Biological Role of Pazopanib and Sunitinib Aldehyde Derivatives in Drug-Induced Liver Injury.

Tyrosine kinase inhibitors pazopanib and sunitinib are both used to treat advanced renal cell carcinoma but expose patients to an increased risk of hepatotoxicity. We have previously identified two aldehyde derivatives for pazopanib and sunitinib (P-CHO and S-CHO, respectively) in liver microsomes. In this study, we aimed to decipher their role in hepatotoxicity by treating HepG2 and HepaRG hepatic cell lines with these derivatives and evaluating cell viability, mitochondrial dysfunction, and oxidative stress accumulation. Additionally, plasma concentrations of P-CHO were assessed in a cohort of patients treated with pazopanib. Results showed that S-CHO slightly decreased the viability of HepG2, but to a lesser extent than sunitinib, and affected the maximal respiratory capacity of the mitochondrial chain. P-CHO decreased viability and ATP production in HepG2. Traces of P-CHO were detected in the plasma of patients treated with pazopanib. Overall, these results showed that P-CHO and S-CHO affect hepatocyte integrity and could be involved in the pazopanib and sunitinib hepatotoxicity.

Model-Based Quantification of Impact of Genetic Polymorphisms and Co-Medications on Pharmacokinetics of Tamoxifen and Six Metabolites in Breast Cancer.

Variations in clinical response to tamoxifen (TAM) may be related to polymorphic cytochromes P450 (CYPs) involved in forming its active metabolite endoxifen (ENDO). We developed a population pharmacokinetic (PopPK) model for tamoxifen and six metabolites to determine clinically relevant factors of ENDO exposure. Concentration-time data for TAM and 6 metabolites come from a prospective, multicenter, 3-year follow-up study of adjuvant TAM (20 mg/day) in patients with breast cancer, with plasma samples drawn every 6 months, and genotypes for 63 genetic polymorphisms (PHACS study, NCT01127295). Concentration data for TAM and 6 metabolites from 928 patients (n = 27,433 concentrations) were analyzed simultaneously with a 7-compartment PopPK model. CYP2D6 phenotype (poor metabolizer (PM), intermediate metabolizer (IM), normal metabolizer (NM), and ultra-rapid metabolizer (UM)), CYP3A4*22, CYP2C19*2, and CYP2B6*6 genotypes, concomitant CYP2D6 inhibitors, age, and body weight had a significant impact on TAM metabolism. Formation of ENDO from N-desmethyltamoxifen was decreased by 84% (relative standard error (RSE) = 14%) in PM patients and by 47% (RSE = 9%) in IM patients and increased in UM patients by 27% (RSE = 12%) compared with NM patients. Dose-adjustment simulations support an increase from 20 mg/day to 40 and 80 mg/day in IM patients and PM patients, respectively, to reach ENDO levels similar to those in NM patients. However, when considering Antiestrogenic Activity Score (AAS), a dose increase to 60 mg/day in PM patients seems sufficient. This PopPK model can be used as a tool to predict ENDO levels or AAS according to the patient's CYP2D6 phenotype for TAM dose adaptation.

Involvement of Pazopanib and Sunitinib Aldehyde Reactive Metabolites in Toxicity and Drug-Drug Interactions in Vitro and in Patient Samples.

Tyrosine kinase inhibitors (TKI) are targeted anticancer drugs that have been successfully developed over the past 2 decades. To date, many of them (around 70%) require warnings for liver injury and five of them, including pazopanib and sunitinib, have Black Box Warning (BBW) labels. Although TKI-induced hepatotoxicity is the first cause of drug failures in clinical trials, BBW labels, and market withdrawals, the underlying mechanisms remain unclear. However, the recent discovery of new reactive metabolites (RM) with aldehyde structures during pazopanib and sunitinib metabolism offers new perspectives for investigating their involvement in the toxicity of these two TKI. These hard electrophiles have a high reactivity potential toward proteins and are thought to be responsible for cytochrome P450 inactivation, drug-drug interactions (DDI), and liver toxicity. We report here, for the first time, the presence of these aldehyde RM in human plasma samples obtained during drug monitoring. Docking experiments in the CYP3A4 active site were performed and showed that pazopanib and sunitinib fitting in the catalytic site are in accordance with their regioselective oxidation to aldehydes. They also suggested that aldehyde RM may react with lysine and arginine residues. Based on these results, we studied the reactivity of the aldehyde RM toward lysine and arginine residues as potential targets on the protein framework to better understand how these RM could be involved in liver toxicity and drug-drug interactions. Adduct formation with different hepatic and plasma proteins was investigated by LC-MS/MS, and adducts between pazopanib or sunitinib aldehyde derivatives and lysine residues on both CYP3A4 and plasma proteins were indeed shown for the first time.

