Survey of Pharmaceutical Industry's Best Practices around In Vitro Transporter Assessment and Implications for Drug Development: Considerations from the International Consortium for Innovation and Quality for Pharmaceutical Development Transporter Working Group.

The International Consortium for Innovation and Quality in Pharmaceutical Development Transporter Working Group had a rare opportunity to analyze a crosspharma collation of in vitro data and assay methods for the evaluation of drug transporter substrate and inhibitor potential. Experiments were generally performed in accordance with regulatory guidelines. Discrepancies, such as not considering the impact of preincubation for inhibition and free or measured in vitro drug concentrations, may be due to the retrospective nature of the dataset and analysis. Lipophilicity was a frequent indicator of crosstransport inhibition (P-gp, BCRP, OATP1B, and OCT1), with high molecular weight (MW ≥500 Da) also common for OATP1B and BCRP inhibitors. A high level of overlap in in vitro inhibition across transporters was identified for BCRP, OATP1B1, and MATE1, suggesting that prediction of DDIs for these transporters will be common. In contrast, inhibition of OAT1 did not coincide with inhibition of any other transporter. Neutrals, bases, and compounds with intermediate-high lipophilicity tended to be P-gp and/or BCRP substrates, whereas compounds with MW <500 Da tended to be OAT3 substrates. Interestingly, the majority of in vitro inhibitors were not reported to be followed up with a clinical study by the submitting company, whereas those compounds identified as substrates generally were. Approaches to metabolite testing were generally found to be similar to parent testing, with metabolites generally being equally or less potent than parent compounds. However, examples where metabolites inhibited transporters in vitro were identified, supporting the regulatory requirement for in vitro testing of metabolites to enable integrated clinical DDI risk assessment. SIGNIFICANCE STATEMENT: A diverse dataset showed that transporter inhibition often correlated with lipophilicity and molecular weight (>500 Da). Overlapping transporter inhibition was identified, particularly that inhibition of BCRP, OATP1B1, and MATE1 was frequent if the compound inhibited other transporters. In contrast, inhibition of OAT1 did not correlate with the other drug transporters tested.

Clinical Relevance of Hepatic and Renal P-gp/BCRP Inhibition of Drugs: An International Transporter Consortium Perspective.

The role of P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) in drug-drug interactions (DDIs) and limiting drug absorption as well as restricting the brain penetration of drugs with certain physicochemical properties is well known. P-gp/BCRP inhibition by drugs in the gut has been reported to increase the systemic exposure to substrate drugs. A previous International Transporter Consortium (ITC) perspective discussed the feasibility of P-gp/BCRP inhibition at the blood-brain barrier and its implications. This ITC perspective elaborates and discusses specifically the hepatic and renal P-gp/BCRP (referred as systemic) inhibition of drugs and whether there is any consequence for substrate drug disposition. This perspective summarizes the clinical evidence-based recommendations regarding systemic P-gp and BCRP inhibition of drugs with a focus on biliary and active renal excretion pathways. Approaches to assess the clinical relevance of systemic P-gp and BCRP inhibition in the liver and kidneys included (i) curation of DDIs involving intravenously administered substrates or inhibitors; (ii) in vitro-to-in vivo extrapolation of P-gp-mediated DDIs at the systemic level; and (iii) curation of drugs with information available about the contribution of biliary excretion and related DDIs. Based on the totality of evidence reported to date, this perspective supports limited clinical DDI risk upon P-gp or BCRP inhibition in the liver or kidneys.

Predicting Oral Absorption for Compounds Outside the Rule of Five Property Space.

The estimation of the extent of absorption of drug candidates intended for oral drug delivery is an important selection criteria in drug discovery. The use of cell-based transwell assays examining flux across cell-monolayers (e.g., Caco-2 or MDCK cells) usually provide satisfactory predictions of the extent of absorption in vivo. These predictions often fall short of expection for molecules outside the traditional low molecular weight property space. In this manuscript the transwell permeability assay was modified to circumvent potential issues that can be encountered when evaluating the aforementioned drug molecules. Particularly, the addition of albumin in the acceptor compartment to reduce potential binding to cells and the acceptor compartment, improved the predictive power of the assay. Cellular binding and lysosomal trapping effects are significantly reduced for larger molecules, particularly lipophilic bases under these more physiological conditions, resulting in higher recovery values and a better prediction power. The data indicate that lysosomal trapping does not impact the rate of absorption of lipophilic bases in general but is rather an exception. Finally, compounds believed to permeate by passive mechanisms were used in a calibration curve for the effective prediction of the fraction absorbed of molecules of interest in current medicinal chemistry efforts.

