Abstract
We aim to establish an in vivo preclinical model to enable simultaneous assessment of inhibition potential of an investigational drug on clinically relevant drug transporters, organic anion-transporting polypeptide (OATP)1B, breast cancer resistance protein (BCRP), P-glycoprotein (P-gp), and organic anion transporter (OAT)3. Pharmacokinetics of substrate cocktail consisting of pitavastatin (OATP1B substrate), rosuvastatin (OATP1B/BCRP/OAT3), sulfasalazine (BCRP), and talinolol (P-gp) were obtained in cynomolgus monkey—alone or in combination with transporter inhibitors. Single-dose rifampicin (30 mg/kg) significantly (P < 0.01) increased the plasma exposure of all four drugs, with a marked effect on pitavastatin and rosuvastatin [area under the plasma concentration–time curve (AUC) ratio ∼21–39]. Elacridar, BCRP/P-gp inhibitor, increased the AUC of sulfasalazine, talinolol, as well as rosuvastatin and pitavastatin. An OAT1/3 inhibitor (probenecid) significantly (P < 0.05) impacted the renal clearance of rosuvastatin (∼8-fold). In vitro, rifampicin (10 µM) inhibited uptake of pitavastatin, rosuvastatin, and sulfasalazine by monkey and human primary hepatocytes. Transport studies using membrane vesicles suggested that all probe substrates, except talinolol, are transported by cynoBCRP, whereas talinolol is a cynoP-gp substrate. Elacridar and rifampicin inhibited both cynoBCRP and cynoP-gp in vitro, indicating potential for in vivo intestinal efflux inhibition. In conclusion, a probe substrate cocktail was validated to simultaneously evaluate perpetrator impact on multiple clinically relevant transporters using the cynomolgus monkey. The results support the use of the cynomolgus monkey as a model that could enable drug–drug interaction risk assessment, before advancing a new molecular entity into clinical development, as well as providing mechanistic insights on transporter-mediated interactions.
Introduction
Prediction of drug–drug interaction (DDI) liability of a new molecular entity (NME) is an important factor to consider during drug design/discovery and development, as such DDIs could impact patient safety or diminish drug efficacy (Bjornsson et al., 2003; Zhang et al., 2007; Bloomer et al., 2013). Transporters of the solute carrier and ATP-binding cassette (ABC) superfamilies are expressed in a variety of organs, including intestine, liver, and kidney, and are increasingly recognized for their key role in the absorption, distribution, clearance, and elimination of drugs (Begley, 2004; Lee and Kim, 2004; Shitara et al., 2006; Koepsell et al., 2007; Feng et al., 2010; Giacomini et al., 2010; Varma et al., 2010; El-Kattan and Varma, 2018). On the basis of established evidences, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), organic anion-transporting polypeptides (OATPs), organic anion and cation transporters (OATs and OCTs), and multidrug and toxic compound extrusion (MATE) transporters are suggested to be of clinical relevance (Giacomini et al., 2010; Varma et al., 2017a). Regulatory guidance from agencies, including European Medical Agency, the Pharmaceuticals and Medical Devices Agency (Japan), and United States Food and Drug Administration, now recommends appropriate in vitro and/or in vivo assessment of investigational drugs for potential transporter DDIs (http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2012/2007/WC500129606.pdf; Saito et al., 2014; http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf). For instance, NME with a potential to inhibit OATP1B1, in which in vitro measured inhibition potency (IC50 or Ki) ≤10 times the unbound maximum plasma liver inlet concentration at the clinical dose, is predicted to increase systemic exposure of substrate drugs (e.g., statins, sartans, and glinides) (http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf).
Although the unbound maximum plasma liver inlet concentration cannot be physically measured and thus carries some uncertainty, accurate determination of IC50 or Ki is a key to the reliable prediction using these methods. Intra- and interlaboratory variability in the in vitro IC50 or Ki values and considerable discrepancy in the in vitro and the estimated in vivo inhibitory potency have been noted previously (Varma et al., 2012; Bentz et al., 2013; Li et al., 2014; Vaidyanathan et al., 2016). For instance, substantial variability was observed in the in vitro P-gp IC50 values of the 16 inhibitors examined at 23 different laboratories (Bentz et al., 2013). Similarly, we previously noted large interlaboratory variability in the in vitro IC50 values for OATP inhibitors; and the geometric mean of the reported in vitro Ki values are several folds greater than the estimated apparent in vivo Ki for OATP inhibitors (Varma et al., 2012; Li et al., 2014). Retrospectively, this disconnect may be explained by the time-dependent inhibition (Amundsen et al., 2010; Gertz et al., 2013) or substrate-dependent inhibition (Noé et al., 2007; Izumi et al., 2013) or contribution of the circulating metabolites (e.g., gemfibrozil 1-O-β-glucuronide) (Shitara et al., 2004; Varma et al., 2015a; Shen et al., 2016). The differences in Ki values with different probe substrates and/or varied experimental settings raise uncertainty in DDI assessment. Overall, compared with cytochrome P450–mediated DDIs, the risk of underestimating potentially hazardous drug interactions involving transporters or transporter–enzyme interplay is relatively high.
