P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine

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P-glycoprotein, CYP3A and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice Seng Chuan Tang, Anita Kort, Ka Lei Cheung, Hilde Rosing, Tatsuki Fukami, Selvi Durmas, Els Wagenaar, Jeroen J.M.A. Hendrikx, Miki Nakajima, Bart J.M. van Vlijmen, Jos H Beijnen, and Alfred H. Schinkel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00470 • Publication Date (Web): 28 Aug 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Molecular Pharmaceutics

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

Seng Chuan Tang1†, Anita Kort1,2†, Ka Lei Cheung3, Hilde Rosing2, Tatsuki Fukami4, Selvi Durmus1, Els Wagenaar1, Jeroen J.M.A. Hendrikx2, Miki Nakajima4, Bart J.M. van Vlijmen3, Jos H. Beijnen2,5, and Alfred H. Schinkel1*

†

) These authors contributed equally to this study.

1

Department of Molecular Oncology, Netherlands Cancer Institute, Amsterdam, the

Netherlands; 2

Department of Pharmacy & Pharmacology, Netherlands Cancer Institute - Antoni van

Leeuwenhoek, Amsterdam, the Netherlands; 3

Department of Thrombosis & Hemostasis, Einthoven Laboratory for Experimental Vascular

Medicine, Leiden University Medical Center, Leiden, the Netherlands; 4

Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa

University, Kakuma-machi, Kanazawa, Japan; 5

Division of Pharmacoepidemiology & Clinical Pharmacology, Department of Pharmaceutical

Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands.

Running title: Ces1, Abcb1 and Cyp3a control cabazitaxel pharmacokinetics

Number of words in abstract: 250 words Number of words in text: 5059 words Number of text pages: 34 Number of tables: 0 + 5 Supporting Tables Number of figures: 5 + 6 Supporting Figures Number of references: 42 Page 1 of 34 ACS Paragon Plus Environment


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Abstract Graphic

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Abstract

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 (6-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.

Keywords:

cabazitaxel,

carboxylesterase,

pharmacokinetics,

P-glycoprotein,

brain

accumulation



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Introduction

Taxanes are chemotherapeutic agents widely used in the clinic as single agents or in combination with other cytotoxic drugs for the treatment of a broad range of cancers such as locally advanced or metastasized head and neck cancer, breast cancer, non-small cell lung cancer, gastric cancer, bladder cancer and metastatic castration resistant prostate cancer.1 For this last type, docetaxel was the first cytotoxic agent to show an overall survival benefit for hormone-refractory patients. However, eventually patients develop resistance to docetaxel via various pathways, one of which may be the upregulation of the multidrug efflux protein P-glycoprotein (P-gp/ABCB1).2-4 For this reason pharmaceutical companies developed an interest in novel third-generation taxanes which are not, or only poorly, transported by P-gp. Cabazitaxel (Jevtana®) is such a semi-synthetic taxane with reduced transport by ABCB1 P-gp. Cabazitaxel, co-administered with predniso(lo)ne, received approval from FDA and EMA to treat patients with metastasized castration-resistant prostate cancer who progressed after docetaxel treatment.5,6 Even though the molecular structure of cabazitaxel is very similar to that of docetaxel, simply replacing two hydroxy groups with two methoxy groups (Supplemental Figure 1), cabazitaxel is able to exert cytotoxic effects in tumors that developed resistance to docetaxel. It has also been found to penetrate the blood-brain barrier (BBB) at high plasma concentrations.7-9 It remains unclear why cabazitaxel is less of an ABCB1 P-gp substrate than its structural analogs. Still, short-term brain perfusion experiments suggested a modest effect of ABCB1 in reducing cabazitaxel brain concentrations.7 Upon intravenous infusion, cabazitaxel is extensively metabolized in the liver, mainly (80-90%) by cytochrome P450 3A4/5 (CYP3A4/5) and to a lesser extent by CYP2C8. From the approximately 20 metabolites formed in vivo, there are 3 metabolites that are pharmacodynamically active (Supplemental Figure 1). These metabolites are docetaxel and two O-demethylated derivatives of cabazitaxel, further on referred to as DM1 and DM2, each Page 4 of 34 ACS Paragon Plus Environment

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with similar cytotoxic activity as cabazitaxel in vitro.5,6 The conversions of cabazitaxel to DM1, DM2, and docetaxel are all thought to be primarily mediated by CYP3A4/5.5,10 Our research group is interested in the development of oral taxanes, as oral drug administration is more patient-friendly and less costly, and more easily allows metronomic therapy.11-14 A drawback is that the oral availability of drugs can be seriously hampered by ABC transporters in the intestine and liver that rapidly efflux the drug out of the blood back to the intestinal lumen or into the bile, respectively. First-pass metabolism by CYP3A4/5 in the same organs is often also a major hindrance to oral drug administration. Several ABC transporters, especially ABCB1, ABCG2, and ABCC2, are highly expressed in the epithelial membranes of a number of organs which are pivotal for absorption and elimination of drugs like liver, small intestine, and kidney, and in the blood-brain barrier (BBB). Cabazitaxel interacts with ABCB1 and is metabolized by CYP3A4/5. We previously demonstrated that the oral availability of both paclitaxel and docetaxel is drastically restricted by ABCB1 and CYP3A.14-19 Furthermore, we have found that Abcc2 plays a role in the disposition of paclitaxel and docetaxel.20,21 We therefore wanted to investigate to what extent cabazitaxel pharmacokinetics, especially oral availability and brain accumulation, is affected by ABCB1 and possibly ABCC2 and ABCG2, and if so, whether a pharmacological inhibitor of one or more of these transporters could reverse these pharmacokinetic effects. We further wanted to study the in vivo impact of CYP3A. To investigate these questions, we analyzed the in vivo behavior of oral cabazitaxel in ABC transporter and CYP3A knockout and humanized transgenic mouse models.



