Bill J. Smith

Prediction of Human Pharmacokinetics From Preclinical Information: Comparative Accuracy of Quantitative Prediction Approaches

Quantitative prediction of human pharmacokinetics is critical in assessing the viability of drug candidates and in determining first‐in‐human dosing. Numerous prediction methodologies, incorporating both in vitro and preclinical in vivo data, have been developed in recent years, each with advantages and disadvantages. However, the lack of a comprehensive data set, both preclinical and clinical, has limited efforts to evaluate the optimal strategy (or strategies) that results in quantitative predictions of human pharmacokinetics. To address this issue, the authors conducted a retrospective analysis using 50 proprietary compounds for which in vitro, preclinical pharmacokinetic data and oral single‐dose human pharmacokinetic data were available. Five predictive strategies, involving either allometry or use of unbound intrinsic clearance from microsomes or hepatocytes, were then compared for their ability to predict human oral clearance, half‐life through predictions of systemic clearance, volume of distribution, and bioavailability. Use of a single‐species scaling approach with rat, dog, or monkey was as accurate as or more accurate than using multiple‐species allometry. For those compounds cleared almost exclusively by P450‐mediated pathways, scaling from human liver microsomes was as predictive as single‐species scaling of clearance based on data from rat, dog, or monkey. These data suggest that use of predictive methods involving either single‐species in vivo data or in vitro human liver microsomes can quantitatively predict human in vivo pharmacokinetics and suggest the possibility of streamlining the predictive methodology through use of a single species or use only of human in vitro microsomal preparations.

Comparative gene expression profiles of ABC transporters in brain microvessel endothelial cells and brain in five species including human

While P-glycoprotein (PGP, ABCB1) is known to play an important role in drug exclusion at the blood brain barrier (BBB), less is known about the contribution of other members in the ATP-binding cassette (ABC) transporter family to BBB drug efflux, or whether these transporters are expressed differently in humans and in mammalian species of pharmacological interest. We used quantitative real-time PCR to determine mRNA expression levels for the majority of ABC family members in brain and in isolated brain microvessel endothelial capillary cells (BMEC) from human, rat, mouse, pig and cow. We confirmed BBB expression of several well-characterized ABC family members that are implicated in xenobiotic exclusion from the brain, including ABCB1 (PGP), ABCG2 (BCRP), ABCC1 (MRP1), ABCC4 (MRP4), and ABCC5 (MRP5). In addition, we detected high expression and enrichment in BMEC of several less well-characterized ABC transporters in one or more species, including ABCA2-4, ABCB4, ABCB6-8, ABCB10, ABCC3, ABCC6, ABCC10, and ABCE1. We also uncovered species differences in the expression of a number of transporters, including ABCG2 and ABCC4. This study identifies several additional ABC family members that may contribute to xenobiotic efflux at the human BBB, and compares the expression of a broad array of efflux transporters between human and four other species relevant to pharmacological research.

Progress in Brain Penetration Evaluation in Drug Discovery and Development

This review discusses strategies to optimize brain penetration from the perspective of drug discovery and development. Brain penetration kinetics can be described by the extent and time to reach brain equilibrium. The extent is defined as the ratio of free brain concentration to free plasma concentration at steady state. For all central nervous system (CNS) drug discovery programs, optimization of the extent of brain penetration should focus on designing and selecting compounds having low efflux transport at the blood-brain barrier (BBB). The time to reach brain equilibrium is determined by both BBB permeability and brain tissue binding. Rapid brain penetration can be achieved by increasing passive permeability and reducing brain tissue binding. Although many drug transporters have been identified at the BBB, the available literature demonstrates only the in vivo functional importance of P-glycoprotein (P-gp) in limiting brain penetration of its substrates. Drug-drug interactions mediated by P-gp at the BBB are possible due to inhibition or induction of P-gp. For newly identified drug transporters at the BBB, more research is needed to reveal their in vivo significance. We propose the following strategies for addressing drug transporters at the BBB. 1) Drug discovery screens should be used to eliminate good P-gp substrates for CNS targets. Special consideration could be given to moderate P-gp substrates as potential CNS drugs based on a high unmet medical need and the presence of a large safety margin. 2) Selection of P-gp substrates as drug candidates for non-CNS targets can reduce their CNS-mediated side effects.

