Publications by authors named "Cornelis E C A Hop"

101 Publications

Development of Robust Quantitative Structure-Activity Relationship Models for CYP2C9, CYP2D6, and CYP3A4 Catalysis and Inhibition.

Drug Metab Dispos 2021 Sep 28;49(9):822-832. Epub 2021 Jun 28.

Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), Rockville, Maryland (E.G., S.J., P.S., N.T.-K., A.Z., D.-T.N., S.S., R.H., M.X. A.S., X.X.); Discovery Technology Laboratories, Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, Yokohama-shi, Japan (N.T.-K.); Pfizer Inc. Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer, Groton, Connecticut (R.S.O.); and Genentech Inc. Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., San Francisco, California (C.E.C.A.H.)

Cytochrome P450 enzymes are responsible for the metabolism of >75% of marketed drugs, making it essential to identify the contributions of individual cytochromes P450 to the total clearance of a new candidate drug. Overreliance on one cytochrome P450 for clearance levies a high risk of drug-drug interactions; and considering that several human cytochrome P450 enzymes are polymorphic, it can also lead to highly variable pharmacokinetics in the clinic. Thus, it would be advantageous to understand the likelihood of new chemical entities to interact with the major cytochrome P450 enzymes at an early stage in the drug discovery process. Typical screening assays using human liver microsomes do not provide sufficient information to distinguish the specific cytochromes P450 responsible for clearance. In this regard, we experimentally assessed the metabolic stability of ∼5000 compounds for the three most prominent xenobiotic metabolizing human cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4, and used the data sets to develop quantitative structure-activity relationship models for the prediction of high-clearance substrates for these enzymes. Screening library included the NCATS Pharmaceutical Collection, comprising clinically approved low-molecular-weight compounds, and an annotated library consisting of drug-like compounds. To identify inhibitors, the library was screened against a luminescence-based cytochrome P450 inhibition assay; and through crossreferencing hits from the two assays, we were able to distinguish substrates and inhibitors of these enzymes. The best substrate and inhibitor models (balanced accuracies ∼0.7), as well as the data used to develop these models, have been made publicly available (https://opendata.ncats.nih.gov/adme) to advance drug discovery across all research groups. SIGNIFICANCE STATEMENT: In drug discovery and development, drug candidates with indiscriminate cytochrome P450 metabolic profiles are considered advantageous, since they provide less risk of potential issues with cytochrome P450 polymorphisms and drug-drug interactions. This study developed robust substrate and inhibitor quantitative structure-activity relationship models for the three major xenobiotic metabolizing cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4. The use of these models early in drug discovery will enable project teams to strategize or pivot when necessary, thereby accelerating drug discovery research.
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http://dx.doi.org/10.1124/dmd.120.000320DOI Listing
September 2021

Intestinal Excretion, Intestinal Recirculation, and Renal Tubule Reabsorption Are Underappreciated Mechanisms That Drive the Distribution and Pharmacokinetic Behavior of Small Molecule Drugs.

J Med Chem 2021 06 19;64(11):7045-7059. Epub 2021 May 19.

Aranmore Pharma Consulting, 11 Andrew Drive, Lawrenceville, New Jersey 08648, United States.

Drug reabsorption following biliary excretion is well-known as enterohepatic recirculation (EHR). Renal tubular reabsorption (RTR) following renal excretion is also common but not easily assessed. Intestinal excretion (IE) and enteroenteric recirculation (EER) have not been recognized as common disposition mechanisms for metabolically stable and permeable drugs. IE and intestinal reabsorption (IR:EHR/EER), as well as RTR, are governed by dug concentration gradients, passive diffusion, active transport, and metabolism, and together they markedly impact disposition and pharmacokinetics (PK) of small molecule drugs. Disruption of IE, IR, or RTR through applications of active charcoal (AC), transporter knockout (KO), and transporter inhibitors can lead to changes in PK parameters. The impacts of intestinal and renal reabsorption on PK are under-appreciated. Although IE and EER/RTR can be an intrinsic drug property, there is no apparent strategy to optimize compounds based on this property. This review seeks to improve understanding and applications of IE, IR, and RTR mechanisms.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01720DOI Listing
June 2021

Prediction of Transporter-Mediated Rosuvastatin Hepatic Uptake Clearance and Drug Interaction in Humans Using Proteomics-Informed REF Approach.

Drug Metab Dispos 2021 02 13;49(2):159-168. Epub 2020 Oct 13.

Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., M.Y., K.I., J.D.U.); Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, California (L.S., C.E.C.A.H.); DMPK, Biogen Idec, Cambridge, Massachusetts (C.R., G.X.); Clinical Pharmacology (A.M.) and Drug Metabolism (Y.L.), Gilead Sciences, Inc., Foster City, California; Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co. Inc., Kenilworth, New Jersey (X.C.); Bristol-Myers Squibb Company, Princeton, New Jersey (W.G.H.); Takeda Pharmaceuticals International Co., Cambridge, Massachusetts (M.L.); SOLVO Biotechnology, Budaörs, Hungary (Z.N., N.S.); and BioIVT, Baltimore, Maryland (S.H.)

Suspended, plated, or sandwich-cultured human hepatocytes are routinely used for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated hepatic clearance (CL) of drugs. However, these hepatocyte models have been reported to underpredict transporter-mediated in vivo hepatic uptake CL ( ) of some drugs. Therefore, we determined whether transporter-expressing cells (TECs) can accurately predict the of drugs. To do so, we determined the uptake CL ( ) of rosuvastatin (RSV) by TECs (organic anion transporting polypeptides/Na-taurocholate cotransporting polypeptide) and then scaled it to that in vivo by relative expression factor (REF) (the ratio of transporter abundance in human livers and TEC) determined by liquid chromatography tandem mass spectrometry-based quantitative proteomics. Both the TEC and hepatocyte models did not meet our predefined success criteria of predicting within 2-fold the RSV value obtained from our positron emission tomography (PET) imaging. However, the TEC performed better than the hepatocyte models. Interestingly, using REF, TECs successfully predicted RSV obtained by the hepatocyte models, suggesting that the underprediction of RSV by TECs and hepatocytes is due to endogenous factor(s) not present in these in vitro models. Therefore, we determined whether inclusion of plasma (or albumin) in TEC uptake studies improved IVIVE of RSV It did, and our predictions were close to or just fell above our lower 2-fold acceptance boundary. Despite this success, additional studies are needed to improve transporter-mediated IVIVE of hepatic uptake CL of drugs. However, using REF and TEC, we successfully predicted the magnitude of PET-imaged inhibition of RSV by cyclosporine A. SIGNIFICANCE STATEMENT: We showed that the in vivo transporter-mediated hepatic uptake CL of rosuvastatin, determined by PET imaging, can be predicted (within 2-fold) from in vitro studies in transporter-expressing cells (TECs) (scaled using REF), but only when plasma proteins were included in the in vitro studies. This conclusion did not hold when plasma proteins were absent in the TEC or human hepatocyte studies. Thus, additional studies are needed to improve in vitro to in vivo extrapolation of transporter-mediated drug CL.
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http://dx.doi.org/10.1124/dmd.120.000204DOI Listing
February 2021

Inter-individual and inter-regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donors.

