Harnessing Preclinical Data as a Predictive Tool for Human Brain Tissue Targeting.

One of the objectives within the medicinal chemistry discipline is to design tissue targeting molecules. The objective of tissue specificity can be either to gain drug access to the compartment of interest (e.g., the CNS) for Neuroscience targets or to restrict drug access to the CNS for all other therapeutic areas. Both neuroscience and non-neuroscience therapeutic areas have struggled to quantitatively estimate brain penetration or the lack thereof with compounds that are substrates of efflux transport proteins such as P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP) that are key components of the blood-brain barrier (BBB). It has been well established that drug candidates with high efflux ratios (ER) of these transporters have poor penetration into brain tissue. In the current work, we outline a parallel analysis to previously published models for the prediction of brain penetration that utilize an alternate MDR1-MDCK cell line as a better predictor of brain penetration and whether a correlation between in vitro, rodent data, non-human primate (NHP), and human in vivo brain penetration data could be established. Analysis of structural and physicochemical properties in conjunction with in vitro parameters and preclinical in vivo data has been highlighted in this manuscript as a continuation of the previously published work.

In Vitro-In Vivo Extrapolation of Key Transporter Activity at the Blood-Brain Barrier.

Understanding the quantitative implications of P-glycoprotein and breast cancer resistance protein efflux is a key hurdle in the design of effective, centrally acting or centrally restricted therapeutics. Previously, a comprehensive physiologically based pharmacokinetic model was developed to describe the in vivo unbound brain-to-plasma concentration ratio as a function of efflux activity measured in vitro. In the present work, the predictive utility of this framework was examined through application to in vitro and in vivo data generated on 133 unique compounds across three preclinical species. Two approaches were examined for the scaling of efflux activity to in vivo, namely relative expression as determined by independent proteomics measurements and relative activity as determined via fitting the in vivo neuropharmacokinetic data. The results with both approaches indicate that in vitro efflux data can be used to accurately predict the degree of brain penetration across species within the context of the proposed physiologically based pharmacokinetic framework.

Insights from mathematical modeling for convection-enhanced intraputamenal delivery of GDNF.

Glial cell line-derived neurotrophic factor (GDNF) is a potential therapy for Parkinson's disease (PD) promoting survival and functional recovery of dopaminergic neurons when delivered to the degenerated striatum. To study the aspects of intraputamenal delivery of GDNF, a mathematical model of recombinant methionyl human GDNF (r-metHuGDNF) convection in the human putamen has been developed. The convection-enhanced delivery infusions of r-metHuGDNF were simulated at rates up to 5 µL/min. The high-rate infusions (≥1 µL/min) permit rapid and uniform distribution of drug with up to 75% of the distribution volume having a concentration within 5% of the infusate concentration. No relevant differences in distribution at infusion rates of 3 and 5 µL/min were found. The patterns of GDNF distribution were analyzed in relation to the anatomy of the posterior dorsal putamen, and a cylindrical shape was found to be preferable considering risks of target overflow. A magnetic resonance (MR) tracer Gd-DTPA (Magnevist®) was evaluated as a surrogate in clinical studies, and the most accurate prediction of GDNF distribution was calculated immediately after infusion. The clearance of GDNF from the striatum is confirmed to be slow, with a half-life of ca. 19 h.

In Vitro-In Vivo Extrapolation of Intestinal Availability for Carboxylesterase Substrates Using Portal Vein-Cannulated Monkey.

Prediction of intestinal availability (FaFg) of carboxylesterase (CES) substrates is of critical importance in designing oral prodrugs with optimal properties, projecting human pharmacokinetics and dose, and estimating drug-drug interaction potentials. A set of ester prodrugs were evaluated using in vitro permeability (parallel artificial membrane permeability assay and Madin-Darby canine kidney cell line-low efflux) and intestinal stability (intestine S9) assays, as well as in vivo portal vein-cannulated cynomolgus monkey. In vitro-in vivo extrapolation (IVIVE) of FaFg was developed with a number of modeling approaches, including a full physiologically based pharmacokinetic (PBPK) model as well as a simplified competitive-rate analytical solution. Both methods converged as in the PBPK simulations enterocyte blood flow behaved as a sink, a key assumption in the competitive-rate analysis. For this specific compound set, the straightforward analytical solution therefore can be used to generate in vivo predictions. Strong IVIVE of FaFg was observed for cynomolgus monkey with R2 of 0.71-0.93. The results suggested in vitro assays can be used to predict in vivo FaFg for CES substrates with high confidence.