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Material and Methods Chemicals and Reagents

Clinically available cabazitaxel (Jevtana®) was used for in vitro and in vivo experiments, Ko143 was from Tocris Bioscience (Bristol, UK) and zosuquidar (Eli Lilly; Indianapolis, USA) was a kind gift of Dr. O. van Tellingen. The origin of other standard chemicals is specified in the Supplementary Materials and Methods section.

Transport Assays

Polarized Madin-Darby Canine Kidney (MDCKII) cell lines transduced with human (h)ABCB1, hABCG2 or murine (m)Abcg2 cDNA were used to assess bidirectional transport of cabazitaxel across epithelial monolayers formed in Transwell plates.22 Further details are presented in the Supplementary Materials and Methods section.

Animals

Animals were housed and handled according to institutional guidelines in compliance with Dutch legislation. Male wild-type, Abcb1a/1b-/-,23 Abcg2-/-,24 Abcb1a/1b;Abcg2-/-,25 Abcc2-/-26 and Abcb1a/1b;Abcc2-/-27 mice were used. Additionally we used Cyp3a-/- mice and Cyp3a knockout mice with specific expression of human CYP3A4 in liver (Cyp3a-/-Tg-3A4Hep), in intestine (Cyp3a-/-Tg-3A4Int) or both (Cyp3a-/-Tg-3A4Hep/Int).17 All mice were of a >99% FVB genetic background and between 8 and 14 weeks of age. Animals were kept in a temperature-controlled environment with a 12-h light/dark cycle and received a standard diet (AM-II, Hope Farms, Woerden, The Netherlands) and acidified water ad libitum.


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Drug Solutions

Cabazitaxel drug solutions were prepared freshly on the day of experiment. We used Jevtana® solution for infusion (10 mg/ml) containing 0.26 mg/ml polysorbate 80 and 15% (w/v) ethanol, adjusted to pH 2-6 with citric acid. This was diluted 10-fold with saline to obtain a cabazitaxel concentration of 1 mg/ml. Elacridar hydrochloride was dissolved in DMSO at 150 mg/ml and further diluted with 50 mM sodium acetate buffer (pH 4.6) to 15 mg/ml. The elacridar vehicle solution contains 10% DMSO in 50 mM sodium acetate buffer (pH 4.6).

Plasma Pharmacokinetics and Tissue Disposition of Cabazitaxel

To minimize variation in absorption, mice were fasted 4 hours prior to cabazitaxel administration. Mice received 10 mg/kg (10 µl/g) cabazitaxel by oral gavage using a bluntended needle. Multiple 50 µl blood samples were collected from the tail vein at 0.25, 0.5, 1, 2 and 4 hours using heparin-coated capillaries (Sarstedt, Numbrecht, Germany). Blood samples were centrifuged at 2,100 g for 6 minutes at 4˚C and plasma was collected and stored at -30˚C. At 8 hours, mice were anesthetized using isoflurane and blood was collected by cardiac puncture. Immediately thereafter, mice were terminated by cervical dislocation and organs were rapidly removed, weighed and stored at -30˚C. Prior to analysis, organs were thawed and homogenized in appropriate volumes of 4% (w/v) BSA in Milli-Q water using a FastPrep®-24 device (MP Biomedicals, SA, California, USA). Cabazitaxel is roughly equally distributed between blood and plasma.6 Thus we corrected the brain concentrations for the amount of blood in the brain vascular space established in FVB mice (1.4%).28

Tissue Accumulation of Cabazitaxel in Combination with Elacridar

Mice were fasted 4 hours prior to intravenous administration of either elacridar or the vehicle solution, followed by an oral dose of 10 mg/kg cabazitaxel fifteen minutes later. One hour after cabazitaxel administration, mice were anesthetized with isoflurane and blood was Page 7 of 34 ACS Paragon Plus Environment


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collected by cardiac puncture. Immediately thereafter, mice were terminated by cervical dislocation and organs were removed and processed as described above.

Inhibitory Effect of Cabazitaxel on Irinotecan Hydrolase Activity by Mouse Plasma In

Vitro The conversion of irinotecan to SN-38 by plasma Ces1c was measured as described.29 Briefly, fresh aliquots of murine plasma (120 µl) were pre-incubated with 15 µl of cabazitaxel solution, cabazitaxel vehicle containing 0.1% ethanol and 0.2% polysorbate 80, or BNPP (an irreversible CES inhibitor) in 1.5 ml Eppendorf tubes for 15 minutes. Addition of 15 µl irinotecan to a final concentration of 5 µM in the mixtures initiated the experiment and the mixtures were incubated at 37°C for 30 minutes. The concentrations of irinotecan and SN-38 were quantified by HPLC with fluorescence detection.30

Hepatic Knockdown of Ces1c in Mice

Hepatic knockdown of Ces1c was performed as described previously in male Abcb1a/1b-/mice, which have constitutively high plasma Ces1c levels.29 Further details are provided in the Supplementary Materials and Methods section.

Inhibitory Effects of Cabazitaxel on Hydrolysis by Recombinant Human CES1 and CES2

Cabazitaxel concentrations that inhibit 50% of p-nitrophenyl acetate hydrolase activities by recombinant human CES1 and CES2 were determined as described previously.29


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RNA Isolation, cDNA Synthesis and RT-PCR

RNA isolation from mouse liver, followed by cDNA synthesis and RT-PCR were performed as described.29 The expression levels of mouse carboxylesterase genes Ces1b, Ces1c, Ces1d, Ces1e, Ces1f, Ces1g and Ces2a were quantified using specific primers (Qiagen, Hilden, Germany).

