Agnès Poirier

Design, Data Analysis, and Simulation of in Vitro Drug Transport Kinetic Experiments Using a Mechanistic in Vitro Model

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, Vmax and Km, as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (Pdif). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for Vmax and Km were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5–6-fold higher permeability at 37°C compared with that at 4°C), for fexofenadine a 16-fold higher passive permeability was seen at 37°C. Therefore, Pdif was better predicted if it was evaluated under the same experimental conditions as Vmax and Km, i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37°C, instead of a parallel control evaluation at 4°C.

Specific Correction of Alternative Survival Motor Neuron 2 Splicing by Small Molecules: Discovery of a Potential Novel Medicine To Treat Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is the leading genetic cause of infant and toddler mortality, and there is currently no approved therapy available. SMA is caused by mutation or deletion of the survival motor neuron 1 (SMN1) gene. These mutations or deletions result in low levels of functional SMN protein. SMN2, a paralogous gene to SMN1, undergoes alternative splicing and exclusion of exon 7, producing an unstable, truncated SMNΔ7 protein. Herein, we report the identification of a pyridopyrimidinone series of small molecules that modify the alternative splicing of SMN2, increasing the production of full-length SMN2 mRNA. Upon oral administration of our small molecules, the levels of full-length SMN protein were restored in two mouse models of SMA. In-depth lead optimization in the pyridopyrimidinone series culminated in the selection of compound 3 (RG7800), the first small molecule SMN2 splicing modifier to enter human clinical trials.

The Need for Human Breast Cancer Resistance Protein Substrate and Inhibition Evaluation in Drug Discovery and Development: Why, When, and How?

Although the multiplicity in transport proteins assessed during drug development is continuously increasing, the clinical relevance of the breast cancer resistance protein (BCRP) is still under debate. Here, our aim is to rationalize the need to consider BCRP substrate and inhibitor interactions and to define optimum selection and acceptance criteria between cell-based and vesicle-based assays in vitro. Information on the preclinical and clinical pharmacokinetics (PK), drug-drug interactions, and pharmacogenomics data was collated for 13 marketed drugs whose PK is reportedly associated with BCRP interaction. Clinical examples where BCRP impacts drug PK and efficacy appear to be rare and confounded by interactions with other transporters. Thirty-seven compounds were selected to be tested as BCRP substrates in a cell-based assay using MDCKII cells (Madin-Darby canine kidney cells) and 18 in membrane vesicles. Depending on the physicochemical compound properties, we observed both in vitro systems to give false-negative readouts. In addition, the inhibition potential of 19 compounds against BCRP was assessed in vesicles and in MDCKII cells, where we observed significant system and substrate-dependent IC50 values. Therefore, neither of the two test systems is superior to the other. Instead, one system may offer advantages under certain situations (e.g., low permeability) and thus should be selected based on the physicochemical compound properties. Finally, given the clinical relevance of BCRP, we propose that its evaluation should remain issue-driven: for low permeable, low bioavailable drugs, in particular when other more common processes do not allow a mechanistic understanding of any unexpected absorption or brain disposition, and for drugs with a low therapeutic window.

Calibration of in vitro multidrug resistance protein 1 substrate and inhibition assays as a basis to support the prediction of clinically relevant interactions in vivo.