Pharmacol Res Perspect 2020 10;8(5):e00645

Genentech Inc., South San Francisco, CA, USA.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12-inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non-P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway-selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12-inch segments of the proximal small intestine shows that the segments immediately after the first 12-inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.
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http://dx.doi.org/10.1002/prp2.645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449955PMC
October 2020

Strategy for Determining the Free Fraction of Labile Covalent Modulators in Plasma Using Equilibrium Dialysis.

J Pharm Sci 2020 10 12;109(10):3181-3189. Epub 2020 Jul 12.

Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, CA 94080. Electronic address:

Determination of free drug fraction (f) in plasma can be challenging for labile covalent modulators due to the off-target reactivity of chemical warheads to matrix proteins. The resulting poor drug recovery yields low confidence in f. Two approaches using diluted plasma and low temperature (4 & 20 °C) for equilibrium dialysis (ED) have been investigated using covalent modulators including osimertinib, ibrutinib, rociletinib, afatinib, neratinib and acalabrutinib. Our data indicate that stability of covalent modulators in plasma varies in different species, and drug depletion may lead to overestimation of f if true equilibrium is not reached. Additionally, although ED at low temperature improves the recovery of covalent modulators, the impact of low temperature may lead to underestimate of f. Overall, ED using diluted plasma is a preferred method because of its faster equilibrium, improved recovery and free of temperature effect on f. If low temperature ED must be used for extremely labile compounds, precaution must be taken to ensure no temperature dependence of f in plasma. Nevertheless, an orthogonal ED approach is recommended for labile covalent modulators to confirm the true equilibrium and impact of temperature on f. Additionally, this strategy can be used for determining f of other liable compounds.
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http://dx.doi.org/10.1016/j.xphs.2020.06.029DOI Listing
October 2020

Characterization of Antineovascularization Activity and Ocular Pharmacokinetics of Phosphoinositide 3-Kinase/Mammalian Target of Rapamycin Inhibitor GNE-947.

Drug Metab Dispos 2020 05 4;48(5):408-419. Epub 2020 Mar 4.

Genentech, Inc., South San Francisco, California (X.Liu., X.Lia., J.L., S.U., J.Chen, J.Cheng, T.L., J.L., J.N., S.S.-L., C.Q., E.S., M.W., C.E.C.A.H., T.P.H.) and QPS, Delaware Technology Park, Newark, Delaware (E.S.)

The objectives of the present study were to characterize GNE-947 for its phosphoinositide 3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitory activities, in vitro anti-cell migration activity in human umbilical vein endothelial cells (HUVECs), in vivo antineovascularization activity in laser-induced rat choroidal neovascular (CNV) eyes, pharmacokinetics in rabbit plasma and eyes, and ocular distribution using matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) and autoradioluminography. Its PI3K and mTOR were 0.0005 and 0.045 µM, respectively, and its HUVEC IC was 0.093 µM. GNE-947 prevented neovascularization in the rat CNV model at 50 or 100 µg per eye with repeat dosing. After a single intravenous injection at 2.5 and 500 μg/kg in rabbits, its plasma terminal half-lives ( ) were 9.11 and 9.59 hours, respectively. After a single intravitreal injection of a solution at 2.5 μg per eye in rabbits, its apparent values were 14.4, 16.3, and 23.2 hours in the plasma, vitreous humor, and aqueous humor, respectively. After a single intravitreal injection of a suspension at 33.5, 100, 200 μg per eye in rabbits, the were 29, 74, and 219 days in the plasma and 46, 143, and 191 days in the eyes, respectively. MALDI-IMS and autoradioluminography images show that GNE-947 did not homogenously distribute in the vitreous humor and aggregated at the injection sites after injection of the suspension, which was responsible for the long of the suspension because of the slow dissolution process. This hypothesis was supported by pharmacokinetic modeling analyses. In conclusion, the PI3K/mTOR inhibitor GNE-947 prevented neovascularization in a rat CNV model, with up to approximately 6 months after a single intravitreal injection of the suspension in rabbit eyes. SIGNIFICANCE STATEMENT: GNE-947 is a potent phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor and exhibits anti-choroidal neovascular activity in rat eyes. The duration of GNE-947 in the rabbit eyes after intravitreal injection in a solution is short, with a half-life ( ) of less than a day. However, the duration after intravitreal dose of a suspension is long, with up to 6 months due to low solubility and slow dissolution. These results indicate that intravitreal injection of a suspension for low-solubility drugs can be used to achieve long-term drug exposure.
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http://dx.doi.org/10.1124/dmd.119.089763DOI Listing
May 2020

Exposure-Efficacy Analysis of Antibody-Drug Conjugates Delivering an Excessive Level of Payload to Tissues.

Drug Metab Dispos 2019 10 29;47(10):1146-1155. Epub 2019 Jul 29.

Drug Metabolism & Pharmacokinetics (D.Z., Y.M., D.S., W.W., S.C.K., C.E.C.A.H.), Discovery Chemistry (P.S.D., T.H.P.), Translational Oncology (S.-F.Y., A.P.), and Protein Chemistry (J.D.S.), Genentech, South San Francisco, California

Antibody-drug conjugates (ADCs) contain a disease-receptor antibody and a payload drug connected via a linker. The payload delivery depends on both tumor properties and ADC characteristics. In this study, we used different linkers, attachment sites, and doses to modulate payload delivery of several ADCs bearing maytansinoids (e.g., DM1), auristatins (e.g., MMAE), and DNA alkylating agents [e.g., pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD)] as payloads in HER2- or CD22-expressing xenograft models. The tumor growth inhibition and ADC stability and exposure data were collected and analyzed from these dosed animals. The trend analysis suggests that intratumoral payload exposures that directly related the combination of conjugate linker and dose correlate with the corresponding efficacies of three payload types in two antigen-expressing xenograft models. These preliminary correlations also suggest that a minimal threshold concentration of intratumoral payload is required to support sustained efficacy. In addition, an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor payload concentrations, the assessments of systemic exposures of total antibody (Tab) as well as the linker, dose, site of attachment, plasma stability, and drug-to-antibody ratio changes of these ADCs did not consistently rationalize the observed ADC efficacies. The requirement of a threshold payload concentration for efficacy is further supported by dose fractionation studies with DM1-, MMAE-, and PBD-containing ADCs, which demonstrated that single-dose regimens showed better efficacies than fractionated dosing. Overall, this study demonstrates that 1) the linker and dose together determine the tissue payload concentration that correlates with the antitumor efficacy of ADCs and 2) an ADC can deliver an unnecessary level of payload to tumors in xenograft models.
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http://dx.doi.org/10.1124/dmd.119.087023DOI Listing
October 2019

Drug Concentration Asymmetry in Tissues and Plasma for Small Molecule-Related Therapeutic Modalities.

