The Interplay of Permeability, Metabolism, Transporters, and Dosing in Determining the Dynamics of the Tissue/Plasma Partition Coefficient and Volume of Distribution-A Theoretical Investigation Using Permeability-Limited, Physiologically Based Pharmacokinetic Modeling.

A permeability-limited physiologically based pharmacokinetic (PBPK) model featuring four subcompartments (corresponding to the intracellular and extracellular water of the tissue, the residual plasma, and blood cells) for each tissue has been developed in MATLAB/SimBiology and applied to various what-if scenario simulations. This model allowed us to explore the complex interplay of passive permeability, metabolism in tissue or residual blood, active uptake or efflux transporters, and different dosing routes (intravenous (IV) or oral (PO)) in determining the dynamics of the tissue/plasma partition coefficient (Kp) and volume of distribution (Vd) within a realistic pseudo-steady state. Based on the modeling exercise, the permeability, metabolism, and transporters demonstrated significant effects on the dynamics of the Kp and Vd for IV bolus administration and PO fast absorption, but these effects were not as pronounced for IV infusion or PO slow absorption. Especially for low-permeability compounds, uptake transporters were found to increase both the Kp and Vd at the pseudo-steady state (Vdss), while efflux transporters had the opposite effect of decreasing the Kp and Vdss. For IV bolus administration and PO fast absorption, increasing tissue metabolism was predicted to elevate the Kp and Vdss, which contrasted with the traditional derivation from the steady-state perfusion-limited PBPK model. Moreover, metabolism in the residual blood had more impact on the Kp and Vdss compared to metabolism in tissue. Due to its ability to offer a more realistic description of tissue dynamics, the permeability-limited PBPK model is expected to gain broader acceptance in describing clinical PK and observed Kp and Vdss, even for certain small molecules like cyclosporine, which are currently treated as perfusion-limited in commercial PBPK platforms.

When special populations intersect with drug-drug interactions: Application of physiologically-based pharmacokinetic modeling in pregnant populations.

Pregnancy results in significant physiological changes that vary across trimesters and into the postpartum period, and may result in altered disposition of endogenous substances and drug pharmacokinetics. Pregnancy represents a unique special population where physiologically-based pharmacokinetic modeling (PBPK) is well suited to mechanistically explore pharmacokinetics and dosing paradigms without subjecting pregnant women or their fetuses to extensive clinical studies. A critical review of applications of pregnancy PBPK models (pPBPK) was conducted to understand its current status for prediction of drug exposure in pregnant populations and to identify areas of further expansion. Evaluation of existing pPBPK modeling efforts highlighted improved understanding of cytochrome P450 (CYP)-mediated changes during pregnancy and identified knowledge gaps for non-CYP enzymes and the physiological changes of the postpartum period. Examples of the application of pPBPK beyond simple dose regimen recommendations are limited, particularly for prediction of drug-drug interactions (DDI) or differences between genotypes for polymorphic drug metabolizing enzymes. A raltegravir pPBPK model implementing UGT1A1 induction during the second and third trimesters of pregnancy was developed in the current work and verified against clinical data. Subsequently, the model was used to explore UGT1A1-related DDI risk with atazanavir and rifampicin along with the effect of enzyme genotype on raltegravir apparent clearance. Simulations of pregnancy-related induction of UGT1A1 either exacerbated UGT1A1 induction by rifampicin or negated atazanavir UGT1A1 inhibition. This example illustrated the advantages of pPBPK modeling for mechanistic evaluation of complex interplays of pregnancy- and drug-related effects in support of model-informed approaches in drug development.

Antibody Subunit LC-MS Analysis for Pharmacokinetic and Biotransformation Determination from In-Life Studies for Complex Biotherapeutics.

