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During its fourth transporter workshop in 2021, the International Transporter Consortium (ITC) provided updates on emerging clinically relevant transporters for drug development. Previously highlighted and new transporters were considered based on up-to-date clinical evidence of their importance in drug-drug interactions and potential for altered drug efficacy and safety, including drug-nutrient interactions leading to nutrient deficiencies. For the first time, folate transport pathways (PCFT, RFC, and FRα) were examined in-depth as a potential mechanism of drug-induced folate deficiency and related toxicities (e.g., neural tube defects and megaloblastic anemia). However, routine toxicology studies conducted in support of drug development appear sufficient to flag such folate deficiency toxicities, whereas prospective prediction from in vitro folate metabolism and transport inhibition is not well enough established to inform drug development. Previous suggestion of a retrospective study of intestinal OATP2B1 inhibition to explain unexpected decreases in drug exposure were updated. Furthermore, when the absorption of a new molecular entity is more rapid and extensive than can be explained by passive permeability, evaluation of the OATP2B1 transport may be considered. Emerging research on hepatic and renal OAT2 is summarized, but current understanding of the importance of OAT2 was deemed insufficient to justify specific consideration for drug development. Hepatic, renal, and intestinal MRPs (MRP2, MRP3, and MRP4) were revisited. MRPs may be considered when they are suspected to be the major determinant of drug disposition (e.g., direct glucuronide conjugates); MRP2 inhibition as a mechanistic explanation for drug-induced hyperbilirubinemia remains justified. There were no major changes in recommendations from previous ITC whitepapers.

P-glycoprotein (P-gp) is an efflux transporter that plays an important role in the pharmacokinetics of its substrate, and P-gp activities can be altered by induction and inhibition effects of rifampicin. This study aimed to establish a physiologically based pharmacokinetic (PBPK) model of rifampicin to predict the P-gp-mediated drug-drug interactions (DDIs) and assess the DDI impact in the intestine, liver, and kidney. The induction and inhibition parameters of rifampicin for P-gp were estimated using two of seven DDI cases of rifampicin and digoxin and incorporated into our previously constructed PBPK model of rifampicin. The constructed rifampicin model was verified using the remaining five DDI cases with digoxin and five DDI cases with other P-gp substrates (talinolol and quinidine). Based on the established PBPK model, following repeated dosing of 600 mg rifampicin, the deduced net effect was an approximately threefold induction in P-gp activities in the intestine, liver, and kidney. Furthermore, in all 12 cases the predicted area under the plasma concentration-time curve ratios of the P-gp substrates were within the predefined acceptance criteria with various dosing regimens. Intestinal effects of P-gp-mediated DDIs had their greatest impact on the pharmacokinetics of digoxin and talinolol, with a minimal impact on the liver and kidney. For quinidine, predicted intestinal P-gp/cytochrome P450 3A-mediated DDIs were slightly underestimated because of the complexity of nonlinearity and transporter-enzyme interplay. These findings demonstrate that our rifampicin model can be applicable to quantitatively predict the net impact of P-gp induction and/or inhibition on diverse P-gp substrates and investigate the magnitude of DDIs in each tissue.

This study was designed to assess the quantitative performance of endogenous biomarkers for organic anion transporting polypeptide (OATP) 1B1/1B3-mediated drug-drug interactions (DDIs). Ten healthy volunteers orally received OATP1B1/1B3 probe cocktail (0.2 mg pitavastatin, 1 mg rosuvastatin, and 2 mg valsartan) and an oral dose of cyclosporin A (CysA, 20 mg and 75 mg) separated by a 1-hour interval (20 mg (-1 hour), and 75 mg (-1 hour)). CysA 75 mg was also given with a 3-hour interval (75 mg (-3 hours)) to examine the persistence of OATP1B1/1B3 inhibition. The area under the plasma concentration-time curve ratios (AUCRs) were 1.63, 3.46, and 2.38 (pitavastatin), 1.39, 2.16, and 1.81 (rosuvastatin), and 1.42, 1.77, and 1.85 (valsartan), at 20 mg, 75 mg (-1 hour) and 75 mg (-3 hours) of CysA, respectively. CysA effect on OATP1B1/1B3 was unlikely to persist at the dose examined. Among 26 putative OATP1B1/1B3 biomarkers evaluated, AUCR and maximum concentration ratio (C

