Lansoprazole Exacerbates Pemetrexed-Mediated Hematologic Toxicity by Competitive Inhibition of Renal Basolateral Human Organic Anion Transporter 3 S

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Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/07/27/dmd.116.070722.DC1

1521-009X/44/10/1543–1549$25.00 http://dx.doi.org/10.1124/dmd.116.070722 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 44:1543–1549, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Lansoprazole Exacerbates Pemetrexed-Mediated Hematologic Toxicity by Competitive Inhibition of Renal Basolateral Human Organic Anion Transporter 3 s

Kenji Ikemura, Yugo Hamada, Chinatsu Kaya, Tomoyuki Enokiya, Yuichi Muraki, Hiroki Nakahara, Hajime Fujimoto, Tetsu Kobayashi, Takuya Iwamoto, and Masahiro Okuda Department of Clinical Pharmacy and Biopharmaceutics, Mie University Graduate School of Medicine, Tsu (K.I., Y.H., T.I., M.O.); Department of Pharmacy, Mie University Hospital, Tsu (K.I., T.E., Y.M., T.I., M.O.); Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Suzuka (C.K.); Department of Pulmonary and Critical Care Medicine, Mie University Graduate School of Medicine, Tsu (H.N., H.F., T.K.), Mie, Japan

Received March 30, 2016; accepted July 18, 2016 Downloaded from

ABSTRACT Pemetrexed, a multitargeted antifolate, is eliminated by tubular effect of lansoprazole was much greater than those of other PPIs secretion via human organic anion transporter 3 (hOAT3). Although and the apparent IC50 value of lansoprazole against pemetrexed proton pump inhibitors (PPIs) are frequently used in cancer patients, transport via hOAT3 was 0.57 6 0.17 mM. The inhibitory type of the drug interaction between PPIs and pemetrexed remains to be lansoprazole was competitive. In a retrospective study, multivariate dmd.aspetjournals.org clarified. In this study, we examined the drug interaction between analysis revealed that coadministration of lansoprazole, but not pemetrexed and PPIs in hOAT3-expressing cultured cells, and other PPIs, with pemetrexed and carboplatin was an independent retrospectively analyzed the impact of PPIs on the development of risk factor significantly contributing to the development of hemato- hematologic toxicity in 108 patients who received pemetrexed and logic toxicity (odds ratio: 10.004, P = 0.005). These findings demon- carboplatin treatment of nonsquamous non–small cell lung cancer strated that coadministration of lansoprazole could exacerbate the for the first time between January 2011 and June 2015. We hematologic toxicity associated with pemetrexed, at least in part, by established that pemetrexed was transported via hOAT3 (K =68.36 competitive inhibition of hOAT3. Our results would aid clinicians to

m at ASPET Journals on September 24, 2021 11.1 mM). Lansoprazole, rabeprazole, pantoprazole, esomeprazole, make decisions of coadministration drugs to avoid drug interaction- omeprazole, and vonoprazan inhibited hOAT3-mediated uptake of induced side effects for achievement of safe and appropriate pemetrexed in a concentration-dependent manner. The inhibitory chemotherapy with pemetrexed.

Introduction encoded by SLC22A genes have been characterized (Sweet and Pemetrexed, a potent multitargeted antifolate, is a key drug in the Pritchard, 1999; Inui et al., 2000; Sekine et al., 2000). Both hOAT1 treatment of nonsquamous non–small cell lung cancer (NSCLC) (Adjei, (SLC22A6) and hOAT3 (SLC22A8) mediate organic anion/a-ketoglu- 2004). It is increasingly used as first-line treatment in combination with tarate exchange at the basolateral membrane of the proximal tubules platinum compounds (Scagliotti et al., 2008) and as maintenance (Sekine et al., 1997; Sweet and Pritchard, 1999). monotherapy for nonsquamous NSCLC (Hanna et al., 2004). Previous studies investigating the characteristics of pemetrexed Pemetrexed is eliminated mainly from the kidney and has a tubular transport via various solute carrier transporters revealed that pemetrexed secretion rate approximately 2.5-fold greater than the glomerular is transported primarily by hOAT3 (Kurata et al., 2014; Posada et al., filtration rate (GFR) in advanced cancer patients with normal renal 2015) and that hOAT3 substrates, such as nonsteroidal anti- function (Rinaldi et al., 1999; Mita et al., 2006). In the renal proximal inflammatory drugs (NSAIDs) and cephalosporin antibiotics, inhibit tubules, membrane transport proteins expressed specifically at the pemetrexed transport via hOAT3 (Kurata et al., 2014; Posada et al., basolateral membranes are responsible for the urinary secretion of 2015). Posada et al. (2015) reported that hOAT4 (SLC22A11) expressed various drugs. The structures and functions of human organic anion at the brush-border membrane is predominantly involved in the transport transporters (hOATs) and human organic cation transporters (hOCTs) of pemetrexed from the renal tubular cells to urine; however, inhibition of hOAT4 had no effect on the pemetrexed plasma clearance in the experiment using physiologic based pharmacokinetic models (Posada et al., 2015). Therefore, hOAT3-mediated uptake, but not hOAT4- This work was supported by a Grant-in-Aid for Scientific Research (C) [Grant 26460195 and 26460196] from the Japan Society for the Promotion of Science mediated efflux, should contribute to plasma clearance of pemetrexed. and Mie University Hospital Seed Grant Program 2015. These findings suggested that particular care should be taken during the dx.doi.org/10.1124/dmd.116.070722. coadministration of inhibitors and/or substrates of hOAT3 in patients s This article has supplemental material available at dmd.aspetjournals.org. receiving pemetrexed.

