Hans Peter Grimm

Design, Data Analysis, and Simulation of in Vitro Drug Transport Kinetic Experiments Using a Mechanistic in Vitro Model

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, Vmax and Km, as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (Pdif). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for Vmax and Km were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5–6-fold higher permeability at 37°C compared with that at 4°C), for fexofenadine a 16-fold higher passive permeability was seen at 37°C. Therefore, Pdif was better predicted if it was evaluated under the same experimental conditions as Vmax and Km, i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37°C, instead of a parallel control evaluation at 4°C.

Challenges and opportunities with modelling and simulation in drug discovery and drug development

The benefits of modelling and simulation at the pre-clinical stage of drug development can be realized through formal and realistic integration of data on physicochemical properties, pharmacokinetics, pharmacodynamics, formulation and safety. Such data integration and the powerful combination of physiologically based pharmacokinetic (PBPK) with pharmacokinetic–pharmacodynamic relationship (PK/PD) models provides the basis for quantitative outputs allowing comparisons across compounds and resulting in improved decision-making during the selection process. Such PBPK/PD evaluations provide crucial information on the potency and safety of drug candidates in vivo and the bridging of the PK/PD concept established during the pre-clinical phase to clinical studies. Modelling and simulation is required to address a number of key questions at the various stages of the drug-discovery and -development process. Such questions include the following. (1) What is the expected human PK profile for potential clinical candidate(s)? (2) Is this profile and its associated PD adequate for the given indication? (3) What is the optimal dosing schedule with respect to safety and efficacy? (4) Is a food effect expected? (5) How can formulation be improved and what is the potential benefit? (6) What is the expected variability and uncertainty in the predictions?

Minipig as a potential translatable model for monoclonal antibody pharmacokinetics after intravenous and subcutaneous administration

Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.

Gaining insights into the consequences of target-mediated drug disposition of monoclonal antibodies using quasi-steady-state approximations

Target-mediated drug disposition (TMDD) is frequently reported for therapeutic monoclonal antibodies and is linked to the high affinity and high specificity of antibody molecules for their target. Understanding TMDD of a monoclonal antibody should go beyond the empirical description of its non-linear PK since valuable insights on the antibody-target interaction itself can be gained. This makes its mechanistic understanding precious for the drug development process, in particular for the optimization of new antibody molecules, for the design and interpretation of pharmacokinetic studies, and possibly even for the evaluation of efficacy and dose selection of drug candidates. Using the observation that the molecular (microscopic) processes are usually much more rapid than the pharmacokinetic (macroscopic) processes, a series of quasi-steady-state conditions on the microscopic level is proposed to bridge the gap between simple empirical and complex mechanistic descriptions of TMDD. These considerations show the impact of parameters such as target turnover, target expression, and target accessibility on the pharmacokinetics and pharmacodynamics of monoclonal antibodies.

The dynamics of Aβ distribution after γ-secretase inhibitor treatment, as determined by experimental and modelling approaches in a wild type rat.

Inhibition of the enzyme(s) that produce the Amyloid beta (Aβ) peptide, namely BACE and γ-secretase, is considered an attractive target for Alzheimer’s disease therapy. However, the optimal pharmacokinetic–pharmacodynamic modelling method to describe the changes in Aβ levels after drug treatment is unclear. In this study, turnover models were employed to describe Aβ levels following treatment with the γ-secretase inhibitor RO5036450, in the wild type rat. Initially, Aβ level changes in the brain, cerebral spinal fluid (CSF) and plasma were modeled as separate biological compartments, which allowed the estimation of a compound IC50 and Aβ turnover. While the data were well described, the model did not take into consideration that the CSF pool of Aβ most likely originates from the brain via the CSF drainage pathway. Therefore, a separate model was carried out, with the assumption that CSF Aβ levels originated from the brain. The optimal model that described the data involved two brain Aβ 40 sub-compartments, one with a rapid turnover, from which CSF Aβ 40 is derived, and a second quasi-static pool of ~20%. Importantly, the estimated in vivo brain IC50 was in a good range of the in vitro IC50 (ratio, 1.4). In conclusion, the PK/PD models presented here are well suited for describing the temporal changes in Aβ levels that occur after treatment with an Aβ lowering drug, and identifying physiological parameters.