The multidrug resistance protein 1 (MDR1) is known to limit brain penetration of drugs and play a key role in drug-drug interactions (DDIs). Theoretical cut-offs from regulatory guidelines are used to extrapolate MDR1 interactions from in vitro to in vivo. However, these cut-offs do not account for interlaboratory variability. Our aim was to calibrate our experimental system to allow better in vivo predictions. We selected 166 central nervous system (CNS) and non-CNS drugs to calibrate the MDR1 transport screening assay using Lewis lung cancer porcine kidney 1 epithelial cells overexpressing MDR1 (L-MDR1). A threshold efflux ratio (ER) of 2 was established as one parameter to assess brain penetration in lead optimization. The inhibitory potential of 57 molecules was evaluated using IC50 values based on the digoxin ER—IC50(ER)—or apparent permeability—IC50(Papp)—in L-MDR1 cells. Published clinical data for 68 DDIs involving digoxin as the victim drug were collected. DDI risk assessments were based on intestinal concentrations ([I2]) as well as unbound [I1u] and total plasma [I1T] concentrations. A receiver operating characteristic analysis identified an [I2]/IC50(ER) of 6.5 as the best predictor of a potential interaction with digoxin in patients. The model was further evaluated with a test set of 11 digoxin DDIs and 16 nondigoxin DDIs, resulting in only one false negative for each test set, no false positives among the digoxin DDIs, and two among the nondigoxin DDIs. Future refinements might include using cerebrospinal fluid to unbound plasma concentration ratios rather than therapeutic class, better estimation of [I2], and dynamic modeling of MDR1-mediated DDIs.

PK/PD assessment in CNS drug discovery: Prediction of CSF concentration in rodents for P-glycoprotein substrates and application to in vivo potency estimation

The unbound drug concentration in brain parenchyma is considered to be the relevant driver for interaction with central nervous system (CNS) biological targets. Drug levels in cerebrospinal fluid (C_CSF) are frequently used surrogates for the unbound concentrations in brain. For drugs actively transported across the blood-brain barrier (BBB), C_CSF differs from unbound plasma concentration (Cu_p) to an extent that is commonly unknown. In this study, the relationship between CSF-to-unbound plasma drug partitioning in rats and the mouse Pgp (Mdr1a) efflux ratio (ER) obtained from in vitro transcellular studies has been investigated for a set of 61 CNS compounds exhibiting substantial diversity in chemical structure and physico-chemical properties. In order to understand the in vitro-in vivo extrapolation of Pgp efflux, a mechanistic model was derived relating in vivo CNS distribution kinetics to in vitro active transport. The model was applied to predict C_CSF from Cu_p and ER data for 19 proprietary Roche CNS drug candidates. The calculated CSF concentrations were correlated with CNS pharmacodynamic responses observed in rodent models. The correlation between in vitro and in vivo potency for different pharmacological endpoints indicated that the predicted C_CSF is a valuable surrogate of the concentration at the target site. Overall, C_CSF proved superior description of PK/PD data than unbound plasma or total brain concentration for Mdr1a substrates. Predicted C_CSF can be used as a default approach to understand the PK/PD relationships in CNS efficacy models and can support the extrapolation of efficacious brain exposure for new drug candidates from rodent to man.

Prediction of pharmacokinetic profile of valsartan in humans based on in vitro uptake-transport data.

The aim of this study was to evaluate a physiologically based pharmacokinetic (PBPK) model for predicting PK profiles in humans based on a model refined in rats and humans in vitro uptake‐transport data using valsartan as a probe substrate. Valsartan is eliminated unchanged, mostly through biliary excretion, both in humans and rats. It was, therefore, chosen as model compound to predict in vivo elimination based on in vitro hepatic uptake‐transport data using a fully mechanistic PBPK model. Plated rat and human hepatocytes, and cell lines overexpressing human OATP1B1 and OATP1B3 were used for in vitro uptake experiments. A mechanistic two‐compartment model was used to derive the active and passive transport parameters, namely uptake Michaelis–Menten parameters (Vmax and Km,u) together with passive diffusion (Pdif). These transport parameters were then used as input in a whole body physiologically based pharmacokinetic (PBPK) model. The uptake rate of valsartan was higher for rat hepatocytes (Km,u=28.4±3.7 μM, Vmax=1320±180 pmol/mg/min, and Pdif =1.21±0.42 μl/mg/min) compared to human hepatocytes (Km,u=44.4±14.6 μM, Vmax=304±85 pmol/mg/min, and Pdif=0.724±0.271 μl/mg/min). OATP1B1 and ‐1B3 parameters were correlated to human hepatocyte data, using experimentally established relative activity factors (RAF). Resulting PBPK simulations were compared for plasma‐ (humans and rats) and bile‐ (rats) concentration–time profiles following iv bolus administration of valsartan. Plasma clearances (CLP) for rats and humans were predicted within twofold relative to predictions based on respective in vitro data. The simulations were extended to simulate the impact of either OATP1B1 or ‐1B3 inhibition on plasma profile. The limited data set indicates that the mechanistic model allowed for accurate evaluation of in vitro transport data; and the resulting hepatic uptake transport kinetic parameters enabled the prediction of in vivo PK profiles and plasma clearances, using PBPK modelling. Moreover, the interspecies difference in elimination rate observed in vivo was correctly reflected in the transport parameters determined in vitro.