ABBREVIATIONS: AST, aspartate aminotransferase; CrCl, creatinine clearance; CTCAE, Common Terminology Criteria for Adverse Events; GFR, glomerular filtration rate; Hb, hemoglobin; hOAT, human organic anion transporter; hOCT, human organic cation transporter; NSAIDs, nonsteroidal anti-inflammatory drugs; NSCLC, non-small cell lung cancer; PLT, platelet; PPI, proton pump inhibitor; WBC, white blood cell.

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Proton pump inhibitors (PPIs) are the most commonly prescribed i.d.; Tosho, Tokyo, Japan) and a Waters 2996 photodiode array detector (Waters, drugs for the treatment of gastroesophageal hyperacidity (Targownik Milford, MA). The column temperature was set at 40C. Pemetrexed was eluted et al., 2007). An estimated 20% of cancer patients have been treated with with 0.2% formic acid (pH 3.08 adjusted with 1 M NaOH): acetonitrile = 20: 80 at PPIs for alleviating the symptoms of gastroesophageal reflux (Smelick 1 ml/min. The detection wavelength was 254 nm. et al., 2013), highlighting the importance of investigating the drug Kinetic Analyses. Kinetic analyses were performed with GraphPad Prism version 6.0 (GraphPad Software Inc., San Diego, CA). The apparent Michaelis- interaction between PPIs and anticancer agents to provide safe and Menten constant (K ) and maximal velocity (V ) values were calculated using appropriate chemotherapy. Recent studies have reported that PPIs are m max the Michaelis-Menten equation: V = Vmax × S/(Km + S), where V is the transport inhibitors of hOATs and hOCTs (Nies et al., 2011; Chioukh et al., 2014). velocity, S is the concentration of pemetrexed, Vmax is the maximal velocity, and Since hematologic toxicity as a serious side effect of pemetrexed has Km is Michaelis-Menten constant by nonlinear regression analysis. The apparent been correlated with drug exposure (Rollins and Lindley, 2005), IC50 values were calculated from the inhibition plots according to the equation: n decreased clearance results in greater systemic exposure, which may V = Vbottom +(Vtop 2 Vbottom)/[1 + (log I/IC50) ] by nonlinear least square be associated with increased side effects. Taking these findings into regression analysis, where V is the transport velocity, Vbottom is the transport consideration, we hypothesized that coadministration of PPIs may velocity at the highest concentration of inhibitor, Vtop is the transport velocity exacerbate the hematologic toxicity of pemetrexed by inhibiting the without inhibitor, I is the concentration of the inhibitor, and n is the Hill coefficient. renal elimination of pemetrexed via hOAT3; however, the drug Patients and Data Collection. A retrospective study was conducted in 116 hospitalized patients who received combination therapy of pemetrexed and interaction between pemetrexed and PPIs and the impact of PPIs on carboplatin in the absence or presence of bevacizumab for the treatment of the development of hematologic toxicity of pemetrexed remain to be nonsquamous NSCLC for the first time at Mie University Hospital between Downloaded from explored in clinical situations. January 2011 and June 2015. Eligible patients received an i.v. infusion of In the present study, drug interaction between PPIs and pemetrexed pemetrexed (500 mg/m2) for 10 minutes, followed by a 60-minute i.v. infusion of was examined in hOAT3-expressing cultured cells, and the impact of carboplatin at a dose calculated to produce an area under the concentration-time PPIs on the development of hematologic toxicity was retrospectively curve of 5 or 6 mg×min/ml and an i.v. infusion of bevacizumab at a dose of analyzed in hospitalized patients who received combination therapy of 15 mg/kg for 90 minutes. Patients were excluded if they had missing data, pemetrexed and carboplatin for the treatment of nonsquamous NSCLC. creatinine clearance (CrCl) ,45 ml/min as estimated by the Cockcroft-Gault equation (Cockcroft and Gault, 1976), did not receive vitamin B12 and folic acid dmd.aspetjournals.org supplements for preventing the adverse effects of pemetrexed, showed alanine Material and Methods aminotransferase and aspartate aminotransferase (AST) $100 IU/liter, had Materials. Pemetrexed disodium, pantoprazole, and rabeprazole were Eastern Cooperative Oncology Group performance status $2, and hematologic obtained from LKT Laboratories, Inc. (St. Paul, MN). Probenecid, lansoprazole, parameters $grade 2 before chemotherapy, including white blood cell (WBC) and omeprazole were purchased from WAKO Pure Chemical (Osaka, Japan). count, platelet (PLT) count, or hemoglobin (Hb) level, defined as the Common Esomeprazole was purchased from Sigma-Aldrich (St. Louis, MO), and Terminology Criteria for Adverse Events version 