This white paper provides updated International Transporter Consortium (ITC) recommendations on transporters that are important in drug development following the 3rd ITC workshop. New additions ...include prospective evaluation of organic cation transporter 1 (OCT1) and retrospective evaluation of organic anion transporting polypeptide (OATP)2B1 because of their important roles in drug absorption, disposition, and effects. For the first time, the ITC underscores the importance of transporters involved in drug‐induced vitamin deficiency (THTR2) and those involved in the disposition of biomarkers of organ function (OAT2 and bile acid transporters).
Probe drug cocktails are used clinically to assess the potential for drug-drug interactions (DDIs), and in particular, DDIs resulting from coadministration of substrates and inhibitors of cytochrome ...P450 enzymes. However, a probe drug cocktail has not been identified to assess DDIs involving inhibition of drug transporters. We propose a cocktail consisting of the following substrates to explore the potential for DDIs caused by inhibition of key transporters: digoxin (P-glycoprotein, P-gp), rosuvastatin (breast cancer resistance protein, BCRP; organic anion transporting polypeptides, OATP), metformin (organic cation transporter, OCT; multidrug and toxin extrusion transporters, MATE), and furosemide (organic anion transporter, OAT). Furosemide was evaluated in vitro, and is a substrate of OAT1 and OAT3, with Km values of 38.9 and 21.5 μM, respectively. Furosemide was also identified as a substrate of BCRP, OATP1B1, and OATP1B3. Furosemide inhibited BCRP (50% inhibition of drug transport: 170 μM), but did not inhibit OATP1B1, OATP1B3, OCT2, MATE1, and MATE2-K at concentrations below 300 μM, and P-gp at concentrations below 2000 μM. Conservative approaches for the estimation of the likelihood of in vivo DDIs indicate a remote chance of in vivo transporter inhibition by these probe drugs when administered at low single oral doses. This four component probe drug cocktail is therefore proposed for clinical evaluation.
Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine ...5′-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency (Vmax/Km) of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.
Background and Objective
A novel cocktail containing four substrates of key drug transporters was previously optimized to eliminate mutual drug–drug interactions between the probes digoxin ...(P-glycoprotein substrate), furosemide (organic anion transporter 1/3), metformin (organic cation transporter 2, multidrug and toxin extrusion protein 1/2-K), and rosuvastatin (organic anion transporting polypeptide 1B1/3, breast cancer resistance protein). This clinical trial investigated the effects of four commonly employed drug transporter inhibitors on cocktail drug pharmacokinetics.
Methods
In a randomized open-label crossover trial in 45 healthy male subjects, treatment groups received the cocktail with or without single oral doses of rifampin, verapamil, cimetidine or probenecid. Concentrations of the probe drugs in serial plasma samples and urine fractions were measured by validated liquid chromatography-tandem mass spectrometry assays to assess systemic exposure.
Results
The results were generally in accordance with known in vitro and/or clinical drug–drug interaction data. Single-dose rifampin increased rosuvastatin area under the plasma concentration–time curve up to the last quantifiable concentration (AUC
0–tz
) by 248% and maximum plasma concentration (
C
max
) by 1025%. Probenecid increased furosemide AUC
0–tz
by 172% and
C
max
by 23%. Cimetidine reduced metformin renal clearance by 26%. The effect of single-dose verapamil on digoxin systemic exposure was less than expected from multiple-dose studies (AUC
0–tz
unaltered,
C
max
+ 22%).
Conclusions
Taking all the interaction results together, the transporter cocktail is considered to be validated as a sensitive and specific tool for evaluating transporter-mediated drug–drug interactions in drug development.
Clinical Trial Registration
EudraCT number 2017-001549-29.
Objective
An
in vitro
relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of ...different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair.
Methods
In vitro
studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations.
Results
The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin’s
in vivo
intrinsic hepatic clearance (CL
int,h,
in
vivo
) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CL
int,h,
in
vivo
predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77–1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR.
Conclusions
This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.
