Jeroen J.M.A. Hendrikx

P‐glycoprotein and cytochrome P450 3A act together in restricting the oral bioavailability of paclitaxel

Paclitaxel is avidly transported by P‐glycoprotein (P‐gp/MDR1/ABCB1). This results in low oral bioavailability, which can be boosted by coadministration of P‐gp inhibitors. Unlike paclitaxel, docetaxel is extensively metabolized by CYP3A4 and its oral bioavailability can be enhanced in mice and humans by coadministration of the potent CYP3A inhibitor ritonavir. Unexpectedly, ritonavir also enhances the oral bioavailability of paclitaxel in humans. We aimed to resolve the mechanism underlying this enhancement. Using mice lacking Cyp3a and/or P‐gp, we investigated the combined and separate restricting roles of Cyp3a and P‐gp in the oral bioavailability of paclitaxel, and the boosting effect of ritonavir. CYP3A4‐humanized mice were used for translation to the human situation. P‐gp had a dominant effect (11.6‐fold, p < 0.001) over Cyp3a (<1.5‐fold, n.s.) in limiting plasma concentrations of oral paclitaxel. However, in the absence of P‐gp, Cyp3a decreased paclitaxel plasma concentrations twofold (p < 0.001). Coadministered ritonavir inhibited Cyp3a‐mediated metabolism, but not P‐gp‐mediated transport of paclitaxel. Owing to the dominant effect of P‐gp, ritonavir enhanced only paclitaxel plasma concentrations in P‐gp‐deficient mice. Mouse liver microsomes metabolized paclitaxel far less efficiently than human or CYP3A4‐transgenic liver microsomes, revealing much lower efficiency of paclitaxel metabolism by mouse than by human CYP3As. Accordingly, ritonavir could enhance the oral bioavailability of paclitaxel in CYP3A4‐humanized mice, despite the fact that these mice are P‐gp‐proficient. Our results show that CYP3A4 inhibition most likely underlies the boosting effect of ritonavir on oral paclitaxel bioavailability in humans. Furthermore, CYP3A4‐humanized mice allow improved understanding of CYP3A4‐mediated paclitaxel metabolism in humans.

Human OATP1B1, OATP1B3 and OATP1A2 can mediate the in vivo uptake and clearance of docetaxel.

Organic anion transporting polypeptides (human: OATPs and mouse: Oatps) are uptake transporters with important roles in drug pharmacokinetics and toxicity. We aimed to study the in vivo impact of mouse and human OATP1A/1B transporters on docetaxel plasma clearance and liver and intestinal uptake. Docetaxel was administered to Oatp1a/1b knockout and liver‐specific humanized OATP1B1, OATP1B3 and OATP1A2 transgenic mice. Experiments were conducted with a low polysorbate 80 (2.8%) formulation, as 8% polysorbate somewhat inhibited docetaxel plasma clearance after intravenous administration. After intravenous administration (10 mg/kg), Oatp1a/1b knockout mice had an approximately threefold higher plasma area under the curve (AUC). Impaired liver uptake was evident from the significantly reduced (approximately threefold) liver‐to‐plasma AUC ratios. Absence of mouse Oatp1a/1b transporters did not affect the intestinal absorption of orally administered docetaxel (10 mg/kg), while the systemic exposure of docetaxel was again substantially increased owing to impaired liver uptake. Most importantly, liver‐specific expression of each of the human OATP1B1, OATP1B3 and OATP1A2 transporters provided a nearly complete rescue of the increased plasma levels of docetaxel in Oatp1a/1b‐null mice after intravenous administration. Our data show that one or more of the mouse Oatp1a/1b transporters and each of the human OATP1A/1B transporters can mediate docetaxel uptake in vivo. This might be clinically relevant for OATP1A/1B‐mediated tumor uptake of docetaxel and for docetaxel clearance in patients in whom the transport activity of OATP1A/1B transporters is reduced owing to genetic variation or pharmacological inhibition, leading to potentially altered toxicity and therapeutic efficacy of this drug.

