RESUMO
Monoclonal antibodies (mAbs) are a rapidly growing drug class for which great efforts have been made to optimize certain molecular features to achieve the desired pharmacokinetic (PK) properties. One approach is to engineer the interactions of the mAb with the neonatal Fc receptor (FcRn) by introducing specific amino acid sequence mutations, and to assess their effect on the PK profile with in vivo studies. Indeed, FcRn protects mAbs from intracellular degradation, thereby prolongs antibody circulation time in plasma and modulates its systemic clearance. To allow more efficient and focused mAb optimization, in vitro input that helps to identify and quantitatively predict the contribution of different processes driving non-target mediated mAb clearance in vivo and supporting translational PK modeling activities is essential. With this aim, we evaluated the applicability and in vivo-relevance of an in vitro cellular FcRn-mediated transcytosis assay to explain the PK behavior of 25 mAbs in rat or monkey. The assay was able to capture species-specific differences in IgG-FcRn interactions and overall correctly ranked Fc mutants according to their in vivo clearance. However, it could not explain the PK behavior of all tested IgGs, indicating that mAb disposition in vivo is a complex interplay of additional processes besides the FcRn interaction. Overall, the transcytosis assay was considered suitable to rank mAb candidates for their FcRn-mediated clearance component before extensive in vivo testing, and represents a first step toward a multi-factorial in vivo clearance prediction approach based on in vitro data.
Assuntos
Anticorpos Monoclonais Murinos/farmacocinética , Bioensaio/métodos , Antígenos de Histocompatibilidade Classe I/imunologia , Receptores Fc/imunologia , Transcitose/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Macaca fascicularis , Camundongos , Ratos , Ratos WistarRESUMO
The multidrug resistance protein 1 (MDR1) is known to limit brain penetration of drugs and play a key role in drug-drug interactions (DDIs). Theoretical cut-offs from regulatory guidelines are used to extrapolate MDR1 interactions from in vitro to in vivo. However, these cut-offs do not account for interlaboratory variability. Our aim was to calibrate our experimental system to allow better in vivo predictions. We selected 166 central nervous system (CNS) and non-CNS drugs to calibrate the MDR1 transport screening assay using Lewis lung cancer porcine kidney 1 epithelial cells overexpressing MDR1 (L-MDR1). A threshold efflux ratio (ER) of 2 was established as one parameter to assess brain penetration in lead optimization. The inhibitory potential of 57 molecules was evaluated using IC50 values based on the digoxin ER-IC50(ER)-or apparent permeability-IC50(Papp)-in L-MDR1 cells. Published clinical data for 68 DDIs involving digoxin as the victim drug were collected. DDI risk assessments were based on intestinal concentrations ([I2]) as well as unbound [I1u] and total plasma [I1T] concentrations. A receiver operating characteristic analysis identified an [I2]/IC50(ER) of 6.5 as the best predictor of a potential interaction with digoxin in patients. The model was further evaluated with a test set of 11 digoxin DDIs and 16 nondigoxin DDIs, resulting in only one false negative for each test set, no false positives among the digoxin DDIs, and two among the nondigoxin DDIs. Future refinements might include using cerebrospinal fluid to unbound plasma concentration ratios rather than therapeutic class, better estimation of [I2], and dynamic modeling of MDR1-mediated DDIs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Interações Medicamentosas/fisiologia , Preparações Farmacêuticas/metabolismo , Animais , Bioensaio/métodos , Transporte Biológico/fisiologia , Calibragem , Linhagem Celular Tumoral , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Digoxina/metabolismo , Humanos , Técnicas In Vitro/métodos , Permeabilidade , SuínosRESUMO
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, V(max) and K(m), 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 (P(dif)). 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 V(max) and K(m) 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 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees C. Therefore, P(dif) was better predicted if it was evaluated under the same experimental conditions as V(max) and K(m), i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37 degrees C, instead of a parallel control evaluation at 4 degrees C.
Assuntos
Simulação por Computador , Hepatócitos/metabolismo , Modelos Biológicos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Algoritmos , Animais , Transporte Biológico Ativo , Células CHO , Cricetinae , Cricetulus , Difusão , Estrona/análogos & derivados , Estrona/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Fluvastatina , Indóis/metabolismo , Cinética , Masculino , Membranas Artificiais , Naftalenos/metabolismo , Oligopeptídeos/metabolismo , Transportadores de Ânions Orgânicos/genética , Permeabilidade , Piperidinas/metabolismo , Quinolinas/metabolismo , Ratos , Ratos Wistar , Sincalida/metabolismo , Temperatura , Terfenadina/análogos & derivados , Terfenadina/metabolismoRESUMO
Hepatic uptake carriers of the organic anion-transporting peptide (OATP) family of solute carriers are more and more recognized as being involved in hepatic elimination of many drugs and potentially associated drug-drug interactions. The gemfibrozil-statin interaction was studied at the level of active hepatic uptake as a model for such drug-drug interactions. Active, temperature-dependent uptake of fluvastatin into primary human hepatocytes was shown. Multiple transporters are involved in this uptake as Chinese hamster ovary or HEK293 cells expressing either OATP1B1 (K(m) = 1.4-3.5 microM), OATP2B1 (K(m) = 0.7-0.8 microM), or OATP1B3 showed significant fluvastatin uptake relative to control cells. For OATP1B1 the inhibition by gemfibrozil was substrate-dependent as the transport of fluvastatin (IC(50) of 63 microM), pravastatin, simvastatin, and taurocholate was inhibited by gemfibrozil, whereas the transport of estrone-3-sulfate and troglitazone sulfate (both used at 3 microM) was not affected. The OATP1B1- but not OATP2B1-mediated transport of estrone-3-sulfate displayed biphasic saturation kinetics, with two distinct affinity components for estrone-3-sulfate (0.23 and 45 microM). Only the high-affinity component was inhibited by gemfibrozil. Recombinant OATP1B1-, OATP2B1-, and OATP1B3-mediated fluvastatin transport was inhibited to 97, 70, and 62% by gemfibrozil (200 microM), respectively, whereas only a small inhibitory effect by gemfibrozil (200 microM) on fluvastatin uptake into primary human hepatocytes was observed (27% inhibition). The results indicate that the in vitro engineered systems can not always predict the behavior in more complex systems such as freshly isolated primary hepatocytes. Therefore, selection of substrate, substrate concentration, and in vitro transport system are critical for the conduct of in vitro interaction studies involving individual liver OATP carriers.