Journal of pharmacological and toxicological methods最新文献

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) present an attractive in vitro platform to model safety and toxicity assessments—notably screening pro-arrhythmic compounds. The utility of the platform is stymied by a hiPSC-CM contractile apparatus and calcium handling mechanism akin to fetal phenotypes, evidenced by a negative force-frequency relationship. As such, hiPSC-CMs are limited in their ability to assess compounds that modulate contraction mediated by ionotropic compounds (Robertson, Tran, & George, 2013). To address this limitation, we utilize Agilent's xCELLigence Real-Time Cell Analyzer ePacer (RTCA ePacer) to enhance hiPSC-CM functional maturity. A continuous, progressive increase of electrical pacing is applied to hiPSC-CMs for up to 15 days. Contraction and viability are recorded by measurement of impedance using the RTCA ePacer. Our data confirms hiPSC-CMs inherently demonstrate a negative impedance amplitude frequency that is reversed after long-term electrical pacing. The data also indicate positive inotropic compounds increase the contractility of paced cardiomyocytes and calcium handling machinery is improved. Increased expression of genes critical to cardiomyocyte maturation further underscores the maturity of paced cells. In summary, our data suggest the application of continuous electrical pacing can functionally mature hiPSC-CMs, enhancing cellular response to positive inotropic compounds and improving calcium handling.

Summary

Long-term electrical stimulation of hiPSC-CM leads to functional maturation enabling predictive assessment of inotropic compounds.

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca²⁺ dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach. We performed a study to evaluate primary human CM and have them compared to freshly isolated dog cardiomyocytes for their basic function and responses to positive inotropes with well-known mechanisms. Our data showed that simultaneous assessment of sarcomere shortening and Ca²⁺-transient can be performed with both myocytes using the IonOptix system. Amplitude of sarcomere shortening and Ca²⁺-transient (CaT) were significantly higher in dog compared to human CM in the basic condition (absence of treatment), while longer duration of sarcomere shortening and CaT were observed in human cells. We observed that human and dog CMs have similar pharmacological responses to five inotropes with different mechanisms, including dobutamine and isoproterenol (β-adrenergic stimulation), milrinone (PDE3 inhibition), pimobendan and levosimendan (increase of Ca²⁺sensitization as well as PDE3 inhibition). In conclusion, our study suggests that myocytes obtained from both human donor hearts and dog hearts can be used to simultaneously assess drug-induced effects on sarcomere shortening and CaT using the IonOptix platform.

Microsampling, a reduced volume sampling method, has successfully gained attention at the International Conference on Harmonization (ICH) level and established benefits support its use in Toxicokinetic (TK) studies. These improved sampling techniques are less invasive and in large animal species improve animal welfare (refinement). To evaluate if the plasma concentrations of drugs were influenced by the blood sampling method, the traditional method from femoral vein and microsampling from tail vein in Cynomolgus monkeys were compared. The pharmacokinetic parameters (C_max, T_max and AUC) of four drugs (selected based on acid-base and volume of distribution properties) in non-human primate were correlated. The plasma samples were quantified using standard LC-MS/MS methods, qualified to evaluate the precision and accuracy before the analysis of real samples.

The results reported in this work demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. The data show that the exposure of drugs tested after blood collection using standard procedure from femoral vein and microsampling from tail vein is correlated and is not influenced by acid-base characteristics and volume of distribution.

The ICH E14/S7B Questions and Answers (Q&As) guideline introduces the concept of a “double negative” nonclinical scenario (negative hERG assay and negative in vivo QTc study) to demonstrate that a drug does not produce a clinically relevant QT prolongation (i.e., no QT liability). This nonclinical “double negative” data package, along with negative Phase 1 clinical QTc data, may be sufficient to substitute for a clinical Thorough QT (TQT) study in some specific cases. While standalone GLP in vivo cardiovascular studies in non-rodent species are standard practice during nonclinical drug development for small molecule programs, a variety of approaches to the design, conduct, analysis and interpretation are utilized across pharmaceutical companies and contract research organizations (CROs) that may, in some cases, negatively impact the stringent sensitivity needed to fulfill the new Q&As. Subject matter experts from both Pharma and CROs have collaborated to recommend best practices for more robust nonclinical cardiovascular telemetry studies in non-rodent species, with input from clinical and regulatory experts. The aim was to increase consistency and harmonization across the industry and to ensure delivery of high quality nonclinical QTc data to meet the proposed sensitivities defined within the revised ICH E14/S7B Q&As guideline (Q&As 5.1 and 6.1). The detailed best practice recommendations presented here cover the design and execution of the safety pharmacology cardiovascular study, including optimal methods for acquiring, analyzing, reporting, and interpreting the resulting QTc and pharmacokinetic data to allow for direct comparison to clinical exposures and assessment of safety margin for QTc prolongation.

The IMI project ConcePTION was launched to fill the knowledge gap of using medicines during pregnancy and lactation. To achieve this goal, several studies are being conducted, including the bioanalysis of amoxicillin in minipig plasma and milk. A high-throughput, robust and reliable liquid chromatography tandem mass spectrometry method was developed and validated according to FDA and EMA guidelines to determine the concentrations of amoxicillin in a large number of minipig plasma and milk samples. Chromatographic separation was achieved on a Luna® Omega Polar C18, 1.6 μm, 100 × 2.1 mm column, with a mobile phase consisting of 0.1% formic acid in water and acetonitrile. Mass spectrometry used in a positive ionization mode and the transitions m/z 366.1 → 349.2 was selected to monitor amoxicillin, while m/z 370.1 → 114.15 was selected for the stable isotope labelled internal standard. This method features a linear quantification range of 10 ng/mL - 10 μg/mL, recovery of not less than 94.1%, a single sample extraction method for both plasma and milk matrices, and an analysis runtime of 5 min.