[Image: see text] While a drug treatment is unavailable, the global incidence of Dengue virus (DENV) infections and its associated severe manifestations continues to rise. We report the construction of the first physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model that predicts viremia levels in relevant target organs based on preclinical data with the broad spectrum antiviral soraphen A (SorA), an inhibitor of the host cell target acetyl-CoA-carboxylase. SorA was highly effective against DENV in vitro (EC(50) = 4.7 nM) and showed in vivo efficacy by inducing a significant reduction of viral load in the spleen and liver of IFNAR(–/–) mice infected with DENV-2. PBPK/PD predictions for SorA matched well with the experimental infection data. Transfer to a human PBPK/PD model for DENV to mimic a clinical scenario predicted a reduction in viremia by more than one log(10) unit for an intravenous infusion regimen of SorA. The PBPK/PD model is applicable to any DENV drug lead and, thus, represents a valuable tool to accelerate and facilitate DENV drug discovery and development.

Previous reports suggest that the null genotype (*0/*0) of glutathione S-transferase (GST) M1 and/or GSTT1 could be risk factors for drug-induced liver injury (DILI). However, multi-institutional pharmacogenetic research with various suspected drugs has rarely been performed in Japan. Therefore, the aim of this study was to investigate the role of GSTM1 and GSTT1 null genotype in the occurrence of DILI in Japanese patients.Blood samples of 270 DILI patients from 23 hospitals throughout Japan collected between 2010 and 2018 were subjected to genotyping of null genotypes of GSTM1 and GSTT1 using the SmartAmp-2 method. We also collected information on DILI types, time to onset of DILI, pharmacological classification of suspected drugs and Digestive Disease Week-Japan score, as well as genotypes of GSTM1 and GSTT1 in each patient with DILI.The distribution of a combination of null genotypes of GSTM1 and GSTT1 in Japanese patients with DILI was significantly different from that reported in the general Japanese population. Notably, the incidence of the GSTM1 null genotype in patients with DILI was significantly higher than that of the control population. A significant relationship between the frequency of GSTM1 and GSTT1 null genotypes and pharmacological classification of suspected drugs, clinical laboratory data for liver function, time to onset of DILI, and Digestive Disease Week-Japan scores was not observed.The GSTM1 null genotype was associated with an increased incidence of DILI in Japanese patients.

Hepatic uptake clearance has been measured in suspended human hepatocytes (SHH). Plated human hepatocytes (PHH) after short-term culturing are increasingly employed to study hepatic transport driven mainly by its higher throughput. To know pros/cons of both systems, the hepatic uptake clearances of several organic anion transporting polypeptide 1B substrates were compared between PHH and SHH by determining the initial uptake velocities or through dynamic model-based analyses. For cerivastatin, pitavastatin and rosuvastatin, initial uptake clearances (PSinf) obtained using PHH were comparable to those using SHH, while cell-to-medium concentration (C/M) ratios were 2.7- to 5.4-fold higher. For pravastatin and dehydropravastatin, hydrophilic compounds with low uptake/cellular binding, their PSinf and C/M ratio in PHH were 1.8- to 3.2-fold lower than those in SHH. These hydrophilic substrates are more prone to wash-off during the uptake study using PHH, which may explain the apparently lower uptake than SHH. The C/M ratios obtained using PHH were stable over an extended time, making PHH suitable to estimate the C/M ratios and hepatocyte-to-medium unbound concentration ratios (Kp,uu). In conclusion, PHH is useful in evaluating hepatic uptake/efflux clearances and Kp,uu of OATP1B substrates in a high-throughput manner, however, a caution is warranted for hydrophilic drugs with low uptake/cellular binding.

