Development and implementation of urinary transporter biomarkers to facilitate assessment of drug-drug interaction.

Aim: Novel urinary biomarker evaluation approaches to support inhibition assessment for renal transporters (e.g., OCT2, multidrug and toxin extrusion proteins [MATEs]). Methods: Highly sensitive and robust hydrophilic interaction chromatography-MS/high-resolution MS assays, for urine and plasma, were developed and characterized to evaluate transporter biomarkers including N1-methyladenosine and N1-methylnicotinamide. Results: The assays were simple and reliable with good selectivity and sensitivity, and successfully supported a clinical drug-drug interaction study with a drug candidate that presented in vitro inhibition of OCT2 and MATEs. Conclusion: The multiplexed assays enable a performance comparison, including biomarker specificity and sensitivity, that should increase the confidence in early clinical OCT2/MATEs drug-drug interaction risk assessment.

Effect of a Ketohexokinase Inhibitor (PF-06835919) on In Vivo OATP1B Activity: Integrative Risk Assessment Using Endogenous Biomarker and a Probe Drug.

PF-06835919 is a first-in-class ketohexokinase inhibitor (KHKi), recently under development for the treatment of metabolic and fatty liver diseases, which inhibited organic anion transporting polypeptide (OATP)1B1 in vitro and presented drug-drug interaction (DDI) risk. This study aims to investigate the dose-dependent effect of KHKi on OATP1B in vivo activity. We performed an open-label study comparing pharmacokinetics of atorvastatin (OATP1B probe) dosed alone (20 mg single dose) and coadministered with two dose strengths of KHKi (50 and 280 mg once daily) in 12 healthy participants. Additionally, changes in exposure of coproporphyrin-I (CP-I), an endogenous biomarker for OATP1B, were assessed in the atorvastatin study (1.12-fold and 1.49-fold increase in area under the plasma concentration-time profile (AUC) with once-daily 50 and 280 mg, respectively), and a separate single oral dose study of KHKi alone (100-600 mg, n = 6 healthy participants; up to a 1.80-fold increase in AUC). Geometric mean ratios (90% confidence interval) of atorvastatin AUC following 50 and 280 mg KHKi were 1.14 (1.00-1.30) and 1.54 (1.37-1.74), respectively. Physiologically-based pharmacokinetic modeling of CP-I plasma exposure following a single dose of KHKi predicted in vivo OATP1B inhibition from about 13% to 70% over the 100 to 600 mg dose range, while using the in vitro inhibition potency (1.9 µM). Model-based analysis correctly predicted "no-effect" (AUC ratio < 1.25) at the low dose range and "weak" effect (AUC ratio < 2) on atorvastatin pharmacokinetics at the high dose range of KHKi. This study exemplified the utility of biomarker-informed model-based approach in discerning even small effects on OATP1B activity in vivo, and to project DDI risk at the clinically relevant doses.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K. In addition to the previously reported endogenous biomarker candidates (creatinine and N1 -methylnicotinamide (1-NMN)), N1 -methyladenosine (m1 A) was included as novel biomarkers. 1-NMN and m1 A presented as superior MATE1/2-K biomarkers since changes in their renal clearance (CLr ) along with pyrimethamine dose were well-correlated with metformin CLr changes. The CLr of creatinine was reduced by pyrimethamine, however, its changes poorly correlated with metformin CLr changes. Nonlinear regression analysis (CLr vs. mean total concentration of pyrimethamine in plasma) yielded an estimate of the inhibition constant (Ki ) of pyrimethamine and the fraction of the clearance pathway sensitive to pyrimethamine. The in vivo Ki value thus obtained was further converted to unbound Ki using plasma unbound fraction of pyrimethamine, which was comparable to the in vitro Ki for MATE1 (1-NMN) and MATE2-K (1-NMN and m1 A). It is concluded that 1-NMN and m1 A CLr can be leveraged as quantitative MATE1/2-K biomarkers for DDI risk assessment in healthy volunteers.