Recent clinical studies showed encouraging data regarding the utility of endogenous biomarkers for assessing investigational drugs as an inhibitor, particularly against OATP1B1/1B3, OAT1/3, and OCT2/MATEs (Lai et al., 2016; Rodrigues et al., 2018). Also, clinical studies employing multiple probe substrate drug cocktails have been proposed to allow simultaneous assessment of inhibition risk (Stopfer et al., 2016; Prueksaritanont et al., 2017). Although these studies show promising results, risk assessment prior to first-in-man studies—needed to nominate the best candidate into clinical development—still remains a major challenge. It is therefore desirable to establish a preclinical animal model that can project the clinical DDI risk. In this regard, some recent studies suggested utility of the cynomolgus monkey to assess inhibition of drug transporters, including OATP, P-gp, BCRP, and OAT1/3 (Tahara et al., 2006; Shen et al., 2013, 2016; Karibe et al., 2015).
In this study, we evaluated a cocktail of probe substrates for early assessment of transporter-mediated DDIs using cynomolgus monkey as a preclinical model. The cocktail consisted of probe substrates of OATPs (pitavastatin and rosuvastatin), P-gp (talinolol), BCRP (sulfasalazine), and OAT3 (rosuvastatin); and validated against a single-dose oral treatment of an OATP inhibitor (rifampicin), P-gp and BCRP inhibitor (elacridar), or OAT1/3 inhibitor (probenecid). In vitro transport (substrate and inhibition) studies were carried out using membrane vesicle systems and primary monkey and human hepatocytes to corroborate the interaction mechanisms.
Materials and Methods
Chemicals and Reagents.
Rifampicin (rifampin) was purchased from Sigma-Aldrich (St. Louis, MO). Pitavastatin and rosuvastatin were purchased from Sequoia Research Products (Oxford, UK). Probenecid and sulfasalazine were purchased from USP (Rockville, MD), and elacridar and talinolol were purchased from eNovation Chemicals (Bridgewater, NJ). InVitroGro-HT and CP and hepatocyte media were purchased from Celsis IVT (Baltimore, MD). Collagen I–coated 24-well plates were obtained from BD Biosciences (Franklin Lakes, NJ). Cryopreserved cynomolgus monkey hepatocytes (10106012, female) were purchased from In Vitro ADMET Laboratories (Columbia, MD). Cryopreserved human hepatocytes (Hu8246, female, Caucasian, 37 years old) were purchased from Thermo Fisher Scientific (Carlsbad, CA). Bicinchoninic acid protein assay kit was purchased from Pierce (Rockford, IL). Cynomolgus monkey P-gp vesicles (cynoP-gp; gene information, Macaca fascicularis ABCB1, GenBank accession: NM_001287322.1) and BCRP (cynoBCRP; gene information, M. fascicularis ABCG2, GenBank accession: XM_005555388.1) were provided by Solvo Biotechnology (Budapest, Hungary).
In Vivo Studies with Cynomolgus Monkeys.
All procedures performed on these animals were in accordance with regulations and established guidelines, were reviewed and approved by Pfizer Institutional Animal Care and Use Committee, and were conducted at Pfizer (Groton, CT). Male cynomolgus macaque Mauritian monkeys (6–9 years of age) were used, and the procedures similar to those previously reported were adopted with some modifications (Varma et al., 2017b; de Bruyn et al., 2018; Ufuk et al., 2018). A crossover study design was employed, in which the same four animals were dosed over all studies, following a minimum 1-week washout period between each study. Animals were provided a normal food schedule the day before the study (meals at 8:00 AM and 11:00 AM, with one treat daily) and were allowed free access to water. Animals were housed in metabolism cages during sample collection. On the day of the study, monkeys were fed at approximately 1 and 3 hours postdose and allowed water ad libitum. Blank vehicle, rifampicin (30 mg/kg), elacridar (30 mg/kg), or probenecid (30 mg/kg) was administered via oral gavage in a 0.5% w/v methylcellulose (in water) suspension 30 minutes prior to oral cassette dosing of pitavastatin (1 mg/kg), rosuvastatin (1 mg/kg), talinolol (1 mg/kg), and sulfasalazine (0.5 mg/kg). Sulfasalazine (in 2% v/v dimethylsulfoxide: 98% v/v phosphate-buffered saline) and talinolol (in 2% v/v dimethylsulfoxide: 10% v/v polyethylene glycol 400: 88% v/v of 12% w/v sulfobutyl ether β-cyclodextrin in water) were also dosed individually at 1 and 0.2 mg/kg, respectively, via i.v. administration at a dose volume of 0.5 ml/kg in separate studies to calculate oral bioavailability. Serial blood samples were collected via the femoral vein into K2EDTA tubes prior to dosing and then at 0.083, 0.25, 0.5, 0.75, 1, 2, 3, 5, 6, and 24 hours postoral cassette dosing.