4.0. vonoprazan fumarate (TAK-438) was obtained from ChemScene, LLC (Mon- Demographic data were extracted from the electronic medical records. In mouth Junction, NJ). All other chemicals used were of the highest purity addition to the use of PPIs (lansoprazole 15 or 30 mg/day, omeprazole 10 mg/day, at ASPET Journals on September 24, 2021 available. esomeprazole 20 mg/day, and rabeprazole 10 mg/day), periodic coadministration Cell Culture. The hOAT3-expressing human embryonic kidney cell line drugs, which may cause potential interactions, was identified by Lexicomp Lexi- HEK293 (HEK-hOAT3) and mock-transfectants obtained by transfecting pBK- Interact Online (Lexi-Comp, Inc., Hudson, OH). Hematologic toxicity was CMV vector into HEK293 cells (HEK-pBK), were kind gifts from Prof. Ken-ichi defined as $grade 3 of WBC, PLT, or Hb by the Common Terminology Criteria Inui (Department of Pharmacy, Kyoto University Hospital, Japan). HEK293 cells for Adverse Events within 21 days of pemetrexed administration. This study was were cultured in Medium 199 (Life Technologies, Carlsbad, CA) supplemented conducted in accordance with the Declaration of Helsinki and was approved by with 10% fetal bovine serum containing G418 (0.5 mg/ml) (Sigma-Aldrich) and the Ethics Committee of Mie University Graduate School of Medicine and were used between passage numbers 88 and 107. These cells were maintained at Faculty of Medicine. 37C under 5% CO2 in a humidified atmosphere. For the uptake study, cells (12 Â Statistical Analyses. The in vitro experimental data are expressed as the 105 cells/dish) were seeded in 3.5-cm dishes with culture medium in the absence mean 6 S.E. Statistical comparisons between the two groups were performed of G418. The cell monolayers were used for the uptake study after 48 hours of using the unpaired t test with GraphPad Prism version 6.0. Multivariate logistic culture. regression analysis was performed to identify the impact of coadministration of Uptake Experiments of Pemetrexed. Cellular uptake of pemetrexed was lansoprazole on the development of hematologic toxicity after administration measured with monolayer cultures of HEK-hOAT3 and HEK-pBK cells. The of pemetrexed and carboplatin, with the following variables: age; WBC; PLT; composition of the incubation medium was as follows: 145 mM NaCl, 3 mM KCl, CrCl; Hb; coadministration of lansoprazole, other PPIs, or NSAIDs; pemetrexed 1mMCaCl2, 0.5 mM MgCl2,5mMD-glucose, and 5 mM HEPES (pH 7.4). After dose; and carboplatin dose. Statistical analyses of the clinical data were performed the culture medium was removed, the cells were washed once with incubation with IBM SPSS statistics for windows version 22.0 (Armonk, NY). Significance medium and preincubated with 1 ml of incubation medium for 10 minutes at was established at a P , 0.05. 37C. After preincubation, the medium was replaced with 1 ml of incubation medium containing pemetrexed in the absence or presence of various concentrations of probenecid or PPI. The medium was aspirated at the end of the incubation, Results and the monolayers were rapidly rinsed three times with ice-cold incubation medium. To evaluate the accumulation of pemetrexed into the cells, pemetrexed Time Course of Pemetrexed Uptake by HEK293 Cells Express- was eluted with 0.5 ml of extraction solution (30 mM phosphate buffer, pH 7.0: ing hOAT3. The uptake of pemetrexed (100 mM) by HEK293 cells methanol = 50:50) and then subjected to high-performance liquid chromatography transfected with hOAT3 and pBK was evaluated. As shown in Fig. 1, the (HPLC). The cells were solubilized in 1 M NaOH, and the protein contents of the uptake of pemetrexed in HEK-hOAT3 cells increased in a time- cells were measured using the Bradford method (Bradford, 1976) by using a dependent manner and reached equilibrium state after 5 minutes. Coomassie Brilliant Blue protein assay kit (Nacalai Tesque, Kyoto, Japan) with Moreover, the uptake of pemetrexed was significantly higher in HEK- bovine g-globulin as a standard. Determination of Pemetrexed in Cells. The concentrations of pemetrexed in hOAT3 cells than in HEK-pBK cells, the corresponding controls, at all cells were determined by HPLC according to the method in previous studies time points. (Respaud et al., 2011; Kurata et al., 2014) with slight modifications. HPLC Concentration-Dependent Uptake of Pemetrexed by HEK293 analysis was performed using a Waters Alliance 2695 HPLC system (Waters, Cells Expressing hOAT3. To examine the characteristics of pemetrexed Milford, MA) connected to a TSKgel ODS-80Tm 5-mm column (150 Â 4.6 mm transport via hOAT3, concentration-dependent uptake studies for 2 minutes Lansoprazole Inhibits hOAT3-Mediated Pemetrexed Transport 1545