Genetic Diversity in Drug Transporters: Impact in African Populations.

Polymorphisms in drug transporters, like the adenosine triposphate-binding cassette (ABC) and solute carrier (SLC) superfamilies, may contribute to the observed diversity in drug response in African patients. This review aims to provide a comprehensive summary and analysis of the frequencies and distributions in African populations of ABC and SLC variants that affect drug pharmacokinetics (PK) and pharmacodynamics (PD). Of polymorphisms evaluated in African populations, SLCO1B1 rs4149056 and SLC22A6 rs1158626 were found at markedly higher frequencies than in non-African populations. SLCO1B1 rs4149056 was associated with reduction in rifampin exposure, which has implications for dosing this important anti-tuberculosis therapy. SLC22A6 rs1158626 was associated with increased affinity for antiretroviral drugs. Genetic diversity in SLC and ABC transporters in African populations has implications for conventional therapies, notably in tuberculosis and HIV. More PK and PD data in African populations are needed to assess potential for a different response to drugs compared with other global populations.

Disposition of asciminib, a potent BCR-ABL1 tyrosine kinase inhibitor, in healthy male subjects.

Asciminib is a potent, specific BCR-ABL1 inhibitor being developed for the treatment of patients with chronic myelogenous leukemia (CML) and Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL).Here, we present the results of human oral absorption, distribution, metabolism, excretion (ADME) and in vitro studies that together provide an overall understanding of the metabolism, distribution and clearance of asciminib in humans.Asciminib was rapidly absorbed with a maximum plasma concentration at two hours post-dose. Total radioactivity and asciminib showed similar terminal half-lives in plasma.Oral asciminib absorption ranged between a minimum of 33%, and a maximum of 57% based on the metabolite profiles of late time-point feces collections.Asciminib was eliminated mainly through feces via unchanged asciminib excretion and metabolism.Direct glucuronidation and oxidation were major metabolic pathways in human that were catalyzed predominantly by UDP-glucuronosyltransferase (UGT)2B7 and cytochrome P450 (CYP)3A4, respectively.The relative contribution of the glucuronidation pathway to the total clearance of asciminib via metabolism is estimated to range ∼28-58%, whereas the relative contribution of the oxidative pathway is estimated to range ∼37-64%, based upon the maximum oral absorption in humans.

Intestinal Transporter-Associated Drug Absorption and Toxicity.

Oral drug administration is the most favorable route of drug administration in the clinic. Intestinal transporters have been shown to play a significant role in the rate and extent of drug absorption of some, but not all, drug molecules. Due to the heterogeneous expression of multiple transporters along the intestine, the preferential absorption sites for drugs may vary significantly. In this chapter, we aim to summarize the current research on the expression, localization, function, and regulation of human intestinal transporters implicated in altering the absorption of low to medium molecular weight drug molecules. The role played by bile acid transport proteins (e.g., ASBT and OST-α/ß) is included in the discussion. The synergistic action of intestinal drug metabolism and transport is also discussed. Despite the complicated regulatory factors, the biopharmaceutics drug disposition classification system (BDDCS) put forward by Wu and Benet may help us better predict the effect of transporters on drug absorption. The drug-induced toxicity in the intestine, which may result from drug-drug interaction, gut microbiota, and bile salt toxicity, is also discussed.

A Systematic In Vitro Investigation of the Inhibitor Preincubation Effect on Multiple Classes of Clinically Relevant Transporters.