Drug Metab Dispos 2019 10 2;47(10):1122-1135. Epub 2019 Jul 2.

Genentech, South San Francisco, California (D.Z., C.E.C.A.H., J.R.K., K.N., M.Z., M.R.W., R.Z., S.C.K.); Department of Medicine, Division of Clinical Pharmacology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (H.K.S., N.N.B.); Brain Barriers Research Center, Department of Pharmaceutics, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota (G.G.); Department of Pharmaceutics, University of Washington, Seattle, Washington (G.P.-V., J.D.U.); Biogen, Cambridge, Massachusetts (X.L.); Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., Eastern Point Road, Groton, Connecticut (L.D.); Drug Disposition, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana (L.Z.); and Drug Metabolism, Gilead Sciences, Foster City, California (Y.L.)

The well accepted "free drug hypothesis" for small-molecule drugs assumes that only the free (unbound) drug concentration at the therapeutic target can elicit a pharmacologic effect. Unbound (free) drug concentrations in plasma are readily measurable and are often used as surrogates for the drug concentrations at the site of pharmacologic action in pharmacokinetic-pharmacodynamic analysis and clinical dose projection in drug discovery. Furthermore, for permeable compounds at pharmacokinetic steady state, the free drug concentration in tissue is likely a close approximation of that in plasma; however, several factors can create and maintain disequilibrium between the free drug concentration in plasma and tissue, leading to free drug concentration asymmetry. These factors include drug uptake and extrusion mechanisms involving the uptake and efflux drug transporters, intracellular biotransformation of prodrugs, membrane receptor-mediated uptake of antibody-drug conjugates, pH gradients, unique distribution properties (covalent binders, nanoparticles), and local drug delivery (e.g., inhalation). The impact of these factors on the free drug concentrations in tissues can be represented by , the ratio of free drug concentration between tissue and plasma at steady state. This review focuses on situations in which free drug concentrations in tissues may differ from those in plasma (e.g., > or <1) and discusses the limitations of the surrogate approach of using plasma-free drug concentration to predict free drug concentrations in tissue. This is an important consideration for novel therapeutic modalities since systemic exposure as a driver of pharmacologic effects may provide limited value in guiding compound optimization, selection, and advancement. Ultimately, a deeper understanding of the relationship between free drug concentrations in plasma and tissues is needed.
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http://dx.doi.org/10.1124/dmd.119.086744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756291PMC
October 2019

Toward a Consensus on Applying Quantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research: A White Paper.

Clin Pharmacol Ther 2019 09 26;106(3):525-543. Epub 2019 Jul 26.

Centre for Applied Pharmacokinetic Research, University of Manchester, Manchester, UK.

Quantitative translation of information on drug absorption, disposition, receptor engagement, and drug-drug interactions from bench to bedside requires models informed by physiological parameters that link in vitro studies to in vivo outcomes. To predict in vivo outcomes, biochemical data from experimental systems are routinely scaled using protein quantity in these systems and relevant tissues. Although several laboratories have generated useful quantitative proteomic data using state-of-the-art mass spectrometry, no harmonized guidelines exit for sample analysis and data integration to in vivo translation practices. To address this gap, a workshop was held on September 27 and 28, 2018, in Cambridge, MA, with 100 experts attending from academia, the pharmaceutical industry, and regulators. Various aspects of quantitative proteomics and its applications in translational pharmacology were debated. A summary of discussions and best practices identified by this expert panel are presented in this "White Paper" alongside unresolved issues that were outlined for future debates.
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http://dx.doi.org/10.1002/cpt.1537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692196PMC
September 2019

Positron Emission Tomography Imaging of [ C]Rosuvastatin Hepatic Concentrations and Hepatobiliary Transport in Humans in the Absence and Presence of Cyclosporin A.

Clin Pharmacol Ther 2019 11 22;106(5):1056-1066. Epub 2019 Jul 22.

Department of Pharmaceutics, University of Washington, Seattle, Washington, USA.

Using positron emission tomography imaging, we determined the hepatic concentrations and hepatobiliary transport of [ C]rosuvastatin (RSV; i.v. injection) in the absence (n = 6) and presence (n = 4 of 6) of cyclosporin A (CsA; i.v. infusion) following a therapeutic dose of unlabeled RSV (5 mg, p.o.) in healthy human volunteers. The sinusoidal uptake, sinusoidal efflux, and biliary efflux clearance (CL; mL/minute) of [ C]RSV, estimated through compartment modeling were 1,205.6 ± 384.8, 16.2 ± 11.2, and 5.1 ± 1.8, respectively (n = 6). CsA (blood concentration: 2.77 ± 0.24 μM), an organic-anion-transporting polypeptide, Na -taurocholate cotransporting polypeptide, and breast cancer resistance protein inhibitor increased [ C]RSV systemic blood exposure (45%; P < 0.05), reduced its biliary efflux CL (52%; P < 0.05) and hepatic uptake (25%; P > 0.05) but did not affect its distribution into the kidneys. CsA increased plasma concentrations of coproporphyrin I and III and total bilirubin by 297 ± 69%, 384 ± 102%, and 81 ± 39%, respectively (P < 0.05). These data can be used in the future to verify predictions of hepatic concentrations and hepatobiliary transport of RSV.
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http://dx.doi.org/10.1002/cpt.1506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6777999PMC
November 2019

Catalytic Cleavage of Disulfide Bonds in Small Molecules and Linkers of Antibody-Drug Conjugates.

Drug Metab Dispos 2019 10 13;47(10):1156-1163. Epub 2019 May 13.