Blood samples were stored on wet ice prior to being centrifuged to obtain plasma (3000 rpm, 10 minutes at 4°C). Urine was also collected on wet ice, predose and at intervals of 0–6 and 6–24 hours postdose. Due to the potential instability of rifampicin and possible interconversion of lactone to pitavastatin or rosuvastatin, all plasma and urine samples were equally divided into two aliquots prior to being stored frozen. The first aliquot was untreated matrix, whereas the second aliquot was added to an equal volume of 0.1 M sodium acetate buffer (pH 4). All urine and plasma samples, treated and untreated, were kept cold during collection, after which they were stored frozen at −20°C.
Bioanalysis of In Vivo Samples.
The concentrations of elacridar, probenecid, rifampicin, talinolol, pitavastatin, rosuvastatin, and sulfasalazine were measured in plasma, and urine samples treated with 0.1 M sodium acetate buffer (pH 4) using liquid chromatography–tandem mass spectrometry (LC-MS/MS). All standards were prepared in blank monkey plasma mixed with an equal volume of 0.1 M sodium acetate buffer (pH 4). Aliquots of 20 µl standards or plasma samples were prepared by protein precipitation with 100 µl acetonitrile containing an internal standard (IS) mixture of verapamil (2.5 ng/ml), terfenadine (30 ng/ml), propranolol (12.5 ng/ml), and tolbutamide (5 ng/ml). Urine samples were diluted with nine volumes of a 50/50 mix of blank monkey plasma/0.1 M sodium acetate buffer (pH 4) and analyzed with the plasma samples. The plates were vortexed for 2 minutes and centrifuged at 3000 rpm for 5 minutes, and 50 µl supernatants of the mixture were transferred to a new sample block, diluted with 100 µl water containing 0.1% formic acid, and analyzed via LC-MS/MS.
Chromatography was performed on a Waters Acquity iClass UPLC System (Milford, MA). The autosampler and column were kept at 10°C and 40°C, respectively. Separation was achieved with an Acquity UPLC HSS T3 column (2.1 × 50 mm, 1.8 µm) and a gradient of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) at a flow rate of 0.600 ml/min. An initial mobile-phase composition of 5% B was held for 0.3 minute, ramped to 95% over 2 minutes, held at 95% for 0.3 minute, and then returned to initial 5% B over 0.1 minute for re-equilibration. A Valco VICI valve (Valco Instruments, Houston, TX) was used to divert the first 0.3 minute and the last 0.3 minute of high-performance liquid chromatography effluent to waste. The total run time for each injection was 3 minutes. Data were collected on an AB Sciex API5500 mass spectrometer (Applied Biosystems, Foster City, CA) using either positive or negative Turbo IonSpray electrospray ionization and multiple reaction monitoring mode. Typical source conditions (heated capillary temperature, gas1, gas2, and curtain gas) were set at 500°C, 40°C, 60°C, and 20°C, respectively. Multiple reaction monitoring transitions for each analyte, along with the IS used, are included in a Supplemental Table 1.
Uptake Studies Using Cryopreserved Plateable Monkey Hepatocytes (PMH).
The hepatic uptake assay was performed using short-term culture format, as described previously, with some modification (Bi et al., 2017, 2018). Briefly, cryopreserved cynomolgus hepatocytes and human hepatocytes were thawed in the InVitro-HT media at 37°C at a ratio of one or two vials/50 ml in a conical tube. The cells were centrifuged at 50g for 3 minutes and resuspended in In VitroGro-CP medium. The cells were seeded into 24-well collagen I–coated plates with 0.35 × 106 cells/well in a volume of 0.5 ml/well. The cells were cultured in the InVitro-CP media overnight (∼18 hours). Cell culture was first rinsed twice with Hanks’ balanced salt solution (HBSS) buffer and then preincubated for 30 minutes at 37°C with HBSS with or without inhibitors. The preincubation buffer was aspirated, and the uptake and inhibition reaction were initiated by addition of prewarmed buffer containing substrates with or without inhibitors. The reactions were terminated at designated time points (0.5 and 2 minutes) by adding ice-cold HBSS immediately after removal of the incubation buffer. The cells were washed three times with ice-cold HBSS and lysed with 100% methanol containing IS, and the samples were analyzed by LC-MS/MS (Supplemental Material). Uptake rates were estimated from the initial time course (0.5–2 minutes) by linear regression.
In Vitro Transport Studies Using Membrane Vesicles.