Role of the Intestinal Peptide Transporter PEPT1 in Oseltamivir Absorption: In Vitro and In Vivo Studies

It was reported that oseltamivir (Tamiflu) absorption was mediated by human peptide transporter (hPEPT) 1. Understanding the exact mechanism(s) of absorption is important in the context of drug-drug and diet-drug interactions. Hence, we investigated the mechanism governing the intestinal absorption of oseltamivir and its active metabolite (oseltamivir carboxylate) in wild-type [Chinese hamster ovary (CHO)-K1] and hPEPT1-transfected cells (CHO-PEPT1), in pharmacokinetic studies in juvenile and adult rats, and in healthy volunteers. In vitro cell culture studies showed that the intracellular accumulation of oseltamivir and its carboxylate into CHO-PEPT1 and CHO-K1 was always similar under a variety of experimental conditions, demonstrating that these compounds are not substrates of hPEPT1. Furthermore, neither oseltamivir nor its active metabolite was capable of inhibiting Gly-Sar uptake in CHO-PEPT1 cells. In vivo pharmacokinetic studies in juvenile and adult rats showed that the disposition of oseltamivir and oseltamivir carboxylate, after oral administration of oseltamivir, was sensitive to the feed status but insensitive to the presence of milk and Gly-Sar. Moreover, oseltamivir and oseltamivir carboxylate exhibited significantly higher exposure in rats under fasted conditions than under fed conditions. In humans, oral dosing after a high-fat meal resulted in a statistically significant but moderate lower exposure than after an overnight fasting. This change has no clinical implications. Taken together, the results do not implicate either rat Pept1 or hPEPT1 in the oral absorption of oseltamivir.

In vitro profiling of the metabolism and drug–drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP

Abstract 1. The metabolism and drug–drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

Discovery of Risdiplam, a Selective Survival of Motor Neuron-2 (SMN2) Gene Splicing Modifier for the Treatment of Spinal Muscular Atrophy (SMA)

SMA is an inherited disease that leads to loss of motor function and ambulation and a reduced life expectancy. We have been working to develop orally administrated, systemically distributed small molecules to increase levels of functional SMN protein. Compound 2 was the first SMN2 splicing modifier tested in clinical trials in healthy volunteers and SMA patients. It was safe and well tolerated and increased SMN protein levels up to 2-fold in patients. Nevertheless, its development was stopped as a precautionary measure because retinal toxicity was observed in cynomolgus monkeys after chronic daily oral dosing (39 weeks) at exposures in excess of those investigated in patients. Herein, we describe the discovery of 1 (risdiplam, RG7916, RO7034067) that focused on thorough pharmacology, DMPK and safety characterization and optimization. This compound is undergoing pivotal clinical trials and is a promising medicine for the treatment of patients in all ages and stages with SMA.

A Phase 1 healthy male volunteer single escalating dose study of the pharmacokinetics and pharmacodynamics of risdiplam (RG7916, RO7034067), a SMN2 splicing modifier: Risdiplam healthy volunteer Phase 1

Risdiplam (RG7916, RO7034067) is an orally administered, centrally and peripherally distributed, survival of motor neuron 2 (SMN2) mRNA splicing modifier for the treatment of spinal muscular atrophy (SMA). The objectives of this entry‐into‐human study were to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of risdiplam, and the effect of the strong CYP3A inhibitor itraconazole on the PK of risdiplam in healthy male volunteers.