Drug Metabolism & Pharmacokinetics (D.Z., R.T.C., L.L., Y.D., C.E.C.A.H., S.C.K.), Biochemical and Cellular Pharmacology (A.F.-O., K.R.K., Y.L.), Discovery Chemistry (P.S.D., T.H.P.), and Protein Chemistry (J.D.S.), Genentech, Inc., South San Francisco, California

In cells, catalytic disulfide cleavage is an essential mechanism in protein folding and synthesis. However, detailed enzymatic catalytic mechanism relating cleavage of disulfide bonds in xenobiotics is not well understood. This study reports an enzymatic mechanism of cleavage of disulfide bonds in xenobiotic small molecules and antibody conjugate (ADC) linkers. The chemically stable disulfide bonds in substituted disulfide-containing pyrrolobenzodiazepine (PBD, pyrrolo[2,1-c][1,4]benzodiazepine) monomer prodrugs in presence of glutathione or cysteine were found to be unstable in incubations in whole blood of humans and rats. It was shown the enzymes involved were thioredoxin (TRX) and glutaredoxin (GRX). For a diverse set of drug-linker conjugates, we determined that TRX in the presence of TRX-reductase and NADPH generated the cleaved products that are consistent with catalytic disulfide cleavage and linker immolation. GRX was less rigorously studied; in the set of compounds studied, its role in the catalytic cleavage was also confirmed. Collectively, these in vitro experiments demonstrate that TRX as well as GRX can catalyze the cleavage of disulfide bonds in both small molecules and linkers of ADCs.
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http://dx.doi.org/10.1124/dmd.118.086132DOI Listing
October 2019

Exposure-Effect Relationships in Established Rat Adjuvant-Induced and Collagen-Induced Arthritis: A Translational Pharmacokinetic-Pharmacodynamic Analysis.

J Pharmacol Exp Ther 2019 06 2;369(3):406-418. Epub 2019 Apr 2.

Departments of Drug Metabolism and Pharmacokinetics (H.W., L.L., Y.D., M.R.W., C.E.C.A.H.) and Immunology (W.O.), Genentech Inc., South San Francisco, California.

The ability of rodent immune-mediated arthritis models to quantitatively predict therapeutic activity of antiarthritis agents is poorly understood. Two commonly used preclinical models of arthritis are adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in rats. The objective of the current study is to investigate the relationship between efficacy in AIA and CIA in rats, and clinical efficacy in rheumatoid arthritis patients using translational pharmacokinetic-pharmacodynamic (PK-PD) analysis. A range of doses of indomethacin (a nonsteroidal anti-inflammatory drug), and three disease-modifying antirheumatic drugs (DMARDs), methotrexate, etanercept, and tofacitinib, were evaluated in AIA and CIA rats. Dexamethasone was included in this study as a positive control. The area under the ankle diameter-time profile (AUC) and ankle histopathology summed scores (AHSS) were used as efficacy endpoints for activity against disease symptoms (joint inflammation) and disease progression (joint damage), respectively. Translational PK-PD analysis was performed to rank order preclinical efficacy endpoints at clinically relevant concentrations. For each drug tested, inhibition of AUC and AHSS scores was generally comparable in both magnitude and rank order. Overall, based on both AUC and the AHSS inhibition, the rank ordering of preclinical activity for the DMARDs evaluated was tofacitinib > etanercept ≥ methotrexate. This ranking of preclinical efficacy was consistent with reported clinical efficacy. Of interest, indomethacin showed equal or often better efficacy than the three DMARDs evaluated on inhibiting AHSS despite having limited ability to prevent joint damage clinically in patients. The translational value of performing PK-PD analysis of arthritis models in rats is discussed.
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http://dx.doi.org/10.1124/jpet.118.255562DOI Listing
June 2019

Evaluation of the predictive performance of physiologically based pharmacokinetic models for intramuscular injections of therapeutic proteins.

Xenobiotica 2019 Dec 22;49(12):1423-1433. Epub 2019 Feb 22.

Faculty of Pharmaceutical Sciences, The University of British Columbia , Vancouver , BC , Canada.

Several physiologically-based pharmacokinetic (PBPK) models have been reported for intravenous (IV) and subcutaneous (SC) injections, but there has been a paucity of work for intramuscular (IM) injections. The primary objective of this work was a wide-scale evaluation of the predictive performance of IM PBPK models of therapeutic proteins. PBPK models for all administration routes available in the literature have regarded muscle as the total muscle (TM) in the body; however, anatomically, the body is composed of discrete muscle groups. Clinically, IM is administered to a specific muscle (SM). We explored the predictive performance of IM PBPK models with an SM or TM dosing site. The plasma concentration-time profiles of seven therapeutic proteins after an IM dose in humans served as the clinically observed data for model evaluation - this was a diverse group ranging from 30 to 149 kDa from six protein classes. Pharmacokinetic parameters , , AUC, and ka were estimated. SM and TM IM PBPK approaches were compared using Average Fold Error (AFE) and Pearson Chi-Square LineShape analyses. This work represents the first wide-scale validation of IM PBPK models and suggests that these models predict IM PBPK reasonably well. The SM and TM approach provided comparable performance.
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http://dx.doi.org/10.1080/00498254.2019.1571651DOI Listing
December 2019

Strategies to optimize drug half-life in lead candidate identification.

Expert Opin Drug Discov 2019 03 24;14(3):221-230. Epub 2019 Jan 24.

a Department of Drug Metabolism and Pharmacokinetics , Genentech , South San Francisco , CA , USA.

Introduction: The PK optimization of drug candidates is one of the most resource-intensive tasks in pharmaceutical research and development. With the increasing availability of in silico, in vitro and mechanistic in vivo ADME models, drug discovery scientists have progressively learned to recognize common SAR patterns and engineer data-driven strategies to accelerate the resolution of ADME issues in lead optimization. Many of these strategies gravitate toward the concept of drug-likeness, which defines a number of optimal holistic physicochemical parameters (such as lipophilicity) that idealized oral drugs possess. Areas covered: Herein, the authors discuss the interplay of lipophilicity with in vitro and in vivo ADME data in order to refine existing thought around drug half-life optimization. Strategies to prolong the half-life of oral drugs via formulation are beyond the scope of this review. Expert opinion: Optimizing active properties such as potency, selectivity, and intrinsic metabolic clearance is an unambiguously beneficial strategy for small molecules within or beyond the Lipinski rule of five chemical space. The data that we present in this work suggests that emphasis should be primarily placed on optimizing active properties such as potency, selectivity, and metabolic stability.
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http://dx.doi.org/10.1080/17460441.2019.1569625DOI Listing
March 2019

Interindividual and Regional Variability in Drug Transporter Abundance at the Human Blood-Brain Barrier Measured by Quantitative Targeted Proteomics.

Clin Pharmacol Ther 2019 07 18;106(1):228-237. Epub 2019 Mar 18.

Department of Pharmaceutics, University of Washington, Seattle, Washington, USA.