Drug Analysis

Concentrations of cabazitaxel and its three active metabolites in DMEM, plasma and tissue homogenates were determined using a validated liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) assay using deuterated internal standards for cabazitaxel and docetaxel. Details are presented in the Supplementary Materials and Methods section.

Pharmacokinetic Calculations and Statistics

Pharmacokinetic parameters were estimated by non-compartmental analysis using the software package PK Solutions 2.0.2 (Summit Research Services, Ashland, OH, USA). The area under the curve (AUC) of the plasma concentration-time curve was calculated using the trapezoidal rule, without extrapolating to infinity. The peak plasma concentration (Cmax) and the time to reach peak plasma concentration (Tmax) were determined directly from individual concentration-time data. Ordinary one-way analysis of variance was used to determine significant differences between groups and individual groups were compared after performing post-hoc tests with Bonferroni correction. When variances in the groups were not homogeneously distributed, data were log-transformed before applying statistical tests. Differences were considered statistically significant when P < 0.05. Data are presented as mean ± SD.



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Results

In Vitro Transport of Cabazitaxel by ABCB1, but not ABCG2 Transepithelial cabazitaxel transport was assessed using polarized monolayers of MDCKII subclones overexpressing human (h)ABCB1, hABCG2 or mouse (m)Abcg2. In MDCKII parental cells, there was modest but significant apically directed transport of cabazitaxel (transport ratio r = 1.7, Figure 1A), which was completely inhibited by the ABCB1 inhibitor zosuquidar (r = 1.1, Figure 1B), suggesting some transport by the low-level endogenous canine ABCB1. In MDCKII-hABCB1 cells, there was pronounced apically directed transport of cabazitaxel, with an r of 11.8 (Figure 1C), which was strongly inhibited by zosuquidar (Figure 1D). In subsequent experiments with MDCKII-hABCG2 and MDCKII-mAbcg2 cells, zosuquidar was added to inhibit the endogenous canine ABCB1. Ko143 was used to inhibit hABCG2 or mAbcg2. There was no substantial polarized transport of cabazitaxel by hABCG2 or mAbcg2 (Figure 1E-H). Cabazitaxel thus appears to be a good transported substrate of hABCB1, but not of hABCG2 or mAbcg2.

Cabazitaxel Plasma Pharmacokinetics in ABC Transporter Knockout Strains

Although cabazitaxel is clinically registered for intravenous administration, the development of oral formulations of taxane anticancer drugs is a main focus of research in our groups. We therefore determined the separate and combined effects of Abcb1 and Abcg2 on the oral availability and tissue disposition of cabazitaxel. We administered cabazitaxel (10 mg/kg) orally to wild-type, Abcb1a/1b-/-, Abcg2-/- and Abcb1a/1b;Abcg2-/- mice, and measured plasma and tissue levels. Absorption of cabazitaxel was rapid, with a Tmax occurring at or before 30 minutes in all strains (Figure 2A). Four out of five wild-type mice had low plasma levels of cabazitaxel, but one clear outlier mouse displayed a ~5-fold higher oral plasma AUC and strongly reduced clearance. We therefore separately present data for the “low” and “high” cabazitaxel wild-type mice. Surprisingly, the Abcb1a/1b and Abcg2 single and combination


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knockout mice all showed very similar, ~11-fold increases in plasma AUC relative to the “low” wild-type mice (Figure 2A and Supplementary Table 1), even though cabazitaxel is not an mAbcg2 substrate in vitro. These results, including the incidental “high” wild-type values, reminded us of previous findings with the drug everolimus, for which the plasma level is strongly influenced by its binding to plasma carboxylesterase (Ces) 1c.29 Ces1c is markedly upregulated in liver and blood of some wild-type and all Abcb1a/1b and/or Abcg2 knockout mice.29,31 Relative to “low” wild-type mice, Ces1c RNA levels in livers of the “high” wild-type mice are ~50-fold increased and in the Abcb1a/1b-/-, Abcg2-/- and Abcb1a/1b;Abcg2-/- strains even ~100-fold. As mouse Ces1c protein lacks an endoplasmic reticulum retention signal, it is largely secreted from the liver into the blood. We therefore hypothesized that cabazitaxel, like everolimus, may bind to plasma Ces1c, thus causing a markedly increased plasma retention in “high” wild-type mice and all the knockout strains with Ces1c upregulation. The strongly increased total plasma levels of cabazitaxel in the knockout strains might then not be primarily caused by increased oral absorption of cabazitaxel due to ABC transporter deficiency.

ABCB1 Restricts Brain Accumulation of Cabazitaxel

In spite of the large differences in total plasma cabazitaxel levels, the brain concentrations in “low” and “high” wild-type mice were quite similar 8 hr after oral cabazitaxel administration (Figure 2B), and the same was true for liver and kidney concentrations of cabazitaxel in wildtype and Abcb1 and/or Abcg2 knockout strains 1 hr after drug administration (Figure 3C, Supplementary Figure 2A). This suggests that the free (as opposed to total) cabazitaxel concentrations in the plasma of these mouse strains with different Ces1c blood levels were quite similar. Despite the virtually identical high cabazitaxel plasma levels seen between all three knockout strains (Figure 2A), 8 hr after drug administration Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice showed 22- and 25-fold higher brain concentrations, respectively, than Abcg2-/- mice, and similar increases relative to the brain concentrations in both “low” and Page 11 of 34 ACS Paragon Plus Environment


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“high” wild-type mice (Figure 2B). This pronounced increase in brain concentrations in both Abcb1-deficient strains appears solely due to the absence of Abcb1, as there was no further increase in brain concentration in the combination Abcb1a/1b;Abcg2 knockout strain (Figure 2B). It thus appears that Abcb1, but not Abcg2, strongly restricts the brain accumulation of cabazitaxel.