Inhibition of hOAT3-Mediated Transport of Pemetrexed by Probenecid. To verify whether pemetrexed is specifically transported by hOAT3, the cellular uptake of pemetrexed (25 mM) was measured for 2 minutes in the absence or presence of various concentrations of probenecid, a typical inhibitor of hOATs (Fig. 3). Probenecid inhibited hOAT3-mediated uptake of pemetrexed in a concentration-dependent manner. The apparent IC50 values for the probenecid were calculated from the inhibition plot (Fig. 3). The apparent IC50 value of probenecid against pemetrexed transport via hOAT3 was 1.97 6 1.31 mM. Inhibition of hOAT3-Mediated Transport of Pemetrexed by PPIs. To assess whether PPIs inhibit hOAT3-mediated transport of pemetrexed, the cellular uptake of pemetrexed (25 mM) was measured for 2 minutes in the absence or presence of various concentrations of PPIs (Fig. 4). All investigated PPIs inhibited hOAT3-mediated uptake of pemetrexed in a concentration-dependent manner. The apparent IC50 values for the PPIs were calculated from the inhibition plot (Fig. 4). Lansoprazole demonstrated potent inhibitory effect (IC50 =0.576 Downloaded from 0.17 mM) against pemetrexed transport via hOAT3. The rank order of inhibitory effect on hOAT3-mediated transport of pemetrexed was as . . . . Fig. 1. Time course of pemetrexed uptake by HEK-hOAT3 cells. HEK-hOAT3 follows: lansoprazole rabeprazole pantoprazole esomeprazole (closed circles) or pBK (open circles) cells were incubated for specified durations omeprazole . vonoprazan. (2, 3, 5, and 15 minutes) at 37C with 100 mM pemetrexed (pH 7.4). Each point Dixon Plot of the Inhibitory Effect of Lansoprazole against represents mean 6 S.E. of three separate experiments using three monolayers. **P , 0.01, ***P , 0.001 compared with HEK-pBK cells. When the standard hOAT3-Mediated Transport of Pemetrexed. A Dixon plot was dmd.aspetjournals.org errors of the means are small, they are contained within the symbols. constructed to clarify the type of inhibition of lansoprazole against hOAT3-mediated transport of pemetrexed (Fig. 5). Cellular uptake of pemetrexed (12.5, 25, and 50 mM) was measured for 2 minutes in the were conducted. Figure 2 shows the concentration-dependent uptake of absence and presence of lansoprazole (0.2, 0.5, and 1.0 mM). The Dixon pemetrexed via hOAT3 by subtracting the uptake in HEK-pBK cells from plot clearly indicated that the inhibitory type of lansoprazole against that in HEK-hOAT3 cells. The uptake of pemetrexed mediated by hOAT3 hOAT3-mediated transport of pemetrexed was competitive; the in- was saturated at high concentrations. Figure 2 shows the results: the hibitory constant value was 0.42 6 0.08 mM. apparent Km and Vmax values of hOAT3-mediated uptake of pemetrexed Patients’ Characteristics. According to the exclusion criteria, at ASPET Journals on September 24, 2021 were 68.3 6 11.1 mM and 157 6 9 pmol/mg protein/min, respectively. 108 of 116 patients were enrolled in the retrospective study. Patients’ Moreover, the Eadie-Hofstee plots were linear (insert, Fig. 2). characteristics are summarized in Table 1. The median age of patients was 68 (range, 39–82 years). Seventy-nine patients (73%) were men. Hematologic toxicity $grade 3 after administration of pemetrexed and carboplatin was observed in 22 patients (20%). Among the 22 patients with hematologic toxicity, leukopenia (n = 9), thrombocytopenia

Fig. 2. Concentration dependent uptake of pemetrexed transport mediated by hOAT3. HEK293 cells were incubated at 37C for 2 minutes with various concentrations (5, 10, 20, 50, 100, 200, and 500 mM) of pemetrexed (pH 7.4). The Fig. 3. Inhibition of pemetrexed uptake by probenecid in HEK-hOAT3 cells. uptake mediated by hOAT3 was obtained by subtracting the uptake in HEK-pBK HEK293-hOAT3 cells were incubated at 37C for 2 minutes with 25 mM cells from that in HEK-hOAT3 cells. Each point represents mean 6 S.E. of three pemetrexed (pH 7.4) in the absence or presence of various concentrations of separate experiments using three monolayers. When the standard errors of the means probenecid. Each point represents mean 6 S.E. of three separate experiments using are small, they are contained within the symbols. Insert: Eadie-Hofstee plot of three monolayers. When the standard errors of the means are small, they are pemetrexed uptake after subtraction of nonsaturable components. V is the uptake contained within the symbols. The apparent IC50 values were calculated by fitting velocity (pmol/mg protein/min), and S is the concentration of pemetrexed (mM). the data to a sigmoidal dose-response regression curve. 1546 Ikemura et al.