For in vitro to in vivo extrapolation (IVIVE) of brain distribution of drugs that are transported at the human blood-brain barrier (BBB), it is important to quantify the interindividual and regional variability of drug transporter abundance at this barrier. Therefore, using quantitative targeted proteomics, we compared the abundance of adenosine triphosphate-binding cassette and solute carrier transporters in brain microvascular endothelial cells (BMECs) isolated from postmortem specimens of two matched brain regions, the occipital (Brodmann Area (BA)17) and parietal (BA39) lobe, from 30 adults. Of the quantifiable transporters, the abundance ranked: glucose transporter (GLUT)1 > breast cancer resistance protein > P-glycoprotein (P-gp) > equilibrative nucleoside transporter (ENT)1 > organic anion-transporting polypeptide (OATP)2B1. The abundance of multidrug resistance protein 1/2/3/4, OATP1A2, organic anion transporter (OAT)3, organic cation transporter (OCT)1/2, OCTN1/2, or ENT2 was below the limit of quantification. Transporter abundance per gram of tissue (scaled using GLUT1 abundance in BMEC vs. brain homogenate) in BA17 was 30-42% higher than BA39. The interindividual variability in transporter abundance (percentage of coefficient of variation (%CV)) was 35-57% (BA17) and 27-46% (BA39). These data can be used in proteomics-informed bottom-up IVIVE to predict human brain drug distribution.
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http://dx.doi.org/10.1002/cpt.1373DOI Listing
July 2019

A Comparison of Total and Plasma Membrane Abundance of Transporters in Suspended, Plated, Sandwich-Cultured Human Hepatocytes Versus Human Liver Tissue Using Quantitative Targeted Proteomics and Cell Surface Biotinylation.

Drug Metab Dispos 2019 04 8;47(4):350-357. Epub 2019 Jan 8.

Department of Pharmaceutics, University of Washington, Seattle, Washington (V.K., J.D.U.); Drug Metabolism and Pharmacokinetics, Genentech, South San Francisco, California (L.S., C.E.C.A.H.); Drug Metabolism and Pharmacokinetics, Biogen Idec, Cambridge, Massachusetts (G.X.); Departments of Clinical Research, Clinical Pharmacology, and Drug Metabolism and Pharmacokinetics, Gilead Sciences, Foster City, California (Y.L., A.M.); Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, Merck & Co., Kenilworth, New Jersey (X.C.); Bristol-Myers Squibb, Princeton, New Jersey (W.G.H.); Takeda Pharmaceuticals International, Cambridge, Massachusetts (M.L.); and BioIVT, Baltimore, Maryland (S.H.)

Suspended (SH), plated (PH), and sandwich-cultured hepatocytes (SCH) are commonly used models to predict in vivo transporter-mediated hepatic uptake (SH or PH) or biliary (SCH) clearance of drugs. When doing so, the total and the plasma membrane abundance (PMA) of transporter are assumed not to differ between hepatocytes and liver tissue (LT). This assumption has never been tested. In this study, we tested this assumption by measuring the total and PMA of the transporters in human hepatocyte models versus LT (total only) from which they were isolated. Total abundance of OATP1B1/2B1/1B3, OCT1, and OAT2 was not significantly different between the hepatocytes and LT. The same was true for the PMA of these transporters across the hepatocyte models. In contrast, total abundance of the sinusoidal efflux transporter, MRP3, and the canalicular efflux transporters, MRP2 and P-gp, was significantly greater ( < 0.05) in SCH versus LT. Of the transporters tested, only the percentage of PMA of OATP1B1, P-gp, and MRP3, in SCH (82.8% ± 7.3%, 57.5% ± 10.9%, 69.3% ± 5.7%) was significantly greater ( < 0.05) than in SH (73.3% ± 6.4%, 27.4% ± 6.4%, 53.6% ± 4.1%). If the transporters measured in the plasma membrane are functional and the PMA in SH is representative of that in LT, these data suggest that SH, PH, and SCH will result in equal prediction of hepatic uptake clearance of drugs mediated by the transporters tested above. However, SCH will predict higher sinusoidal efflux and biliary clearance of drugs if the change in PMA of these transporters is not taken into consideration.
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http://dx.doi.org/10.1124/dmd.118.084988DOI Listing
April 2019

Improving Confidence in the Determination of Free Fraction for Highly Bound Drugs Using Bidirectional Equilibrium Dialysis.

J Pharm Sci 2019 03 14;108(3):1296-1302. Epub 2018 Oct 14.

Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., South San Francisco, California 94080. Electronic address:

Equilibrium dialysis has been widely used for the measurement of the fraction of unbound drug (f) in plasma, but it suffers from the accuracy and reliability for low f values. To address this concern, an orthogonal approach, called the bidirectional equilibrium dialysis, is described to simultaneously measure a pair of f values for each drug based on equilibration in 2 opposite dialysis directions: from plasma to buffer (f) and from buffer to plasma (f). Hypothetically, if true equilibrium is attained in both dialysis directions, the measured f and f values for a given drug should converge, and thus, the ratio of f to f becomes unity (1.0). Thus, the ratio can be used as a tangible readout for data reliability. This methodology has been extensively tested in the present study using various drugs with distinct plasma binding characteristics. Our results clearly showed that low f values (<0.01) could be reliably determined and verified using either the standard or dilution bidirectional equilibrium dialysis method for some known highly bound drugs; for extensively bound drugs with high logD, such as montelukast, bedaquiline, and venetoclax, only a range of f can be reported with confidence because of uncertainty in the true equilibrium.
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http://dx.doi.org/10.1016/j.xphs.2018.10.011DOI Listing
March 2019

Correction to: Strategies of Drug Transporter Quantitation by LC-MS: Importance of Peptide Selection and Digestion Efficiency.

AAPS J 2018 06 6;20(4):75. Epub 2018 Jun 6.

Genentech Inc, 1 DNA Way, South San Francisco, California, 94080, USA.

Liling Liu was not noted as the co-first author in the original article. Buyun Chen and Liling Liu contributed equally to the article.
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http://dx.doi.org/10.1208/s12248-018-0236-3DOI Listing
June 2018

Abundance of Phase 1 and 2 Drug-Metabolizing Enzymes in Alcoholic and Hepatitis C Cirrhotic Livers: A Quantitative Targeted Proteomics Study.

Drug Metab Dispos 2018 07 25;46(7):943-952. Epub 2018 Apr 25.

University of Washington, Seattle, Washington (B.P., D.K.B., K.J., R.C., J.D.U.); Merck Sharp & Dohme Corporation, Kenilworth, New Jersey (X.C.); Gilead Sciences, Inc., Foster City, California (A.S.R., A.M.); Genentech, South San Francisco, California (L.S., C.E.C.A.H.); Biogen, Cambridge, Massachusetts (G.X.); Ardea Biosciences, Inc., San Diego, California (C.L.); Bristol-Myers Squibb Company, Princeton, New Jersey (Y.L., W.H.); Takeda Pharmaceuticals International Co., Cambridge, Massachusetts (M.L.); and University of Kansas Medical Center, Kansas City, Kansas (S.C.K.)