Evaluation of Cerebrospinal Fluid Concentration and Plasma Free Concentration As a Surrogate Measurement for Brain Free Concentration

This study was designed to evaluate the use of cerebrospinal fluid (CSF) drug concentration and plasma unbound concentration (Cu,plasma) to predict brain unbound concentration (Cu,brain). The concentration-time profiles in CSF, plasma, and brain of seven model compounds were determined after subcutaneous administration in rats. The Cu,brain was estimated from the product of total brain concentrations and unbound fractions, which were determined using brain tissue slice and brain homogenate methods. For theobromine, theophylline, caffeine, fluoxetine, and propranolol, which represent rapid brain penetration compounds with a simple diffusion mechanism, the ratios of the area under the curve of Cu,brain/CCSF and Cu,brain/Cu,plasma were 0.27 to 1.5 and 0.29 to 2.1, respectively, using the brain slice method, and were 0.27 to 2.9 and 0.36 to 3.9, respectively, using the brain homogenate method. A P-glycoprotein substrate, CP-141938 (methoxy-3-[(2-phenyl-piperadinyl-3-amino)-methyl]-phenyl-N-methyl-methane-sulfonamide), had Cu,brain/CCSF and Cu,brain/Cu,plasma ratios of 0.57 and 0.066, using the brain slice method, and 1.1 and 0.13, using the brain homogenate method, respectively. The slow brain-penetrating compound, N[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl-]sarcosine, had Cu,brain/CCSF and Cu,brain/Cu,plasma ratios of 0.94 and 0.12 using the brain slice method and 0.15 and 0.018 using the brain homogenate method, respectively. Therefore, for quick brain penetration with simple diffusion mechanism compounds, CCSF and Cu,plasma represent Cu,brain equally well; for efflux substrates or slow brain penetration compounds, CCSF appears to be equivalent to or more accurate than Cu,plasma to represent Cu,brain. Thus, we hypothesize that CCSF is equivalent to or better than Cu,plasma to predict Cu,brain. This hypothesis is supported by the literature data.

Single-Dose Pharmacokinetics of Varenicline, a Selective Nicotinic Receptor Partial Agonist, in Healthy Smokers and Nonsmokers

Varenicline is a novel and selective α4β2 nicotinic receptor partial agonist that is under development for smoking cessation. The primary objectives of this double‐blind, placebo‐controlled, single‐dose, dose‐escalation study were to determine the clinical pharmacology of single doses of varenicline in healthy smokers and nonsmokers under fed and fasted conditions and to determine the clinical pharmacology of varenicline administered in the morning and in the evening to smokers. Within each subject group, 4 subjects were randomized to varenicline and 2 subjects to placebo. Subjects received one single oral administration of varenicline or placebo: 6 doses (0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg) were investigated in nonsmokers and 7 doses in smokers (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10.0 mg). Varenicline was well tolerated after single doses up to 3.0 mg in smokers and 1.0 mg in nonsmokers. Nausea and vomiting at doses above 3.0 mg in smokers and 1.0 mg in nonsmokers were dose limiting. Systemic exposure to varenicline and pharmacokinetic variability were similar between smokers and nonsmokers. Coadministration with food, smoking restriction, and time‐of‐day dosing did not affect the pharmacokinetics of varenicline.

Pharmacokinetic-Pharmacodynamic Modeling of Biomarker Response and Tumor Growth Inhibition to an Orally Available cMet Kinase Inhibitor in Human Tumor Xenograft Mouse Models