A Multiplexed HILIC-MS/HRMS Assay for the Assessment of Transporter Inhibition Biomarkers in Phase I Clinical Trials: Isobutyryl-Carnitine as an Organic Cation Transporter (OCT1) Biomarker.

There is a growing interest in using endogenous compounds as drug transporter biomarkers to facilitate drug-drug interaction (DDI) risk assessment in early phase I clinical trials. Compared to other drug transporters, however, no valid biomarker for hepatic organic cation transporter (OCT) 1 has been described to date. The present work represents the first report of an endogenous compound, isobutyryl-l-carnitine (IBC), as a potential clinical OCT1 biomarker for DDI assessment. A hydrophilic interaction chromatography (HILIC)-mass spectrometry/high resolution mass spectrometry (MS/HRMS) assay with a simple sample preparation method was developed. The assay is capable of simultaneously quantifying multiple endogenous compounds, including IBC, thiamine, N1-methylnicotinamide (1-NMN), creatinine, carnitine, and metformin, which is a probe for OCT1 and OCT2 and MATE1 and MATE2K (multidrug and toxin extrusion proteins) in clinical studies. The HRMS assay was fit-for-purpose validated in human plasma and demonstrated good linearity, accuracy, and precision for all analytes. It was further applied to two phase I clinical trials to evaluate potential biomarkers for OCT1 and additional cation transporters (renal OCT2, MATE1, and MATE2K). The clinical data demonstrated that plasma IBC changes correlated well with in vitro data and supported its use as a liver OCT1 biomarker. The described HILIC-MS/HRMS assay can be used as a "biomarker cocktail" to simultaneously assess clinical DDI risk for the inhibition of OCT1/2 and MATEs in clinical studies with new drug candidates.

Simultaneous Assessment of Transporter-Mediated Drug-Drug Interactions Using a Probe Drug Cocktail in Cynomolgus Monkey.

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.

Validation of 4ß-hydroxycholesterol and evaluation of other endogenous biomarkers for the assessment of CYP3A activity in healthy subjects.

AIMS: This study aimed to assess changes in the plasma concentrationss of 4ß-hydroxycholesterol (4ßHC) against intravenous (i.v.) and oral midazolam (MDZ) pharmacokinetics (PK) after administration of a potent CYP3A inhibitor [ketoconazole (KETO)] and inducer [rifampicin (RIF)]. METHODS: Thirty-two healthy subjects (HS) were allocated into three groups of 12 each in KETO and RIF and 10 in a placebo group (PLB). All HS were randomized to receive oral and i.v. MDZ on day 1 or 2 and on day 15 or 16 after receiving RIF (600 mg once daily), KETO (400 mg once daily) or PLB for 2 weeks. Subjects were followed until day 30. The effect of treatments on 4ßHC was assessed by analyzing % change from baseline using a linear spline mixed effects model. RESULTS: Compared with PLB, KETO decreased 4ßHC mean values up to 13% (P = 0.003) and RIF increased 4ßHC mean values up to 220% (P < 0.001). Within 14 days of stopping KETO and RIF, 4ßHC had either returned to baseline (KETO) or was still returning to baseline (RIF). Compared with baseline, mean oral MDZ AUC increased by 11-fold (90% CI ranging from 9-fold to 13-fold increase) and decreased by 92% (90% CI ranging from 90% to 95% decrease) after KETO and RIF, respectively. Similar trends were observed for 6ß-hydroxycortisol : cortisol (6ßHCL : CL) urinary ratios. CONCLUSIONS: Changes in plasma 4ßHC can be utilized as a surrogate for MDZ PK after multiple doses of potent CYP3A inducers. There is a more limited dynamic range for 4ßHC for assessment of potential CYP3A inhibitors. 4ßHC is a valuable tool for the assessment of potential CYP3A inducers in early drug development.