Complex biotherapeutics present challenges from drug discovery, screening, and development perspectives. While monoclonal antibody drugs are not monitored for metabolites in the same manner as small molecules, biotherapeutics such as fusion proteins, antibody-drug conjugates, or bispecific antibodies may undergo biotransformation (such as clipping, deamidation, or oxidation) in vivo, resulting in catabolites that can have a direct impact on drug safety or efficacy. Here antibody subunit LC-MS is utilized for evaluation of two classes of complex biotherapeutics: an antibody-drug conjugate and a mAb-fusion biotherapeutic. Pharmacokinetic concentration, biotransformation, and DAR data are collectively presented using the subunit LC-MS approach for the two molecules, and the methods shared in detail can be applied to any humanized IgG1 mAb biotherapeutic for preclinical study support. Overall, the data generated from antibody LC-MS analyses can provide key information in early phase development and deliver multiple study end points with a single data set.

Hepatobiliary Disposition of Atovaquone: A Case of Mechanistically Unusual Biliary Clearance.

Atovaquone, an antiprotozoal and antipneumocystic agent, is predominantly cleared by biliary excretion of unchanged parent drug. Atovaquone is ≥10,000-fold concentrated in human bile relative to unbound plasma. Even after correcting for apparent nonspecific binding and incomplete solubility in bile, atovaquone is still concentrated ≥100-fold in bile, consistent with active biliary excretion. Mechanisms of atovaquone hepatobiliary disposition were studied using a multiexperimental in vitro and in vivo approach. Atovaquone uptake was not elevated in HEK293 cells singly overexpressing OATP1B1, OATP1B3, OATP2B1, OCT1, NTCP, or OAT2. Hepatocyte uptake of atovaquone was not impaired by OATP and OCT inhibitor cocktail (rifamycin and imipramine). Atovaquone liver-to-blood ratio at distributional equilibrium was not reduced in Oatp1a/1b and Oct1/2 knockout mice. Atovaquone exhibited efflux ratios of approximately unity in P-gp and BCRP overexpressing MDCK cell monolayers and did not display enhanced uptake in MRP2 vesicles. Biliary and canalicular clearance were not decreased in P-gp, Bcrp, Mrp2, and Bsep knockout rats. In the present study, we rule out the involvement of major known basolateral uptake and bile canalicular efflux transporters in the hepatic uptake and biliary excretion of atovaquone. This is the first known example of a drug cleared by biliary excretion in humans, with extensive biliary concentration, which is not transported by the mechanisms investigated herein.

Effect of Activated Charcoal on Mavacamten Pharmacokinetics in Healthy Participants.

OBJECTIVE: To assess the effect of activated charcoal on the single-dose pharmacokinetics of mavacamten when administered 2 h or 6 h after mavacamten dosing. METHODS: In this open-label, randomized, parallel-group study, healthy adults were randomized into three groups to receive mavacamten 15 mg alone or mavacamten 15 mg plus activated charcoal 50 g administered either 2 h or 6 h after mavacamten dosing. Pharmacokinetic parameters were derived from plasma concentration-time data using noncompartmental methods. RESULTS: Of the 45 participants randomized, 37 completed the study. When activated charcoal was administered 2 h after mavacamten dosing, mavacamten absorption and exposure were reduced compared with when mavacamten was administered alone: the area under the concentration-time curve from 0 to 72 h (AUC0-72) and area under the concentration-time curve from time 0 extrapolated to infinity (AUCINF) were reduced by 14% and 34%, respectively. The maximum plasma concentration (Cmax) was also slightly lower when activated charcoal was administered 2 h after mavacamten dosing than with mavacamten alone. Pharmacokinetic profiles were similar for mavacamten alone and mavacamten plus activated charcoal administered 6 h after mavacamten dosing. CONCLUSIONS: Activated charcoal was successful in reducing mavacamten absorption and exposure when administered as soon as possible after identification of a need for adsorption (2 h after mavacamten dosing). No change in exposure was observed when activated charcoal was administered 6 h after mavacamten dosing. CLINICAL TRIAL REGISTRATION: NCT05320094.

Bioavailability, metabolism and disposition of oral pazopanib in patients with advanced cancer.