Vesicle assay buffer (10 mM Tris base, 250 mM sucrose, 10 mM magnesium chloride) and stop buffer (assay buffer plus 100 mM sodium chloride) were prepared at pH 7.4. For substrate assays, HEK293-cynoBCRP, HEK293-cynoP-gp, and HEK293-mock vesicles were diluted to 50 µg/well in assay buffer and treated with 5 mM ATP and 1 µM test compound (pitavastatin, rosuvastatin, sulfasalazine, talinolol, or n-methyl quinidine). Assays were incubated at 22°C while shaking. Reactions were stopped at 0.25, 0.5, 1, 2.5, 5, and 10 minutes by the addition of 200 µl ice-cold stop buffer. The entire reaction was quickly removed from the assay plate and filtered on a vacuum filter plate (Multiscreen; Millipore Burlington, MA) and then washed four times with ice-cold stop buffer. For inhibition assays, HEK293-cynoBcrp and HEK293-cynoP-gp vesicles were diluted to 50 µg/well in assay buffer and were treated with 5 mM ATP, 0.2 µM rosuvastatin (BCRP), or 0.2 µM n-methyl quinidine (P-gp) and varied concentrations of inhibitor (elacridar, probenecid, rifampicin, or control inhibitors Ko143 and PCS833). BCRP and P-gp assays were incubated for 1 and 2 minutes, respectively, at 22°C, while shaking, and were stopped, as described above.
Intravesicle samples for BCRP and P-gp incubations were extracted by treating the vesicles with 0.1 ml IS solution in 100% methanol. Vesicles were shaken for 15 minutes at room temperature. Vesicle extracts were transferred to 96-well polypropylene deep-well plates by centrifugation and dried down under nitrogen. Samples were reconstituted in 50:50 methanol:water prior to injection onto a LC-MS/MS system. Samples were analyzed for probe and IS peak areas. LC-MS/MS methods are described in Supplemental Material.
Data for BCRP and P-gp assays were acquired using Analyst 1.6.2, and peak area ratios (analyte:IS) were exported into Microsoft Excel. For substrate assays, the amount of analyte uptake was calculated as transfected:mock peak area ratio. For inhibition assays, percent transporter activity was calculated by the following equation:Percent activity values were exported into GraphPad Prism to estimate IC50 values.
Pharmacokinetic Analysis and Statistics.
LC-MS/MS data acquisition and peak integration of plasma and urine samples were performed using Analyst 1.6.2 software (Applied Biosystems/MDS Sciex, Ontario, Canada). Peak area values were imported into Watson LIMS version 7.5 (Thermo Fisher Scientific, Waltham, MA) for standard curve regression, quantitation, and for calculation of model independent, noncompartmental pharmacokinetic parameters: area under the plasma concentration–time curve (AUC), maximum plasma concentration (Cmax), and half-life (t1/2). One-way analysis of variance was applied to test statistical significance across groups with Tukey’s test for multiple comparisons (GraphPad Software, San Diego, CA).
Results
Effect of Inhibitors on the Oral Pharmacokinetics of Probe Substrates in Cynomolgus Monkey.
Oral pharmacokinetics of four probe substrate drugs (pitavastatin, rosuvastatin, talinolol, and sulfasalazine) in a cocktail dosed after treatment with vehicle control or inhibitor drugs (rifampicin, elacridar, probenecid) were studied in cynomolgus monkeys (n = 4) (Fig. 1). Dose-normalized pharmacokinetics of the probe drugs obtained following cocktail dose in the control arm of this study are in good agreement with the data reported following individual oral dosing (Karibe et al., 2015, 2018; Shen et al., 2015; Varma et al., 2017). Single-dose oral rifampicin (30 mg/kg) significantly (one-way analysis of variance, P < 0.01) increased the plasma exposure of all four probe drugs (Fig. 2; Tables 1–4). Particularly, rifampicin showed marked effects on OATP substrates, pitavastatin and rosuvastatin—with AUC ratios (ratio of AUC in the presence of inhibitor to vehicle control) of about 39 ± 22 (geometric mean ± S.D.) and 21 ± 35, respectively. Interindividual variability in AUC was relatively high (CV ∼50%) for rosuvastatin in each arm, resulting in %CVs up to 100% in AUC ratios (±rifampicin). Variability in AUC of other probes is generally low (25%–40% CV).
Rifampicin also significantly increased the plasma Cmax of all probe drugs (Fig. 2; Tables 1–4). P-gp and BCRP inhibitor, elacridar, significantly increased plasma AUC of talinolol and sulfasalazine. Although statistical significance was not reached (P > 0.05), the plasma exposure of pitavastatin and rosuvastatin was increased by ∼2.8- and 4.4-fold, respectively, with elacridar. Probenecid, an OAT1/3 inhibitor, did not present a significant effect on the plasma exposure of all the drugs tested, although an increasing trend was apparent for pitavastatin and rosuvastatin (∼twofold). No significant change (P > 0.05) in the terminal t1/2 of the probe drugs was observed. This reflects expected decrease in both systemic clearance and volume of distribution of statins due to OATP1B inhibition by rifampicin (Ufuk et al., 2018). For talinolol and sulfasalazine, lack of t1/2 change may suggest limited effect of the inhibitors on their systemic clearance. The time at maximum plasma concentration (Tmax) of pitavastatin, talinolol, and sulfasalazine was not affected by any inhibitor. However, rifampicin and elacridar reduced the Tmax of rosuvastatin from about 3.5 to 0.8–1.8 hours.