Breast cancer resistance protein (BCRP; ABCG2) limits intestinal absorption of low-permeability substrate drugs and mediates biliary excretion of drugs and metabolites. Based on clinical evidence of ...BCRP-mediated drug-drug interactions (DDIs) and the c.421C>A functional polymorphism affecting drug efficacy and safety, both the US Food and Drug Administration and European Medicines Agency recommend preclinical evaluation and, when appropriate, clinical assessment of BCRP-mediated DDIs. Although many BCRP substrates and inhibitors have been identified in vitro, clinical translation has been confounded by overlap with other transporters and metabolic enzymes. Regulatory recommendations for BCRP-mediated clinical DDI studies are challenging, as consensus is lacking on the choice of the most robust and specific human BCRP substrates and inhibitors and optimal study design. This review proposes a path forward based on a comprehensive analysis of available data. Oral sulfasalazine (1000 mg, immediate-release tablet) is the best available clinical substrate for intestinal BCRP, oral rosuvastatin (20 mg) for both intestinal and hepatic BCRP, and intravenous rosuvastatin (4 mg) for hepatic BCRP. Oral curcumin (2000 mg) and lapatinib (250 mg) are the best available clinical BCRP inhibitors. To interrogate the worst-case clinical BCRP DDI scenario, study subjects harboring the BCRP c.421C/C reference genotype are recommended. In addition, if sulfasalazine is selected as the substrate, subjects having the rapid acetylator phenotype are recommended. In the case of rosuvastatin, subjects with the organic anion-transporting polypeptide 1B1 c.521T/T genotype are recommended, together with monitoring of rosuvastatin's cholesterol-lowering effect at baseline and DDI phase. A proof-of-concept clinical study is being planned by a collaborative consortium to evaluate the proposed BCRP DDI study design.
Aims
Previous pharmacokinetic characterization of a transporter probe cocktail containing digoxin (P‐gp), furosemide (OAT1, OAT3), metformin (OCT2, MATE1, MATE2‐K) and rosuvastatin (OATP1B1, OATP1B3, ...BCRP) in healthy subjects showed increases in rosuvastatin systemic exposure compared to rosuvastatin alone. In this trial, the doses of metformin and furosemide as putative perpetrators were reduced to eliminate their drug–drug interaction (DDI) with rosuvastatin.
Methods
In a randomized, open‐label, single‐centre, five‐treatment, five‐period crossover trial, 30 healthy male subjects received as reference treatments separately 0.25 mg digoxin, 1 mg furosemide, 10 mg metformin and 10 mg rosuvastatin, and as test treatment all four drugs administered together as a cocktail. Primary pharmacokinetic endpoints were AUC0‐tz (area under the plasma concentration–time curve from time zero to the last quantifiable concentration) and Cmax (maximum plasma concentration) of each probe drug.
Results
Geometric mean ratios and 90% confidence intervals of test (cocktail) to reference (single drug) for AUC0‐tz were 96.4% (88.2–105.3%) for digoxin, 102.6% (93.8–112.3%) for furosemide, 97.5% (93.5–101.6%) for metformin and 105.0% (96.4–114.4%) for rosuvastatin, indicating lack of interaction. The same analysis for Cmax and for pharmacokinetic parameters of urinary excretion of all cocktail components also indicated no DDI.
Conclusions
Digoxin (0.25 mg), furosemide (1 mg), metformin (10 mg) and rosuvastatin (10 mg) exhibit no mutual pharmacokinetic interactions and are well tolerated administered as a cocktail. The cocktail is thus optimized and has the potential to be used as a screening tool for clinical investigation of transporter‐mediated DDI.
Purpose
To provide whole-body physiologically based pharmacokinetic (PBPK) models of the potent clinical organic anion transporter (OAT) inhibitor probenecid and the clinical OAT victim drug ...furosemide for their application in transporter-based drug-drug interaction (DDI) modeling.
Methods
PBPK models of probenecid and furosemide were developed in PK-Sim®. Drug-dependent parameters and plasma concentration-time profiles following intravenous and oral probenecid and furosemide administration were gathered from literature and used for model development. For model evaluation, plasma concentration-time profiles, areas under the plasma concentration–time curve (AUC) and peak plasma concentrations (C
max
) were predicted and compared to observed data. In addition, the models were applied to predict the outcome of clinical DDI studies.
Results
The developed models accurately describe the reported plasma concentrations of 27 clinical probenecid studies and of 42 studies using furosemide. Furthermore, application of these models to predict the probenecid-furosemide and probenecid-rifampicin DDIs demonstrates their good performance, with 6/7 of the predicted DDI AUC ratios and 4/5 of the predicted DDI C
max
ratios within 1.25-fold of the observed values, and all predicted DDI AUC and C
max
ratios within 2.0-fold.
Conclusions
Whole-body PBPK models of probenecid and furosemide were built and evaluated, providing useful tools to support the investigation of transporter mediated DDIs.
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