1. Pazopanib (Votrient) is an oral tyrosine kinase inhibitor that was recently approved for the treatment of renal cell carcinoma and soft tissue sarcoma. 2. In this two-part study, we investigated the metabolism, disposition of [(14)C]pazopanib, and the oral bioavailability of pazopanib tablets in patients with advanced cancer. 3. In part A, three men each received a single oral dose of [(14)C]pazopanib in suspension (400 mg, 70 µCi). Pazopanib was the predominant drug-related component in circulation. Two metabolites derived from hydroxylation and one from N-demethylation were also circulating, but were minor, each accounting for <5% of plasma radioactivity. Faecal elimination predominated, accounting for 82.2% of the administered radio-dose, with negligible renal elimination (2.6% of dose). Pazopanib was primarily excreted as the unchanged drug in faeces (67% of dose). 4. In part B, seven additional patients received a single intravenous administration of 5 mg pazopanib (day 1) followed by oral administration of 800 mg pazopanib tablet once daily for 26 days (days 3 or 5-28). In the three evaluable patients from part B, pazopanib had a slow plasma clearance and a small volume of distribution. The absolute oral bioavailability of the 800 mg pazopanib tablet ranged from 14% to 39%.

Physiologically Based Pharmacokinetic Modeling and Simulation of Mavacamten Exposure with Drug-Drug Interactions from CYP Inducers and Inhibitors by CYP2C19 Phenotype.

Mavacamten is a first-in-class, oral, selective, allosteric, reversible cardiac myosin inhibitor approved by the US Food and Drug Administration for the treatment of adults with symptomatic New York Heart Association functional class II-III obstructive hypertrophic cardiomyopathy. Mavacamten is metabolized in the liver, predominantly via cytochrome P450 (CYP) enzymes CYP2C19 (74%), CYP3A4 (18%), and CYP2C9 (8%). A physiologically-based pharmacokinetic (PBPK) model was developed using Simcyp version 19 (Certara, Princeton, NJ). Following model verification, the PBPK model was used to explore the effects of strong CYP3A4 and CYP2C19 inducers, and strong, moderate, and weak CYP2C19 and CYP3A4 inhibitors on mavacamten pharmacokinetics (PK) in a healthy population, with the effect of CYP2C19 phenotype predicted for poor, intermediate, normal, and ultrarapid metabolizers. The PBPK model met the acceptance criteria for all verification simulations (> 80% of model-predicted PK parameters within 2-fold of those observed clinically). A weak induction effect was predicted when mavacamten was administered with a strong CYP3A4 inducer in poor metabolizers. Moderate reductions in mavacamten exposure were predicted with a strong CYP2C19/CYP3A4 inducer in all CYP2C19 phenotypes. Except for the effect of strong CYP2C19 inhibitors on ultrarapid metabolizers, steady-state area under plasma concentration-time curve and maximum plasma concentration values were weakly affected (< 2-fold) or not affected (< 1.25-fold), regardless of CYP2C19 phenotype. In conclusion, a fit-for-purpose PBPK model was developed and verified, which accurately predicted the available clinical data and was used to simulate the potential impact of CYP induction and inhibition on mavacamten PKs, stratified by CYP2C19 phenotype.

Drug-Drug Interaction Potential of Mavacamten with Oral Contraceptives: Results from a Clinical Pharmacokinetic Study and a Physiologically Based Pharmacokinetic Model.

Mavacamten is a potential inducer of cytochrome P450 (CYP) 3A4 and, as such, could reduce the exposure of the active components of oral contraceptives, ethinyl estradiol (EE) and norethindrone (NOR), where CYP3A4 is involved in metabolism. This study assessed if repeat doses of mavacamten led to a drug-drug interaction with EE and/or NOR. This was an open-label study in healthy women. In Period 1, participants received 35 mcg of EE and 1 mg of NOR. In Period 2, participants received oral loading doses of mavacamten 25 mg on Days 1-2, 15 mg/day on Days 3-17, and 35 mcg of EE and 1 mg of NOR on Day 15. Plasma concentrations of mavacamten, EE, and NOR were obtained before dosing and up to 72 hours after dosing. For EE only, a physiologically based pharmacokinetic model was used to simulate mavacamten-mediated CYP3A4 induction with EE for various CYP2C19 phenotypes. In total, 13 women were enrolled (mean age, 38.9 [standard deviation, 9.65] years). After mavacamten administration, modest increases in area under the concentration-time curves were observed for both EE and NOR. The maximum concentrations and half-lives for EE and NOR were not affected by coadministration with mavacamten. Criteria for bioequivalence were met or nearly met for EE and NOR exposure with geometric mean ratios between 0.8 and 1.25. All adverse events were mild. The physiologically based pharmacokinetic model predicted a less than 15% decrease in EE exposure across CYP2C19 phenotypes. Coadministration of mavacamten at a therapeutically relevant dose with EE and NOR did not decrease the exposure to either EE or NOR to a level that may lead to reduced effectiveness.