Probenecid, but not rifampicin or elacridar, markedly decreased (P < 0.05) the renal clearance of rosuvastatin–renal clearance ratio (i.e., CLrenal,+inh/CLrenal,control) of ∼7.8 (Table 2). However, talinolol renal clearance was not inhibited by any inhibitor. In contrast, urinary excretion of pitavastatin and sulfasalazine is negligible or often undetectable.
Plasma samples from these studies were measured to obtain concentration–time profiles of inhibitor drugs (Fig. 3). The unbound Cmax of rifampicin was about 10.7 µM (total Cmax, 50.8 µM; measured monkey fu,plasma, 0.21). In the case of elacridar, unbound Cmax is less than 0.1 nM (total Cmax, ∼100 nM; fu,plasma, 0.001). In contrast, unbound Cmax of probenecid at this given dose is about 76 µM (total Cmax, 588 µM; fu,plasma, 0.13). Through the time course of 24 hours, rifampicin plasma-unbound concentrations are well above the in vitro IC50 (0.54 µM) (Ufuk et al., 2018) for uptake inhibition in monkey hepatocytes (Fig. 3). Similarly, probenecid plasma-unbound concentrations are several fold higher than its in vitro IC50 (3.0 µM) (Tahara et al., 2006) obtained in monkey OAT3-transfected cells. Monkey plasma-unbound concentrations of elacridar are, however, negligible. Additionally, unbound concentrations reached in the present monkey studies are within the range observed in humans: 2–6 µM for 600 mg rifampicin (Varma et al., 2012; Prueksaritanont et al., 2014; Yoshikado et al., 2017); <1 nM for 1000 mg elacridar (Sawicki et al., 2017); and 6–51 µM for 500–1500 mg probenecid (Mathialagan et al., 2017).
Effect of Inhibitors on the In Vitro Uptake of Probe Substrates by Cynomolgus Monkey and Human Hepatocytes.
Uptake of each of the four probe substrate drugs was evaluated in monkey and human hepatocytes (short-term culture format) in the absence and presence of the inhibitors—at concentrations close to their corresponding unbound Cmax for rifampicin and probenecid. Elacridar was incubated at 0.1 µM, which is much above its unbound Cmax. Rifampicin (10 µM) significantly inhibited uptake of pitavastatin, rosuvastatin, and sulfasalazine, in both monkey and human hepatocytes (Fig. 4). Elacridar (0.1 µM) did not show inhibition of hepatic uptake for any substrate. Interestingly, probenecid (50 µM) significantly inhibited uptake of statins and sulfasalazine by cynomolgus monkey hepatocytes; however, only a marginal inhibition (10%–15%) was noted in human hepatocytes. Apart from this difference, interaction mechanism(s) involving hepatic uptake for each substrate–inhibitor pair is comparable across the two species.
Effect of Inhibitors on the In Vitro Transport of Probe Substrates in cynoBCRP and cynoP-gp Membrane Vesicles.
We first evaluated pitavastatin, rosuvastatin, sulfasalazine, and talinolol for their substrate affinity to cynoBCRP and cynoP-gp using membrane vesicles prepared from transfected HEK293 cells (Fig. 5). Time-dependent uptake was observed with cynoBCRP vesicles for pitavastatin, rosuvastatin, and sulfasalazine. Talinolol showed significant uptake by cynoP-gp vesicles compared with mock vesicles. Interestingly, P-gp–mediated transport was also seen for rosuvastatin, although the activity is notably small compared with cynoBCRP activity (Fig. 5, B and F).
Using rosuvastatin and N-methyl-quinidine (NMQ) as substrates (validated for the intended use with control inhibitors Ko143 and PSC833, respectively), cynoBCRP and cynoP-gp inhibition was evaluated for elacridar, rifampicin, and probenecid. Elacridar and rifampicin showed concentration-dependent inhibition of both cynoBCRP- and cynoP-gp–mediated transport, whereas probenecid had a minimal effect up to 300 μM (Fig. 6). Inhibition potency (IC50) estimates are provided in Table 5. Additionally, IC50 of the inhibitor drugs against human BCRP and P-gp were extracted from the literature and compared with the cynomolgus monkey. Based on the ratio of gut concentration (IGut; calculated as a ratio of dose to gut volume, in which gut volume was assumed to be 250 ml for humans and 10 ml/kg for monkey) (Amidon et al., 1995; Kararli, 1995) to IC50, elacridar and rifampicin show high potential for in vivo intestinal efflux inhibition in both cynomolgus monkey and human (i.e., IGut/IC50 > > 10) (Table 5).