Effect of the ABCB1 Inhibitor Elacridar on Plasma and Brain Exposure of Oral Cabazitaxel

As Abcb1 markedly restricted the brain accumulation of cabazitaxel, we assessed whether this could be reversed by the Abcb1 inhibitor elacridar. We therefore intravenously administered elacridar (10 mg/kg) 15 minutes prior to oral cabazitaxel administration (10 mg/kg) to the wild-type and Abcb1a/1b;Abcg2-/- strains, and measured plasma and brain cabazitaxel levels at 1 hr, i.e., not long after the cabazitaxel Tmax (Figure 3A). Coincidentally, no “high” cabazitaxel wild-type mice were present in this experiment. In the absence of elacridar, cabazitaxel plasma concentrations were approximately 5.2- to 5.6-fold higher in all the single and double knockout strains than in the “low” wild-type mice (Figure 3A). Pretreatment with elacridar had no impact on the plasma levels of cabazitaxel in “low” wildtype and Abcb1a/1b;Abcg2-/- mice (Figure 3A), further supporting that the increased plasma levels in the knockout strains were not directly caused by the absence of Abcb1 and/or Abcg2 activity (Figure 3A). In the absence of elacridar, Abcb1a/1b-/- and Abcb1a/1b;Abcg2-/- mice had 8.1- and 12-fold increased brain concentrations relative to “low” wild-type mice, whereas the Abcg2-/brain concentrations were not significantly increased (Figure 3B). In contrast to the lack of effect on plasma concentrations, elacridar strongly increased the brain concentration of cabazitaxel in wild-type mice by 9.6-fold, resulting in similar brain concentrations as observed for Abcb1a/1b;Abcg2-/- mice treated with or without elacridar (Figure 3B).


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Despite the large differences in plasma total cabazitaxel levels, the liver and kidney concentrations in “low” wild-type mice and knockout strains were quite similar, regardless of coadministration of elacridar (Figure 3C and Supplementary Figure 2A). This suggests that the free plasma cabazitaxel concentrations were quite similar between the “low” wild-type mice and knockout strains, with or without elacridar. We therefore plotted both the brain-toliver and brain-to-kidney concentration ratios in the different strains (Figure 3D and Supplementary Figure 2B), rather than the brain-to-plasma ratios. In the absence of elacridar, brain-to-liver and brain-to-kidney concentration ratios were approximately 10- to 14-fold higher in both Abcb1-deficient strains relative to the wild-type and Abcg2-/- mice (P < 0.001). Elacridar coadministration increased the brain-to-liver and brain-to-kidney ratios in wild-type mice to similar levels as seen in the Abcb1a/1b;Abcg2-/- mice (Figure 3D and Supplementary Figure 2B). Thus, intravenous elacridar coadministration could completely inhibit the activity of mouse Abcb1 in the BBB, leading to highly increased cabazitaxel concentrations in the brain.

Cabazitaxel Inhibits the Conversion of Irinotecan to SN-38 by Carboxylesterase 1c in Mouse Plasma

High plasma Ces1c in mice can hydrolyze the anticancer prodrug irinotecan to its active metabolite SN-38.29,32 We hypothesized that if cabazitaxel binds to plasma Ces1c, it might also inhibit its hydrolytic activity towards irinotecan. We therefore tested the conversion of spiked irinotecan to SN-38 in individual wild-type and knockout plasmas in vitro, and the effect of preincubation of these plasmas with cabazitaxel or the irreversible carboxylesterase inhibitor BNPP. Cabazitaxel itself was quite stable in mouse plasma (data not shown), and thus not noticeably hydrolyzed by Ces1c itself. There was little conversion of irinotecan to SN-38 by “low” wild-type plasma, but extensive conversion by “high” wild-type and especially knockout plasmas (Supplementary Figure 3A and B). The extensive irinotecan hydrolysis in “high” wild-type and knockout plasmas was weakly inhibited by the cabazitaxel vehicle (0.1% Page 13 of 34 ACS Paragon Plus Environment


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ethanol and 0.2% polysorbate 80), but virtually completely by cabazitaxel or BNPP (Supplementary Figure 3A and B). Cabazitaxel thus appears to be an effective Ces1c inhibitor, consistent with its potentially tight binding to this protein.

In Vivo Knockdown Confirms the Impact of Ces1c on Cabazitaxel Pharmacokinetics To demonstrate more directly that upregulated plasma Ces1c is responsible for the altered plasma pharmacokinetics of cabazitaxel, we performed an in vivo Ces1c knockdown experiment in Abcb1a/1b-/- mice, which show a consistent upregulation of Ces1c RNA.29 Three days after intravenous administration of the siRNA formulation, a very extensive knockdown of hepatic Ces1c RNA relative to the negative control siRNA was observed, as judged by RT-PCR (∆Ct 1.15 ± 0.46 vs. -4.19 ± 0.43, a 40.8-fold linear decrease; P < 0.001; Supplementary Table 2). Pharmacokinetic analysis of orally administered cabazitaxel on this day 3 showed that Ces1c siRNA-treated Abcb1a/1b-/- mice had a 4.5-fold lower plasma AUC relative to the negative control siRNA-treated mice, not much above the AUC seen in “low” wild-type mice (Figures 4A and 2A, Supplementary Table 3). In contrast, cabazitaxel liver and brain concentrations and brain-to-liver ratios at 2 hr after cabazitaxel administration were not affected by the Ces1c knockdown (Figure 4B-D), illustrating that the free cabazitaxel plasma concentrations were similar between these two groups. These data confirm that Ces1c upregulation was the main cause of the anomalous total cabazitaxel plasma pharmacokinetics seen in the knockout strains.