Pemafibrate (PMF) is highly albumin-bound (>99.8%) and a substrate for hepatic uptake transporters (OATP1B) and CYP enzymes. Here, we developed a PBPK model of PMF to capture drug-drug interactions (DDI) incurred by cyclosporine (CsA) and rifampicin (RIF), the two OATP1B inhibitors. Initial PBPK modeling of PMF utilized in vitro hepatic uptake clearance (PSinf) obtained in the absence of albumin, but failed in capturing the blood PMF pharmacokinetic (PK) profiles. Based on the results that in vitro PSinf of unbound PMF was enhanced in the presence of albumin, we applied the albumin-facilitated dissociation model and the resulting PSinf parameters improved the prediction of the blood PMF PK profiles. In refining our PBPK model toward improved prediction of the observed DDI data (PMF co-administered with single dosing of CsA or RIF; PMF following multiple RIF dosing), we adjusted the previously obtained in vivo OATP1B inhibition constants (Ki,OATP1B) of CsA or RIF for pitavastatin by correcting for substrate-dependency. We also incorporated the induction of OATP1B and CYP enzymes after multiple RIF dosing. Sensitivity analysis informed that the higher gastrointestinal absorption rate constant could further improve capturing the observed DDI data, suggesting the possible inhibition of intestinal ABC transporter(s) by CsA or RIF.

To identify oral drugs that likely display nonlinear pharmacokinetics due to saturable metabolism by intestinal CYP3A, our previous report using CYP3A substrate drugs proposed an approach using thresholds for the linear index number (LIN3A = dose/Km; Km, Michaelis-Menten constant for CYP3A) and the intestinal availability (FaFg). Here, we aimed to extend the validity of the previous approach using both CYP3A substrate and non-substrate drugs and to devise a decision tree suited for early drug candidates using in vitro metabolic intrinsic clearance (CLint, vitro) instead of FaFg. Out of 152 oral drugs (including 136 drugs approved in Japan, US or both), type I nonlinearity (in which systemic drug exposure increases in a more than dose-proportional manner) was noted with 82 drugs (54%), among which 58 drugs were identified as CYP3A substrates based on public information. Based on practical feasibility, 41 drugs were selected from CYP3A substrates and subjected to in-house metabolic assessment. The results were used to determine the thresholds for CLint, vitro (0.45 μL/min/pmol CYP3A4) and LIN3A (1.0 L). For four drugs incorrectly predicted, potential mechanisms were looked up. Overall, our proposed decision tree may aid in the identification of early drug candidates with intestinal CYP3A-derived type I nonlinearity.

In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation.

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.

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.

Phase 0 approaches — which include microdosing — evaluate subtherapeutic exposures of new drugs in first-in-human studies known as exploratory clinical trials. Recent progress extends phase 0 benefits beyond assessment of pharmacokinetics to include understanding of mechanism of action and pharmacodynamics. Phase 0 approaches have the potential to improve preclinical candidate selection and enable safer, cheaper, quicker and more informed developmental decisions. Here, we discuss phase 0 methods and applications, highlight their advantages over traditional strategies and address concerns related to extrapolation and developmental timelines. Although challenges remain, we propose that phase 0 approaches be at least considered for application in most drug development scenarios. Phase 0 approaches, including microdosing, evaluate subtherapeutic exposures to novel drugs, potentially enabling safer, cheaper and quicker first-in-human studies. Here, Burt et al. discuss the fundamentals and applications of phase 0 approaches, highlight the potential advantages of their application in drug development and address the associated limitations.

There is an increasing interest in transporter induction (i.e., decreased systemic drug exposure due to increased efflux-limited absorption or transporter-mediated clearance) as a mechanism of drug-drug interactions (DDIs), although evidence of clinical relevance is still evolving. Intestinal P-glycoprotein (P-gp) and hepatic organic anion transporting polypeptides 1B (OATP1B) can be important determinants of drug absorption and disposition, as well as targets for DDIs. Current data indicate that intestinal P-gp protein levels can be induced up to threefold to fourfold in humans primarily with pregnane X receptor (PXR) activators, and that this induction can decrease the systemic exposure of drugs with P-gp efflux-limited absorption (e.g., ≤ 67% decrease in the exposure of total dabigatran following rifampin multiple oral dosing). Evaluation of the clinical relevance of P-gp induction as a DDI mechanism must consider the induction potential of the perpetrator drug for P-gp and attenuation of exposure of the victim drug in the context of its therapeutic window. Practical drug development recommendations are provided herein. Reports are contradictory on OATP1B induction by PXR activators in human hepatocytes and liver biopsies. Some clinical investigations demonstrated that rifampin pretreatment decreased exposure of OATP1B substrates, while other studies found no differences, and the potential involvement of other mechanisms in these observed DDIs cannot be definitively ruled out. Thus, further studies are needed to understand hepatic OATP1B induction and potential involvement of other mechanisms contributing to reduced exposure of OATP1B substrates. This review critically summarizes the state-of-the-art on intestinal P-gp and hepatic OATP1B induction, and highlights implications for drug development.