Discussion
De-risking DDIs involving membrane transporters is an essential part of medicinal chemistry design and drug development. However, translation of in vitro IC50 (or Ki) values to assess DDI potential is challenged with poorly understood substrate-, incubation time–, and in vitro system–dependent measurements. In this study, we evaluated a cocktail of probe substrates to simultaneously assess the in vivo inhibition potency of NMEs against clinically relevant transporters, OATP1B1/1B3, P-gp, BCRP, and OAT3, using cynomolgus monkey as a preclinical animal model. The proposed cocktail of four probe drugs has been validated in vivo using relatively selective inhibitor drugs. In vitro studies using primary hepatocytes and membrane vesicles verified the interaction mechanisms in the cynomolgus monkey, and further implied similarities in the transporter-mediated disposition in this preclinical model and humans. Although the concept of a transporter probe drug cocktail has been tested clinically (Stopfer et al., 2016; Prueksaritanont et al., 2017), to our knowledge this is the first report of a study employing such a cocktail in cynomolgus monkeys. This approach can thus be valuable in assessing the transporter inhibition potential of NMEs before advancing into first-in-man studies, in rational staging of clinical DDI studies during drug development, and to substantiate in vitro findings in the regulatory submissions.
The probe substrates selected for the proposed cocktail are shown to be associated with the drug transporters of interest based on the clinical pharmacokinetics, DDI, and pharmacogenomics studies. Pitavastatin and rosuvastatin represent extended clearance classification system (ECCS) class 1B and class 3B, respectively, in which OATP-mediated hepatic uptake plays a rate-determining role in the systemic clearance in humans (Shitara et al., 2013; Varma et al., 2015b; El-Kattan et al., 2016). In vitro, both statins are transported by human OATP1B1/1B3/2B1 and Na+-taurocholate cotransporting polypeptide (NTCP), whereas rosuvastatin is also a substrate to BCRP (Shitara et al., 2013). Renal clearance of pitavastatin in humans is low, but rosuvastatin shows up to 30% dose excretion in urine and involves OAT3-mediated renal secretion (Mathialagan et al., 2017). OATP1B1/1B3 inhibition by a single-dose rifampicin perpetrates ∼3- to sevenfold AUC increase for rosuvastatin and pitavastatin in humans (Prueksaritanont et al., 2014, 2017). In contrast, sulfasalazine (ECCS class 3A) and talinolol (ECCS class 4) are low permeability drugs with limited oral absorption in humans (El-Kattan et al., 2016). Consequently, clinical pharmacokinetics of sulfasalazine and talinolol are considerably altered when coadministered with BCRP and P-gp inhibitors, respectively (Gramatté et al., 1996; Gramatté and Oertel, 1999). Although sulfasalazine is a preferred drug to probe BCRP inhibition in clinical studies (Lee et al., 2015), digoxin is commonly used to assess intestinal P-gp activity (Fenner et al., 2009; http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm292362.pdf). In our preliminary studies, a combination of single-dose digoxin (0.25 mg/kg) and sulfasalazine (1 mg/kg) caused minor adverse events in the cynomolgus monkeys (i.e., emesis in the first 24 hours and lack of appetite for up to 96 hours) (data not shown). We speculated that digoxin is the major source for these effects on the basis of its toxicity profile in humans (MacLeod-Glover et al., 2016), and thus considered talinolol as an alternative to measure P-gp function. Talinolol is very similar to digoxin in regard to the disposition attributes in human. For instance, P-gp inhibitors such as curcumin, erythromycin, and verapamil increase oral exposure of both drugs in humans (Gramatté et al., 1996; Gramatté and Oertel, 1999; Fenner et al., 2009). Additionally, clinical pharmacokinetics of both talinolol and digoxin are influenced by rifampicin-induced P-gp–mediated secretion (Greiner et al., 1999; Westphal et al., 2000). Finally, the four probe drugs are minimally metabolized in the human and monkey (Elsby et al., 2012; Shitara et al., 2013; Takahashi et al., 2013; Karibe et al., 2015, 2018; El-Kattan et al., 2016; Shen et al., 2016), making them suitable candidates to reliably probe transporter activity.