Cabazitaxel Inhibits Human CES1 and CES2 Weakly

As cabazitaxel inhibited Ces1c hydrolytic activity in mouse plasma, we also tested the inhibitory effect of cabazitaxel on recombinant human CES1 and CES2, using p-nitrophenyl acetate as substrate at 100 µM, close to the Km values of CES1 and CES2 for this compound. Cabazitaxel only inhibited approximately 30% of the hydrolase activities for both


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enzymes at 50 µM, the highest concentration that could be tested without the vehicle compounds starting to inhibit CES1 and CES2 (Supplementary Figure 4). Thus, cabazitaxel only weakly inhibits human CES1 and CES2.

Cyp3a and CYP3A4, but not Abcc2, Limit the Oral Availability of Cabazitaxel in Mice

We know that similar levels of Ces1c upregulation occur in liver and plasma of Abcb1a/1b, Abcg2, Cyp3a and various combination knockout strains, but not in Abcc2 knockout mice.29,31 Notwithstanding this Ces1c upregulation and likely increased cabazitaxel binding, we aimed to assess the separate and combined impact of Abcb1 and Cyp3a on the oral availability of cabazitaxel in mice. Cabazitaxel was orally administered at 10 mg/kg to wild-type, Abcb1a/1b, Cyp3a and Abcb1a/1b;Cyp3a knockout strains, and plasma cabazitaxel concentrations were measured (Figure 5A). The ~5-fold increase in cabazitaxel plasma levels in Abcb1a/1b-/- mice was primarily caused by increased plasma Ces1c binding, as documented above, but the further ~6-fold increase in plasma AUC in Cyp3a-/- and Abcb1a/1b;Cyp3a-/- mice indicates a prominent role of Cyp3a in restricting cabazitaxel oral availability (Figure 5A and Supplementary Table 4). As cabazitaxel is O-demethylated primarily by CYP3A at two different O-methyl groups to yield metabolites DM1, DM2, and docetaxel (Supplemental Figure 1), we also measured the concentration of these metabolites and plotted their respective metabolite/cabazitaxel ratios (Supplemental Figure 5). As expected, far less of each of these metabolites relative to cabazitaxel was found in both the Cyp3a-deficient strains than in the wild-type and Abcb1a/1b-/- strains. Given the similar (upregulated) Ces1c levels in Abcb1a/1b, Cyp3a, and Abcb1a/1b;Cyp3a knockout mice, the data indicate a strong impact of Cyp3a-mediated metabolism in reducing the oral availability of cabazitaxel. The lack of significant effect of the additional Abcb1a/1b knockout in Abcb1a/1b;Cyp3a-/- mice relative to Cyp3a-/- mice (Figure 5A) further confirms that there was no substantial role of Abcb1 in restricting the oral availability of cabazitaxel.



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To qualitatively assess whether human CYP3A4 would have a similar effect on cabazitaxel oral availability as mouse Cyp3a, we measured the plasma concentrations of cabazitaxel after oral administration at 10 mg/kg to wild-type mice and Cyp3a knockout mice transgenic for liver or intestinal CYP3A4, and a combination of both transgenes. As shown in Supplementary Figure 6, panel A, hepatic or intestinal CYP3A4 expression each reversed the ~29-fold increase in cabazitaxel plasma AUC seen in Cyp3a-/- mice extensively, albeit not completely to wild-type levels. The combination of hepatic and intestinal CYP3A4 expression, however, reduced the plasma cabazitaxel AUC to well below wild-type levels. Human CYP3A4 can thus have a substantial impact on cabazitaxel oral availability. Although complex, the metabolite/cabazitaxel ratios for this experiment (Supplementary Figure 6, panels B-D) indicate that both hepatic and intestinal CYP3A4 produced at least as much of the DM1, DM2, and docetaxel metabolites from cabazitaxel as the wild-type mouse Cyp3a complex, and probably substantially more. This likely reflects a somewhat differential catalytic efficacy towards cabazitaxel of the human CYP3A4 enzyme versus the mixture of different Cyp3a enzymes normally expressed in mouse gut and liver. In the absence of Abcb1a/1b activity, Abcc2 has a marked impact on oral paclitaxel plasma pharmacokinetics, whereas the effect of Abcc2 on oral plasma docetaxel pharmacokinetics was only noticeable when both Cyp3a and Abcb1 were absent.20,21 Since cabazitaxel resembles docetaxel, we orally administered cabazitaxel at 10 mg/kg to wildtype, Abcb1a/1b, Abcc2 and combination Abcb1a/1b;Abcc2 knockout strains, and measured the plasma levels (Figure 5B). As expected, the cabazitaxel plasma AUC was increased 5.1fold in Abcb1a/1b-/- mice relative to “low” wild-type mice, likely due to Ces1c upregulation (Figure 5B and Supplementary Table 5). However, the cabazitaxel plasma AUC in Abcc2-/mice, which do not have Ces1c upregulation,29 was similar to that in the “low” wild-type mice, and that in Abcb1a/1b;Abcc2-/- mice was similar to that in Abcb1a/1b-/- mice (Figure 5B and Supplementary Table 5). Oral cabazitaxel pharmacokinetics was thus not substantially


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affected by Abcc2 deficiency in either the presence or absence of Abcb1a/1b and/or Ces1c upregulation.