We developed a practical synthetic method for fluorine-18 (18F)-labeled pitavastatin ([18F]PTV) as a positron emission tomography (PET) tracer to assess hepatobiliary transporter activity and condu...

To address the most appropriate endogenous biomarker for drug–drug interaction risk assessment, eight healthy subjects received an organic anion transporting polypeptide 1B (OATP1B) inhibitor (rifampicin, 150, 300, and 600 mg), and a probe drug cocktail (atorvastatin, pitavastatin, rosuvastatin, and valsartan). In addition to coproporphyrin I, a widely studied OATP1B biomarker, we identified at least 4 out of 28 compounds (direct bilirubin, glycochenodeoxycholate‐3‐glucuronide, glycochenodeoxycholate‐3‐sulfate, and hexadecanedioate) that presented good sensitivity and dynamic range in terms of the rifampicin dose‐dependent change in area under the plasma concentration‐time curve ratio (AUCR). Their suitability as OATP1B biomarkers was also supported by the good correlation of AUC0‐24h between the endogenous compounds and the probe drugs, and by nonlinear regression analysis (AUCR−1 vs. rifampicin plasma Cmax (maximum total concentration in plasma)) to yield an estimate of the inhibition constant of rifampicin. These endogenous substrates can complement existing OATP1B‐mediated drug–drug interaction risk assessment approaches based on agency guidelines in early clinical trials.

Parameter estimation problems of mathematical models can often be formulated as nonlinear least squares problems. Typically these problems are solved numerically using iterative methods. The local minimiser obtained using these iterative methods usually depends on the choice of the initial iterate. Thus, the estimated parameter and subsequent analyses using it depend on the choice of the initial iterate. One way to reduce the analysis bias due to the choice of the initial iterate is to repeat the algorithm from multiple initial iterates (i.e. use a multi-start method). However, the procedure can be computationally intensive and is not always used in practice. To overcome this problem, we propose the Cluster Gauss–Newton (CGN) method, an efficient algorithm for finding multiple approximate minimisers of nonlinear-least squares problems. CGN simultaneously solves the nonlinear least squares problem from multiple initial iterates. Then, CGN iteratively improves the approximations from these initial iterates similarly to the Gauss–Newton method. However, it uses a global linear approximation instead of the Jacobian. The global linear approximations are computed collectively among all the iterates to minimise the computational cost associated with the evaluation of the mathematical model. We use physiologically based pharmacokinetic (PBPK) models used in pharmaceutical drug development to demonstrate its use and show that CGN is computationally more efficient and more robust against local minima compared to the standard Levenberg–Marquardt method, as well as state-of-the art multi-start and derivative-free methods.

Cyclosporine A (CsA) and rifampin are potent inhibitors of organic anion transporting polypeptide (OATP) 1B1 and are widely used to assess the risk for drug-drug interactions. CsA displays preincubation time-dependent, long-lasting inhibition of OATP1B1 in vitro and in rats in vivo, and a proposed mechanism is the

It is widely accepted that uptake and efflux transporters on clearance organs play crucial roles in drug disposition. Although in vitro transporter assay system can identify the intrinsic properties of the target transporters, it is not so easy to precisely predict in vivo pharmacokinetic parameters from in vitro data. Positron emission tomography (PET) imaging is a useful tool to directly assess the activity of drug transporters in humans. We recently developed a practical synthetic method for fluorine-18-labeled pitavastatin ([

Introduction: Statins are prescribed widely for cholesterol-lowering therapy, but it is known that their efficacy and safety profiles vary, despite the shared pharmacophore and pharmacological target. The immense body of related clinical and preclinical data offers a unique opportunity to explore the possible factors underlying inter-statin and inter-individual variabilities.Area covered: Clinical and preclinical data from various statins were compiled with regard to the efficacy (cholesterol-lowering effect) and safety (muscle toxicity). Based on the compiled data, dose- and exposure-response relationships were explored to obtain mechanistic and quantitative insights into the variations in the efficacy and safety profiles of statins.Expert opinion: Our analyses indicated that the inter-statin variability in the cholesterol-lowering effect may be mainly attributable to variations in potency of inhibition of the pharmacological target, rather than variations in drug exposure at the site of drug action. However, the drug exposure at the sites of drug action (i.e., the liver for efficacy and the muscle for safety) may contribute to the differences in the efficacy and safety observed in individual patients.