Preincubation of a drug transporter with its inhibitor in a cell-based assay may result in the apparent enhancement of the inhibitory potency. Currently, limited data are available on potentiation of transporter inhibition by preincubation (PTIP) for clinically relevant solute-carrier transporters other than OATP1B1 and OATP1B3. Therefore, PTIP was examined systematically using OATP1B1, OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, and MATE2-K cell lines. IC50 values of 30 inhibitors were determined with or without 3 hours of preincubation, and compounds with a PTIP ≥2.5× were further characterized by assessing the time course of transport inhibition potency and cellular concentration. For each compound, correlations were calculated between highest observed PTIP and physicochemical properties. PTIP was prevalent among organic cation transporters (OCTs) and organic anion-transporting polypeptides (OATPs) but not among organic anion transporters (OATs) or multidrug and toxin extrusion transporters (MATEs), and most instances of PTIP persisted after controlling for toxicity and nonspecific binding. Occasionally, preincubation in excess of 2 hours was required to attain full inhibitory potency. For four drugs examined, preincubation had the potential to change the in vitro drug-drug interaction risk prediction from "no risk" to "risk" on the basis of current regulatory criteria. Molecular weight and LogD7.4, as well as the ratio of passive cellular accumulation and cellular uptake rate correlated with PTIP; thus, low cellular permeation and a slow build-up of unbound intracellular inhibitor concentration may contribute to PTIP. Taken together, our data suggest that PTIP is partly determined by the physicochemical properties of the perpetrator drug, and preincubation may affect the in vitro predicted drug-drug interaction risk for OCTs as well as OATPs. SIGNIFICANCE STATEMENT: During the development of a novel pharmaceutical drug, in vitro studies are conducted to assess the risk of potential adverse interactions between existing medications a patient may already be taking and the novel compound. The exact way these in vitro assays are performed may influence the outcome of risk assessment. Here we suggest that the interaction risk may be underestimated unless specific assay protocols are modified to include an additional incubation step that allows the test drug to accumulate inside the cells, and demonstrate that adding this step is particularly important for large and hydrophobic drug molecules.

Clinical Probes and Endogenous Biomarkers as Substrates for Transporter Drug-Drug Interaction Evaluation: Perspectives From the International Transporter Consortium.

Drug transporters can govern the absorption, distribution, metabolism, and excretion of substrate drugs and endogenous substances. Investigations to examine their potential impact to pharmacokinetic (PK) drug-drug interactions (DDIs) are an integral part of the risk assessment in drug development. To evaluate a new molecular entity as a potential perpetrator of transporters, use of well characterized and/or clinically relevant probe substrates with good selectivity and sensitivity are critical for robust clinical DDI assessment that could inform DDI management strategy in the product labeling. The availability of endogenous biomarkers to monitor transporter-mediated DDIs in early phases of clinical investigations would greatly benefit downstream clinical plans. This article reviews the state-of-the-art in transporter clinical probe drugs and emerging biomarkers, including current challenges and limitations, delineates methods and workflows to identify and validate novel endogenous biomarkers to support clinical DDI evaluations, and proposes how these probe drugs or biomarkers could be used in drug development.

Transport properties of valsartan, sacubitril and its active metabolite (LBQ657) as determinants of disposition.

1. The potential for drug-drug interactions of LCZ696 (a novel, crystalline complex comprising sacubitril and valsartan) was investigated in vitro. 2. Sacubitril was shown to be a highly permeable P-glycoprotein (P-gp) substrate and was hydrolyzed to the active anionic metabolite LBQ657 by human carboxylesterase 1 (CES1b and 1c). The multidrug resistance-associated protein 2 (MRP2) was shown to be capable of LBQ657 and valsartan transport that contributes to the elimination of either compound. 3. LBQ657 and valsartan were transported by OAT1, OAT3, OATP1B1 and OATP1B3, whereas no OAT- or OATP-mediated sacubitril transport was observed. 4. The contribution of OATP1B3 to valsartan transport (73%) was appreciably higher than that by OATP1B1 (27%), Alternatively, OATP1B1 contribution to the hepatic uptake of LBQ657 (∼70%) was higher than that by OATP1B3 (∼30%). 5. None of the compounds inhibited OCT1/OCT2, MATE1/MATE2-K, P-gp, or BCRP. Sacubitril and LBQ657 inhibited OAT3 but not OAT1, and valsartan inhibited the activity of both OAT1 and OAT3. Sacubitril and valsartan inhibited OATP1B1 and OATP1B3, whereas LBQ657 weakly inhibited OATP1B1 but not OATP1B3. 6. Drug interactions due to the inhibition of transporters are unlikely due to the redundancy of the available transport pathways (LBQ657: OATP1B1/OAT1/3 and valsartan: OATP1B3/OAT1/3) and the low therapeutic concentration of the LCZ696 analytes.

Evaluation of Drug-Drug Interaction Potential Between Sacubitril/Valsartan (LCZ696) and Statins Using a Physiologically Based Pharmacokinetic Model.