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Discussion

This study demonstrates that Abcb1 P-gp profoundly restricts the brain accumulation of cabazitaxel, but not its oral availability. This is consistent with the clear hABCB1-mediated transport of cabazitaxel in vitro, which is however not as pronounced as that of docetaxel or paclitaxel. mAbcg2 and hABCG2 have little or no transport capacity for cabazitaxel in vivo and/or in vitro, and mAbcc2 deficiency does not alter the oral availability of cabazitaxel. The restricted brain accumulation of cabazitaxel due to BBB P-gp activity can be virtually completely reversed by coadministration of the ABCB1 inhibitor elacridar. Unlike Abcb1, the drug-metabolizing Cyp3a complex does drastically restrict the oral availability of cabazitaxel, with an oral plasma AUC about 6 times higher in Cyp3a-/- and Abcb1a/1b;Cyp3a-/- mice than in Abcb1a/1b-/- mice. Human CYP3A4 activity in both liver and intestine appears to contribute to similar extents to this reduction in oral availability. Finally, binding of cabazitaxel to plasma Ces1c carboxylesterase that is strongly upregulated in several knockout mouse strains plays a major role in its retention in plasma of these mice. Cabazitaxel markedly inhibits mouse Ces1c, but the human CES1 and CES2 enzymes only weakly. Cabazitaxel was developed as a docetaxel derivative that is less efficiently transported by ABCB1 than docetaxel and paclitaxel, to reduce the likelihood of the development of ABC transporter-mediated multidrug resistance.33,34 Our data show that cabazitaxel is still substantially transported by ABCB1 in vitro and in vivo, but not as efficiently as paclitaxel and docetaxel. Accordingly, at 10 mg/kg, the oral availability of cabazitaxel in mice was not substantially reduced by Abcb1a/1b activity, whereas that of docetaxel and paclitaxel is about 3-fold and 6- to 12-fold reduced, in line with their respective efficiencies of transport by Abcb1a/1b.15,16,19 On the other hand, we found that BBB Abcb1a/1b still strongly reduces the brain accumulation of cabazitaxel, by a factor of 10 or more as judged by brain-to-liver ratios (Figure 3D). Elacridar coadministration could virtually completely reverse this protective BBB effect, without affecting the cabazitaxel plasma concentration. Collectively, these data suggest that the oral availability of cabazitaxel is not


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restricted by Abcb1, but that Abcb1 may still contribute to resistance against cabazitaxel in tumors that express appreciable amounts of Abcb1. A recent in vitro study also concluded that ABCB1 can confer significant resistance to cabazitaxel, albeit less than to docetaxel, in ABCB1-overexpressing cell lines.35 Moreover, Abcb1 in the BBB may substantially reduce the cabazitaxel exposure of primary brain tumors or (micro-)metastases that are functionally positioned behind an intact BBB, either in whole or in part, and thus restrict therapeutic efficacy against such tumors. Thus, coadministration of elacridar may be considered to improve cabazitaxel efficacy against such tumors. Microtubule-stabilizing agents including taxanes

have

recently

been

identified

as

potential

candidates

to

treat

some

neurodegenerative diseases of the brain. Taxanes thought to be capable of overcoming ABCB1-mediated transport, including cabazitaxel, have been suggested as potentially promising candidates for this as they might more readily cross the BBB.36 Our results suggest that cabazitaxel may be less suitable for such CNS indications, given the limiting effect of ABCB1 on brain uptake. We found that both mouse Cyp3a and human CYP3A4 in liver and intestine are a major factor in reducing the oral availability of cabazitaxel. Correcting for the Ces1c upregulation, the Cyp3a knockout caused at least a 6-fold increase in the oral AUC of cabazitaxel, and this was extensively reversed by human CYP3A4 in liver and intestine (Figure 5A and Supplemental Figures 5 and 6 and Table 4). This suggests that also in humans the oral availability of cabazitaxel may be strongly restricted by CYP3A. In the perspective of our current clinical and preclinical programs to improve the oral availability of taxanes like docetaxel and paclitaxel by coadministration of efficient CYP3A inhibitors like ritonavir,14,37,38 it will be of great interest to investigate this for cabazitaxel as well. Based on in vitro studies, it was expected that cabazitaxel clearance in patients might be affected by CYP3A inhibitors.5 A recent clinical study found a modestly reduced cabazitaxel clearance (by 20%) due to oral coadministration of the CYP3A inhibitor ketoconazole with intravenously infused cabazitaxel.10 We expect that after oral administration of cabazitaxel a CYP3A