The pharmacokinetics of telmisartan are nonlinear within the clinical dose range. To identify the underlying mechanism of this nonlinearity, we conducted a PET study in healthy subjects using [11C]telmisartan. Eight healthy male subjects were enrolled in a 2-way crossover study. PET imaging was performed after intravenous administration of [11C]telmisartan with or without a 1-h oral predose of two 40 mg Micardis® tablets. About 60% of the injected [11C]telmisartan accumulated in the liver within 10 min after injection. With predosing of 80 mg telmisartan, the systemic elimination of [11C]telmisartan was slightly delayed, but the liver exposure started to decrease earlier and biliary excretion was greatly enhanced. Hepatic uptake clearance of the radioactivity was not changed by telmisartan predosing, whereas the biliary clearance of radioactivity from the liver was significantly increased. Thus, the alteration in the pharmacokinetics of the radioactivity could not be explained simply by the saturation of hepatic uptake. Therefore, other mechanisms, such as the saturation of intracellular binding of telmisartan and/or its glucuronide, and the glucuronidation of telmisartan by uridine 5′-diphospho-glucuronosyltransferases, should be considered. This is the first reported human PET study using [11C]telmisartan, the results of which can assist understanding of the hepatobiliary transport of telmisartan in humans.

As rifampicin can cause the induction and inhibition of multiple metabolizing enzymes and transporters, it has been challenging to accurately predict the complex drug-drug interactions (DDIs). We previously constructed a physiologically-based pharmacokinetic (PBPK) model of rifampicin accounting for the components for the induction of cytochrome P450 (CYP) 3A/CYP2C9 and the inhibition of organic anion transporting polypeptide 1B (OATP1B). This study aimed to expand and verify the PBPK model for rifampicin by incorporating additional components for the induction of OATP1B and CYP2C8 and the inhibition of multidrug resistance protein 2. The established PBPK model was capable of accurately predicting complex rifampicin-induced alterations in the profiles of glibenclamide, repaglinide, and coproporphyrin I (an endogenous biomarker of OATP1B activities) with various dosing regimens. Our comprehensive rifampicin PBPK model may enable quantitative prediction of DDIs across diverse potential victim drugs and endogenous biomarkers handled by multiple metabolizing enzymes and transporters.

Metformin is an important antidiabetic drug and often used as a probe for drug-drug interactions (DDIs) mediated by renal transporters. Despite evidence supporting the inhibition of multidrug and toxin extrusion proteins as the likely DDI mechanism, the previously reported physiologically-based pharmacokinetic (PBPK) model required the substantial lowering of the inhibition constant values of cimetidine for multidrug and toxin extrusion proteins from those obtained in vitro to capture the clinical DDI data between metformin and cimetidine.1 We constructed new PBPK models in which the transporter-mediated uptake of metformin is driven by a constant membrane potential. Our models successfully captured the clinical DDI data using in vitro inhibition constant values and supported the inhibition of multidrug and toxin extrusion proteins by cimetidine as the DDI mechanism upon sensitivity analysis and data fitting. Our refined PBPK models may facilitate prediction approaches for DDI involving metformin using in vitro inhibition constant values.