(R)-3-[1-(2,6-Dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine (PF02341066) was identified as an orally available, ATP-competitive small molecule inhibitor of cMet receptor tyrosine kinase. The objectives of the present studies were to characterize 1) the pharmacokinetic-pharmacodynamic relationship of the plasma concentrations of PF02341066 to cMet phosphorylation in tumor (biomarker) and 2) the relationship of cMet phosphorylation to antitumor efficacy (pharmacological response). Athymic mice implanted with GTL16 gastric carcinoma or U87MG glioblastoma xenografts were treated with PF02341066 once daily at doses selected to encompass ED50 values. Plasma concentrations of PF02341066 were best described by a one-compartment pharmacokinetic model. A time-delay (hysteresis) was observed between the plasma concentrations of PF02341066 and the cMet phosphorylation response. A link model was therefore used to account for this hysteresis. The model fitted the time courses of cMet phosphorylation well, suggesting that the main reason for the hysteresis is a rate-limiting distribution from plasma into tumor. The EC50 and EC90 values were estimated to be 19 and 167 ng/ml, respectively. For tumor growth inhibition, the exponential tumor growth model fitted the time courses of individual tumor growth inhibition well. The EC50 for the GTL16 tumor growth inhibition was estimated to be 213 ng/ml. Thus, the EC90 for the inhibition of cMet phosphorylation corresponded to the EC50 for the tumor growth inhibition, suggesting that near-complete inhibition of cMet phosphorylation (>90%) is required to significantly inhibit tumor growth (>50%). The present results will be helpful in determining the appropriate dosing regimen and in guiding dose escalation to rapidly achieve efficacious systemic exposure in the clinic.

Utility of Mdr1-gene deficient mice in assessing the impact of P-glycoprotein on pharmacokinetics and pharmacodynamics in drug discovery and development.

Since the generation of the multi-drug resistance 1 (mdr1) gene knockout (KO) mice in the early 90s, these animals have been instrumental to our understanding of the physiological roles of mdr1 gene product P-glycoprotein. Located in crucial organs such as brain, intestine, liver, and kidney, P-glycoprotein-mediated transport has been shown to affect both the pharmacokinetics and pharmacodynamics of endogenous compounds and xenobiotics. It appears that P-glycoprotein may not be essential for the maintenance of normal body function as suggested by the similarity in life span and serum chemistry values of mdr1 gene KO mice compared to their genetically competent littermates. However, numerous studies have demonstrated that P-glycoprotein limits the brain penetration of many drug substrates. The reduced central nervous system (CNS) access of these compounds has been linked to decreased pharmacological or toxicological effects. In contrast to the critical role that P-glycoprotein plays in the brain, the extent of P-glycoprotein involvement in oral absorption and hepatobiliary or renal excretion of xenobiotics appears more variable. In addition to the mdr1 gene KO model, in vitro cell lines that over-express P-glycoprotein, and clinical trials using P-glycoprotein modulators have allowed for the comparison of in vitro-in vivo and species related difference in P-glycoprotein activity. For the most part, studies have shown reasonable in vitro-in vivo correlations, modest species-related differences, and comparable human-mouse in vivo P-glycoprotein effects on systemic drug disposition. Therefore, the mdr1 gene KO mouse model, when used appropriately, may allow for prediction of CNS drug access and certain drug-drug interaction.

Pharmacokinetic/Pharmacodynamic Modeling of Crizotinib for Anaplastic Lymphoma Kinase Inhibition and Antitumor Efficacy in Human Tumor Xenograft Mouse Models

Crizotinib [Xalkori; PF02341066; (R)-3-[1-(2,6-dichloro-3-fluoro-phenyl)-ethoxy]-5-(1-piperidin-4-yl-1H-pyrazol-4-yl)-pyridin-2-ylamine] is an orally available dual inhibitor of anaplastic lymphoma kinase (ALK) and hepatocyte growth factor receptor. The objectives of the present studies were to characterize: 1) the pharmacokinetic/pharmacodynamic relationship of crizotinib plasma concentrations to the inhibition of ALK phosphorylation in tumors, and 2) the relationship of ALK inhibition to antitumor efficacy in human tumor xenograft models. Crizotinib was orally administered to athymic nu/nu mice implanted with H3122 non–small-cell lung carcinomas or severe combined immunodeficient/beige mice implanted with Karpas299 anaplastic large-cell lymphomas. Plasma concentration-time courses of crizotinib were adequately described by a one-compartment pharmacokinetic model. A pharmacodynamic link model reasonably fit the time courses of ALK inhibition in both H3122 and Karpas299 models with EC50 values of 233 and 666 ng/ml, respectively. A tumor growth inhibition model also reasonably fit the time course of individual tumor growth curves with EC50 values of 255 and 875 ng/ml, respectively. Thus, the EC50 for ALK inhibition approximately corresponded to the EC50 for tumor growth inhibition in both xenograft models, suggesting that >50% ALK inhibition would be required for significant antitumor efficacy (>50%). Furthermore, based on the observed clinical pharmacokinetic data coupled with the pharmacodynamic parameters obtained from the present nonclinical xenograft mouse model, >70% ALK inhibition was projected in patients with non–small-cell lung cancer who were administered the clinically recommended dosage of crizotinib, twice-daily doses of 250 mg (500 mg/day). The result suggests that crizotinib could sufficiently inhibit ALK phosphorylation for significant antitumor efficacy in patients.