Apical ABC Transporters and Cancer Chemotherapeutic Drug Disposition

ATP-binding cassette (ABC) transporters are transmembrane efflux transporters that mediate cellular extrusion of a broad range of substrates ranging from amino acids, lipids, and ions to xenobiotics including many anticancer drugs. ABCB1 (P-GP) and ABCG2 (BCRP) are the most extensively studied apical ABC drug efflux transporters. They are highly expressed in apical membranes of many pharmacokinetically relevant tissues such as epithelial cells of the small intestine and endothelial cells of the blood capillaries in brain and testis, and in the placental maternal-fetal barrier. In these tissues, they have a protective function as they efflux their substrates back to the intestinal lumen or blood and thus restrict the intestinal uptake and tissue disposition of many compounds. This presents a major challenge for the use of many (anticancer) drugs, as most currently used anticancer drugs are substrates of these transporters. Herein, we review the latest findings on the role of apical ABC transporters in the disposition of anticancer drugs. We discuss that many new, rationally designed anticancer drugs are substrates of these transporters and that their oral availability and/or brain disposition are affected by this interaction. We also summarize studies that investigate the improvement of oral availability and brain disposition of many cytotoxic (e.g., taxanes) and rationally designed (e.g., tyrosine kinase inhibitor) anticancer drugs, using chemical inhibitors of these transporters. These findings provide a better understanding of the importance of apical ABC transporters in chemotherapy and may therefore advance translation of promising preclinical insights and approaches to clinical studies.

A sensitive combined assay for the quantification of paclitaxel, docetaxel and ritonavir in human plasma using liquid chromatography coupled with tandem mass spectrometry

A combined assay for the determination of paclitaxel, docetaxel and ritonavir in human plasma is described. The drugs were extracted from 200 μL human plasma using liquid-liquid extraction with tertiar-butylmethylether, followed by high performance liquid chromatography analysis using 10 mM ammonium hydroxide pH 10:methanol (3:7, v/v) as mobile phase. Chromatographic separation was obtained using a Zorbax Extend C(18) column. Labelled analogues of the analytes are used as internal standards. For detection, positive ionization electrospray tandem mass spectrometry was used. Method development including optimisation of the mass transitions and response, mobile phase optimisation and column selection are discussed. The method was validated according to FDA guidelines and the principles of Good Laboratory Practice (GLP). The validated range was 0.5-500 ng/mL for paclitaxel and docetaxel and 2-2000 ng/mL for ritonavir. For quantification, quadratic calibration curves were used (r(2)>0.99). The total runtime of the method is 9 min and the assay combines analytes with differences in ionisation and desired concentration range. Inter-assay accuracy and precision were tested at four concentration levels and were within 10% and less than 10%, respectively, for all analytes. Carry-over was less than 6% and endogenous interferences or interferences between analytes and internal standards were less than 20% of the response at the lower limit of quantification level. The matrix factor and recovery were determined at low, mid and high concentration levels. The matrix factor was around 1 for all analytes and total recovery between 77.5 and 104%. Stability was investigated in stock solutions, human plasma, dry extracts, final extracts and during 3 freeze/thaw cycles. The described method was successfully applied in clinical studies with oral administration of docetaxel or paclitaxel in combination with ritonavir.

Oral co-administration of elacridar and ritonavir enhances plasma levels of oral paclitaxel and docetaxel without affecting relative brain accumulation

Background:The intestinal uptake of the taxanes paclitaxel and docetaxel is seriously hampered by drug efflux through P-glycoprotein (P-gp) and drug metabolism via cytochrome P450 (CYP) 3A. The resulting low oral bioavailability can be boosted by co-administration of P-gp or CYP3A4 inhibitors.Methods:Paclitaxel or docetaxel (10 mg/kg) was administered to CYP3A4-humanised mice after administration of the P-gp inhibitor elacridar (25 mg kg−1) and the CYP3A inhibitor ritonavir (12.5 mg kg−1). Plasma and brain concentrations of the taxanes were measured.Results:Oral co-administration of the taxanes with elacridar increased plasma concentrations of paclitaxel (10.7-fold, P<0.001) and docetaxel (four-fold, P<0.001). Co-administration with ritonavir resulted in 2.5-fold (paclitaxel, P<0.001) and 7.3-fold (docetaxel, P<0.001) increases in plasma concentrations. Co-administration with both inhibitors simultaneously resulted in further increased plasma concentrations of paclitaxel (31.9-fold, P<0.001) and docetaxel (37.4-fold, P<0.001). Although boosting of orally applied taxanes with elacridar and ritonavir potentially increases brain accumulation of taxanes, we found that only brain concentrations, but not brain-to-plasma ratios, were increased after co-administration with both inhibitors.Conclusions:The oral availability of taxanes can be enhanced by co-administration with oral elacridar and ritonavir, without increasing the brain penetration of the taxanes.

Genetically modified mouse models for oral drug absorption and disposition.