This study aimed to elucidate the impact of OATP1B1 genotype (*1b/*1b, *1b/*15, and *15/*15) on plasma concentrations of endogenous OATP1B1 substrates. Healthy volunteers with OATP1B1 *1b/*1b (n = 10), *1b/*15 (n = 7), or *15/*15 (n = 2) received oral administration of a cocktail of statins (atorvastatin, pitavastatin, rosuvastatin, and fluvastatin). Mean area under the plasma concentration of atorvastatin, pitavastatin, and rosuvastatin in OATP1B1 *15/*15 were 2.2, 1.7 and 1.58-times greater than the corresponding values in OATP1B1 *1b/*1b, respectively, whereas that of fluvastatin was identical to those in other OATP1B1 genotypes. OATP1B1 *15/*15 also showed higher mean plasma concentrations of OATP1B1 endogenous substrates compared with the other OATP1B1 genotypes, such as coproporphyrin I, glycochenodeoxycholate sulfate (GCDCA-S), lithocholate sulfate (LCA-S), glycolithocholate sulfate (GLCA-S) and taurolithocholate sulfate (TLCA-S), but not total or direct bilirubin, chenodeoxycholate-24-glucuronide, or ω-dicarboxylic long-chain fatty acids. Area under the plasma concentration-time curves of plasma coproporphyrin I and GLCA-S discriminated OATP1B1 genotype *15/*15 from the other genotypes. In combination with previously published clinical studies, these results support the notion that coproporphyrin I, and GLCA-S and GCDCA-S could be a surrogate probe for assessing human in vivo OATP1B1 activities.

The antidiabetic drugs glibenclamide, repaglinide, and nateglinide are well-known substrates for hepatic uptake transporters of the organic anion transporting polypeptide (OATP) family and metabolizing enzymes of the cytochrome P450 (CYP) 2C subfamily. The systemic exposure of these drugs varies substantially among individuals, impacted by genetic polymorphisms of transporters and metabolizing enzymes as well as drug-drug interactions. The use of the conventional in vitro-in vivo extrapolation (IVIVE) method was found to underestimate their hepatic intrinsic clearance (CLint,all); the clinically observed CLint,all values were ≥10-fold higher than the predicted values from in vitro data. In order to improve the accuracy in predicting CLint,all of these drugs, the following modifications were implemented; i) the extended clearance concept was applied during IVIVE processes, ii) albumin was added to metabolic assays using human liver microsomes (to minimize the impact of intrinsic inhibitors on kinetic parameters for CYP2C-mediated metabolism) and to hepatic uptake assays (to accommodate the enhanced hepatic uptake observed with albumin-bound drugs), and iii) differing rates of efflux and influx via diffusion were used. The IVIVE method with these modifications yielded the predicted CLint,all values from in vitro data in closer agreement with the CLint,all values observed in vivo; the fold differences between the predicted and observed CLint,all values reduced from 13-15 to 5.9-6.7. Our current approach offers an improvement in the prediction of CLint,all and further investigations are warranted to enhance the prediction accuracy of IVIVE.

Phase 0 approaches, including microdosing, involve the use of sub-therapeutic exposures to the tested drugs, thus enabling safer, more-relevant, quicker and cheaper first-in-human (FIH) testing. These approaches also have considerable potential to limit the use of animals in human drug development. Recent years have witnessed progress in applications, methodology, operations, and drug development culture. Advances in applications saw an expansion in therapeutic areas, developmental scenarios and scientific objectives, in, for example, protein drug development and paediatric drug development. In the operational area, the increased sensitivity of Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS), expansion of the utility of Positron Emission Tomography (PET) imaging, and the introduction of Cavity Ring-Down Spectroscopy (CRDS), have led to the increased accessibility and utility of Phase 0 approaches, while reducing costs and exposure to radioactivity. PET has extended the application of microdosing, from its use as a predominant tool to record pharmacokinetics, to a method for recording target expression and target engagement, as well as cellular and tissue responses. Advances in methodology include adaptive Phase 0/Phase 1 designs, cassette and cocktail microdosing, and Intra-Target Microdosing (ITM), as well as novel modelling opportunities and simulations. Importantly, these methodologies increase the predictive power of extrapolation from microdose to therapeutic level exposures. However, possibly the most challenging domain in which progress has been made, is the culture of drug development. One of the main potential values of Phase 0 approaches is the opportunity to terminate development early, thus not only applying the principle of ‘kill-early-kill-cheap’ to enhance the efficiency of drug development, but also obviating the need for the full package of animal testing required for therapeutic level Phase 1 studies. Finally, we list developmental scenarios that utilised Phase 0 approaches in novel drug development.