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inhibitor such as ketoconazole or ritonavir will have a much more pronounced impact on the AUC due to more pronounced first-pass metabolism effects. The plasma AUC of cabazitaxel is strongly increased by upregulation of the Ces1c protein in plasma of several knockout strains and some wild-type mice. This behavior is very similar to what we previously found for the drug everolimus,29 and can be explained by extensive binding of cabazitaxel to plasma Ces1c. This is supported by the inhibitory effect that cabazitaxel has on plasma Ces1c catalytic activity (Supplemental Figure 3). Although cabazitaxel itself contains several carboxylester bonds (Supplemental Figure 1), the strongly increased rather than decreased plasma levels upon Ces1c upregulation, as well as ex vivo plasma stability assays (not shown), indicate that cabazitaxel is not substantially hydrolyzed by Ces1c. In spite of the highly increased total plasma levels, it appears that the concentration of free cabazitaxel was not much altered upon Ces1c upregulation, considering the very similar cabazitaxel distribution to organs such as liver and kidney in wild-type, Abcb1a/1b-/-, and Abcg2-/- mice (Figure 3 and Supplemental Figure 2). This suggests quite tight binding of cabazitaxel to plasma Ces1c. Although cabazitaxel is known to also bind to plasma albumin,6 its binding to Ces1c must be far tighter, considering that the plasma concentration of albumin in mice (~35 g/l) is far higher than that of upregulated Ces1c in the knockout strains (estimated at maximally 0.3 g/l based on everolimus pharmacokinetic data).29 On the other hand, as cabazitaxel was still cleared reasonably well from plasma in Abcb1a/1b and Abcg2 knockout mice (Figure 2A and 5A), this binding is not irreversible. Clearly, Ces1c upregulation in the various knockout strains, if not properly recognized, could be a major confounder in pharmacokinetic studies of cabazitaxel, and caution should be exercised in interpreting such studies. Unlike mice, humans generally have very little CES in plasma,39,40 and this confounder is therefore unlikely to play a role for cabazitaxel in patients. Furthermore, cabazitaxel is at best a weak inhibitor of the two main pharmacologically relevant human CESs, CES1 and CES2, making it unlikely that there


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would be significant CES-mediated drug-drug interactions between cabazitaxel and other CES-substrate drugs in patients. Cabazitaxel is, after everolimus, the second documented example of a drug of which the pharmacokinetics is strongly affected by binding to upregulated plasma Ces1c.29 As there is very little structural resemblance between these two drugs (except for being fairly large, hydrophobic compounds), one can infer that many other structurally unrelated drugs may be similarly affected by binding to plasma Ces1c. Since cabazitaxel is an O-dimethylated derivative of docetaxel, this raises the question whether docetaxel itself (and possibly paclitaxel) might also be affected by plasma Ces1c binding. If so, this would considerably impact interpretation of our previous studies with docetaxel or paclitaxel in Abcb1a/1b, Cyp3a, and Oatp1a/1b knockout mice, which all show Ces1c upregulation.15-17,41,42 To test this, we compared the plasma pharmacokinetics of oral docetaxel and paclitaxel (at 10 mg/kg) in wild-type and Abcg2-/- mice, as Abcg2 itself does not transport docetaxel or paclitaxel, and Ces1c is highly upregulated in Abcg2-/- mice. We did not find any significant difference for either docetaxel or paclitaxel plasma AUCs (data not shown), indicating that the pharmacokinetics of these taxanes are not substantially affected by Ces1c binding. Our previous studies with these drugs in various knockout strains are therefore unlikely to be affected by Ces1c upregulation. These findings further suggest that even the modest structural differences between cabazitaxel and docetaxel can have a major impact on the relative efficiency of binding to Ces1c, underscoring the difficulty in predicting which drugs will interact with Ces1c and which not. Future studies with drugs in these knockout mouse strains will therefore have to be carefully checked for this possible confounder. Collectively, our data show that cabazitaxel, like other clinically used taxanes, is substantially affected in vivo by both ABCB1 (P-gp) and CYP3A activity. These impair oral availability, clearance, and tissue distribution of cabazitaxel, and, by implication, also tumor distribution. It will therefore be of great interest to consider judicious coadministration of effective CYP3A and/or ABCB1 inhibitors to improve one or more of these pharmacokinetic



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characteristics of cabazitaxel and its metabolites and hence its therapeutic efficacy in patients.

Acknowledgement of research support: This work was supported in part by an academic

staff training scheme fellowship from the Malaysian Ministry of Science, Technology and Innovation to S.C.T., and by Dutch Cancer Society grant NKI 2007-3764 to A.H.S. and J.H.B. We thank Olaf van Tellingen for his assistance with high performance liquid chromatography analysis of irinotecan and SN-38.


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Figures and figure captions



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Figure 1. Transepithelial transport of cabazitaxel (5 µM) assessed using either MDCK-II parental cells (A and B) or MDCK-II cells transduced with hABCB1 (C and D), hABCG2 (E and F) or mAbcg2 (G and H) cDNA. At t = 0 hr, cabazitaxel was applied in the donor compartment and the concentration in the acceptor compartment at t = 2 hr was measured and plotted as total amount of transport (ng) in the graphs (n = 3). Zosuquidar (5 µM) was applied to inhibit endogenous canine ABCB1 or hABCB1 (B, D, E-H). Ko143 (5 µM) was applied to inhibit hABCG2 or mAbcg2 (F and H). □, translocation from basolateral to apical compartment; ∆, translocation from apical to basolateral compartment. Points, mean (n = 3); bars, SD.

Figure 2. Plasma concentration-time curves (A) and brain concentration at t = 8 hr (B) of cabazitaxel in male wild-type, Abcb1a/1b-/-, Abcg2-/- and Abcb1a/1b;Abcg2-/- mice after oral administration of 10 mg/kg cabazitaxel. The inset shows a semi logarithmic representation of the same data. Points, mean (n = 4-5, except for “high” wild-type mice n = 1); bars, SD. Columns, mean (n = 1-5); bars, SD. ***, P < 0.001 when compared with wild-type mice with low cabazitaxel plasma levels.


