Survey of pharmaceutical industry's best practices around in vitro transporter assessment and Implications for Drug Development: Considerations from the IQ Transporter Working Group.

The IQ Transporter Working Group had a rare opportunity to analyse a cross-pharma 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 pre-incubation 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 cross-transport inhibition (P-gp, BCRP, OATP1B and OCT1) with high molecular weight ({greater than or equal to}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 whilst 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, whilst 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 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.

Pharmacokinetics of Staccato® alprazolam in healthy adult participants in two phase 1 studies: An open-label smoker study and a randomized, placebo-controlled ethnobridging study.

OBJECTIVE: Staccato® alprazolam is a single-use, drug-device combination delivering alprazolam to the deep lung that is being evaluated as treatment for rapid and early seizure termination. This article reports pharmacokinetic (PK) data from two phase 1 studies of Staccato alprazolam in healthy adult participants. METHODS: The smoker study (EPK-002/NCT03516305) was an open-label, nonrandomized, single-dose, PK study in smokers and nonsmokers aged 21-50 years, administered a single inhaled dose of 1 mg Staccato alprazolam. The ethnobridging study (UP0101/NCT04782388) was a double-blind, placebo-controlled study in Japanese, Chinese, and Caucasian participants aged 18-55 years randomized 4:1 to a single inhaled dose of Staccato alprazolam 2 mg or Staccato placebo. RESULTS: In the smoker study, 36 participants (18 smokers, 18 nonsmokers) were enrolled and received Staccato alprazolam. Following Staccato administration, alprazolam was rapidly absorbed, with a median time to peak drug plasma concentration (Tmax) of 2 min in both smokers (range = 2-30 min) and nonsmokers (range = 2-60 min). Staccato alprazolam was rapidly absorbed to a similar extent in both smokers and nonsmokers. The most commonly reported treatment-emergent adverse events (TEAEs) were somnolence and dizziness. In the ethnobridging study, 10 participants each of Japanese, Chinese, and Caucasian ethnicities were randomized 4:1 to Staccato alprazolam or Staccato placebo. Following Staccato administration, alprazolam was rapidly absorbed and distributed, with a median Tmax of 1.5-2 min in Japanese (range = 1-2 min), Chinese (range = 1-34 min), and Caucasian (range = 1-120 min) participants. Somnolence and sedation were the most commonly reported TEAEs. In both studies, there were no deaths, and no participants reported serious or severe TEAEs, or discontinued due to TEAEs. SIGNIFICANCE: Alprazolam was rapidly absorbed, and therapeutic drug levels were achieved within 2 min postdose when administered to the lung with the Staccato device. Staccato alprazolam was generally well tolerated and displayed a safety profile consistent with that known from other alprazolam applications. No new safety signals were identified.

Clinical Bridging Studies and Modeling Approach for Implementation of a Patient Centric Sampling Technique in Padsevonil Clinical Development.

Volumetric absorptive microsampling (VAMS) techniques have gained popularity these last years as innovative tool for collection of blood pharmacokinetic (PK) samples in clinical trials as they offer many advantages over dried blood spot and conventional venous blood sampling. The use of Mitra®, a blood collection device based on volumetric absorptive microsampling (VAMS) technology, was implemented during clinical development of padsevonil (PSL), an anti-seizure medication (ASM) candidate. The present study describes the approach used to bridge plasma (obtained from conventional venous blood sampling) and blood exposures (obtained with Mitra®) to support the use of Mitra as sole blood PK sampling method in clinical trials. Paired blood (using Mitra®) and plasma samples (using conventional venous blood sampling) were collected in healthy volunteers as well as in patients with epilepsy. PSL concentration in plasma and blood were analyzed using different approaches which included evaluation of blood-to-plasma ratios (B/P) over time, linear regression, Bland-Altman analysis as well as development of a linear-mixed effect model based on clinical pharmacology studies. Results showed that the observed in vivo B/P and the measured bias between the 2 collection methods were consistent with the measured in vitro B/P. Graphical analysis demonstrated a clear time effect on the B/P which was confirmed in the linear mixed effect model with sampling time identified as significant covariate. Finally, the built-in model was validated using independent datasets and was shown to adequately predict plasma concentration based on blood concentration with a mean bias of less than 9% (predicted versus observed plasma concentration).