Sacubitril/valsartan (LCZ696) has been approved for the treatment of heart failure. Sacubitril is an in vitro inhibitor of organic anion-transporting polypeptides (OATPs). In clinical studies, LCZ696 increased atorvastatin Cmax by 1.7-fold and area under the plasma concentration-time curve by 1.3-fold, but had little or no effect on simvastatin or simvastatin acid exposure. A physiologically based pharmacokinetics modeling approach was applied to explore the underlying mechanisms behind the statin-specific LCZ696 drug interaction observations. The model incorporated OATP-mediated clearance (CLint,T) for simvastatin and simvastatin acid to successfully describe the pharmacokinetic profiles of either analyte in the absence or presence of LCZ696. Moreover, the model successfully described the clinically observed drug effect with atorvastatin. The simulations clarified the critical parameters responsible for the observation of a low, yet clinically relevant, drug-drug interaction DDI between sacubitril and atorvastatin and the lack of effect with simvastatin acid. Atorvastatin is administered in its active form and rapidly achieves Cmax that coincide with the low Cmax of sacubitril. In contrast, simvastatin requires a hydrolysis step to the acid form and therefore is not present at the site of interactions at sacubitril concentrations that are inhibitory. Similar models were used to evaluate the drug-drug interaction risk for additional OATP-transported statins which predicted to maximally result in a 1.5-fold exposure increase.

Clinical disposition, metabolism and in vitro drug-drug interaction properties of omadacycline.

1. Absorption, distribution, metabolism, transport and elimination properties of omadacycline, an aminomethylcycline antibiotic, were investigated in vitro and in a study in healthy male subjects. 2. Omadacycline was metabolically stable in human liver microsomes and hepatocytes and did not inhibit or induce any of the nine cytochrome P450 or five transporters tested. Omadacycline was a substrate of P-glycoprotein, but not of the other transporters. 3. Omadacycline metabolic stability was confirmed in six healthy male subjects who received a single 300 mg oral dose of [14C]-omadacycline (36.6 µCi). Absorption was rapid with peak radioactivity (∼610 ngEq/mL) between 1-4 h in plasma or blood. The AUClast of plasma radioactivity (only quantifiable to 8 h due to low radioactivity) was 3096 ngEq h/mL and apparent terminal half-life was 11.1 h. Unchanged omadacycline reached peak plasma concentrations (∼563 ng/mL) between 1-4 h. Apparent plasma half-life was 17.6 h with biphasic elimination. Plasma exposure (AUCinf) averaged 9418 ng h/mL, with high clearance (CL/F, 32.8 L/h) and volume of distribution (Vz/F 828 L). No plasma metabolites were observed. 4. Radioactivity recovery of the administered dose in excreta was complete (>95%); renal and fecal elimination were 14.4% and 81.1%, respectively. No metabolites were observed in urine or feces, only the omadacycline C4-epimer.

Assessment of pharmacokinetic drug-drug interaction between pradigastat and atazanavir or probenecid.

Pradigastat, a novel diacylglycerol acyltransferase-1 inhibitor, has activity in common metabolic diseases associated with abnormal accumulation of triglycerides. In vitro studies suggest that glucuronidation is the predominant metabolism pathway for elimination of pradigastat in humans and confirmed the role of uridine 5'-diphosphoglucuronosyltransferase (UGT) enzymes, UGT1A1, -1A3, and -2B7. The in vitro studies using atazanavir as a selective inhibitor of UGT1A1 and -1A3 indicated that these enzymes contribute ∼55% toward the overall glucuronidation pathway. Therefore, a clinical study was conducted to assess the potential for drug interaction between pradigastat and probenecid (purported general UGT inhibitor) or atazanavir (selective UGT1A1, -1A3 inhibitor). The study included 2 parallel cohorts, each with 3 sequential treatment periods and 22 healthy subjects per cohort. The 90%CI of the geometric mean ratios for Cmax,ss and AUCτ,ss of pradigastat were within 0.80-1.25 when administered in combination with probenecid. However, the Cmax,ss and AUCτ,ss of pradigastat decreased by 31% (90%CI: 0.62-0.78) and 26% (0.67-0.82), respectively, when administered in combination with atazanavir. This magnitude of decrease in pradigastat steady-state exposure is not considered clinically relevant. Pradigastat was well tolerated by all subjects, either alone or in combination with atazanavir or probenecid.

Evaluation of a potential transporter-mediated drug interaction between rosuvastatin and pradigastat, a novel DGAT-1 inhibitor.