To predict the impact of liver cirrhosis on hepatic drug clearance using physiologically based pharmacokinetic (PBPK) modeling, we compared the protein abundance of various phase 1 and phase 2 drug-metabolizing enzymes (DMEs) in S9 fractions of alcoholic ( = 27) or hepatitis C (HCV, = 30) cirrhotic versus noncirrhotic (control) livers ( = 25). The S9 total protein content was significantly lower in alcoholic or HCV cirrhotic versus control livers (i.e., 38.3 ± 8.3, 32.3 ± 12.8, vs. 51.1 ± 20.7 mg/g liver, respectively). In general, alcoholic cirrhosis was associated with a larger decrease in the DME abundance than HCV cirrhosis; however, only the abundance of UGT1A4, alcohol dehydrogenase (ADH)1A, and ADH1B was significantly lower in alcoholic versus HCV cirrhotic livers. When normalized to per gram of tissue, the abundance of nine DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, CYP1A2, ADH1A, ADH1B, aldehyde oxidase (AOX)1, and carboxylesterase (CES)1) in alcoholic cirrhosis and five DMEs (UGT1A6, UGT1A4, CYP3A4, UGT2B7, and CYP1A2) in HCV cirrhosis was <25% of that in control livers. The abundance of most DMEs in cirrhotic livers was 25% to 50% of control livers. CES2 abundance was not affected by cirrhosis. Integration of UGT2B7 abundance in cirrhotic livers into the liver cirrhosis (Child Pugh C) model of Simcyp improved the prediction of zidovudine and morphine PK in subjects with Child Pugh C liver cirrhosis. These data demonstrate that protein abundance data, combined with PBPK modeling and simulation, can be a powerful tool to predict drug disposition in special populations.
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http://dx.doi.org/10.1124/dmd.118.080523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987995PMC
July 2018

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

Drug Metab Dispos 2018 Jun 27;46(6):865-878. Epub 2018 Feb 27.

Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland (S.S., C.F.); Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach a.d. Riß, Germany (B.B., F.R.); Drug Metabolism and Pharmacokinetics Department, Takeda Pharmaceutical International Co., Cambridge, Massachusetts (S.K.C., J.Y.); Genentech, Inc., Drug Metabolism and Pharmacokinetics, South San Francisco, California (C.E.C.A.H., S.C.K.); Bristol-Myers Squibb Pharmaceutical Co., Princeton, New Jersey (W.G.H.); Research Division, Chugai Pharmaceutical Co., Ltd., Gotemba, Japan (F.I.); PK Sciences (ADME), Novartis Institutes for Biomedical Research, Basel, Switzerland (A.D.J.); PK Sciences (ADME), Novartis Institutes for Biomedical Research, One Health Plaza, East Hanover, New Jersey (M.K.); Unilabs York Bioanalytical Solutions, Discovery Park House, Discovery Park, Sandwich, Kent, United Kingdom (A.N.R.N); Drug Metabolism, Pharmacokinetics and Clinical Pharmacology, Agios, Cambridge, Massachusetts (C.P.); Merck Biopharma, Quantitative Pharmacology and Drug Disposition, NCE Drug Disposition, Darmstadt, Germany (H.S., P.S.); and Pfizer, Pharmacokinetics, Dynamics and Metabolism, Groton, Connecticut (D.K.S., S.T., R.S.O.).

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry.
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http://dx.doi.org/10.1124/dmd.117.079848DOI Listing
June 2018

Prediction of the Pharmacokinetics of Pravastatin as an OATP Substrate Using Plateable Human Hepatocytes With Human Plasma Data and PBPK Modeling.

CPT Pharmacometrics Syst Pharmacol 2018 04 13;7(4):251-258. Epub 2018 Feb 13.

Department of Drug Metabolism and Pharmacokinetics, Genentech, A Member of the Roche Group, South San Francisco, California, USA.

Plateable human hepatocytes with human plasma were utilized to generate the uptake transporter kinetic data for pravastatin, an organic anion-transporting polypeptide (OATP) transporter substrate. The active hepatic uptake of pravastatin was determined with a J value of 134.4 pmol/min/million cells and K of 76.77 µM in plateable human hepatocytes with human plasma. The physiologically-based pharmacokinetic (PBPK) model with incorporation of these in vitro kinetic data successfully simulated the i.v. pharmacokinetic profile of pravastatin without applying scaling factor (the mean predicted area under the curve (AUC) is within 1.5-fold of the observed). Furthermore, the PBPK model also adequately described the oral plasma concentration-time profiles of pravastatin at different dose levels. The current investigation demonstrates an approach allowing us to build upon the translation of in vitro OATP uptake transporter data to in vivo, with a hope of utilizing the in vitro data for the prospective human pharmacokinetic (PK) prediction.
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http://dx.doi.org/10.1002/psp4.12283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5915609PMC
April 2018

Immolation of p-Aminobenzyl Ether Linker and Payload Potency and Stability Determine the Cell-Killing Activity of Antibody-Drug Conjugates with Phenol-Containing Payloads.

Bioconjug Chem 2018 02 7;29(2):267-274. Epub 2018 Feb 7.

Drug Metabolism & Pharmacokinetics, ‡Biochemical and Cellular Pharmacology, §Molecular Oncology, ⊥Discovery Chemistry, and ∥Protein Chemistry, Genentech , South San Francisco, California 94080, United States.

The valine-citrulline (Val-Cit) dipeptide and p-aminobenzyl (PAB) spacer have been commonly used as a cleavable self-immolating linker in ADC design including in the clinically approved ADC, brentuximab vedotin (Adcetris). When the same linker was used to connect to the phenol of the cyclopropabenzindolone (CBI) (P1), the resulting ADC1 showed loss of potency in CD22 target-expressing cancer cell lines (e.g., BJAB, WSU-DLCL2). In comparison, the conjugate (ADC2) of a cyclopropapyrroloindolone (CPI) (P2) was potent despite the two corresponding free drugs having similar picomolar cell-killing activity. Although the corresponding spirocyclization products of P1 and P2, responsible for DNA alkylation, are a prominent component in buffer, the linker immolation was slow when the PAB was connected as an ether (PABE) to the phenol in P1 compared to that in P2. Additional immolation studies with two other PABE-linked substituted phenol compounds showed that electron-withdrawing groups accelerated the immolation to release an acidic phenol-containing payload (to delocalize the negative charge on the anticipated anionic phenol oxygen during immolation). In contrast, efficient immolation of LD4 did not result in an active ADC4 because the payload (P4) had a low potency to kill cells. In addition, nonimmolation of LD5 did not affect the cell-killing potency of its ADC5 since immolation is not required for DNA alkylation by the center-linked pyrrolobenzodiazepine. Therefore, careful evaluation needs to be conducted when the Val-Cit-PAB linker is used to connect antibodies to a phenol-containing drug as the linker immolation, as well as payload potency and stability, affects the cell-killing activity of an ADC.
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http://dx.doi.org/10.1021/acs.bioconjchem.7b00576DOI Listing
February 2018

Intratumoral Payload Concentration Correlates with the Activity of Antibody-Drug Conjugates.