Assessment of vandetanib as an inhibitor of various human renal transporters: inhibition of multidrug and toxin extrusion as a possible mechanism leading to decreased cisplatin and creatinine clearance.

Vandetanib was evaluated as an inhibitor of human organic anion transporter 1 (OAT1), OAT3, organic cation transporter 2 (OCT2), and multidrug and toxin extrusion (MATE1 and MATE2K) transfected (individually) into human embryonic kidney 293 cells (HEK293). Although no inhibition of OAT1 and OAT3 was observed, inhibition of OCT2-mediated uptake of 1-methyl-4-phenylpyridinium (MPP(+)) and metformin was evident (IC(50) of 73.4 ± 14.8 and 8.8 ± 1.9 µM, respectively). However, vandetanib was an even more potent inhibitor of MATE1- and MATE2K-mediated uptake of MPP(+) (IC(50) of 1.23 ± 0.05 and 1.26 ± 0.06 µM, respectively) and metformin (IC(50) of 0.16 ± 0.05 and 0.30 ± 0.09 µM, respectively). Subsequent cytotoxicity studies demonstrated that transport inhibition by vandetanib (2.5 µM) significantly decreased the sensitivity [right shift in concentration of cisplatin giving rise to 50% cell death; IC(50(CN))] of MATE1-HEK and MATE2K-HEK cells to cisplatin [IC(50(CN)) of 1.12 ± 0.13 versus 2.39 ± 0.44 µM; 0.85 ± 0.09 versus 1.99 ± 0.16 µM; P < 0.05), but not OCT2-HEK cells (1.36 ± 0.19 versus 1.47 ± 0.24 µM) versus vandetanib untreated cells and Mock-HEK cells [IC(50(CN)) of 2.34 ± 0.31 µM]. In summary, the results show that vandetanib is a potent inhibitor of MATE1 and MATE2K (versus OCT2). Inhibition of the two transporters may explain why there are reports of decreased creatinine clearance, and increased cisplatin nephrotoxicity (reduced cisplatin clearance), in some subjects receiving vandetanib therapy.

Development of novel, 384-well high-throughput assay panels for human drug transporters: drug interaction and safety assessment in support of discovery research.

Transporter proteins are known to play a critical role in affecting the overall absorption, distribution, metabolism, and excretion characteristics of drug candidates. In addition to efflux transporters (P-gp, BCRP, MRP2, etc.) that limit absorption, there has been a renewed interest in influx transporters at the renal (OATs, OCTs) and hepatic (OATPs, BSEP, NTCP, etc.) organ level that can cause significant clinical drug-drug interactions (DDIs). Several of these transporters are also critical for hepatobiliary disposition of bilirubin and bile acid/salts, and their inhibition is directly implicated in hepatic toxicities. Regulatory agencies took action to address transporter-mediated DDI with the goal of ensuring drug safety in the clinic and on the market. To meet regulatory requirements, advanced bioassay technology and automation solutions were implemented for high-throughput transporter screening to provide structure-activity relationship within lead optimization. To enhance capacity, several functional assay formats were miniaturized to 384-well throughput including novel fluorescence-based uptake and efflux inhibition assays using high-content image analysis as well as cell-based radioactive uptake and vesicle-based efflux inhibition assays. This high-throughput capability enabled a paradigm shift from studying transporter-related issues in the development space to identifying and dialing out these concerns early on in discovery for enhanced mechanism-based efficacy while circumventing DDIs and transporter toxicities.

Quantification of 4-beta-hydroxycholesterol in human plasma using automated sample preparation and LC-ESI-MS/MS analysis.