Drug clearance by aldehyde oxidase: can we avoid clinical failure?

Despite increased awareness of aldehyde oxidase (AO) as a major drug-metabolising enzyme, predicting the pharmacokinetics of its substrates remains challenging. Several drug candidates have been terminated due to high clearance, which were subsequently discovered to be AO substrates. Even retrospective extrapolation of human clearance, from models more sensitive to AO activity, often resulted in underprediction.The questions of the current work thus were: Is there an acceptable degree of in vitro AO metabolism that does not result in high in vivo human clearance? And, if so, how can this be predicted?We built an in vitro/in vivo correlation using known AO substrates, combining multiple in vitro parameters to calculate the blood metabolic clearance mediated by AO (CLbAO). This value was compared with observed blood clearance (CLb-obs), establishing cut-off CLbAO values, to discriminate between low and high CLb-obs. The model was validated using additional literature compounds, and CLb-obs was predicted in the correct category.This simple, categorical, semi-quantitative yet multi-factorial model is readily applicable in drug discovery. Further, it is valuable for high-clearance compounds, as it predicts the CLb group, rather than an exact CLb value, for the substrates of this poorly-characterised enzyme.

Current Practices, Gap Analysis, and Proposed Workflows for PBPK Modeling of Cytochrome P450 Induction: An Industry Perspective.

The International Consortium for Innovation and Quality (IQ) Physiologically Based Pharmacokinetic (PBPK) Modeling Induction Working Group (IWG) conducted a survey across participating companies around general strategies for PBPK modeling of induction, including experience with its utility to address various questions, regulatory interactions, and regulatory acceptance. The results highlight areas where PBPK modeling is used with high confidence and identifies opportunities where confidence is lower and further evaluation is needed. To enhance the survey results, the PBPK-IWG also collected case studies and analyzed recent literature examples where PBPK models were applied to predict CYP3A induction-mediated drug-drug interactions. PBPK modeling of induction has evolved and progressed significantly, proving to have great potential to accelerate drug discovery and development. With the aim of enabling optimal use for new molecular entities that are either substrates and/or inducers of CYP3A, the PBPK-IWG proposes initial workflows for PBPK application, discusses future trends, and identifies gaps that need to be addressed.

Designing Out PXR Activity on Drug Discovery Projects: A Review of Structure-Based Methods, Empirical and Computational Approaches.

This perspective discusses the role of pregnane xenobiotic receptor (PXR) in drug discovery and the impact of its activation on CYP3A4 induction. The use of structural biology to reduce PXR activity on drug discovery projects has become more common in recent years. Analysis of this work highlights several important molecular interactions, and the resultant structural modifications to reduce PXR activity are summarized. The computational approaches undertaken to support the design of new drugs devoid of PXR activation potential are also discussed. Finally, the SAR of empirical design strategies to reduce PXR activity is reviewed, and the key SAR transformations are discussed and summarized. In conclusion, this perspective demonstrates that PXR activity can be greatly diminished or negated on active drug discovery projects with the knowledge now available. This perspective should be useful to anyone who seeks to reduce PXR activity on a drug discovery project.

Use of a physiologically based pharmacokinetic-pharmacodynamic model for initial dose prediction and escalation during a paediatric clinical trial.

AIMS: To build and verify a physiologically based pharmacokinetic (PBPK) model for radiprodil in adults and link this to a pharmacodynamic (PD) receptor occupancy (RO) model derived from in vitro data. Adapt this model to the paediatric population and predict starting and escalating doses in infants based on RO. Use the model to guide individualized dosing in a clinical trial in 2- to 14-month-old children with infantile spasms. METHODS: A PBPK model for radiprodil was developed to investigate the systemic exposure of the drug after oral administration in fasted and fed adults; this was then linked to RO via a PD model. The model was then expanded to include developmental physiology and ontogeny to predict escalating doses in infants that would result in a specific RO of 20, 40 and 60% based on average unbound concentration following a twice daily (b.i.d.) dosing regimen. Dose progression in the clinical trial was based on observed concentration-time data against PBPK predictions. RESULTS: For paediatric predictions, the elimination of radiprodil, based on experimental evidence, had no ontogeny. Predicted b.i.d. doses ranged from 0.04 mg/kg for 20% RO, 0.1 mg/kg for 40% RO to 0.21 mg/kg for 60% RO. For all infants recruited in the study, observed concentration-time data following the 0.04 mg/kg and subsequent doses were within the PBPK model predicted 5th and 95th percentiles. CONCLUSION: To our knowledge, this is the first time a PBPK model linked to RO has been used to guide dose selection and escalation in the live phase of a paediatric clinical trial.