Mol Cancer Ther 2018 03 18;17(3):677-685. Epub 2018 Jan 18.

Drug Metabolism & Pharmacokinetics, Genentech Inc., South San Francisco, California.

Antibody-drug conjugates (ADC) have become important scaffolds for targeted cancer therapies. However, ADC exposure-response correlation is not well characterized. We demonstrated that intratumor payload exposures correlated well with the corresponding efficacies of several disulfide-linked ADCs, bearing an DNA alkylating agent, pyrrolo[2,1-c][1,4]benzodiazepine-dimer (PBD), in HER2-expressing xenograft models. The correlation suggests that a threshold concentration of intratumor payload is required to support sustained efficacy and an ADC can deliver an excessive level of payload to tumors that does not enhance efficacy ("Plateau" effect). In contrast to tumor PBD concentrations, related assessments of systemic exposures, plasma stability, and drug-to-antibody ratio changes of related ADCs did not consistently rationalize the observed ADC efficacies. A minimal efficacious dose could be determined by ADC dose-fractionation studies in the xenograft models. Mechanistic investigations revealed that both linker immolation and linker disulfide stability are the key factors that determine intratumor PBD concentrations. Overall, this study demonstrates how a linker design can impact ADC efficacy and that the intratumor exposure of a payload drug as the molecular mechanism quantitatively correlate with and predict the antitumor efficacy of ADCs. .
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http://dx.doi.org/10.1158/1535-7163.MCT-17-0697DOI Listing
March 2018

Transporter Expression in Noncancerous and Cancerous Liver Tissue from Donors with Hepatocellular Carcinoma and Chronic Hepatitis C Infection Quantified by LC-MS/MS Proteomics.

Drug Metab Dispos 2018 02 14;46(2):189-196. Epub 2017 Nov 14.

Department of Pharmaceutics, University of Washington, Seattle, Washington (S.B., E.J.K., B.P., J.D.U.); Departments of Clinical Research, Clinical Pharmacology, and Drug Metabolism and Pharmacokinetics, Gilead Sciences, Inc., Foster City, California (A.S.R., A.M., Y.L.); Drug Metabolism and Pharmacokinetics, Genentech, Inc., South San Francisco, California (L.S., C.E.C.A.H.); DMPK, Biogen Idec, Cambridge, Massachusetts (G.X., C.R.); Pharmacokinetics, Pharmacodynamics and Drug Metabolism, Merck & Co., Rahway, New Jersey (X.C., R.E.); Bristol-Myers Squibb Company, Princeton, New Jersey (W.G.H.); Takeda Pharmaceuticals International Co., Cambridge, Massachusetts (M.L.); and Translational Sciences, Ardea Biosciences, Inc., San Diego, California (C.A.L.)

Protein expression of major hepatobiliary drug transporters (NTCP, OATPs, OCT1, BSEP, BCRP, MATE1, MRPs, and P-gp) in cancerous (C, = 8) and adjacent noncancerous (NC, = 33) liver tissues obtained from patients with chronic hepatitis C with hepatocellular carcinoma (HCV-HCC) were quantified by LC-MS/MS proteomics. Herein, we compare our results with our previous data from noninfected, noncirrhotic (control, = 36) and HCV-cirrhotic ( = 30) livers. The amount of membrane protein yielded from NC and C HCV-HCC tissues decreased (31%, 67%) relative to control livers. In comparison with control livers, with the exception of NTCP, MRP2, and MATE1, transporter expression decreased in NC (38%-76%) and C (56%-96%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP expression increased (113%), MATE1 expression decreased (58%), and MRP2 expression was unchanged relative to control livers. In C HCV-HCC tissues, NTCP and MRP2 expression decreased (63%, 56%) and MATE1 expression was unchanged relative to control livers. Compared with HCV-cirrhotic livers, aside from NTCP, OCT1, BSEP, and MRP2, transporter expression decreased in NC (41%-71%) and C (54%-89%) HCV-HCC tissues. In NC HCV-HCC tissues, NTCP and MRP2 expression increased (362%, 142%), whereas OCT1 and BSEP expression was unchanged. In C HCV-HCC tissues, OCT1 and BSEP expression decreased (90%, 80%) relative to HCV-cirrhotic livers, whereas NTCP and MRP2 expression was unchanged. Expression of OATP2B1, BSEP, MRP2, and MRP3 decreased (56%-72%) in C HCV-HCC tissues in comparison with matched NC tissues ( = 8), but the expression of other transporters was unchanged. These data will be helpful in the future to predict transporter-mediated hepatocellular drug concentrations in patients with HCV-HCC.
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http://dx.doi.org/10.1124/dmd.117.077289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5776333PMC
February 2018

Strategy for CYP3A Induction Risk Assessment from Preclinical Signal to Human: a Case Example of a Late-Stage Discovery Compound.

Pharm Res 2017 Nov 16;34(11):2403-2414. Epub 2017 Aug 16.

Department of Drug Metabolism and Pharmacokinetics, Genentech, A Member of the Roche Group, 1 DNA Way, South San Francisco, California, 94080, USA.

Purpose: The exposure of G2917 decreased by four-fold at oral doses of 100 mg/kg twice daily for seven days in cynomolgus monkeys. Additional investigative work was conducted to understand: (1) the causes for the significant reduction in G2917 exposure in monkeys; (2) the extrapolation of in vitro induction data to in vivo findings in monkeys, and (3) the relevance of this pre-clinical finding to humans at the projected human efficacious dose.

Methods: Pharmacokinetic and induction potency (in vitro and in vivo) of G2917 in monkeys, and the in vitro human induction potency were studied. The hepatic CYP3A biomarkers 4β-hydroxycholesterol (4β-HC) and 6β-hydroxycortisol/cortisol ratio (6β-OHC/C) were monitored in in vivo studies. The static mechanistic model was used to quantitatively understand the in vitro-in vivo extrapolation (IVIVE) on the magnitude of induction retrospectively. Physiologically based pharmacokinetic (PBPK) modeling was used to predict the human pharmacokinetics and induction-based drug-drug interactions (DDI).

Results: All in vitro and in vivo data indicate that the significant reduction in exposure of G2917 in monkeys is caused by auto-induction of CYP3A. The mechanistic understanding of IVIVE of G2917 induction in monkey provides higher confidence in the induction risk prediction in human using the PBPK modeling. PBPK model analysis predicted minimum auto-induction and DDI liability in humans at the predicted efficacious dose.

Conclusions: The learning of this example provided a strategy to address the human CYP3A induction risk prospectively when there is an auto-induction finding in preclinical toxicology study.
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http://dx.doi.org/10.1007/s11095-017-2246-8DOI Listing
November 2017

Mixed Matrix Method Provides A Reliable Metabolite Exposure Comparison for Assessment of Metabolites in Safety Testing (MIST).