Fig. 4. Inhibition of pemetrexed uptake by PPIs in HEK-hOAT3 cells. HEK-hOAT3 cells were incubated at 37C for 2 minutes with Downloaded from 25 mM pemetrexed (pH 7.4) in the absence or presence of various concentrations of lansoprazole (A), rabeprazole (B), pantoprazole (C), esomeprazole (D), omeprazole (E), and vonoprazan (F). Each point represents mean 6 S.E. of three separate experiments using three monolayers. When the standard errors of the means are small, they are contained within the symbols. The apparent IC50 values were calculated by fitting the data to a sigmoidal dose-response regression curve. dmd.aspetjournals.org at ASPET Journals on September 24, 2021

(n = 20), and anemia (n = 3) were identified. On the other hand, the patients who received combination therapy of pemetrexed and carbo- coadministration of PPIs and pemetrexed was reported in 26 patients platin for the treatment of nonsquamous NSCLC (Table 2). The results (24%). These patients received lansoprazole 15 mg/day (n = 13) or revealed that coadministration of lansoprazole with pemetrexed and 30 mg/day (n = 2), esomeprazole 20 mg/day (n = 6), rabeprazole carboplatin was an independent risk factor that significantly contributed 10 mg/day (n = 4), and omeprazole 10 mg/day (n = 1), respectively. The to the development of hematologic toxicity (odds ratio: 10.004, P = coadministrated drugs in 22 patients with hematologic toxicity were 0.005). On the other hand, variables such as age, dose of pemetrexed and listed in Supplemental Table 1. Seven of 15 patients (47%) with carboplatin, CrCl, WBC count, PLT count, Hb level, and coadministra- lansoprazole developed hematologic toxicity. Furthermore, potential tion of NSAIDs and other PPIs were not significant risk factors for drug interactions of pemetrexed were verified using Lexi-Interact for all hematologic toxicity after pemetrexed and carboplatin administration. patients. Drug interaction between pemetrexed and NSAIDs was identified in 23 patients (21%). These patients received celecoxib 200 mg/day (n =3)or400mg/day(n = 1), diclofenac 75 mg/day (n =1), Discussion loxoprofen 180 mg/day (n = 16), meloxicam 10 mg/day (n = 1), and The drug interaction between PPIs and pemetrexed remains to be naproxen 300 mg/day (n = 1), respectively. In addition, 15 patients clarified. To our knowledge, this was the first study to report the effect of (14%) received amlodipine 2.5 mg/day (n = 4) or 5 mg/day (n =11). lansoprazole on the development of hematologic toxicity associated with Interestingly, 8 of 15 patients (53%) who received amlodipine developed pemetrexed by inhibition of pemetrexed transport via hOAT3. hematologic toxicity (Supplemental Table 1). Although some transporters have been previously reported to be Impact of Coadministration of Lansoprazole on the Develop- involved in renal elimination of pemetrexed (Li et al., 2013; Posada ment of Hematologic Toxicity after Pemetrexed and Carboplatin et al., 2015), only hOAT3 has been identified as an active transporter Administration. Considering the results of the in vitro studies, a of pemetrexed uptake from blood to renal tubular cells. As shown in multivariate logistic regression analysis was conducted to investigate the Fig. 2, hOAT3 was confirmed as a high-affinity type transporter for impact of lansoprazole on the development of hematologic toxicity in pemetrexed. Moreover, the inhibition of hOAT3-mediated transport of Lansoprazole Inhibits hOAT3-Mediated Pemetrexed Transport 1547

TABLE 2 Multivariate analysis of the development of hematologic toxicity after pemetrexed and carboplatin administration

Variables Odds Ratio 95% CI P value Lansoprazole 10.004 2.033–49.234 0.005 Other PPIs 1.959 0.310–12.382 0.475 Age 1.056 0.951–1.173 0.305 Pemetrexed dose (mg/m2) 1.049 0.993–1.107 0.087 Carboplatin dose (mg×min/ml) 0.993 0.983–1.004 0.238 CrCl (ml/min) 0.992 0.944–1.042 0.739 WBC (Â109/liter) 0.796 0.598–1.060 0.118 PLT (Â109/liter) 0.997 0.989–1.004 0.406 Hb (g/dl) 0.808 0.539–1.212 0.303 NSAIDs 2.015 0.432–9.407 0.373

CI, confidential interval; CrCl, creatinine clearance; Hb, hemoglobin; NSAIDs, nonsteroidal anti-inflammatory drugs; PLT, platelet; PPI, proton pump inhibitor; WBC, white blood cell. Downloaded from and retrospective studies have demonstrated that coadministration of PPIs delayed the elimination of methotrexate (Suzuki et al., 2009; Santucci et al., 2010; Reeves et al., 2014). It remains unclear, however, Fig. 5. Dixon plot of the inhibition of pemetrexed uptake by lansoprazole in HEK- whether PPIs inhibit pemetrexed transport via hOAT3. As shown in hOAT3 cells. HEK-hOAT3 cells were incubated at 37C for 2 minutes with 12.5 mM (closed squares), 25 mM (open circles), and 50 mM (closed circles) of Figs. 3 and 4, the inhibitory effect of lansoprazole was comparable to pemetrexed (pH 7.4) in the absence or presence of lansoprazole (0.2, 0.5, and that of probenecid and was much higher than those of other PPIs. The 1.0 mM). Each point represents mean 6 S.E. of three separate experiments using dmd.aspetjournals.org apparent IC50 value of lansoprazole against pemetrexed transport via three monolayers. When the standard errors of the means are small, they are 6 contained within the symbols. V is the uptake velocity (pmol/mg protein/min). hOAT3 was 0.57 0.17 mM. Moreover, its inhibitory type of lansoprazole was competitive (Fig. 5). The decision tree defined by the U.S. Food and Drug Administration’s pemetrexed was verified with probenecid (Fig. 3). Therefore, hOAT3 2012 draft guidance on drug-drug interaction concludes that a ratio of plays an important role in the first step of renal tubular secretion of unbound Cmax to IC50 value $0.1 indicates recommendation for the pemetrexed. evaluation of clinical drug interaction. When 30 mg of lansoprazole was