In Vitro Characterization of Axitinib Interactions with Human Efflux and Hepatic Uptake Transporters. Implications for Disposition and Drug Interactions.

Axitinib is an inhibitor of tyrosine kinase vascular endothelin growth factor receptors 1, 2, and 3. The ATP-binding cassette (ABC) and solute carrier (SLC) transport properties of axitinib were determined in selected cellular systems. Axitinib exhibited high passive permeability in all cell lines evaluated (Papp ≥ 6 × 10−6 cm/s). Active efflux was observed in Caco-2 cells, and further evaluation in multidrug resistance gene 1 (MDR1) or breast cancer resistance protein (BCRP) transfected Madin-Darby canine kidney cells type 2 (MDCK) cells indicated that axitinib is at most only a weak substrate for P-glycoprotein (P-gp) but not BCRP. Axitinib showed incomplete inhibition of P-gp-mediated transport of digoxin in Caco-2 cells and BCRP transport of topotecan in BCRP-transfected MDCK cells with IC50 values of 3 μM and 4.4 μM, respectively. Axitinib (10 mg) did not pose a risk for systemic drug interactions with P-gp or BCRP per regulatory guidance. A potential risk for drug interactions through inhibition of P-gp and BCRP in the gastrointestinal tract was identified because an axitinib dose of 10 mg divided by 250 mL was greater than 10-fold the IC50 for each transporter. However, a GastroPlus simulation that considered the low solubility of axitinib resulted in lower intestinal concentrations and suggested a low potential for gastrointestinal interactions with P-gp and BCRP substrates. Organic anion transporting polypeptide 1B1 (OATP1B1) and OATP1B3 transfected human embryonic kidney 293 (HEK293) cells transported axitinib to a minor extent but uptake into suspended hepatocytes was not inhibited by rifamycin SV suggesting that high passive permeability predominates. Mouse whole-body autoradiography revealed that [14C]axitinib-equivalents showed rapid absorption and distribution to all tissues except the brain. This suggests that efflux transport of axitinib may occur at the mouse blood-brain barrier.

Expression and Functional Analysis of Hepatic Cytochromes P450, Nuclear Receptors, and Membrane Transporters in 10- and 25-Week-Old db/db Mice

Proper characterization of animal models used for efficacy and safety assessment is crucial. The present study focuses on characterizing proteins that are important components of the absorption, distribution, metabolism, and elimination of xenobiotics. Hepatic gene expression of Cyp2b10, Cyp2c29, Cyp3a11, Cyp2e1, Cyp4a10, Nr1i2, Nr1i3, slco1a1, slco1a4, slco1b2, abcb1b, abcc2, and abcg2 was examined using the real-time polymerase chain reaction method in male db/db mice, a commonly used type II diabetes model. We evaluated age and disease effects on gene expression and enzymatic activity in 10- and 25-week-old db/db and 25-week-old C57BLKS/J (strain-matched lean control) mice. Functional analysis was conducted in hepatic microsomes for Cyp2b, Cyp2c, and Cyp3a using cytochrome P450-specific substrates. There were no significant age- or disease-dependent changes in the expression of Cyp3a11 and Cyp3a activity in the db/db mice. The mRNA levels and the activities of Cyp2b10 and Cyp2c29 in the 25-week-old db/db mice decreased significantly compared with those of the 10-week-old db/db mice. There was a significant age-dependent increase in Cyp4a10 expression noted. The most marked expression change in db/db mice versus a control was the ∼400-fold reduction of mRNA expression of slco1a1. Slco1a4 and sloc1b2 showed increased expression compared with that in an age-matched control, whereas abcb1b showed decreased expression. No expression changes were observed for Cyp2e1, Nr1i2, Nr1i3, abcc2, and abcg2. Our data demonstrate that significant expression and activity differences exist between the db/db and the lean control mice, which are probably age- and disease-dependent.