Drug Metab Lett 2017 11;11(1):21-28

Department of Drug Metabolism and Pharmacokinetics, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Background: The regulatory guidances on metabolites in safety testing (MIST) by US Food and Drug Administration (FDA) and International Conference on Harmonisation (ICH) describe the necessity to assess exposures of major circulating metabolites in humans at steady state relative to exposures achieved in nonclinical safety studies prior to the initiation of large scale clinical trials. This comparison can be accomplished by measuring metabolite concentrations in animals and humans with validated bioanalytical methods. However, bioanalysis of metabolites in multiple species and multiple studies is resource intensive and may impact the timelines of clinical studies.

Method: A simple, reliable and accurate method has been developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volume of human plasma with blank plasma of animal species and vice versa followed by an analysis using LC-SRM or LC-HRMS. Here, we explored the reliability and accuracy of this method in several development projects at Genentech and compared the results to those obtained from validated bioanalytical methods.

Results: The mixed-matrix method provided comparable accuracy (within ±20%) to those obtained from validated bioanalysis but does not require authentic standards or radiolabeled compounds, which could translate to time and resource savings in drug development.

Conclusion: Quantitative assessment of metabolite coverage in safety species can be made using mixed matrix method with similar accuracy and scientific rigor to those obtained from validated bioanalytical methods. Moving forward, we are encouraging the industry and regulators to consider accepting the mixed matrix method for assessing metabolite exposure comparisons between humans and animal species used in toxicology studies.
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http://dx.doi.org/10.2174/1872312811666170710193229DOI Listing
November 2017

Strategies of Drug Transporter Quantitation by LC-MS: Importance of Peptide Selection and Digestion Efficiency.

AAPS J 2017 09 6;19(5):1469-1478. Epub 2017 Jun 6.

Genentech Inc, 1 DNA Way, South San Francisco, California, 94080, USA.

Huge variation of drug transporter abundance was seen in the literature, making PBPK prediction difficult when transporters play a major role. Among multiple factors such as membrane fraction, digestion, and peptide selection that contributed to such variation, peptide selection is the least discussed. Herein, a strategy was established by using a small amount of purified protein standard to select a peptide with near 100% digestion efficiency for quantitation of a transporter protein MDR1. The impact of native membrane protein's tertiary structure on the digestion efficiency of surrogate peptides of MDR1 was investigated. Peptides in more solvent accessible regions are found to be digested much more efficiently than those in large stretches of helical structures. The concentration of peptide EALDESIPPVSFWR(EAL) in the most solvent accessible linker region of MDR1 was found closest to the true protein concentration. When using EAL for MDR1 quantitation, the abundance is over 10 times higher than previously reported, indicating the importance of peptide selection for transporter quantitation. In addition, this study also proposes a screening strategy to select peptides appropriate for relative quantitation for in vitro-in vivo extrapolation in the absence of any protein standard.
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http://dx.doi.org/10.1208/s12248-017-0106-4DOI Listing
September 2017

Novel Mechanism of Decyanation of GDC-0425 by Cytochrome P450.

Drug Metab Dispos 2017 05 10;45(5):430-440. Epub 2017 Feb 10.

Departments of Drug Metabolism and Pharmacokinetics (R.H.T., J.S.H., Y.C., C.E.C.A.H., S.C.K., S.M.), and Discovery Chemistry (M.S.), Genentech, Inc., 1 DNA Way, South San Francisco, California.

GDC-0425 [5-((1-ethylpiperidin-4-yl)oxy)-9H-pyrrolo[2,3-b:5,4-c']dipyridine-6-carbonitrile] is an orally bioavailable small-molecule inhibitor of checkpoint kinase 1 that was investigated as a novel cotherapy to potentiate chemotherapeutic drugs, such as gemcitabine. In a radiolabeled absorption, distribution, metabolism, and excretion study in Sprague-Dawley rats, trace-level but long-lived C-labeled thiocyanate was observed in circulation. This thiocyanate originated from metabolic decyanation of GDC-0425 and rapid conversion of cyanide to thiocyanate. Excretion studies indicated decyanation was a minor metabolic pathway, but placing C at nitrile magnified its observation. Cytochrome P450s catalyzed the oxidative decyanation reaction in vitro when tested with liver microsomes, and in the presence of O, one atom of O was incorporated into the decyanated product. To translate this finding to a clinical risk assessment, the total circulating levels of thiocyanate (endogenous plus drug-derived) were measured following repeated administration of GDC-0425 to rats and cynomolgus monkeys. No overt increases were observed with thiocyanate concentrations of 121-154 M in rats and 71-110 M in monkeys receiving vehicle and all tested doses of GDC-0425. These findings were consistent with results from the radiolabel rat study where decyanation accounted for conversion of <1% of the administered GDC-0425 and contributed less than 1 M thiocyanate to systemic levels. Further, in vitro studies showed only trace oxidative decyanation for humans. These data indicated that, although cyanide was metabolically released from GDC-0425 and formed low levels of thiocyanate, this pathway was a minor route of metabolism, and GDC-0425-related increases in systemic thiocyanate were unlikely to pose safety concerns for subjects of clinical studies.
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http://dx.doi.org/10.1124/dmd.116.074336DOI Listing
May 2017

Linker Immolation Determines Cell Killing Activity of Disulfide-Linked Pyrrolobenzodiazepine Antibody-Drug Conjugates.

ACS Med Chem Lett 2016 Nov 26;7(11):988-993. Epub 2016 Aug 26.

Drug Metabolism & Pharmacokinetics, Discovery Chemistry, Biochemical and Cellular Pharmacology, Protein Chemistry, and Molecular Oncology, Genentech , South San Francisco, California 94080, United States.

Disulfide bonds could be valuable linkers for a variety of therapeutic applications requiring tunable cleavage between two parts of a molecule (e.g., antibody-drug conjugates). The in vitro linker immolation of β-mercaptoethyl-carbamate disulfides and DNA alkylation properties of associated payloads were investigated to understand the determinant of cell killing potency of anti-CD22 linked pyrrolobenzodiazepine (PBD-dimer) conjugates. Efficient immolation and release of a PBD-dimer with strong DNA alkylation properties were observed following disulfide cleavage of methyl- and cyclobutyl-substituted disulfide linkers. However, the analogous cyclopropyl-containing linker did not immolate, and the associated thiol-containing product was a poor DNA alkylator. As predicted from these in vitro assessments, the related anti-CD22 ADCs showed different target-dependent cell killing activities in WSU-DLCL2 and BJAB cell lines. These results demonstrate how the in vitro immolation models can be used to help design efficacious ADCs.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108036PMC
November 2016
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