A recent study reported that hOAT3-mediated transport of metho- administered, the Cmax of lansoprazole was approximately 2.5–4.9 mM at ASPET Journals on September 24, 2021 trexate, which has a structure and pharmacokinetics similar to peme- (Ieiri et al., 2001). Since the protein binding of lansoprazole is 95.5% trexed, was inhibited by PPIs (Chioukh et al., 2014). Several case reports (McCallum et al., 2014), the unbound Cmax of lansoprazole was estimated to be 0.11–0.22 mM. The ratio of unbound Cmax to IC50 value of lansoprazole was 0.2–0.4 ($0.1), indicating that clinical trial TABLE 1 regarding drug interaction between pemetrexed and lansoprazole should ’ Patients characteristics be performed; however, the ratios of unbound Cmax to IC50 values of the Values are presented as median (range) or number (%). CrCl was estimated by the Cockcroft- PPIs, excluding lansoprazole, were much lower than 0.1 (data not Gault equation. shown). Therefore, these findings suggest that coadministration of Characteristic Patients (n = 108) lansoprazole and pemetrexed could lead to clinical drug interaction. Age (yr) 68 (39–82) Based on these findings, we hypothesized that coadministration of Male 79 (73) lansoprazole may exacerbate the hematologic toxicity of pemetrexed by Height (cm) 164.4 (143.6–188.1) inhibiting the tubular secretion of pemetrexed via hOAT3. The retro- – Body weight (kg) 59.5 (32.4 92.7) spective analysis of clinical data confirmed the impact of lansoprazole Body surface area (m2) 1.63 (1.24–2.19) Clinical disease stage (III/IV) 20 (19)/88 (81) on the development of hematologic toxicity in patients administered History of platinum-based chemotherapy 12 (11) with pemetrexed and carboplatin. As shown in Table 2, the multivariate 2 Pemetrexed dose (mg/m ) 497 (427–552) logistic regression analysis suggested that the coadministration of Carboplatin dose (mg×min/ml) 5.21 (4.21–6.60) Combination of bevacizumab 48 (44) lansoprazole, but not other PPIs, was an independent risk factor that Baseline biologic parameters significantly contributes to the development of hematologic toxicity WBC (Â109/liter) 6.87 (3.37–23.85) after pemetrexed administration (odds ratio: 10.004, P = 0.005). Â 9 – PLT ( 10 /liter) 247 (75 508) Interestingly, among the seven patients who developed hematologic Hb (g/dl) 13.2 (8.8–18) CrCl (ml/min) 73.4 (45.1–156.8) toxicity during coadministration of lansoprazole, one patient did not – Albumin (g/dl) 3.9 (3.0 4.7) develop hematologic toxicity when famotidine (histamine H2 receptor Coadministration drugs antagonist) was administered instead of lansoprazole during the next Lansoprazole 15 (14) Other PPIs (Esomeprazole/rabeprazole/omeprazole) 6 (6)/4 (4)/1 (1) course of chemotherapy with pemetrexed and carboplatin. These NSAIDs 23 (21) findings strongly suggest that coadministration of lansoprazole could Amlodipine 15 (14) exacerbate the hematologic toxicity of pemetrexed. Hematologic toxicity 22 (20) Leukopenia 9 (8) Lansoprazole is metabolized by the cytochrome P450 2C19 Thrombocytopenia 20 (19) (CYP2C19). It is well known that genetic polymorphism exists for this Anemia 3 (3) enzyme, and the pharmacokinetics of lansoprazole differs between

CrCl, creatinine clearance; Hb, hemoglobin; NSAIDs, nonsteroidal anti-inflammatory drugs; extensive and poor metabolizers of CYP2C19 (Katsuki et al., 1997; PLT, platelet; PPI, proton pump inhibitor; WBC, white blood cell. Sohn et al., 1997). The frequency of poor metabolizers of CYP2C19 in 1548 Ikemura et al. the Japanese population is approximately 20% (Kimura et al., 1998). for the development of hematologic toxicity after pemetrexed therapy in Although CYP2C19 polymorphism was not determined in our present patients without severe renal dysfunction. study, it is probable that CYP2C19 polymorphism contributed to the Interestingly, 8 of 22 patients with hematologic toxicity received development of hematologic toxicity by pemetrexed during coadmin- amlodipine (Supplemental Table 1). Multivariate logistic analysis with istration of lansoprazole. variables, including coadministration of amlodipine, revealed that Carboplatin is eliminated primarily from the kidney, and approxi- coadministration of amlodipine was also an independent risk factor that mately 90% of the dose is recovered in the urine as unchanged form significantly contributes to the development of hematologic toxicity by within 24 hours after administration in patients with normal renal pemetrexed (odds ratio: 22.910, P , 0.001) as shown in Supplemental function (Go and Adjei, 1999). Unlike other platinum compounds, Table 2. Two patients who developed hematologic toxicity received carboplatin is excreted mainly via glomerular filtration (Sorensen et al., both lansoprazole and amlodipine (Supplemental Table 1); however, the 1992) and is not transported by organic cation transporters including severity of hematologic toxicity in these two patients with both hOCT1 and hOCT2 (Yonezawa et al., 2006). Moreover, a previous lansoprazole and amlodipine was not greater than those in patients with study reported that the accumulation of p-aminohippurate (a typical either lansoprazole or amlodipine. Moreover, there were no reports substrate of hOATs) was not inhibited by carboplatin in the experiment regarding the inhibitory effect of amlodipine on the activity of hOAT3 or using rat renal cortical slices (Kanou et al., 2004). Thus, it is unlikely that the development of hematologic toxicity by amlodipine. Further study is lansoprazole inhibits tubular secretion of carboplatin. needed to clarify the detailed mechanism. To prevent the occurrence of adverse events after administration of The present study had some limitations. First, the delayed elimination Downloaded from pemetrexed, folic acid and vitamin B12 supplements are recommended of pemetrexed caused by lansoprazole was not evaluated because the during pemetrexed therapy (Molina and Adjei, 2003). Although human plasma concentration of pemetrexed could not be determined. Second, proton-coupled folate transporter plays a key role in the intestinal it was difficult to exclude the potential effects of other unknown absorption of folic acid (Visentin et al., 2014), Urquhart et al. (2010) confounders in the retrospective study. Therefore, a prospective study reported that human proton-coupled folate transporter mRNA levels needs to be conducted to determine the pharmacokinetics of pemetrexed decreased in patients receiving PPIs. Thus, it is likely that the decreased when coadministered with lansoprazole or other PPIs and to assess the oral absorption of folic acid caused by PPIs contributed to the toxicities of pemetrexed during coadministration. dmd.aspetjournals.org development of hematologic toxicity associated with pemetrexed; In conclusion, our study was the first to demonstrate that coadmin- however, we demonstrated that the coadministration of other PPIs was istration of lansoprazole could exacerbate the hematologic toxicity of not a significant risk factor for hematologic toxicity associated with pemetrexed, at least in part, by competitive inhibition of renal hOAT3. pemetrexed (Table 2). This finding suggests that decreased renal Therefore, lansoprazole should be discontinued or switched to other clearance of pemetrexed rather than decreased oral absorption of folic PPIs during chemotherapy with pemetrexed. Alternatively, the dose of acid was primarily accountable for the increased hematologic toxicity by pemetrexed could be reduced during coadministration of lansoprazole. pemetrexed. The present findings provide important information for safe and appropri- at ASPET Journals on September 24, 2021 In the present retrospective study, variables such as CrCl and ate chemotherapy with pemetrexed. coadministration of NSAIDs were not significant risk factors for hematologic toxicity of pemetrexed (Table 2). Mita et al. (2006) Authorship Contributions reported that pemetrexed plasma clearance positively correlated with Participated in research design: Ikemura, Iwamoto, Okuda. GFR, resulting in increased drug exposure in patients with impaired Conducted experiments: Ikemura, Hamada, Kaya. renal function. Pemetrexed was reported to be well tolerated, however, at Performed data analysis: Ikemura, Hamada, Kaya, Enokiya, Nakahara, 2 a dose of 500 mg/m in combination with vitamin supplements in Fujimoto, Kobayashi. patients with GFR $ 40 ml/min (Mita et al., 2006). In the present study, Wrote or contributed to the writing of the manuscript: Ikemura, Enokiya, all patients showed CrCl $ 45 ml/min and were administered Muraki, Iwamoto, Okuda. pemetrexed at a dose of approximately 500 mg/m2 in combination with folic acid and vitamin B12 supplements, implying that the enrolled References patients could tolerate pemetrexed therapy well. Therefore, it was not Adjei AA (2004) Pemetrexed (ALIMTA), a novel multitargeted antineoplastic agent. Clin Cancer relevant to evaluate the influence of CrCl on pemetrexed-mediated Res 10:4276s–4280s. hematologic toxicity. 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(2008) Phase III study comparing cisplatin plus at ASPET Journals on September 24, 2021 Drug Metabolism & Disposition Lansoprazole exacerbates pemetrexed-mediated hematological toxicity by competitive inhibition of renal basolateral human organic anion transporter 3 Kenji Ikemura, Yugo Hamada, Chinatsu Kaya, Tomoyuki Enokiya, Yuichi Muraki, Hiroki Nakahara, Hajime Fujimoto, Tetsu Kobayashi, Takuya Iwamoto, and Masahiro Okuda

Supplemental Table 1. Lists of the co-administrated drugs in 22 patients who developed hematological toxicity after combination therapy of pemetrexed and carboplatin for non-squamous non-small cell lung cancer.