It has recently been proposed that plasma levels of 4ß-hydroxycholesterol (4ßHC) may be indicative of cytochrome P450 3A4 (P450 3A) activity and therefore could be used to probe for P450 3A-mediated drug-drug interactions. With this in mind, we describe a highly sensitive and precise liquid chromatography-electrospray ionization-tandem mass spectrometry method for the measurement of 4ßHC in human plasma with a lower limit of quantification established at 2 ng/mL using 50 µL of plasma. The entire sample preparation scheme including saponification and derivatization of 4ßHC to the corresponding dipicolinyl ester (DPE) was completed in less than 8 h using an automated sample preparation scheme enabling higher-throughput capabilities. Chromatographic resolution of 4ßHC from 4α-hydroxycholesterol and other endogenous isobaric species was achieved in 11-min using an isocratic gradient on a C18 column. Because of endogenous concentrations of 4ßHC in plasma, a stable isotope labeled (SIL) analogue, d7-4ßHC, was used as a surrogate analyte and measured in the standard curve and quality control samples prepared in plasma. A second SIL analogue, d4-4ßHC, was used as the internal standard. The intraday and interday accuracy for the assay was within 6% of nominal concentrations, and the precision for these measurements was less than 5% relative standard deviation. Rigorous stability assessments demonstrated adequate stability of endogenous 4ßHC in plasma and the corresponding DPE derivative for the analysis of clinical study samples. The results from clinical samples following treatment with a potent P450 3A inducer (rifampin) or inhibitor (ketoconazole) are reported and demonstrate the potential future application for this highly precise and robust analytical assay.

Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human liver drug transporters.

17alpha-Ethinylestradiol (EE2), a component of oral contraceptives, is known to undergo considerable first-pass 3-O-sulfation in the intestine and liver. Once formed, the 3-O-sulfate conjugate (EE2-Sul) is detected in circulation at appreciable levels (versus parent EE2) and is present in bile. Therefore, hepatic uptake of EE2-Sul was assessed with suspensions of cryopreserved human primary hepatocytes. In this instance, there was evidence for active (temperature-dependent) uptake, which was described by a two-K(m) (Michaelis constant) model (K(m1) = 220 nM; K(m2) = 15.5 microM). Uptake was inhibited (approximately 90%) by bromosulfophthalein but not by tetraethylammonium or p-aminohippurate. In agreement, EE2-Sul was shown to be a substrate of recombinant organic anion transporter peptides (OATP1B1 and OATP2B1), and Na(+)/taurocholate-cotransporting polypeptide (NTCP), expressed individually in human embryonic kidney (HEK) 293 cells. Transport by OATP1B1 was described by two K(m) values (87 nM and 141 microM), whereas OATP2B1- and NTCP-mediated uptake into HEK-293 cells conformed to single K(m) kinetics (10.7 and 2.6 microM, respectively). EE2-Sul was also assessed as an efflux transporter substrate using membrane vesicles expressing bile salt export pump, breast cancer resistance protein (BCRP), and individual forms of multidrug resistance-associated protein (MRP1, MRP2, and MRP3). Transport studies were also conducted with a cell line expression P-glycoprotein. Only vesicles that contained BCRP exhibited ATP-dependent uptake of EE2-Sul (K(m1) = 2.9 and K(m2) = 307 microM). Collectively, the data show that hepatic uptake of EE2-Sul can be mediated by three transporters (OATP1B1, OATP2B1, and NTCP), whereas biliary excretion of EE2-Sul into bile likely involves BCRP.

Transporter studies with the 3-O-sulfate conjugate of 17alpha-ethinylestradiol: assessment of human kidney drug transporters.

17alpha-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-K(m) (Michaelis constant) kinetics (K(m) = 21.1 microM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-K(m) model (K(m1) = 1.6 microM; K(m2) = 195 microM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.

Further assessment of 17alpha-ethinyl estradiol as an inhibitor of different human cytochrome P450 forms in vitro.