OBJECTIVE: An in vitro drugdrug interaction (DDI) study was performed to assess the potential for pradigastat to inhibit breast cancer resistance protein (BCRP), organic anion-transporting polypeptide (OATP), and organic anion transporter 3 (OAT3) transport activities. To understand the relevance of these in vitro findings, a clinical pharmacokinetic DDI study using rosuvastatin as a BCRP, OATP, and OAT3 probe substrate was conducted. METHODS: The study used cell lines that stably expressed or over-expressed the respective transporters. The clinical study was an open-label, single sequence study where subjects (n = 36) received pradigastat (100 mg once daily x 3 days thereafter 40 mg once daily) and rosuvastatin (10 mg once daily), alone and in combination. RESULTS: Pradigastat inhibited BCRP-mediated efflux activity in a dose-dependent fashion in a BCRP over-expressing human ovarian cancer cell line with an IC(50) value of 5 µM. Similarly, pradigastat inhibited OATP1B1, OATP1B3 (estradiol 17ß glucuronide transport), and OAT3 (estrone 3 sulfate transport) activity in a concentrationdependent manner with estimated IC(50) values of 1.66 ± 0.95 µM, 3.34 ± 0.64 µM, and 0.973 ± 0.11 µM, respectively. In the presence of steady state pradigastat concentrations, AUC(τ, ss) of rosuvastatin was unchanged and its Cmax,ss decreased by 14% (5.30 and 4.61 ng/mL when administered alone and coadministered with pradigastat, respectively). Pradigastat AUC(τ, ss) and C(max, ss) were unchanged when coadministered with rosuvastatin at steady state. Both rosuvastatin and pradigastat were well tolerated. CONCLUSION: These data indicate no clinically relevant pharmacokinetic interaction between pradigastat and rosuvastatin.

Potent nonimmunosuppressive cyclophilin inhibitors with improved pharmaceutical properties and decreased transporter inhibition.

Nonimmunosuppressive cyclophilin inhibitors have demonstrated efficacy for the treatment of hepatitis C infection (HCV). However, alisporivir, cyclosporin A, and most other cyclosporins are potent inhibitors of OATP1B1, MRP2, MDR1, and other important drug transporters. Reduction of the side chain hydrophobicity of the P4 residue preserves cyclophilin binding and antiviral potency while decreasing transporter inhibition. Representative inhibitor 33 (NIM258) is a less potent transporter inhibitor relative to previously described cyclosporins, retains anti-HCV activity in cell culture, and has an acceptable pharmacokinetic profile in rats and dogs. An X-ray structure of 33 bound to rat cyclophilin D is reported.

Physiologically based pharmacokinetic modeling for assessing the clinical drug-drug interaction of alisporivir.

Alisporivir is a novel cyclophilin-binding molecule with potent anti-hepatitis C virus (HCV) activity. In vitro data from human liver microsomes suggest that alisporivir is a substrate and a time-dependent inhibitor (TDI) of CYP3A4. The aim of the current work was to develop a novel physiologically based pharmacokinetic (PBPK) model to quantitatively assess the magnitude of CYP3A4 mediated drug-drug interactions with alisporivir as the substrate or victim drug. Towards that, a Simcyp PBPK model was developed by integrating in vitro data with in vivo clinical findings to characterize the clinical pharmacokinetics of alisporivir and further assess the magnitude of drug-drug interactions. Incorporated with absorption, distribution, elimination, and TDI data, the model accurately predicted AUC, Cmax, and tmax values after single or multiple doses of alisporivir with a prediction deviation within ± 32%. The model predicted an alisporivir AUC increase by 9.4-fold and a decrease by 86% when alisporivir was co-administrated with ketoconazole (CYP3A4 inhibitor) or rifampin (CYP3A4 inducer), respectively. Predictions were within ± 20% of the observed changes. In conclusion, the PBPK model successfully predicted the alisporivir PK and the magnitude of drug-drug interactions.

A plasma concentration of α-ketoglutarate influences the kinetic interaction of ligands with organic anion transporter 1.