Intestinal absorption is an essential step in the therapeutic use of most orally administered drugs and often mediated by enterocyte transmembrane transporters. Here we discuss several of these drug transport systems and knockout mouse models to study them. These studies showed that Multidrug resistance-associated protein 2 (Mrp2) can limit intestinal drug absorption. Organic cation transporter n1 (Octn1) and Octn2 might also facilitate intestinal drug absorption, although direct in vivo evidence is lacking. On the other hand, intestinal uptake of drugs is facilitated by the Equilibrative nucleoside transporter 1 (Ent1), Mrp3 and possibly Mrp4. No significant role in intestinal absorption for Oct1 and Oct2 or for Organic anion-transporting polypeptides (Oatp) 1a and 1b was found so far.

The effect of complementary and alternative medicines on CYP3A4-mediated metabolism of three different substrates: 7-benzyloxy-4-trifluoromethyl-coumarin, midazolam and docetaxel

Concomitant use of complementary and alternative medicine (CAM) and anticancer drugs can affect the pharmacokinetics of anticancer drugs by inhibiting the metabolizing enzyme cytochrome P450 3A4 (CYP3A4) (EC 1.14.13.157). Several in vitro studies determined whether CAM can inhibit CYP3A4, but these studies revealed contradictory results. A plausible explanation for these conflicting results is the use only of a single model CYP3A4 substrate in each study. Therefore, the objective was to determine the potential of selected CAM (β‐carotene, Echinacea, garlic, Ginkgo biloba, ginseng, grape seed extract, green tea extract, milk thistle, saw palmetto, valerian, vitamin B6, B12 and C) to inhibit CYP3A4‐mediated metabolism of different substrates: 7‐benzyloxy‐4‐trifluoromethyl‐coumarin (BFC), midazolam and docetaxel. The effect of CAM on CYP3A4‐mediated metabolism of an anticancer drug has never been determined before in vitro, which makes this study unique. The oncolytic CYP3A4 substrate docetaxel was used to establish the predictive value of the model substrates for pharmacokinetic interactions between CAM and anticancer drugs in vitro, and to more closely predict these interactions in vivo.

Quantification of docetaxel and its metabolites in human plasma by liquid chromatography/tandem mass spectrometry

RATIONALE During drug development accurate quantification of metabolites in biological samples using mass spectrometry is often hampered by the lack of metabolites of chemically pure quality. However, quantification of metabolites can be useful for assessment and interpretation of (pre)clinical data. We now describe an approach to quantify docetaxel metabolites in human plasma by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using docetaxel calibration standards. METHODS Metabolites (M1/M3, M2 and M4) were generated using microsomal incubations. Retention times of docetaxel and its metabolites were assessed using an LC/UV assay and peak identification was performed by LC/MS(n). Samples containing isolated metabolites from human faeces were quantified by LC/UV and used as references for spiking human plasma samples. LC/MS/MS was applied to sensitively quantify docetaxel and its metabolites in human plasma using docetaxel calibration standards in a range of 0.25-500 ng/mL. RESULTS Because ionisation of docetaxel and its metabolites differed, correction factors were established to quantify the metabolites using docetaxel calibration samples. During method validation, accuracy and precision of the metabolites were within ±7.7% and ≤17.6%, respectively, and within ±14.3% and ≤10.1%, respectively, for docetaxel. Metabolites were found to be unstable in human plasma at ambient temperature. After storage up to 1 year at -20 °C, recovered metabolite concentrations were within ±25%. CONCLUSIONS Development and validation of an LC/MS/MS assay for the quantification of docetaxel and its metabolites M1/M3, M2 and M4 using docetaxel calibration standards is described. The same approach may be used for quantification of metabolites of other drugs by LC/MS/MS when chemically pure reference substances are unavailable.

P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice.

We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.

Crizotinib-induced fatal fulminant liver failure

Herein we describe a case of a 62-year-old female in good clinical condition with non-small-cell lung cancer who was treated with crizotinib. After 24 days of crizotinib therapy she presented with acute liver failure. Serum aspartate aminotransferase and alanine aminotransferase levels had increased from normal prior to crizotinib start to 2053 IU/L and 6194 IU/L, respectively. Total bilirubin and prothrombin time (PT-INR) increased up to 443 IU/L and 5.33, respectively, and symptoms of hepatic encephalopathy and hepatorenal syndrome emerged. Despite crizotinib discontinuation and intensive supportive therapy, the patient died 40 days after treatment with crizotinib was initiated due to acute liver failure with massive liver cell necrosis.