Identifying the reactive metabolites of tyrosine kinase inhibitors in a comprehensive approach: Implications for drug-drug interactions and hepatotoxicity.

Tyrosine kinase inhibitors (TKI) are small heterocyclic molecules targeting transmembrane and cytoplasmic tyrosine kinases that have met with considerable success in clinical oncology. TKI are associated with toxicities including liver injury that may be serious and even life-threatening. Many of them require warnings in drug labeling against liver injury, and five of them have Black Box Warning (BBW) labels. Although drug-induced liver injury is a matter of clinical and industrial concern, little is known about the underlying mechanisms that likely involve reactive metabolites (RM). RM are electrophiles or radicals originating from the metabolic activation of particular functional groups, known as structural alerts or toxicophores. RM are able to covalently bind to proteins and macromolecules, causing cellular damage and even cell death. If the adducted protein is the enzyme involved in RM formation, time-dependent inhibition of the enzyme-also called mechanism-based inhibition (MBI) or inactivation-can occur and lead to pharmacokinetic drug-drug interactions. To mitigate RM liabilities, common practice in drug development includes avoiding structural alerts and assessing RM formation via RM trapping screens with soft and hard nucleophiles (glutathione, potassium cyanide, and methoxylamine) in liver microsomes. RM-positive derivatives are further optimized to afford drug candidates with blocked or minimized bioactivation potential. However, different structural alerts are still commonly used scaffolds in drug design, including in TKI structures. This review focuses on the current state of knowledge of the relations among TKI structures, bioactivation pathways, RM characterization, and hepatotoxicity and cytochrome P450 MBI in vitro.

Factors Affecting Tamoxifen Metabolism in Patients With Breast Cancer: Preliminary Results of the French PHACS Study.

In addition to the effect of cytochrome P450 (CYP) 2D6 genetic polymorphisms, the metabolism of tamoxifen may be impacted by other factors with possible consequences on therapeutic outcome (efficacy and toxicity). This analysis focused on the pharmacokinetic (PK)-pharmacogenetic evaluation of tamoxifen in 730 patients with adjuvant breast cancer included in a prospective multicenter study. Plasma concentrations of tamoxifen and six major metabolites, the genotype for 63 single-nucleotide polymorphisms, and comedications were obtained 6 months after treatment initiation. Plasma concentrations of endoxifen were significantly associated with CYP2D6 diplotype (P < 0.0001), CYP3A4*22 genotype (P = 0.0003), and concomitant intake of potent CYP2D6 inhibitors (P < 0.001). Comparison of endoxifen levels showed that the CYP2D6 phenotype classification could be improved by grouping intermediate metabolizer (IM)/IM and IM/poor metabolizer diplotype into IM phenotype for future use in tamoxifen therapy optimization. Finally, the multivariable regression analysis showed that formation of tamoxifen metabolites was independently impacted by CYP2D6 diplotype and CYP3A4*22, CYP2C19*2, and CYP2B6*6 genetic polymorphisms.

Metalloporphyrin-Catalyzed Oxidation of Sunitinib and Pazopanib, Two Anticancer Tyrosine Kinase Inhibitors: Evidence for New Potentially Toxic Metabolites.

Oxidation of two tyrosine kinase inhibitors (TKIs) sunitinib and pazopanib, using a chemical catalytic system able to mimic the cytochrome P450 type oxidation, allowed us to prepare  putative reactive/toxic metabolites of these anticancer drugs. Among these metabolites, aromatic aldehyde derivatives were unambiguously characterized. Such biomimetic oxidation of TKI-type drugs was essential to facilitate the identification of low amounts of aldehydes generated from these TKIs when incubated with human liver microsomes (HLM), which are classical models of human hepatic metabolism. These TKI derivative aldehydes quickly react in vitro with amines. A similar reaction is expected to occur in vivo and may be at the origin of the potentially severe hepatotoxicity of these TKIs.

