Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

Hepatic Organic Anion Transporting Polypeptide-Mediated Clearance in the Beagle Dog: Assessing In Vitro-In Vivo Relationships and Applying Cross-Species Empirical Scaling Factors to Improve Prediction of Human Clearance.

In the present study, the beagle dog was evaluated as a preclinical model to investigate organic anion transporting polypeptide (OATP)-mediated hepatic clearance. In vitro studies were performed with nine OATP substrates in three lots of plated male dog hepatocytes ± OATP inhibitor cocktail to determine total uptake clearance (CLuptake) and total and unbound cell-to-medium concentration ratio (Kpuu). In vivo intrinsic hepatic clearances (CLint,H) were determined following intravenous drug administration (0.1 mg/kg) in male beagle dogs. The in vitro parameters were compared with those previously reported in plated human, monkey, and rat hepatocytes; the ability of cross-species scaling factors to improve prediction of human in vivo clearance was assessed. CLuptake in dog hepatocytes ranged from 9.4 to 135 µl/min/106 cells for fexofenadine and telmisartan, respectively. Active process contributed >75% to CLuptake for 5/9 drugs. Rosuvastatin and valsartan showed Kpuu > 10, whereas cerivastatin, pitavastatin, repaglinide, and telmisartan had Kpuu < 5. The extent of hepatocellular binding in dog was consistent with other preclinical species and humans. The bias (2.73-fold) obtained from comparison of predicted versus in vivo dog CLint,H was applied as an average empirical scaling factor (ESFav) for in vitro-in vivo extrapolation of human CLint,H The ESFav based on dog reduced underprediction of human CLint,H for the same data set (geometric mean fold error = 2.1), highlighting its utility as a preclinical model to investigate OATP-mediated uptake. The ESFav from all preclinical species resulted in comparable improvement of human clearance prediction, in contrast to drug-specific empirical scalars, rationalized by species differences in expression and/or relative contribution of particular transporters to drug hepatic uptake.

Chimeric TK-NOG mice: a predictive model for cholestatic human liver toxicity.

Due to the substantial interspecies differences in drug metabolism and disposition, drug-induced liver injury (DILI) in humans is often not predicted by studies performed in animal species. For example, a drug (bosentan) used to treat pulmonary artery hypertension caused unexpected cholestatic liver toxicity in humans, which was not predicted by preclinical toxicology studies in multiple animal species. In this study, we demonstrate that NOG mice expressing a thymidine kinase transgene (TK-NOG) with humanized livers have a humanized profile of biliary excretion of a test (cefmetazole) drug, which was shown by an in situ perfusion study to result from interspecies differences in the rate of biliary transport and in liver retention of this drug. We also found that readily detectable cholestatic liver injury develops in TK-NOG mice with humanized livers after 1 week of treatment with bosentan (160, 32, or 6 mg/kg per day by mouth), whereas liver toxicity did not develop in control mice after 1 month of treatment. The laboratory and histologic features of bosentan-induced liver toxicity in humanized mice mirrored that of human subjects. Because DILI has become a significant public health problem, drug safety could be improved if preclinical toxicology studies were performed using humanized TK-NOG.

Prediction of renal transporter mediated drug-drug interactions for pemetrexed using physiologically based pharmacokinetic modeling.

Pemetrexed, an anionic anticancer drug with a narrow therapeutic index, is eliminated mainly by active renal tubular secretion. The in vitro to in vivo extrapolation approach used in this work was developed to predict possible drug-drug interactions (DDIs) that may occur after coadministration of pemetrexed and nonsteroidal anti-inflammatory drugs (NSAIDs), and it included in vitro assays, risk assessment models, and physiologically based pharmacokinetic (PBPK) models. The pemetrexed transport and its inhibition parameters by several NSAIDs were quantified using HEK-PEAK cells expressing organic anion transporter (OAT) 3 or OAT4. The NSAIDs were ranked according to their DDI index, calculated as the ratio of their maximum unbound concentration in plasma over the concentration inhibiting 50% (IC50) of active pemetrexed transport. A PBPK model for ibuprofen, the NSAID with the highest DDI index, was built incorporating active renal secretion in Simcyp Simulator. The bottom-up model for pemetrexed underpredicted the clearance by 2-fold. The model we built using a scaling factor of 5.3 for the maximal uptake rate (Vmax) of OAT3, which estimated using plasma concentration profiles from patients given a 10-minute infusion of 500 mg/m(2) of pemetrexed supplemented with folic acid and vitamin B12, recovered the clinical data adequately. The observed/predicted increases in Cmax and the area under the plasma-concentration time curve (AUC0-inf) of pemetrexed when ibuprofen was coadministered were 1.1 and 1.0, respectively. The coadministration of all other NSAIDs was predicted to have no significant impact on the AUC0-inf based on their DDI indexes. The PBPK model reasonably reproduced pemetrexed concentration time profiles in cancer patients and its interaction with ibuprofen.

Membrane transporters in drug development.

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

Development and validation of a sensitive, specific, and rapid hybridization-ELISA assay for determination of concentrations of a ribozyme in biological matrices.

Ribozymes are RNA or modified RNA polymers capable of catalyzing cleavage reactions in target strands RNA, and are under development as human therapeutics. Previous methods used for quantitation of nucleic acid polymers in serum or plasma required extraction of the polymer followed by capillary electrophoresis, HPLC, or gel electrophoresis. These methods are time consuming and lack sensitivity. A bioanalytical method has been developed that does not require extraction of the ribozyme analyte from serum. This technique relies on hybridization of the ribozyme molecule to two complementary biotin and digoxigenin labeled oligonucleotide probes. Serum containing the ribozyme is mixed with the labeled probes, and the mixture is heated at 75 degrees C for 5 min to disrupt the ribozyme secondary structure. Samples are then cooled to permit probe annealing and are added to a streptavidin-coated 96-well plate. The bound complex is detected with an anti-digoxigenin alkaline phosphatase (AP) conjugate using PNPP (p-nitrophenyl phosphate) as a substrate. The amount of colored product is measured on a microtiter plate reader at a wavelength of 405 nm. Concentrations of unknown ribozyme samples are estimated based on a standard curve (0.37-270 ng/ml) prepared in serum. The validated lower and upper limits of quantification are 5.0 and 120 ng/ml, respectively. The assay can be completed in approximately 5h and does not require extraction procedures or electrophoretic/chromatographic separation. It is therefore a simple, sensitive and rapid technique. This assay has been validated and has been used for quantitation of serum levels of the HEPTAZYME ribozyme in mouse, monkey, and human pharmacokinetic studies.