Of the transporter-mediated clinical DDIs, interactions involving OATP1B1/1B3 often result in larger exposure change for victim drugs and are of particular interest for risk mitigation during drug design (Giacomini et al., 2010; Varma et al., 2017a; El-Kattan and Varma, 2018). Rifampicin dramatically increased exposure of OATP substrates pitavastatin and rosuvastatin in cynomolgus monkey (Figs. 1 and 2). Given high oral absorption (60%–80%) (Ufuk et al., 2018) and negligible renal clearance in monkey, pitavastatin interaction may predominantly involve OATP1B1/1B3. Rosuvastatin interaction is most likely driven by multiple mechanisms (i.e., intestinal efflux and hepatic uptake), and thus, could serve to confirm the interactions with pitavastatin (OATP1B) and sulfasalazine (BCRP). Moreover, change in rosuvastatin renal clearance can be used as a surrogate for OAT3 inhibition. However, oral AUC change of about 39- and 21-fold for pitavastatin and rosuvastatin observed in this study in the monkeys is much higher than that noted in humans (∼3- to sevenfold for both statins) (Prueksaritanont et al., 2014, 2017). Although the magnitude change is also considerably higher than reported by others at slightly lower doses of rifampicin in cynomolgus monkey (up to 15 mg/kg) (Shen et al., 2013; Watanabe et al., 2015), the current results are nevertheless in line with our previous report in which a single oral rifampicin dose (30 mg/kg) increased AUC of oral pitavastatin and rosuvastatin by ∼19- and ∼15-fold, respectively (Ufuk et al., 2018). Rifampicin-unbound Cmax achieved in monkey (∼10 µM) at 30 mg/kg dose is relatively higher (∼2- to fivefold) than the unbound Cmax in human (∼2–6 µM) following single 600 mg dose (Varma et al., 2012; Prueksaritanont et al., 2014; Yoshikado et al., 2017). Although this may contribute to the higher exposure change in the monkey study to some degree, other factors such as involvement of OATP2B1 in hepatic uptake clearance in humans—which is not inhibitable by rifampicin at in vivo relevant plasma concentrations (OATP2B1 in vitro IC50 > 60 µM)—may also contribute to difference in AUC ratios of statins. Notably, monkeys express low levels of OATP2B1 in the liver, suggesting that the fraction transported by OATP1B1/1B3 is relatively higher in monkeys than in humans (Wang et al., 2015). In contrast, magnitude of interactions registered in this study with the probe substrates and BCRP/P-gp inhibitor (elacridar) is comparable with previous reports (Karibe et al., 2015, 2018). Although clinical data are limited, the AUC change for sulfasalazine with a BCRP inhibitor (curcumin) is about threefold in humans (Kusuhara et al., 2012), which is comparable to its exposure change noted in the monkey. Collectively, the mechanisms of transporter interactions are well preserved across the two species. However, further diligence via in vitro studies and mechanistic modeling would be needed to quantitatively extrapolate monkey DDIs to human.
Interestingly, probenecid significantly inhibited statin uptake by monkey hepatocytes (Fig. 4A), which may explain ∼2- to threefold change in plasma exposure in our monkey studies (Tables 1 and 2). However, the effect of probenecid on uptake is nominal and not statistically significant in human hepatocytes (Fig. 4B). Previous studies have shown in vitro OATP1B1 inhibition by probenecid at clinically relevant concentrations (Izumi et al., 2016). Clinical data on the inhibitory effect of probenecid on hepatic uptake are not directly available. However, we believe that ∼50%–70% increase in fexofenadine plasma AUC by probenecid is at least partly due to hepatic uptake inhibition (Liu et al., 2008). Fexofenadine bioavailability is ∼35% (Lappin et al., 2010), and therefore, inhibition of OAT3-mediated renal clearance resulting in a change in the amount of dose excreted in urine from ∼8% to 4% alone cannot explain the noted AUC change (Liu et al., 2008). In contrast, sulfasalazine uptake in monkey and human hepatocytes was inhibited by rifampicin in vitro; therefore, contribution of uptake clearance to sulfasalazine AUC change when dosed with rifampicin cannot be ruled out. Further studies are warranted in understanding the role of hepatic uptake in the disposition of sulfasalazine, which is currently recommended as a clinical probe to assess BCRP inhibition.
In our previous study, the AUC ratios of both i.v. and oral pitavastatin with increasing doses of oral rifampicin (1–30 mg/kg) in cynomolgus monkey were well predicted from in vitro IC50 data, assuming inhibition of only OATP1B-mediated hepatic uptake (Ufuk et al., 2018). However, in case of rosuvastatin, AUC ratios were well predicted following i.v. administration of rosuvastatin, but underpredicted following oral dose, particularly at higher doses of rifampicin. The current study suggests that the disconnect in the in vitro–in vivo extrapolation of rosuvastatin–rifampicin DDIs is due to lack of consideration to BCRP-mediated intestinal efflux. Rifampicin increased plasma exposure of BCRP (sulfasalazine) and P-gp (talinolol) substrates, implying that rifampicin inhibited intestinal efflux in cynomolgus monkey. Rifampicin inhibited cynoBCRP and cynoP-gp in our in vitro studies using membrane vesicles (Fig. 6). Simple IGut/IC50 calculations suggested that rifampicin considerably inhibits intestinal efflux at the dose employed (Table 5). A comparison of these findings with IC50 for human BCRP and P-gp and the corresponding IGut/IC50 values indicates that rifampicin can impact clinical pharmacokinetics of BCRP and P-gp substrates at the dose (600 mg single dose) recommended to probe OATP1B activity. Similarly, Imax,u/IC50 values of ∼0.14–0.76 imply potential for rifampicin to inhibit P-gp– and BCRP-mediated biliary secretion in both species. These values are likely an underestimation given rifampicin shows high liver-to-plasma–free concentrations in vivo (Zaher et al., 2008). However, contribution of this mechanism to the observed plasma exposure changes of probe substrates depends on multiple components of extended clearance model (Varma et al., 2014). This in vitro–in vivo analysis collectively supports the premise that rifampicin clinical DDIs should be rationalized by accounting for both OATP1B-mediated hepatic uptake and biliary efflux, as well as intestinal efflux of victim drugs.