Patients Co-administrated drugs bromazepam (2 mg), camostat (100 mg), levothyroxine (50 µg), loxoprofen (60 mg), 1 magnesium oxide (330 mg), mecobalamin (500 µg), naftopidil (50 mg), rabeprazole (10 mg), tizanidine (1 mg) 2 None aspirin (100 mg), atenolol (50 mg), candesartan (8 mg), famotidine (20 mg), nifedipine (20 mg), 3 prochlorperazine (5 mg) 4 betamethasone (0.5 mg), lansoprazole (15 mg), zonisamide (100 mg)

5 helenien (5 mg), kallidinogenase (50 IU), meloxicam (10 mg)

6 levothyroxine (50 µg)

7 amlodipine (5 mg), doxazosin (1 mg), telmisartan (40 mg) alfacalcidol (0.25 µg), amlodipine (2.5 mg), asprin (100 mg), doxazosin (2 mg), ecabet sodium hydrate (66.7%), 8 irbesartan (100 mg), magnesium oxide (330 mg), omeprazole (10 mg), probucol (250 mg), tocopherol (200 mg) 9 l-carbocisteine (500 mg), nifedipine (20 mg), theophylline (200 mg)

10 allopurinol (100 mg), amlodipine (5 mg), bromazepam (2 mg), magnesium oxide (330 mg), naproxen (100 mg)

11 lansoprazole (15 mg) clarithromycin (200mg), diclofenac (25 mg), lansoprazole (15 mg), magnesium oxide (0.67 g), 12 rebamipide (100 mg), sodium ferrous citrate (50 mg) amlodipine (2.5 mg), betamethasone (0.5 mg), lansoprazole (15 mg), magnesium oxide (330 mg), 13 pregabalin (25 mg) allopurinol (100 mg), amlodipine (5 mg), carvedilol (20 mg), celecoxib (100 mg), enalapril (5 mg), 14 etizolam (0.5 mg), pravastatin (10 mg), solifenacin (2.5 mg), tamsulosin (0.2 mg) 15 rabeprazol (10mg)

16 levothyroxine (75 µg) amlodipine (5 mg), hydrochloride (250 mg), lansoprazole (15 mg), candesartan (8 mg), loxoprofen (60 mg), 17 rebamipide (100 mg), sennoside (12 mg) ambroxol (15 mg), betamethasone (2 mg), carbocisteine (500 mg), clarithromycin (100 mg), 18 dextromethorphan (15 mg), rebamipide (100 mg) 19 cholecalciferol (0.005 mg), fentanyl (2 mg), lansoprazole (15 mg), oxycodone (5 mg), prednisolone (5 mg) acetaminophen (200 mg), amlodipine (5 mg), doxazosin (1 mg), magnesium oxide (330 mg), oxycodone (5 mg), 20 prochlorperazine (5 mg), rebamipide (100 mg) lansoprazole (15 mg), loxoprofen (60 mg), magnesium oxide (330 mg), mosapride (5 mg), olanzapine (2.5mg), 21 pregabalin (75 mg) amlodipine (5 mg), carvedilol (1.25 mg), furosemide (20 mg), trichlormethiazide (1 mg), valsartan (40 mg), 22 warfarin (1mg) Supplemental Table 2. Multivariate analysis with variables including coadministration of amlodipine for risk factors of the development of hematological toxicity after pemetrexed and carboplatin

Variables Odds ratio 95% CI P value Lansoprazole 17.002 2.585–111.844 0.003 Other PPIs 3.204 0.326–31.535 0.318 Amlodipine 22.910 4.054–129.479 < 0.001 Age 1.035 0.923–1.161 0.558 Pemetrexed dose (mg/m2) 1.062 0.995–1.132 0.069 Carboplatin dose (mgmin/ml) 0.987 0.975–1.001 0.061 CrCl (ml/min) 0.979 0.925–1.036 0.469 WBC (×109/l) 0.738 0.541–1.008 0.056 PLT (×109/l) 0.996 0.987–1.005 0.350 Hb (g/dl) 0.783 0.488–1.256 0.310 NSAIDs 2.296 0.369–14.271 0.373 Multivariate logistic regression analysis was performed to identify the risk factors on the development of hematological toxicity after administration of pemetrexed and carboplatin, with the following variables: age, WBC, PLT, CrCl, Hb, coadministration of lansoprazole, amlodipine, other PPIs, or NSAIDs, pemetrexed dose, and carboplatin dose. CI: confidential interval, CrCl: creatinine clearance, Hb: hemoglobin, NSAIDs: non-steroidal anti-inflammatory drugs, PLT: Platelet, PPI: proton pump inhibitor, WBC: white blood cell