Target-mediated drug disposition and prolonged liver accumulation of a novel humanized anti-CD81 monoclonal antibody in cynomolgus monkeys

CD81 is an essential receptor for hepatitis C virus (HCV). K21 is a novel high affinity anti-CD81 antibody with potent broad spectrum anti-HCV activity in vitro. The pharmacokinetics (PK), pharmacodynamics and liver distribution of K21 were characterized in cynomolgus monkeys after intravenous (i.v.) administration of K21. Characteristic target-mediated drug disposition (TMDD) was shown based on the PK profile of K21 and a semi-mechanistic TMDD model was used to analyze the data. From the TMDD model, the estimated size of the total target pool at baseline (Vc • Rbase) is 16 nmol/kg and the estimated apparent Michaelis-Menten constant (KM) is 4.01 nM. A simulation using estimated TMDD parameters indicated that the number of free receptors remains below 1% for at least 3 h after an i.v. bolus of 7 mg/kg. Experimentally, the availability of free CD81 on peripheral lymphocytes was measured by immunostaining with anti-CD81 antibody JS81. After K21 administration, a dose- and time-dependent reduction in free CD81 on peripheral lymphocytes was observed. Fewer than 3% of B cells could bind JS81 3 h after a 7 mg/kg dose. High concentrations of K21 were found in liver homogenates, and the liver/serum ratio of K21 increased time-dependently and reached ~160 at 168 h post-administration. The presence of K21 bound to hepatocytes was confirmed by immunohistochemistry. The fast serum clearance of K21 and accumulation in the liver are consistent with TMDD. The TMDD-driven liver accumulation of the anti-CD81 antibody K21 supports the further investigation of K21 as a therapeutic inhibitor of HCV entry.

The Effects of Interleukin-6 Signal Blockade on Immune System, Reproductive and Skeletal Development in Juvenile Mice

Interleukin-6 (IL-6) is involved in the pathogenesis of multiple disorders, including juvenile autoimmune diseases. IL-6 participates in a broad spectrum of physiological events, and the IL-6 receptor (IL-6R) is widely distributed across multiple organs. The interrelationship of development phases in juveniles together with organs involved in IL-6 signaling called for evaluations of anti-IL-6R antibody induced effects in a juvenile mouse model to assess the safety of such an approach in human juvenile arthritis. Here we show that naive mice in which IL-6 signals have been transiently blocked during the juvenile period develop normally. The fatal immunogenic reactions recorded earlier by repeated administration of the chosen rat anti-mouse IL-6R antibody, MR16-1, to mice were avoided successfully by application of a high loading dose followed by lower maintenance doses, with the support of modeling data. The high loading-dose regimen enabled us to conduct assessments without any major interference due to immunogenicity. Transient and complete inhibition of IL-6 signals from postnatal days 22 to 79 in mice exhibited no biologically important changes in sexual maturation or development of immune and skeletal systems. Although tendencies toward reductions of peripheral blood T-cell counts were observed, normal levels of antigen-specific IgG/IgM antibody productions indicating sufficient immunological functions were confirmed. Our results demonstrate that blockage of IL-6R by the neutralizing antibody does not affect juvenile development. This may be in part due to the generation or existence of compensatory pathways in the whole body system.

Hematopoietic cells as site of first-pass catabolism after subcutaneous dosing and contributors to systemic clearance of a monoclonal antibody in mice