A perspective on the current use of the phase distribution model for predicting milk-to-plasma drug concentration ratio.

The phase distribution model, proposed by Atkinson and Begg in 1990, has been widely used for predicting breastmilk-to-plasma drug concentration ratio. However, misrepresentations of the equations have been noted in recent publications. In this perspective, we revisit the derivation of the equations and provide an R/Shiny interface for the model with a view to helping scientists in this field acquire in-depth understanding of the theoretical background and implementation of the model.

Performance Verification of CYP2C19 Enzyme Abundance Polymorphism Settings within the Simcyp Simulator v21.

Physiologically based pharmacokinetic (PBPK) modeling has a number of applications, including assessing drug−drug interactions (DDIs) in polymorphic populations, and should be iteratively refined as science progresses. The Simcyp Simulator is annually updated and version 21 included updates to hepatic and intestinal CYP2C19 enzyme abundance, including addition of intermediate and rapid metabolizer phenotypes and changes to the ultra-rapid metabolizer enzyme abundance, with implications for population clearance and DDI predictions. This work details verification of the updates with sensitive CYP2C19 substrates, omeprazole and lansoprazole, using available clinical data from literature. Multiple assessments were performed, including recovery of areas under the concentration-time curve (AUC) and Cmax from compiled datasets for each drug, recovery of victim DDI ratios with CYP2C19 and/or CYP3A4 inhibition and recovery of relative exposure between phenotypes. Simulated data were within respective acceptance criteria for >80% of omeprazole AUC values, >70% of lansoprazole AUC and Cmax, >60% of AUC and Cmax DDI ratios and >80% of exposure ratios between different phenotypes. Recovery of omeprazole Cmax was lower (>50−70% within 2-fold) and possibly attributed to the variety of formulations used in the clinical dataset. Overall, the results demonstrated that the updated data used to parameterize CYP2C19 phenotypes reasonably described the pharmacokinetics of omeprazole and lansoprazole in genotyped or phenotyped individuals.

Investigations of hydrazine cleavage of eltrombopag in humans.

After oral administration to humans, eltrombopag undergoes extensive cleavage of its hydrazine linkage to metabolites, which are exclusively eliminated in urine. In vitro, the cleavage pathway was not detected in systems using cytochrome P450 enzymes, renal or hepatic microsomes, or hepatocytes but was readily evident after anaerobic incubation with rodent cecal contents or human fecal homogenate. Antibiotic treatment in vitro and in vivo inhibited eltrombopag cleavage, further indicating that cleavage is via gut microbes. Antibiotic treatment did not alter the systemic exposure of eltrombopag in mice. Oral and intravenous pharmacokinetic characterization in the mice with one of the cleavage products indicated that it was readily absorbed, conjugated, and eliminated in urine, consistent with its fate after oral administration of eltrombopag. Variation in this microbial pathway, for example by antibiotic cotherapy, is unlikely to be clinically significant.

Intact mAb LC-MS for drug concentration from pre-clinical studies: bioanalytical method performance and in-life samples.

Background: Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Results/methodology: Here, an intact protein LC-MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC-MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC-MS/MS. Discussion/conclusion: Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC-MS, modification monitoring and evaluation of data processing parameters is demonstrated.

Single ocular injection of a sustained-release anti-VEGF delivers 6months pharmacokinetics and efficacy in a primate laser CNV model.