Simultaneous monitoring of pazopanib and its metabolites by UPLC-MS/MS.

Pazopanib is a multi-targeted tyrosine kinase inhibitor (TKI) approved as first-line treatment for patients with advanced renal cell carcinoma (RCC) and as second-line treatment for patients with advanced soft tissue sarcoma (STS) previously treated with chemotherapy. The most common adverse events, observed during the RCC and STS trials, were gastrointestinal disorders, hypertension, fatigue, elevated ALAT and ASAT, but the molecular mechanisms explaining pazopanib toxicity remain unclear. Therapeutic activity is considered to be mainly dependent on pazopanib exposure as the primary metabolites are inactive or display low plasma concentrations, but metabolites may be involved in toxicity as relationships between metabolite profiles and toxicity have not been evaluated. We report here, for the first time, the validation of a method for the simultaneous quantification of pazopanib and semi-quantification of its metabolites (relative determination). As there are no standards available, pazopanib metabolites were generated with human liver microsomes (HLM) to provide controls in the development of an UPLC-MS/MS method for monitoring both pazopanib and metabolites. The optimised method was validated for specificity, linearity, sensitivity, precision, accuracy, matrix effect and stability. The coefficient of variation (CV%) for intra-day and inter-day precision varied from 2.1% to 7.9% and 5.6% to 13.1% respectively. The biases varied from -12% to 2.3% (intra-day) and 3.8% to 13.1% (inter-day) for accuracy evaluation. Intra-day and inter-day precision CV were respectively 20.1% and 19.6% and accuracy biases were between 20.7% (intra-day) and 3.8% (inter-day) at the limit of quantification. The recoveries from matrix samples spiked with pazopanib were respectively 102.6 ±â€¯12.9% and 102.5 ±â€¯1.2% at low and high levels of calibration range. No matrix effect was evidenced as demonstrated by the normalised matrix factor values: 1.3 ±â€¯0.1 and 1.2 ±â€¯0.2 respectively measured at low and high part of calibration range. A good stability of pazopanib was observed during short term, long term and in process storage conditions and after three freeze/thaw cycles. The method was applied to clinical samples from three patients treated with pazopanib to establish the metabolite profiles (semi-quantitative data) during treatment. The assessment of metabolite profiles could be useful to improve our understanding of the occurrence of adverse events and to improve pazopanib pharmacokinetic-pharmacodynamic relationships.

Circulating oxysterol metabolites as potential new surrogate markers in patients with hormone receptor-positive breast cancer: Results of the OXYTAM study.

Accumulating evidence indicates that cholesterol oxygenation products, also known as oxysterols (OS), are involved in breast cancer (BC) promotion. The impact of Tam, as well as aromatase inhibitors (AI), an alternative BC endocrine therapy (ET), on OS metabolism in patients is currently unknown. We conducted a prospective clinical study in BC patients receiving Tam (n=15) or AI (n=14) in adjuvant or in metastatic settings. The primary end point was the feasibility of detecting and quantifying 11 different OS in the circulation of patients before and after 28days of treatment with Tam or AI. Key secondary end points were the measurements of variations in the concentrations of OS according to differences between patients and treatments. OS profiling in the serum of patients was determined by gas chromatography coupled to mass spectrometry. OS profiling was conducted in all patients both at baseline and during treatment regimens. An important inter-individual variability was observed for each OS. Interestingly 5,6ß-epoxycholesterol relative concentrations significantly increased in the entire population (p=0.0109), while no increase in Cholestane-triol (CT) levels was measured. Interestingly, we found that, in contrast to AI, Tam therapy significantly decreased blood levels of 24-hydroxycholesterol (24-HC), 7α-HC and 25-HC (a tumor promoter) (p=0.0007, p=0.0231 and p=0.0231, respectively), whereas 4ß-HC levels increased (p=0.0010). Interestingly, levels of 27-HC (a tumor promoter) significantly increased in response to AI (p=0.0342), but not Tam treatment. According to these results, specific OS are promising candidate markers of Tam and AI efficacy. Thus, further clinical investigations are needed to confirm the use of oxysterols as biomarkers of both prognosis and/or the efficacy of ET.

An UPLC-MS/MS method for separation and accurate quantification of tamoxifen and its metabolites isomers.