Impact of In Vitro Passive Permeability in a P-gp-transfected LLC-PK1 Model on the Prediction of the Rat and Human Unbound Brain-to-Plasma Concentration Ratio.

PURPOSE: More accurate prediction of the extent of drug brain exposure in early drug discovery and understanding potential species differences could help to guide medicinal chemistry and avoid unnecessary animal studies. Hence, the aim of the current study was to validate the use of a P-gp transfected LLC-PK1 model to predict the unbound brain-to-plasma concentration ratio (Kpuu,brain) in rats and humans. METHODS: MOCK-, Mdr1a- and MDR1-transfected LLC-PK1 monolayers were applied in a transwell setup to quantify the bidirectional transport for 12 specific P-gp substrates, 48 UCB drug discovery compounds, 11 compounds with reported rat in situ brain perfusion data and 6 compounds with reported human Kpuu,brain values. The in vitro transport data were introduced in a minimal PBPK model (SIVA®) to determine the transport parameters. These parameters were combined with the differences between in vitro and in vivo passive permeability as well as P-gp expression levels (as determined by LC-MS/MS), to predict the Kpuu,brain. RESULTS: A 10-fold difference between in vitro and in vivo passive permeability was observed. Incorporation of the differences between in vitro and in vivo passive permeability and P-gp expression levels resulted in an improved prediction of rat (AAFE 2.17) and human Kpuu,brain (AAFE 2.10). CONCLUSIONS: We have succesfully validated a methodology to use a P-gp overexpressing LLC-PK1 cell line to predict both rat and human Kpuu,brain by correcting for both passive permeability and P-gp expression levels.

(-)-N-3-Benzylphenobarbital Is Superior to Omeprazole and (+)-N-3-Benzylnirvanol as a CYP2C19 Inhibitor in Suspended Human Hepatocytes.

Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (-)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (-)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (-)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (-)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19 The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19 Overall, this study confirms the selective CYP2C19 inhibition by (-)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (-)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT: (-)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (-)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.

Application of Azamulin to Determine the Contribution of CYP3A4/5 to Drug Metabolic Clearance Using Human Hepatocytes.

Early determination of CYP3A4/5 contribution to the clearance of new chemical entities is critical to inform on the risk of drug-drug interactions with CYP3A inhibitors and inducers. Several in vitro approaches (recombinant P450 enzymes, correlation analysis, chemical and antibody inhibition in human liver microsomes) are available, but they are usually labor-intensive and/or suffer from specific limitations. In the present study, we have validated the use of azamulin as a specific CYP3A inhibitor in human hepatocytes. Azamulin (3 µM) was found to significantly inhibit CYP3A4/5 (>90%), whereas other P450 enzymes were not affected (less than 20% inhibition). Because human hepatocytes were used as a test system, the effect of azamulin on other key drug-metabolizing enzymes (aldehyde oxidase, carboxylesterase, UGT, flavin monooxygenase, and sulfotransferase) was also investigated. Apart from some UGTs showing minor inhibition (∼20%-30%), none of these non-P450 enzymes were inhibited by azamulin. Use of CYP3A5-genotyped human hepatocyte batches in combination with CYP3cide demonstrated that azamulin (at 3 µM) inhibits both CYP3A4 and CYP3A5 enzymes. Finally, 11 compounds with known in vivo CYP3A4/5 contribution have been evaluated in this human hepatocyte assay. Results showed that the effect of azamulin on the in vitro intrinsic clearance of these known CYP3A4/5 substrates was predictive of the in vivo CYP3A4/5 contribution. Overall, the study showed that human hepatocytes treated with azamulin provide a fast and accurate estimation of CYP3A4/5 contribution in metabolic clearance of new chemical entities. SIGNIFICANCE STATEMENT: Accurate estimation of CYP3A4/5 contribution in drug clearance is essential to anticipate risk of drug-drug interactions and select the appropriate candidate for clinical development. The present study validated the use of azamulin as selective CYP3A4/5 inhibitor in suspended human hepatocytes and demonstrated that this novel approach provides a direct and accurate determination of the contribution of CYP3A4/5 (fraction metabolized by CYP3A4/5) in the metabolic clearance of new chemical entities.