Insights From an Integrated Physiologically Based Pharmacokinetic Model for Brain Penetration.

Central-nervous-system, physiologically based pharmacokinetic (PBPK) models predict exposure profiles in the brain, that is, the rate and extent of distribution. The current work develops one such model and presents improved methods for determining key input parameters. A simple linear regression statistical model estimates the passive permeability at the blood-brain barrier from brain uptake index data and descriptors, and a novel analysis extracts the relative active transport parameter from in vitro assays taking into consideration both paracellular transport and unstirred water layers. The integrated PBPK model captures the concentration profiles of both rate-restricted and effluxed compounds with high passive permeability. In many cases, compounds distribute rapidly into the brain and are, therefore, not rate limited. The PBPK model is then simplified to a straightforward equation to describe brain-to-plasma ratios at steady state. The equation can estimate brain penetration either from in vitro efflux data or from in vivo results from another species and, therefore, is a valuable tool in the discovery setting.

Enhancing ketamine translational pharmacology via receptor occupancy normalization.

Ketamine is used preclinically and clinically to study schizophrenia and depression. Accordingly, it is imperative to understand the temporal relationship between the central concentrations and N-methyl-d-aspartate receptor (NMDAR) interactions of both ketamine and norketamine, its primary active metabolite, across species to assess the translatability of animal models to humans and the back-translation of clinical observations to the preclinical realm. However, such an interspecies normalization of ketamine and norketamine exposures at different clinical and preclinical doses (and their different routes and regimens) is lacking. This work defines the NMDAR occupancy (RO) time course following single doses of ketamine in rats, nonhuman primates (nhp) and humans to allow direct interspecies comparisons of specific ketamine-mediated pharmacodynamics via RO normalization. Total plasma concentration (Cp)-time profiles of ketamine and norketamine were generated from rats and nhp following a single, memory-impairing dose of ketamine; neuropharmacokinetics were determined in rats. [(3)H]MK-801-displacement studies in rats determined estimated mean (95% confidence interval) unbound plasma concentrations (Cp,u) for ketamine and norketamine producing 50% RO (IC50) of 1420 (990, 2140) nM and 9110 (5870, 13700) nM, respectively. Together, these datasets transformed Cp,u-time data to predicted RO (ROpred)-time profiles for rats, nhp and humans at behaviorally relevant ketamine doses. Subsequently, this approach helped determine an infusion paradigm in rats producing a ROpred-time profile mirroring that for a clinically antidepressant infusion. The described indication-independent methodology allows normalization to RO at any time following any ketamine dose (regardless of route or regimen) in any species by simply quantifying the Cp of ketamine and norketamine. Matching temporal RO relationships in animals and humans should allow direct comparisons of specific ketamine-dependent NMDAR-based pharmacodynamics.

Permeability comparison between hepatocyte and low efflux MDCKII cell monolayer.

Determination of passive permeability is not only important for predicting oral absorption and brain penetration, but also for accurately predicting hepatic clearance. High throughput (HT) measurement of passive permeability across hepatocyte cell membrane is technically more challenging than using monolayer cell-based permeability assays. In this study, we evaluated if the HT Madin-Darby canine kidney II-low efflux (MDCKII-LE) cell monolayer permeability assay can be used as a surrogate to predict the passive permeability of hepatocytes. Apparent passive permeability of MDCKII-LE is well correlated to passive diffusion clearance of human and rat hepatocytes, suggesting that the HT MDCKII-LE assay can be used as a surrogate to estimate the passive permeability of hepatocytes. In addition, lipophilicity (Log D determined at pH 7.4) was also found to be well correlated with both MDCKII-LE and hepatocyte permeability for most compounds, hence it may serve as another permeability surrogate.

Impact of recovery on fraction unbound using equilibrium dialysis.

Historically, recovery had been used to evaluate the data quality of plasma protein binding or tissue binding obtained from equilibrium dialysis assays. Low recovery was often indicative of high nonspecific binding, instability, or low solubility. This study showed that, when equilibrium was fully established in the dialysis assay, low recovery due to nonspecific binding had no impact on the determination of fraction unbound. The conclusion was supported by the principles of the equilibrium dialysis assay, experimental data, and mathematic simulations. The results suggested that the use of recovery as an acceptance criterion for the equilibrium dialysis assay in drug discovery was too restrictive, and introduced the additional burden of repeating studies unnecessarily.