A selective and accurate analytical method is needed to quantify tamoxifen and its phase I metabolites in a prospective clinical protocol, for evaluation of pharmacokinetic parameters of tamoxifen and its metabolites in adjuvant treatment of breast cancer. The selectivity of the analytical method is a fundamental criteria to allow the quantification of the main active metabolites (Z)-isomers from (Z)'-isomers. An UPLC-MS/MS method was developed and validated for the quantification of (Z)-tamoxifen, (Z)-endoxifen, (E)-endoxifen, Z'-endoxifen, (Z)'-endoxifen, (Z)-4-hydroxytamoxifen, (Z)-4'-hydroxytamoxifen, N-desmethyl tamoxifen, and tamoxifen-N-oxide. The validation range was set between 0.5ng/mL and 125ng/mL for 4-hydroxytamoxifen and endoxifen isomers, and between 12.5ng/mL and 300ng/mL for tamoxifen, tamoxifen N-desmethyl and tamoxifen-N-oxide. The application to patient plasma samples was performed.

Should therapeutic drug monitoring of the unbound fraction of imatinib and its main active metabolite N-desmethyl-imatinib be developed?

PURPOSE: The European Society for Medical Oncology recommends therapeutic drug monitoring (TDM) for imatinib, based on total plasma concentrations in cases of sub-optimal response, failure, or adverse events. Imatinib is highly bound to alpha-1 acid glycoprotein (AGP) in the plasma. We determined the unbound plasma fraction of both imatinib and its main active metabolite (N-desmethyl-imatinib) in plasma from 44 patients. The objective was to quantify the inter-individual variability of the protein binding of imatinib in order to discuss the potential benefits and limits of TDM of free plasma concentrations. PATIENTS AND METHODS: The quantification of unbound fraction of imatinib and N-desmethyl-imatinib was performed using plasma ultrafiltration coupled with LC-MS/MS measurement. 60 pre-dose plasma samples were obtained at steady state within TDM in 44 chronic myeloid leukemia patients. RESULTS: The mean unbound fractions of imatinib and N-desmethyl-imatinib were 2.94 and 5.10 %, respectively, with inter-individual variability (CV in %) of 57 % for imatinib and 71 % for the metabolite. For 11 patients, repeated blood sampling gave a mean intra-individual variability of 28 % for imatinib and 34 % for N-desmethyl-imatinib. No correlation was observed between these measured individual imatinib unbound fraction values and those obtained using an equation based on AGP levels previously proposed by Widmer et al. The mean N-desmethyl-imatinib/imatinib ratio was determined for both total (0.69) and unbound (1.10) concentrations, with inter-individual variabilities of 71 and 86 %, respectively. CONCLUSION: The large inter-individual variability for the unbound fraction of both imatinib and N-desmethyl-imatinib warrants further evaluation of the pharmacokinetic-pharmacodynamic relationship as a potential relevant marker of imatinib therapeutic outcomes.

Determination of unbound fraction of imatinib and N-desmethyl imatinib, validation of an UPLC-MS/MS assay and ultrafiltration method.

Imatinib is a small-molecule tyrosine kinase inhibitor with large inter-individual but low intra-individual pharmacokinetic variability with consistent concentration-efficacy and concentration-toxicity relationships. For these reasons imatinib therapeutic drug monitoring is based on total plasma concentrations. However, since a significant impact of unbound imatinib concentrations on clinical response and/or toxicity evaluation has been suggested, the quantification of free fraction of imatinib and its active metabolite are of interest for therapeutic monitoring. Hence a reliable method for both separation and assay of the free fraction is needed. Using plasma samples spiked with imatinib (from 1000 to 7500 ng/mL) and its metabolite (from 1000 to 2500 ng/mL), an ultrafiltration procedure and an UPLC assay which give reproductive values for unbound fractions of imatinib (mean 3.0±1.0%) and metabolite N-desmethyl imatinib (3.6±1.8%) have been developed. The validation of the analytical UPLC-MS/MS method associated to ultrafiltration for quantification of imatinib and N-desmethyl imatinib was reported. The LOQ was set at 10 ng/mL for imatinib and 20 ng/mL for N-desmethyl imatinib, intraday CV (%) ranged from 2.7 to 4.8% for imatinib and from 5.4 to 12.4% for N-desmethyl imatinib and interday CV (%) ranged from 5.6 to 6.5% for imatinib and from 5.4 to 16.1% for N-desmethyl imatinib. Methodological modifications were attempted to overcome non specific binding (NSB) on the ultrafiltration device. Two types of devices previously used for unbound determination of drugs were tested. Our results clearly showed that the methodology and the features of devices used for ultrafiltration could totally compromise the determination of unbound concentrations of a drug.