17alpha-Ethinyl estradiol (EE) was systematically evaluated as a reversible and time-dependent inhibitor of 11 human drug-metabolizing cytochromes P450 (P450s) (CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, and CYP3A5) in vitro. When ranked, the lowest IC(50) (concentration of inhibitor required to decrease activity by 50%) values were obtained with recombinant CYP1A1 (rCYP1A1) [IC(50(total)) = IC(50(free)) = 2.7 microM] and CYP2C19 activity in human liver microsomes (HLM) [IC(50(total)) = 4.4 microM; IC(50(free)) = 2.8 microM]. For rCYP1A1, formal inhibition studies revealed that EE was a competitive inhibitor [K(i(free)) = 1.4 microM]. All the other IC(50) values were greater than 8.0 microM, and the weakest inhibition was observed with CYP1A2 activity in HLM (IC(50(free)) > 39 microM). In agreement, the IC(50) characterizing the inhibition of melatonin (MEL) 6-hydroxylation in human intestine microsomes (CYP1A1-catalyzed) was lower than that of HLM (0.91 versus >40 microM). Because EE is known to affect the pharmacokinetics of CYP2C19 probe drugs, this result raises the possibility that the concentration of EE during first pass may exceed 1000 nM, sufficient to affect CYP1A1 and CYP2C19, with less impact on CYP3A4 and other P450s. The results implicate intestinal CYP1A1, and possibly CYP2C19, as the loci of EE drug interactions with highly extracted drugs like MEL. Overall, it is very difficult to rationalize drug interactions involving EE based on direct inhibition of CYP2B6 (e.g., selegiline) and hepatic CYP1A2 (e.g., MEL, tizanidine, caffeine, and theophylline).

Cytochrome P450 reaction-phenotyping: an industrial perspective.

It is now widely accepted that the fraction of the dose metabolized by a given drug-metabolizing enzyme is one of the major factors governing the magnitude of a drug interaction and the impact of a polymorphism on (total) drug clearance. Therefore, most pharmaceutical companies determine the enzymes involved in the metabolism of a new chemical entity (NCE) in vitro, in conjunction with human data on absorption, distribution, metabolism and excretion. This so called reaction-phenotyping, or isozyme-mapping, usually involves the use of multiple reagents (e.g., recombinant proteins, liver subcellular fractions, enzyme-selective chemical inhibitors and antibodies). For the human CYPs, reagents are readily available and in vitro reaction-phenotyping data are now routinely included in most regulatory documents. Ideally, the various metabolites have been definitively identified, incubation conditions have afforded robust kinetic analyses, and well characterized (high quality) reagents and human tissues have been employed. It is also important that the various in vitro data are consistent (e.g., scaled turnover with recombinant CYP proteins, CYP inhibition and correlation data with human liver microsomes) and enable an integrated in vitro CYP reaction-phenotype. Results of the in vitro CYP reaction-phenotyping are integrated with clinical data (e.g., human radiolabel and drug interaction studies) and a complete package is then submitted for regulatory review. If the NCE receives market approval, information on key routes of clearance and their associated potential for drug-drug interactions are included in the product label. The present review focuses on in vitro CYP reaction-phenotyping and the integration of data. Relatively simple strategies enabling the design and prioritization of follow up clinical studies are also discussed.

Prioritization of clinical drug interaction studies using in vitro cytochrome P450 data: proposed refinement and expansion of the "rank order" approach.

For any new chemical entity (NCE), in vitro inhibition constants (K(i)) for the different human cytochrome P450 (CYP) forms can be ranked (lowest to highest). The CYP form with the lowest in vitro K(i) is evaluated first clinically, employing a suitable probe drug like midazolam (CYP3A4), theophylline (CYP1A2), (S)-warfarin (CYP2C9) and desipramine (CYP2D6), and the NCE is classified as a "none", "weak", "moderate", or "strong" inhibitor. In turn, the classification governs the next steps. A two stage strategy, in vitro ranking followed by classification, has the potential to enable decision making within an industrial and regulatory setting. With additional refinement and validation, the approach could be applied to mechanism-based inhibitors, inducers and substrates of CYPs also.