ABSTRACT The neonatal Fc receptor (FcRn) has been demonstrated to contribute to a high bioavailability of monoclonal antibodies (mAbs). In this study, we explored the cellular sites of FcRn-mediated protection after subcutaneous (SC) and intravenous (IV) administration. SC absorption and IV disposition kinetics of a mAb were studied in hFcRn transgenic (Tg) bone marrow chimeric mice in which hFcRn was restricted to radioresistant cells or hematopoietic cells. SC bioavailabilities close to 90% were observed in hFcRn Tg mice and chimeric mice with hFcRn expression in hematopoietic cells, whereas SC bioavailabilities were markedly lower when FcRn was missing in hematopoietic cells. Our study demonstrates: 1) FcRn in radiosensitive hematopoietic cells is required for high SC bioavailability, indicating first-pass catabolism after SC administration by hematopoietic cells; 2) FcRn-mediated transcytosis or recycling by radioresistent cells is not required for high SC bioavailability; and 3) after IV administration hematopoietic and radioresistent cells contribute about equally to clearance of the mAb. A pharmacokinetic model was devised to describe a mixed elimination via radioresistent and hematopoietic cells from vascular and extravascular compartments, respectively. Overall, the study indicates a relevant role of hematopoietic cells for first-pass clearance of mAbs after SC administration and confirms their role in the overall clearance of mAbs.

Prediction of the optimal dosing regimen using a mathematical model of tumour uptake for immunocytokine-based cancer immunotherapy

Purpose: Optimal dosing is critical for immunocytokine-based cancer immunotherapy to maximize efficacy and minimize toxicity. Cergutuzumab amunaleukin (CEA-IL2v) is a novel CEA-targeted immunocytokine. We set out to develop a mathematical model to predict intratumoral CEA-IL2v concentrations following various systemic dosing intensities. Experimental Design: Sequential measurements of CEA-IL2v plasma concentrations in 74 patients with solid tumors were applied in a series of differential equations to devise a model that also incorporates the peripheral concentrations of IL2 receptor–positive cell populations (i.e., CD8+, CD4+, NK, and B cells), which affect tumor bioavailability of CEA-IL2v. Imaging data from a subset of 14 patients were subsequently utilized to additionally predict antibody uptake in tumor tissues. Results: We created a pharmacokinetic/pharmacodynamic mathematical model that incorporates the expansion of IL2R-positive target cells at multiple dose levels and different schedules of CEA-IL2v. Model-based prediction of drug levels correlated with the concentration of IL2R-positive cells in the peripheral blood of patients. The pharmacokinetic model was further refined and extended by adding a model of antibody uptake, which is based on drug dose and the biological properties of the tumor. In silico predictions of our model correlated with imaging data and demonstrated that a dose-dense schedule comprising escalating doses and shortened intervals of drug administration can improve intratumoral drug uptake and overcome consumption of CEA-IL2v by the expanding population of IL2R-positive cells. Conclusions: The model presented here allows simulation of individualized treatment plans for optimal dosing and scheduling of immunocytokines for anticancer immunotherapy. Clin Cancer Res; 24(14); 3325–33. ©2018 AACR. See related commentary by Ruiz-Cerdá et al., p. 3236

Quantification of IgG monoclonal antibody clearance in tissues

ABSTRACT Monoclonal antibodies are an important therapeutic entity, and knowledge of antibody pharmacokinetics has steadily increased over the years. Despite this effort, little is known about the extent of IgG antibody degradation in different tissues of the body. While studies have been published identifying sites of degradation with the use of residualizing and non-residualizing radiolabels, quantitative tissue clearances have not yet been derived. Here, we show that in physiologically-based pharmacokinetic (PBPK) models we can combine mouse data of Indium-111 and Iodine-125 labeled antibodies with prior physiologic knowledge to determine tissue-specific intrinsic clearances. Unspecific total tissue clearance (mL/day) in the mouse was estimated to be: liver = 4.75; brain = 0.02; gut = 0.40; heart = 0.07; kidney = 0.97; lung = 0.20; muscle = 3.02; skin = 3.89; spleen = 0.45; rest of body = 2.16. The highest catabolic activity (per g tissue) was in spleen for an FcRn wild-type antibody, but shifts to the liver for an antibody with reduced FcRn affinity. In the model developed, this shift can be explained by the liver having a greater FcRn-mediated protection capacity than the spleen. The quantification of tissue intrinsic clearances and FcRn salvage capacity increases our understanding of quantitative processes that drive the therapeutic responses of antibodies. This knowledge is critical, for instance to estimate the non-specific cellular uptake and degradation of antibodies used for targeted delivery of payloads.