Unexpected high levels of vorinostat when combined with vinorelbine in patients with advanced cancer.

BACKGROUND: This study was a multi-centre, dose-escalation trial in patients with advanced cancers. Primary objective was to determine maximum tolerated dose (MTD) of vorinostat, a competitive inhibitor of histone deacetylase (HDAC), in combination with vinorelbine. Secondary aims were to determine (1) corresponding pharmacokinetics, (2) safety of this regimen, and (3) impact of UGT1A1 and 2B17 polymorphisms on vorinostat pharmacokinetics. METHODS: Starting dose of once daily oral vorinostat was 200 mg for 7 days every 21 days in combination with a 20-min intraveinous weekly infusion of vinorelbine 25 mg/m2, starting 4 hours after the first vorinostat dose. During cycle 1, blood samples were collected at day 1 for vorinostat and at days 1 and 8 for vinorelbine for pharmacokinetic evaluation. RESULTS: Seven patients were included. Most of adverse events observed were mild (grades 0-2) and reversible after treatment discontinuation (hemotological toxicity, asthenia, diarrhea, dyspnea, fever, hyperglycemia and nausea). Two patients had a dose limiting toxicity at the first dose level that consisted of grade 3 hyperglycemia and vinorelbine administration was delayed. The first dose-level was considered as the MDT and therefore dose escalation was stopped. Mean vorinostat plasma AUC was higher than reported previously at a similar dose when used as single agent or in combination with other cytotoxics. There was no obvious vinorelbine-vorinostat interaction nor any correlation with UGT1A1 or 2B17 polymorphisms. CONCLUSION: MDT of the combination was 200 mg oral vorinostat for 7 days in combination with 25 mg/m2 weekly vinorelbine. Severity of hyperglycemia was most likely related to unexpected high vorinostat exposures.

The use of isolated enterocytes to study Phase I intestinal drug metabolism: validation with rat and pig intestine.

An important step in the development of new drugs is to evaluate the extent of their metabolism during absorption in the small intestine. Reliable in vitro systems to do this can expediate the development process, but the current systems are often unsuitable because they lack the appropriate metabolic enzymes (e.g. Caco-2 cell monolayers) or are not representative of the physiological conditions present in the intact intestinal cells (e.g. isolated microsomes). The aim of this study was to validate the use of isolated intestinal epithelial cells (enterocytes), equivalent to hepatocytes, to evaluate Phase I drug metabolism. A method was developed to prepare enterocytes from rat and pig (as metabolically closer to man) that maintained good viability and activity for up to 90 min as judged by trypan blue exclusion and the release of the cytosolic enzyme lactate dehydrogenase. The Phase I metabolism of the established marker drugs: midazolam, bupropion and dextromethorphan were measured by LC-MS and confirmed the activities of the 3A, 2B and 2D families of CYP isoforms, respectively. The kinetic parameters, K(m) and V(max), were compared between isolated cells and isolated intestinal microsomes from the rat. The use of isolated intestinal cells is a simple and practical method to study the Phase I metabolism of drugs during their absorption and the potential for drug-drug interactions. The method could eventually be modified and usefully applied to human studies.

Quantification of topotecan by liquid chromatography-mass spectrometry (LC-MS). Application to intestinal transport using rat everted gut sacs.

Topetecan is an important anti-cancer drug that has recently become available as an oral formulation. In order to study its intestinal absorption in vitro and a potential drug-drug interaction with the anti-emetic ondansetron, a sensitive and accurate method for the analysis of topotecan in biological media was required. We developed a liquid-liquid extraction method at pH 7.0-7.5 with a recovery of 85% and which took into account the complex chemical behaviour of topotecan related to the lactone opening and the keto-enol tautomerism. This enabled us to validate a new specific and sensitive LC-MS method of analysis, with satisfactory inter- and intra-day repeatability and accuracy. The method was applied to a study of topotecan uptake in rat everted gut sacs that showed that, despite being a P-glycoprotein substrate like topotecan, ondansetron did not interfere with topotecan uptake.