This editorial prefaces the annual themed issue on safety pharmacology (SP) methods published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). We highlight here the content derived from the recent 2022 Safety Pharmacology Society (SPS) and Canadian Society of Pharmacology and Therapeutics (CSPT) joint meeting held in Montreal, Quebec, Canada. The meeting also generated 179 abstracts (reproduced in the current volume of JPTM). As in previous years the manuscripts reflect various areas of innovation in SP including a comparison of the sensitivity of cross-over and parallel study designs for QTc assessment, use of human-induced pluripotent stem cell (hi-PSC) neuronal cell preparations for use in neuropharmacological safety screening, and hiPSC derived cardiac myocytes in assessing inotropic adversity. With respect to the latter, we anticipate the emergence of a large data set of positive and negative controls that will test whether the imperative to miniaturize, humanize and create a high throughput process is offset by any loss of precision and accuracy.

Human induced Pluripotent Stem Cell (hiPSC) derived neural cells offer great potential for modelling neurological diseases and toxicities and have found application in drug discovery and toxicology. As part of the European Innovative Medicines Initiative (IMI2) NeuroDeRisk (Neurotoxicity De-Risking in Preclinical Drug Discovery), we here explore the Ca²⁺ oscillation responses of 2D and 3D hiPSC derived neuronal networks of mixed Glutamatergic/GABAergic activity with a compound set encompassing both clinically as well as experimentally determined seizurogenic compounds. Both types of networks are scored against Ca²⁺ responses of a primary mouse cortical neuronal 2D network model serving as an established comparator assay. Parameters of frequency and amplitude of spontaneous global network Ca²⁺ oscillations and the drug-dependent directional changes to these were assessed, and predictivity of seizurogenicity scored using contingency table analysis. In addition, responses between models were compared between both 2D models as well as between 2D and 3D models. Concordance of parameter responses was best between the hiPSC neurospheroid and the mouse primary cortical neuron model (77% for frequency and 65% for amplitude). Decreases in spontaneous Ca²⁺ oscillation frequency and amplitude were found to be the most basic shared determinants of risk of seizurogenicity between the mouse and the neurospheroid model based on testing of clinical compounds with documented seizurogenic activity. Increases in spontaneous Ca²⁺ oscillation frequency were primarily observed with the 2D hIPSC model, though the specificity of this effect to seizurogenic clinical compounds was low (33%), while decreases to spike amplitude in this model were more predictive of seizurogenicity. Overall predictivities of the models were similar, with sensitivity of the assays typically exceeding specificity due to high false positive rates. Higher concordance of the hiPSC 3D model over the 2D model when compared to mouse cortical 2D responses may be the result of both a longer maturation time of the neurospheroid (84–87 days for 3D vs. 22–24 days for 2D maturation) as well as the 3-dimensional nature of network connections established. The simplicity and reproducibility of spontaneous Ca²⁺ oscillation readouts support further investigation of hiPSC derived neuronal sources and their 2- and 3-dimensional networks for neuropharmacological safety screening.

To market a generic product in the United States, it must be registered in Common Technical Document (CTD) format with the US Food and Drug Administration. The Generic Drug User Fee Act went into force in 2012, to expedite the timely review of Abbreviated New Drug Applications (ANDA) by communicating potential defects in the application to the applicant through deficiency letters at different time intervals during the review cycle. This often delays product approval since these deficiencies must be resolved before the product can be approved. In the present study, a study was performed to analyze the recurrent queries for ANDA applications in the CTD quality module from 2013 to 2020, and the probable corrective and preventive action to be taken was drafted. The most frequently occurring queries were observed in the sections titled “Description of manufacturing process and process controls”, “Controls of critical steps and intermediates”, “Specifications (Control of drug product)”, and “Stability data”.

The cardiovascular safety pharmacology (SP) study conducted to satisfy ICH S7A and S7B has commonly used a cross-over study design where each animal receives all treatments. In an increasing number of cases, cross-over designs are not possible and parallel studies have to be used. These can seldom be as large as 8 animals/treatment to match an n = 8 cross-over. Animals in parallel designs receive only one treatment. Parallel studies will have a different sensitivity to detect changes. This sensitivity is a critical question in using nonclinical QTc evaluations to support an integrated proarrhythmic risk assessment under the newly released ICH E14/S7B Q&As. The current analysis used a study large enough (n = 48) to be analyzed both as a parallel and as a cross-over design to directly compare the performance of the two experimental designs coupled to different statistical models, while all other study conduct aspects were the same.

A total of 48 nonhuman primates (NHP) received 2 different treatments twice: vehicle, moxifloxacin (80 mg/kg), vehicle, moxifloxacin (80 mg/kg). Post-dose QTc interval data were recorded for 48 h for each treatment. Data were analyzed using 12 animals randomly selected for each treatment in a parallel design or as an n = 48 animal cross-over study. Different statistical models were used. The primary endpoint was the residual deviation (sigma) from the models applied to hourly time intervals. The sigma was used to determine the minimal detectable difference (MDD) for the study design-statistical model combination.

Two statistical models were applicable to either study design. They gave similar sigma and resulting MDD values. In cross-over designs, the individual animal identification (ID) can be used in the statistical model. This enabled the smallest MDD value. Simple statistical models for analysis were identified: Treatment + Baseline for parallel designs and Treatment + ID for cross-over designs.

The statistical sensitivity of NHP parallel study designs is reasonable (MDD for n = 6 of 12.7 ms), and in combination with testing exposures higher than likely to be necessary in man could be used in an integrated risk assessment. Where sensitivity of the NHP in vivo QTc assessment is critical, the cross-over design enabled a higher sensitivity (MDD 12.2 ms for n = 4; 8 ms for n = 8).