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Figure 3. Plasma levels and tissue disposition of cabazitaxel. Plasma concentration (ng/ml; A), brain concentration (ng/g; B), liver concentration (ng/g; C) and brain-to-liver concentration ratio (D) of cabazitaxel in male wild-type, Abcb1a/1b-/-, Abcg2-/- and Abcb1a/1b;Abcg2-/- mice 1 hr after oral administration of 10 mg/kg cabazitaxel. All data are presented as mean ± SD (n = 4-5; ***, P < 0.001 when compared with “low” wild-type mice without elacridar pretreatment;

†††

, P < 0.001 when compared with “low” wild-type mice with elacridar

pretreatment). Note that by chance no “high” wild-type mice were present in the 1 hr single time-point experiment.



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Page 26 of 34

Figure 4. Effect of Ces1c knockdown on plasma pharmacokinetics and tissue disposition of cabazitaxel in vivo. Plasma concentration-time curve (ng/ml; A), liver concentration (ng/g; B), brain concentration (ng/g; C) and brain-to-liver ratio (D) of cabazitaxel in negative control siRNA (siNEG)- or siCes1c-treated male Abcb1a/1b-/- mice at 2 hr after oral administration of 10 mg/kg cabazitaxel. All data are presented as mean ± SD (n = 6-7).


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Figure 5. Plasma concentration-time curves of cabazitaxel in male wild-type, Abcb1a/1b-/-, Cyp3a-/- and Abcb1a/1b;Cyp3a-/- mice after oral administration of 10 mg/kg cabazitaxel (ng/ml; A). Points, mean (n = 5-6); bars, SD. Plasma concentration-time curves of cabazitaxel in male wild-type, Abcb1a/1b-/-, Abcc2-/- and Abcb1a/1b;Abcc2-/- mice after oral administration of 10 mg/kg cabazitaxel (ng/ml; B). Points, mean (n = 4); bars, SD. Note that by chance no high Ces wild-type mice were present in these experiments.



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ASSOCIATED CONTENT Supporting Information. Part of the materials and methods are presented in the Supporting Information. Supporting Figure 1 shows molecular structures of cabazitaxel, DM1, DM2, and docetaxel. Supporting Figure 2 depicts cabazitaxel kidney distribution in mice. Supporting Figure 3 depicts the inhibitory effect of cabazitaxel on the conversion of irinotecan to SN-38 by carboxylesterase 1c in mouse plasma. Supporting Figure 4 shows the inhibitory effect of various cabazitaxel concentrations on hydrolysis by recombinant human CES1 and CES2. Supporting Figure 5 depicts plasma concentration-time curves of cabazitaxel in semi logarithmic

presentation,

DM1/cabazitaxel

ratio,

DM2/cabazitaxel

ratio

and

docetaxel/cabazitaxel ratio in male wild-type, Abcb1a/1b-/-, Cyp3a-/- and Abcb1a/1b;Cyp3a-/mice after oral administration of 10 mg/kg cabazitaxel. Supporting Figure 6 depicts plasma concentration-time curves of cabazitaxel in semi logarithmic presentation, DM1/cabazitaxel ratio, DM2/cabazitaxel ratio and docetaxel/cabazitaxel ratio in male wild-type, Cyp3a-/-, Cyp3a-/-Tg-3A4Hep, Cyp3a-/-Tg-3A4Int and Cyp3a-/-Tg-3A4Hep/Int mice after oral administration of 10 mg/kg cabazitaxel. Supporting Table 1 presents pharmacokinetic parameters of cabazitaxel in plasma of wild-type, Abcb1a/1b-/-, Abcg2-/- and Abcb1a/1b;Abcg2-/- mice receiving oral cabazitaxel (10 mg/kg). Supporting Table 2 presents an overview of the corresponding ∆Ct values belonging to the RT-PCR results for the knockdown experiment as shown in Figure 4. Supporting Table 3 presents pharmacokinetic parameters, liver concentration, brain concentration and brain-to-liver ratio of cabazitaxel in siNEG- or siCes1c-injected male Abcb1a/1b-/- mice receiving a single oral dose of cabazitaxel (10 mg/kg). Supporting Table 4 presents pharmacokinetic parameters of cabazitaxel in plasma of wild-type, Abcb1a/1b-/-, Cyp3a-/- and Abcb1a/1b;Cyp3a-/- mice receiving oral cabazitaxel (10 mg/kg). Supporting Table 5 presents pharmacokinetic parameters of cabazitaxel in plasma of wild-type, Abcb1a/1b-/-, Abcc2-/- and Abcb1a/1b;Abcc2-/- mice receiving oral cabazitaxel (10 mg/kg). These materials are available free of charge via the Internet at http://pubs.acs.org.


Page 29 of 34

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AUTHOR INFORMATION Corresponding Author * Corresponding author: Dr. Alfred H. Schinkel Department of Molecular Oncology, the Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands, Phone: +31 20 512 2046, Fax: +31 20 669 1383, Email: [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Disclosure of Potential Conflicts of Interest The group of A.H.S. receives revenue from the commercial distribution of some of the mouse strains used in this study. J.H.B. holds patents on oral taxane formulations. The authors declare no other potential conflicts of interest. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.

Abbreviations AUC, area under the concentration-time curve; BBB, blood-brain barrier; BNPP, bis-pnitrophenyl phosphate; BSA, bovine serum albumin; CES, carboxylesterase; Cmax, peak plasma

concentration;

DMEM,

Dulbecco’s

modified

Eagle’s

medium;

DMSO,

dimethylsulfoxide; FVB, Friend Virus B; HPLC, high-performance liquid chromatography; MDCKII, Madin-Darby Canine Kidney II; P-gp, P-glycoprotein; RT-PCR, real-time polymerase chain reaction; SD, standard deviation; tmax, time to reach peak plasma concentration



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P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine

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