Oral absorption of ampicillin: role of paracellular route vs. PepT1 transporter.

The beta-lactam antibiotic ampicillin has a relatively poor oral bioavailability in animals and man (30-40%), and its widespread agricultural use in livestock may be contributing to the emergence of antibiotic resistance in the environment. The aim of this study was to define the absorption mechanism by which ampicillin crosses the small intestinal epithelium. The improved rat everted gut sac system was used, with an emphasis on the role of the PepT1 transporter. The absorption kinetics, effects of pH and the use of competitive substrates failed to provide any substantive evidence that the transporter played a major role in ampicillin absorption. Ethylenediaminetetraacetic acid enhanced the absorption, and tissue levels remained low, suggesting that paracellular transport was predominant. pH and competition studies with glycylsarcosine, the widely used PepT1 substrate, also failed to show any transporter activity. Despite evidence from studies with Caco-2 cells that beta-lactam antibiotics are transported by the PepT1 transporter in rat small intestine, the results rather suggest that paracellular diffusion is the major mechanism of absorption, at least for beta-lactam antibiotics with poor bioavailability, such as ampicillin. We suggest that the use of Caco-2 cells underestimates the role of the paracellular route in the absorption of hydrophilic drugs in vivo, and may exaggerate the role of influx transporters.

The metabolism of midazolam and comparison with other CYP enzyme substrates during intestinal absorption: in vitro studies with rat everted gut sacs.

PURPOSE: The purpose of this study was to quantify the intestinal metabolism of midazolam, a CYP P450 substrate, usually used as a probe for the activity of the isoform CYP3A4/1 and to compare it with previous results obtained for other P450 substrates such as testosterone, dextromethorphan and bupropion, which show some specificities for different CYP isoforms. The aim was to shed light on the role of metabolism in the intestinal tissues and the relationship with efflux mechanisms, such as by P-glycoprotein (P-gp) and the influence of metabolism on bioavailability. METHODS: We used the improved everted rat gut sac model to study in vitro the absorption and metabolism of the different CYP isoenzyme probes: midazolam, testosterone, bupropion and dextromethorphan. This method enables drug metabolism to be studied during absorption, conditions which mimic the in vivo situation. The drugs and their metabolites were measured by LC-MS in the mucosal and serosal media and in the mucosal tissue, to give a complete picture of the transport and metabolism. RESULTS: Midazolam, as with the other CYP probes, was metabolized in everted gut sacs. The metabolites were detected in the same proportions in both the serosal and mucosal compartments for midazolam, testosterone and bupropion. In the case of dextromethorphan, the metabolite methoxymorphinan was found at a higher concentration in the mucosal compartment, indicating efflux from the cells. The transport of dextromethorphan and its metabolite was not modified in the presence of verapamil, a P-gp inhibitor, thus demonstrating that dextromethorphan and methoxymorphinan were not P-gp substrates. CONCLUSION: Given that the rat is a widely used species for pre-clinical studies, the everted gut sac model provides a useful tool to assess the role of metabolism during drug absorption by the intestine and is also capable of demonstrating P-glycoprotein mediated transport.

Impact of excipients on the absorption of P-glycoprotein substrates in vitro and in vivo.

The efflux transporter, P-glycoprotein (P-gp), located in the apical membranes of intestinal absorptive cells, can reduce the bioavailability of a wide range of orally administered drugs. A number of surfactants/excipients have been shown to inhibit P-gp, and thus potentially enhance drug absorption. In this study, the improved everted gut sac technique was used to screen excipients for their ability to enhance the absorption of digoxin and celiprolol in vitro. The most effective excipients with digoxin were (at 0.5%, w/v): Labrasol > Imwitor 742 > Acconon E = Softigen 767 > Cremophor EL > Miglyol > Solutol HS 15 > Sucrose monolaurate > Polysorbate 20 > TPGS > Polysorbate 80. With celiprolol, Cremophor EL and Acconon E had no effect, but transport was enhanced by Softigen 767 > TPGS > Imwitor 742. In vivo, the excipients changed the pharmacokinetic profile of orally administered digoxin or celiprolol, but without increasing the overall AUC. The most consistent change was an early peak of absorption, probably due to the higher concentration of excipient in the proximal intestine where the expression of P-gp is lower. These studies show that many excipients/surfactants can modify the pharmacokinetics of orally administered drugs that are P-gp substrates.