenly across the basolateral membrane; there was no evidence for colocalization of Oatp1a1 with P-glycoprotein (P-gp) or multidrug resistance-associated protein 2 (Mrp2). In contrast, P-gp and Mrp2 expression was lower than Oatp1a1 and confined to junctions between adjacent cells, intracellular compartments, and "legacy" network structures at or near the cell surface. P-gp and Mrp2 staining was more predominant in regions adjacent to former canalicular spaces, identified by zonula occludens-1 staining. Functional analysis of rat hepatocytes cultured for 1 h demonstrated that the fluorescent anion and Mrp2 substrate, 5-(and-6)-carboxy-2',7'-dichlorofluorescein (CDF), accumulated in cellular compartments; compartmental accumulation of CDF was sensitive to (E)-3-[[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid (MK571, Mrp inhibitor) and was not observed in hepatocytes isolated from Mrp2-deficient rats. Drug efflux from freshly isolated hepatocytes as an estimate of apical efflux/biliary excretion would give an inaccurate assessment of true apical elimination and, as such, should not be used to make in vivo extrapolations.

The impact of plasma protein binding on the renal transport of organic anions.

Drugs and xenobiotics bind to plasma proteins with varying degrees of affinity, and the amount of binding has a direct effect on free drug concentration and subsequent pharmacokinetics. Multiple active and facilitative transport systems regulate the excretion of anionic compounds from the blood in excretory and barrier tissues. Assumptions are made about in vivo substrate affinity and route of elimination based on data from plasma protein-free in vitro assays, particularly following expression of cloned transporters. Ochratoxin A (OTA), a fungal mycotoxin, is a high-affinity substrate for several renal secretory organic anion transporters (OATs), and literature suggests that this elimination pathway is the route of entry leading to proximal tubule-targeted toxicity. However, OTA is known to bind to several plasma proteins with a high affinity, particularly serum albumin, which may impact elimination. In this study, we have systematically examined the handling of OTA and other organic anions, estrone sulfate (ES) and methotrexate (MTX), by OATs in the presence of serum albumin. Increasing concentrations of albumin markedly reduced uptake of OTA by both Xenopus laevis oocytes expressing OATs 1, 3, and 4 and organic anion-transporting polypeptide 1. For all transporters tested, virtually all mediated OTA uptake was eliminated by an albumin concentration equivalent to 10% of that present in the blood plasma. Thus, OTA uptake is dependent on the free substrate concentration and severely limited by binding to human serum albumin. MTX and ES uptake were likewise dependent on free concentration.

Molecular aspects of the transport and toxicity of ochratoxin a.

Ochratoxins are a class of naturally occurring compounds produced by several fungi. The most toxic is ochratoxin A (OTA), and occurrence of some human nephropathies and tumors correlate with enhanced OTA exposure. In this Account, the following areas are examined: molecular details of the binding of OTA to human serum albumin (HSA), the influences of binding to HSA on the trans-port of OTA across epithelial cell membranes by organic anion transport proteins, the oxidative activation of OTA, and the formation of OTA adducts with biological molecules. These studies are beginning to provide a detailed chemical model for the trans-port, accumulation, and genotoxic and carcinogenic effects of OTA.