A review of the drug-drug interactions of the antiepileptic drug brivaracetam.

Brivaracetam is an antiepileptic drug (AED) indicated for the treatment of focal seizures, with improved safety and tolerability vs first-generation AEDs. Brivaracetam binds with high affinity to synaptic vesicle protein 2A in the brain, which confers its antiseizure activity. Brivaracetam is rapidly absorbed and extensively biotransformed, and exhibits linear and dose-proportional pharmacokinetics at therapeutic doses. Brivaracetam does not interact with most metabolizing enzymes and drug transporters, and therefore does not interfere with drugs that use these metabolic routes. The favorable pharmacokinetic profile of brivaracetam and lack of clinically relevant drug-drug interactions with commonly prescribed AEDs or oral contraceptives allows administration without dose adjustment, and avoids potential untoward events from decreased efficacy of an AED or oral contraceptive due to a drug-drug interaction. Few agents have been reported to affect the pharmacokinetics of brivaracetam. The strong enzyme-inducing AEDs carbamazepine, phenytoin and phenobarbital/primidone have been shown to moderately lower brivaracetam plasma concentrations, with no adjustment of brivaracetam dose needed. Dose adjustment should be considered when brivaracetam is coadministered with the more potent CYP inducer, rifampin. Additionally, caution should be used when adding or ending treatment with the strong enzyme inducer, St. John's wort. In summary, brivaracetam (50-200 mg/day) has a favorable pharmacokinetic profile and is associated with few clinically relevant drug-drug interactions.

Role of P-glycoprotein in the brain disposition of seletalisib: Evaluation of the potential for drug-drug interactions.

Seletalisib is an orally bioavailable selective inhibitor of phosphoinositide 3-kinase delta (PI3Kδ) in clinical development for the treatment of immune-mediated inflammatory diseases. The present study investigated the role of P-gp in seletalisib disposition, especially brain distribution, and the associated risks of interactions. Seletalisib was found to be actively transported by rodent and human P-gp in vitro (transfected LLC-PK1 cells; Km of ca. 20 µM), with minimal or no affinity for the other tested transporters. A distribution study in knockout rats (single oral dosing at 750 mg kg-1) showed that P-gp restricts the brain disposition of seletalisib while having minimal effect on its intestinal absorption. Restricted brain penetration was also observed in cynomolgus monkeys (single oral dosing at 30 mg kg-1) using brain microdialysis and cerebrospinal fluid sampling (Kp,uu of 0.09 and 0.24, respectively). These findings opened the question of potential pharmacokinetic interaction between seletalisib and P-gp inhibitors. In vitro, CsA inhibited the active transport of seletalisib with an IC50 of 0.13 µM. In rats, co-administration of high doses of CsA (bolus iv followed by continuous infusion) increased the brain distribution of seletalisib (single oral dosing at 5 mg kg-1). The observed data were found aligned with those predicted by in vitro-in vivo extrapolation. Based on the same extrapolation method combined with literature data, only very few P-gp inhibitors (i.e. CsA, quinine, quinidine) were predicted to increase the brain disposition of seletalisib in the clinical setting (maximal 3-fold changes).

Off-line solid phase extraction and liquid chromatography-tandem mass spectrometry method for the quantitation of brivaracetam acid metabolites: Method validation and application to in vitro metabolism assays.

Brivaracetam (BRV) is a new high affinity synaptic vesicle protein 2A ligand recently approved for adults with partial-onset seizures. As a support to in vitro metabolism assays, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method coupled to off-line solid phase extraction (SPE) was developed to quantify BRV acid metabolites, that is, BRV-AC (carboxylic derivative derived from BRV hydrolysis) and BRV-OHAC (corresponding to hydroxylated BRV-AC). The method was validated for various incubates (liver and kidney tissue homogenates and blood, all from humans) and applied to in vitro metabolism assays. The analytes were isolated from buffered samples using ISOLUTE C8 96-well SPE plates. Chromatographic separation was achieved on a Waters Atlantis T3 C18 analytical column (2.1 mm × 50 mm, 5 µm) with detection accomplished using a Waters Premier tandem mass spectrometer in positive ion electrospray and multiple reaction monitoring (MRM) mode. The standard curves, which ranged from 1.00 to 200 ng/mL for BRV-AC, BRV-OHAC, were fitted to a 1/x2 weighted linear regression model. The intra-assay precision and inter-assay precision (expressed as coefficient of variation -%CV) were <8.5%, and the assay accuracy (deviation - %Dev) was within ±7.1% for the different matrices. This accurate, precise, and selective SPE/LC-MS/MS method has been successfully applied to in vitro assays aimed at characterizing the kinetics of BRV hydrolysis. BRV was found to be a better substrate for hydrolysis than its hydroxylated metabolite BRV-OH. BRV hydrolysis was detected in blood, liver and kidneys, demonstrating the broad distribution of the enzyme catalyzing the reaction.

Role of the OATP Transporter Family and a Benzbromarone-SensitiveEfflux Transporter in the Hepatocellular Disposition of Vincristine.

PURPOSE: Vincristine is known to interfere with OATP-mediated uptake of other compounds, hinting that vincristine itself could be a substrate of OATP transporters. The present study therefore aimed to investigate the role of OATP transporters in the hepatocellular disposition of vincristine. METHODS: Vincristine uptake was studied in suspended rat and human hepatocytes as well as OATP-transfected Chinese hamster ovary (CHO) cells in the absence and presence of OATP transporter inhibitors. Membrane vesicles containing MDR1 or MRP1/2/3 were used to directly assess the role of these efflux transporters in vincristine disposition. RESULTS: Uptake in suspended rat hepatocytes was temperature-dependent and could be inhibited by a range of OATP inhibitors. Furthermore, the MRP-inhibitor benzbromarone, but none of the tested MDR1 inhibitors, reduced vincristine efflux in rat and human suspended hepatocytes. OATP1B1-, OATP1B3- and OATP2B1- transfected CHO cells showed significantly increased vincristine uptake as compared to wild-type cells. Moreover, uptake in OATP-transfected CHO cells was reduced by OATP inhibitors. However, uptake studies in suspended human hepatocytes showed that only 10% of the total vincristine uptake process could be attributed to OATP-mediated transport. Studies with transporter-expressing membrane vesicles confirmed vincristine as an MDR1 substrate, while MRP1/2/3-mediated transport of vincristine could not be observed with this model system. CONCLUSIONS: Our findings show the involvement of OATP transporters in the disposition of vincristine in rat and human hepatocytes. However, in both species, hepatic uptake is overshadowed by a benzbromarone-sensitive efflux mechanism, possibly MRP3.

A Dual-Administration Microtracer Technique to Characterize the Absorption, Distribution, Metabolism, and Excretion of [14 C]Seletalisib (UCB5857) in Healthy Subjects.

Phosphoinositide 3 kinases are targets for development of small-molecule inhibitors to disrupt progression of immune-inflammatory diseases. This phase 1 open-label study (Eudract 2014-005353-39) evaluated the safety and relative bioavailability of 2 new seletalisib (UCB5857) formulations (A and B) compared with a reference formulation. Absolute bioavailability (period 1a, n = 6) and disposition and metabolism (period 1b, n = 6) of the reference formulation were evaluated: healthy subjects received 30 mg orally plus ∼20 µg of a 14 C-labeled microtracer (intravenously in 1a, orally in 1b). New formulations were evaluated: subjects from periods 1a and 1b were pooled and randomly distributed to receive a single oral dose (30 mg) of formulation A (n = 6) or B (n = 6) in periods 2 and 3, using a crossover design. Absolute oral bioavailability of seletalisib was 97% (90% confidence interval 87, 107). Unchanged [14 C]seletalisib was the predominant radioactive component in plasma (94.8%). After oral dosing, the radioactive dose was primarily recovered in feces (74.6%, geometric coefficient of variation [GeoCV] 18.1%), mostly as metabolites. Seletalisib demonstrated a 24-hour terminal half-life, volume of distribution of 60.9 L (GeoCV 23.8%), and a total plasma clearance of 1.7 L/h (GeoCV 35.4%). Formulations A and B displayed similar or even higher exposure compared with reference seletalisib (areas under the concentration-time curves 19 337 [GeoCV 30.8%], 20 380 [GeoCV 37.7%], and 15 932 [GeoCV 36.4%] h·ng/mL, respectively). New formulations A and B were bioequivalent with each other, and all 3 formulations showed acceptable safety profiles. This radiolabeled microtracer approach successfully informed on the absorption, distribution, metabolism, and excretion of seletalisib and further guided the mechanistic pharmacokinetic modeling.

Prediction of drug-drug interactions with carbamazepine-10,11-epoxide using a new in vitro assay for epoxide hydrolase inhibition.

1. Carbamazepine is an antiepileptic drug which is metabolized by CYP3A4 into carbamazepine-10,11-epoxide. This metabolite is then detoxified by epoxide hydrolase. As carbamazepine-10,11-epoxide has been associated with neurotoxicity, it is critical to identify whether a new antiepileptic drug has the potential to inhibit epoxide hydrolase and therefore increase carbamazepine-10,11-epoxide plasma levels. 2. In this study, an in vitro assay was developed to evaluate epoxide hydrolase activity by using carbamazepine-10,11-epoxide as probe substrate. The ability of this assay to predict drug-drug interactions (DDI) at the epoxide hydrolase level was also investigated. 3. To this aim, known inhibitors of epoxide hydrolase for which in vivo data are available were used. Firstly, carbamazepine-10,11-epoxide hydrolase activity was determined in liver microsomes, cytosol and hepatocytes. Thereafter, the IC50 of epoxide hydrolase inhibitors (progabide, valproic acid, valpromide and valnoctamide) was determined in liver microsomes and hepatocytes. Finally, prediction of AUC increase was performed using the in vitro data generated. 4. Interestingly, epoxide hydrolase activity was found to be much higher in human hepatocytes compared to liver microsomes/cytosol. Even though assessed on a limited number of compounds, this study demonstrated that the use of hepatocytes seems to be a more relevant model to assess and predict DDI at the epoxide hydrolase level.

Effect of Rifampin on the Disposition of Brivaracetam in Human Subjects: Further Insights into Brivaracetam Hydrolysis.

Brivaracetam (BRV) is a high-affinity synaptic vesicle protein 2A ligand developed for the treatment of uncontrolled partial-onset seizures. The present phase I, open-label, two-way crossover study was designed to assess the effect of rifampin on the pharmacokinetics of BRV and its hydroxy (BRV-OH), acid (BRV-AC), and hydroxy acid (BRV-OHAC) metabolites. Twenty-six healthy subjects received BRV (150-mg single oral dose) either alone or following 5 days of rifampin 600 mg/day. BRV and its metabolites were examined for their plasma profiles and urinary excretion. Pharmacokinetic modeling was developed to estimate the rate constants of the various metabolic routes. Parallel in vitro assays were conducted to characterize the hydrolysis of BRV to BRV-AC as well as to identify any potential effect of rifampin on the hydrolysis reaction. Rifampin did not significantly affect the maximum plasma concentration (Cmax) of BRV, but decreased its area under the curve (AUC) by 45%. In addition, rifampin significantly increased the AUC of BRV-OH (+109%), decreased the AUC of BRV-AC (-53%), but had little effect on BRV-OHAC (-10%). In vitro assays showed that the major urinary metabolite BRV-AC (33% of the dose) was likely to be formed by amidase EC 3.5.1.4. In vitro data indicated that the enzyme was not significantly inhibited nor induced by rifampin. Modeling confirmed that all of the observed changes in vivo were secondary to the induction of the CYP2C19-mediated hydroxylation of BRV to BRV-OH (3.7-fold increase in the rate constant).

Brivaracetam, a selective high-affinity synaptic vesicle protein 2A (SV2A) ligand with preclinical evidence of high brain permeability and fast onset of action.

OBJECTIVE: Rapid distribution to the brain is a prerequisite for antiepileptic drugs used for treatment of acute seizures. The preclinical studies described here investigated the high-affinity synaptic vesicle glycoprotein 2A (SV2A) antiepileptic drug brivara-cetam (BRV) for its rate of brain penetration and its onset of action. BRV was compared with levetiracetam (LEV). METHODS: In vitro permeation studies were performed using Caco-2 cells. Plasma and brain levels were measured over time after single oral dosing to audiogenic mice and were correlated with anticonvulsant activity. Tissue distribution was investigated after single dosing to rat (BRV and LEV) and dog (LEV only). Positron emission tomography (PET) displacement studies were performed in rhesus monkeys using the SV2A PET tracer [11C]UCB-J. The time course of PET tracer displacement was measured following single intravenous (IV) dosing with LEV or BRV. Rodent distribution data and physiologically based pharmacokinetic (PBPK) modeling were used to compute blood-brain barrier permeability (permeability surface area product, PS) values and then predict brain kinetics in man. RESULTS: In rodents, BRV consistently showed a faster entry into the brain than LEV; this correlated with a faster onset of action against seizures in audiogenic susceptible mice. The higher permeability of BRV was also demonstrated in human cells in vitro. PBPK modeling predicted that, following IV dosing to human subjects, BRV might distribute to the brain within a few minutes compared with approximately 1 h for LEV (PS of 0.315 and 0.015 ml/min/g for BRV and LEV, respectively). These data were supported by a nonhuman primate PET study showing faster SV2A occupancy by BRV compared with LEV. SIGNIFICANCE: These preclinical data demonstrate that BRV has rapid brain entry and fast brain SV2A occupancy, consistent with the fast onset of action in the audiogenic seizure mice assay. The potential benefit of BRV for treatment of acute seizures remains to be confirmed in clinical studies.

Cross-Species Differences in the Preclinical Pharmacokinetics of CT7758, an α4ß1/α4ß7 Integrin Antagonist.

CT7758, a carboxylate containing α4ß1/α4/ß7 integrin antagonist, was characterized for its pharmacokinetic profile in various in vitro and in vivo assays in support of clinical development. The oral bioavailability of CT7758 was 4% in mice, 2% in rats, 7-55% in dogs, and 0.2% in cynomolgus monkeys. The low bioavailability in rodents and monkey results from low intestinal absorption as evidenced by a low fraction absorbed in the rat portal vein model (3%), low-to-medium permeability in Caco-2 cells (≤1.3 × 10(-6) cm/s) with evidences of polarized efflux, and high polar surface area (104 Å). In rodents and cynomolgus monkeys, the total plasma clearance was moderate to high (≥50% hepatic blood flow QH) and associated with a short elimination half-life (≤1 hour). This contrast with the dog data which showed a much lower clearance (6% QH) and a longer t1/2 (2.4 hours). The volume of distribution (Vz) also varied significantly across species with value of 5.5, 2.8, 0.24, and 0.93 l/kg in mouse, rat, dog, and cynomolgus monkey, respectively. In vitro assays demonstrated that active hepatic uptake accounted for most of the in vivo clearance and was the source of the large species variability. In vitro uptake assays predicted a total plasma clearance in humans in the low range (33% QH), a finding subsequently confirmed in the clinic. Assays in OAPT1B1-transfected cells demonstrated active uptake transport through this transporter. The prospect of limited absorption in human prompted the synthesis an ethyl ester prodrug, CDP323, which demonstrated higher in vitro permeability, increased oral bioavailability, as well as efficient in vivo release of its active moiety CT7758.

Brivaracetam and carbamazepine interaction in healthy subjects and in vitro.

This phase I, open-label study investigated the effects of steady-state brivaracetam administration on steady-state pharmacokinetics of carbamazepine, and steady-state carbamazepine administration on single-dose and steady-state pharmacokinetics of brivaracetam, in 14 healthy participants who received brivaracetam 200mg single doses on days 1 and 22, and 200mg twice daily (bid) on days 24-35; and were titrated to carbamazepine 300mg bid on days 4-35. Brivaracetam did not significantly alter carbamazepine area under the plasma concentration-time curve (AUC) over a dosing interval, but resulted in a 2.6-fold increase in carbamazepine-epoxide. Carbamazepine decreased brivaracetam AUC by 29%, while hydroxy-brivaracetam metabolite was increased by 17%. Urinary 6ß-hydroxycortisol/cortisol ratio was unchanged by brivaracetam, but was increased 3-fold by carbamazepine. In vitro hydrolysis of carbamazepine-epoxide in human hepatocytes was inhibited by brivaracetam, with an IC50 of 8.2µM. Brivaracetam 200mg bid was predicted to increase carbamazepine-epoxide by 2.3-fold, in close agreement with the observed value. In conclusion, brivaracetam did not modify carbamazepine exposure but increased carbamazepine-epoxide. Carbamazepine modestly decreased brivaracetam exposure and increased oxidative metabolism.