A potent anti-vascular endothelial growth factor (VEGF) biologic and a compatible delivery system were co-evaluated for protection against wet age-related macular degeneration (AMD) over a 6month period following a single intravitreal (IVT) injection. The anti-VEGF molecule is dimeric, containing two different anti-VEGF domain antibodies (dAb) attached to a human IgG1 Fc region: a dual dAb. The delivery system is based on microparticles of PolyActive™ hydrogel co-polymer. The molecule was evaluated both in vitro for potency against VEGF and in ocular VEGF-driven efficacy models in vivo. The dual dAb is highly potent, showing a lower IC50 than aflibercept in VEGF receptor binding assays (RBAs) and retaining activity upon release from microparticles over 12months in vitro. Microparticles released functional dual dAb in rabbit and primate eyes over 6months at sufficient levels to protect Cynomolgus against laser-induced grade IV choroidal neovascularisation (CNV). This demonstrates proof of concept for delivery of an anti-VEGF molecule within a sustained-release system, showing protection in a pre-clinical primate model of wet AMD over 6months. Polymer breakdown and movement of microparticles in the eye may limit development of particle-based approaches for sustained release after IVT injection.

Interrogating the relationship between rat in vivo tissue distribution and drug property data for >200 structurally unrelated molecules.

The ability to explain distribution patterns from drug physicochemical properties and binding characteristics has been explored for more than 200 compounds by interrogating data from quantitative whole body autoradiography studies (QWBA). These in vivo outcomes have been compared to in silico and in vitro drug property data to determine the most influential properties governing drug distribution. Consistent with current knowledge, in vivo distribution was most influenced by ionization state and lipophilicity which in turn affected phospholipid and plasma protein binding. Basic and neutral molecules were generally better distributed than acidic counterparts demonstrating weaker plasma protein and stronger phospholipid binding. The influence of phospholipid binding was particularly evident in tissues with high phospholipid content like spleen and lung. Conversely, poorer distribution of acidic drugs was associated with stronger plasma protein and weaker phospholipid binding. The distribution of a proportion of acidic drugs was enhanced, however, in tissues known to express anionic uptake transporters such as the liver and kidney. Greatest distribution was observed into melanin containing tissues of the eye, most likely due to melanin binding. Basic molecules were consistently better distributed into parts of the eye and skin containing melanin than those without. The data, therefore, suggest that drug binding to macromolecules strongly influences the distribution of total drug for a large proportion of molecules in most tissues. Reducing lipophilicity, a strategy often used in discovery to optimize pharmacokinetic properties such as absorption and clearance, also decreased the influence of nonspecific binding on drug distribution.

Assessing a novel depot delivery strategy for noninvasive administration of VEGF/PDGF RTK inhibitors for ocular neovascular disease.

PURPOSE: Two noninvasive delivery strategies for VEGF/PDGF receptor tyrosine kinase inhibitors (RTKI) were explored that exploited uveal retention as a means for establishing an ocular drug depot: a single oral "loading" dose and topical administration. METHODS: Melanin binding was confirmed by centrifugation and mass spectrometry. Ocular retention was examined in pigmented and albino rats. Ocular release kinetics were measured 3 to 28 days postdosing in pigmented rats. Microautoradiography was used to demonstrate retention of RTKI in the uveal tract. A uveal drug depot of pazopanib was created by a single oral dose prior to induction of laser choroidal neovascularization (CNV). Choroid/retinal pigmented epithelium (RPE) retention of a related RTKI with enhanced topical bioavailability, GW771806, was confirmed by bioanalytics, and its ability to regress CNV compared with pazopanib. RESULTS: Pazopanib and GW771806 directly bound melanin and were retained within the uveal tract of pigmented rats for weeks following a single oral dose. Pazopanib was undetectable systemically following a single oral administration prior to CNV induction, and reduced CNV as well as twice daily dosing. Topical ocular delivery of GW771806 at 5 mg/mL led to high choroidal/RPE exposure and significantly regressed CNV lesions; 2 mg/mL prevented lesion progression. CONCLUSIONS: Uveal retention of drugs such as pazopanib can be used to create a sustained-release depot. Topical GW771806 regressed CNV. These data indicate that topical or infrequent oral loading dose treatment with VEGF/PDGF RTKI retained in the choroid/RPE might allow noninvasive treatments for ocular neovascular disease.