The purpose of the present study was to determine whether a physiologic plasma concentration of α-ketoglutarate (αKG) influences the kinetic interaction of ligands with organic anion transporter 1 (OAT1). The effect of extracellular αKG on the kinetics of para-aminohippurate (PAH) and cidofovir transport was examined along with its effect on the potency of 10 drugs in five different classes (uricosuric, nonsteroidal anti-inflammatories, loop diuretics, angiotensin II receptor antagonists, and ß-lactam antibiotics) to inhibit OAT1 expressed in Chinese hamster ovary cells. Extracellular αKG competitively inhibited PAH and cidofovir transport with Ki values (∼5 µM) approximating its unbound plasma concentration (determined by equilibrium dialysis). When PAH was the substrate, extracellular αKG (5 µM) significantly increased IC50 values for some inhibitors (up to 4-fold), such as probenecid, but not for others (an inhibitor-dependent effect). For some inhibitors, a significant increase in IC50 value was observed when cidofovir was the substrate, but not PAH (a substrate-dependent effect). A significant increase in IC50 value was also observed for inhibition of PAH transport by probenecid in renal basolateral membrane vesicles (5.2-fold). The substrate- and inhibitor-dependent effect of extracellular αKG on ligand interactions with OAT1 highlights the complexity of the OAT1 ligand-binding surface. The effect of extracellular αKG on the potency of OAT1 inhibition should be considered when assessing drug-drug interaction potential at the transporter.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Application of receiver operating characteristic analysis to refine the prediction of potential digoxin drug interactions.

In the 2012 Food and Drug Administration (FDA) draft guidance on drug-drug interactions (DDIs), a new molecular entity that inhibits P-glycoprotein (P-gp) may need a clinical DDI study with a P-gp substrate such as digoxin when the maximum concentration of inhibitor at steady state divided by IC50 ([I1]/IC50) is ≥0.1 or concentration of inhibitor based on highest approved dose dissolved in 250 ml divide by IC50 ([I2]/IC50) is ≥10. In this article, refined criteria are presented, determined by receiver operating characteristic analysis, using IC50 values generated by 23 laboratories. P-gp probe substrates were digoxin for polarized cell-lines and N-methyl quinidine or vinblastine for P-gp overexpressed vesicles. Inhibition of probe substrate transport was evaluated using 15 known P-gp inhibitors. Importantly, the criteria derived in this article take into account variability in IC50 values. Moreover, they are statistically derived based on the highest degree of accuracy in predicting true positive and true negative digoxin DDI results. The refined criteria of [I1]/IC50 ≥ 0.03 and [I2]/IC50 ≥ 45 and FDA criteria were applied to a test set of 101 in vitro-in vivo digoxin DDI pairs collated from the literature. The number of false negatives (none predicted but DDI observed) were similar, 10 and 12%, whereas the number of false positives (DDI predicted but not observed) substantially decreased from 51 to 40%, relative to the FDA criteria. On the basis of estimated overall variability in IC50 values, a theoretical 95% confidence interval calculation was developed for single laboratory IC50 values, translating into a range of [I1]/IC50 and [I2]/IC50 values. The extent by which this range falls above the criteria is a measure of risk associated with the decision, attributable to variability in IC50 values.

Quantitation of UGT1A1 in human liver microsomes using stable isotope-labelled peptides and mass spectrometry based proteomic approaches.

1. UDP-glucuronosyltransferases (UGTs) are a group of drug-metabolizing enzymes that catalyse the conjugation of endogeonous compounds and xenobiotics to yield hydrophilic glucuronides which subsequently undergo excretion. This report describes an approach for the identification and accurate quantitation of human UGT1A1 in complex biological matrices using liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) analysis of protein digests. 2. A stable isotope-labelled (SIL) peptide of a unique peptide spanning residues 54-69 in exon 1 of the human UGT1A1 protein with the sequence RIYLSADPALVVIEHG was synthesized. The peptide sequence synthesized was in the reverse order of the human peptide with the stable isotope-labels in the amino acid arginine ((13)C6(15)N4) resulting in an increase in the mass of the SIL peptide of 10 amu, from 1753 to 1763. The SIL peptide was quantitated by injecting increasing concentrations of the peptide into the LC-MS to obtain a standard curve. 3. The labelled peptide along with precursor ion monitoring was used to quantify the levels of UGT1A1 in commercial recombinant preparations (supersomes) and individual human liver microsomal samples and pooled human liver micrsomes obtained from BD Biosciences. 4. Glucuronidation activity studies were performed, which demonstrated a positive correlation between enzyme activity levels and the UGT1A1 content in the liver microsomes obtained from individual human donors.