In agreement with others (Tahara et al., 2006; Shen et al., 2013, 2016; Karibe et al., 2015), the results of the present study support the utility of the cynomolgus monkey as a model species for assessing transporter-mediated DDIs involving OATPs, P-gp, BCRP, and OAT1/3. In the case of OATPs and OAT1/3, efforts have been made to identify, characterize, and validate suitable biomarkers to support clinical DDI studies (Lai et al., 2016; Rodrigues et al., 2018). Therefore, it may be possible to use the cynomolgus monkey, integrated with clinically validated biomarkers, to quickly discharge DDI risk or prioritize dedicated (drug probe–based) clinical DDI studies. To date, however, suitable biomarkers for intestinal P-gp and BCRP have not been identified, and DDI predictions as well as in vitro–in vivo extrapolations involving both transporters remain challenging. In this regard, the cynomolgus monkey has potential to serve as a model to drive in vitro–in vivo extrapolations involving P-gp and BCRP inhibition and support human DDI risk assessment. Obviously, one would have to consider, and account for, species differences in perpetrator absorption, exposure, and intrinsic inhibitory potency. As described in this work, the latter can be addressed with in vitro studies using human and monkey vesicles and primary hepatocytes.
Although DDIs involving hepatic OCT1 and renal OCT2/MATEs are also suggested to be clinically relevant, the change in pharmacokinetics of substrate drugs is often marginal (AUC ratio <1.5) (Varma et al., 2017a). Arguably, preclinical DDI risk assessments in such cases do not definitively inform the decision to progress NMEs to clinical development. Therefore, the proposed cocktail did not include OCT1/OCT2/MATE probe substrates. Nevertheless, a separate DDI study in cynomolgus monkey using metformin as a probe substrate could be conducted (Shen et al., 2016). Alternatively, changes in creatinine levels measured in preclinical toxicology studies potentially indicate inhibition of OCT2 and/or MATEs (Fuchs and Hewitt, 2011). Such findings can form the basis for monitoring endogenous biomarkers such as creatinine and N1-methylnicotinamide in first-in-man studies, which in turn can determine the need for a dedicated clinical DDI study with a drug probe (Rodrigues et al., 2018).
Conclusion
In conclusion, the proposed approach for simultaneously assessing major transporter-mediated DDIs using a probe substrate cocktail in cynomolgus monkey can be valuable in drug design and clinical development. Such preclinical in vivo data in conjunction with in vitro interaction assessments can help flag the unforeseen interaction risks or avoid unnecessary clinical DDI studies. Finally, this study confirms similarities in drug handling by major transporters between human and cynomolgus monkey, suggesting the latter is a viable in vivo model for mechanistic investigation of transporter-mediated disposition and pharmacokinetic interaction.
Acknowledgments
We especially thank Solvo Biotechnology (Budapest, Hungary) for providing cynomolgus monkey BCRP and P-gp membrane vesicles. We acknowledge Marko Andric, Emese Kis, and Beáta Tóth (Solvo Biotechnology) for the technical inputs regarding the membrane vesicle assays.
Authorship Contributions
Participated in research design: Kosa, Lazzaro, Rodrigues, Tremaine, Varma.
Conducted experiments: Kosa, Lazzaro, Bi, Tierney, Gates, Modi.
Performed data analysis: Kosa, Lazzaro, Bi, Tierney, Gates, Modi, Costales, Varma.
Wrote or contributed to the writing of the manuscript: Kosa, Lazzaro, Bi, Tierney, Gates, Modi, Costales, Rodrigues, Tremaine, Varma.
Footnotes
- Received April 1, 2018.
- Accepted May 30, 2018.
All authors are full-time employees of Pfizer. The authors have no conflicts of interest that are directly relevant to this study.
↵This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- ABC
- ATP-binding cassette
- AUC
- area under the plasma concentration–time curve
- BCRP
- breast cancer resistance protein
- Cmax
- maximum plasma concentration
- DDI
- drug–drug interaction
- ECCS
- extended clearance classification system
- HBSS
- Hanks’ balanced salt solution
- IGut
- gut concentration
- IS
- internal standard
- LC-MS/MS
- liquid chromatography–tandem mass spectrometry
- MATE
- multidrug and toxin extrusion protein
- NME
- new molecular entity
- OAT
- organic anion transporter
- OATP
- organic anion-transporting polypeptide
- OCT
- organic cation transporter
- P-gp
- P-glycoprotein
- t1/2
- half-life
- Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics