Journal of pharmacological and toxicological methods最新文献

The potential for unintended drug induced changes in cardiac contractility is a major concern in medicines development. Whilst direct left ventricular pressure (LVP) measurement is the gold standard for measuring cardiac contractility in vivo, it is resource intensive and poses a welfare burden on research animals. In contrast, arterial blood pressure (BP) measurement has fewer challenges. Symmetric Projection Attractor Reconstruction (SPAR) is a signal processing technique which transforms physiological time-series signals into a corresponding visual image (‘attractor’), amplifying morphology changes within physiological waveforms. It was hypothesized that SPAR analysis of BP signals would provide a surrogate measure of cardiac contractility by specifically amplifying the maximum slope of the systolic upstroke. BP (abdominal aorta) signals obtained from beagle dogs, treated with positive and negative inotropes, were retrospectively analysed to identify signal features that correlated with the maximum upslope of the LVP signal from simultaneously acquired LVP recordings. SPAR transformation of BP signals quantified drug induced changes in the maximum slope of the systolic upstroke. We identified key SPAR metrics that provided >0.8 correlation with the LVP maximum upslope, outperforming the BP systolic upstroke alone. This was observed for all 4 different drugs, doses and time points evaluated across studies. Thus, we conclude that the SPAR measures derived from the BP signal could be used as a first pass in vivo screen to flag any risk of drug induced changes in cardiac contractility during the conduct of non-clinical medicines development, potentially reducing or replacing the need to perform direct left ventricular measurements.

Background

Accurate and selective LC/ESI-MSMS method development and validation for the quantitation of pacritinib is the primary goal of this study to perform kinetic studies in the healthy rabbit.

Methods

Chromatographic resolution was accomplished with a hypersil/ODS (50 mm × 4.6 mm, 3 μ) analytical C₁₈ column and a mobile phase composition of 0.1% formic acid and ACN in the proportion of 25:75 with a 0.6 ml/min flow of the mobile phasic system from the analytical column. The method was employed by monitoring the established ionic transitions of m/z-473.25/98.09 for Pacritinib and 506.18/57.12 for the internal standard (Amprenavir) in multiple reaction monitoring.

Results

The calibration plot regression line was y = 0.0002× + 0.007, with a correction coefficient (r²) of 0.9989. The CV outcomes for the matrix effect at low-QC and high-QC levels were 4.79% and 4.91%, respectively. The percentage average recoveries for Pacritinib in High-QC (12.70 μg/ml), MQC (8.50 μg/ml), and Low-QC (1.19 μg/ml) were 95.87%, 103.64%, and 94.32%, respectively. The obtained values were found between 2.98 and 5.07% for the QC (1.19, 8.50, and 12.70 μg/ml) samples. The established procedure was subjected to kinetics study of Pacritinib after oral administration in rabbits. C_max, T_max, and T_1/2, of the Pacritinib tablets were 247.25 ± 3.32 ng/ml, 6.0 ± 0.03 h, and 12.24 ± 0.53 h, respectively. AUC_0-∞ infinity for Pacritinib tablets was 1691.74 ± 3.67 ng h/ml.

Conclusion

After oral administration of Pacritinib to healthy rabbits, pharmacokinetic characteristics were presented, and the established technique was effectively verified.

Introduction

Corrected QT interval (QTc)is an established biomarker for drug-induced Torsade de Pointe (TdP), but with concerns for a false positive signal. Clinically, JTpc and TpTec have emerged as ECG sub-intervals to differentiate predominant hERG vs. mixed ion channel blocking drugs that prolong QTc.

Methods

In a multicentric, prospective, controlled study, different proarrhythmic drug effects on QTc, JTpc and TpTec were characterized with cynomolgus monkeys using telemetry in a Lead II configuration for internal and external telemetry.Drugs and vehicle were administered orally (PO) to group size of 4 to 8 animals, in 4 laboratories.

Results

In monkeys, dofetilide (0.03–0.3 mg/kg) was associated with exposure dependent QTc and JTpc increase, but no significant TpTec effect. Similarly, quinidine (2–50 mg/kg) increased QTc and JTpc but did not change TpTec. Mexiletine (1–15 mg/kg) and verapamil (50 mg/kg) did not induce any significant effect on QTc, JTpc or TpTec.

Discussion

Clinically, predominant hERG blockers (dofetilide and quinidine) prolong QTc, JTpc and TpTec and are associated with increased risk for TdP. Results from this study demonstrate that ECG changes after dofetilide and quinidine administration to telemetered monkeys differ from the clinical response, lacking the expected effects on TpTec. Potential explanations for the lack of translation include physio-pharmacology species differences or ECG recording and analysis methodology variations. Mixed ion channel blockers verapamil and mexiletine administered to monkeys showed no significant QTc, JTpc or TpTec prolongation as expected based on the similar clinical response for these agents.

First-order compartment models are common tools for modelling many biological processes, including pharmacokinetics. Given the compartments and the transfer rates, solutions for the time-dependent quantity (or concentration) curves can normally be described by a sum of exponentials. This paper investigates cases that go beyond simple sums of exponentials. With specific relations between the transfer rate constants, two exponential rate constants can be equal, in which case the normal solution cannot be used. The conditions for this to occur are discussed, and advice is provided on how to circumvent these cases. An example of an analytic solution is given for the rare case where an exact equality is the expected result. Furthermore, for models with at least three compartments, cases exist where the solution to a real-valued model involves complex-valued exponential rate constants. This leads to solutions with an oscillatory element in the solution for the tracer concentration, i.e., there are cases where the solution is not a simple sum of (real-valued) exponentials but also includes sine and cosine functions. Detailed solutions for three-compartment cases are given. As a tentative conclusion of the analysis, oscillatory solutions appear to be tied to cases with a cyclic element in the model itself.

X-376 is a novel anaplastic lymphoma kinase (ALK) inhibitor that is capable of penetrating the blood brain barrier. This makes it suitable for use in patients with ALK-positive non-small cell lung cancer (NSCLC) who have metastases in the central nervous system. This study developed a highly sensitive, fast, eco-friendly, and reliable UPLC-MS/MS approach to quantify X-376 in human liver microsomes (HLMs). This approach was used to evaluate X-376's metabolic stability in HLMs in vitro. The UPLC-MS/MS analytical technique validation followed US-FDA bio-analytical method validation guidelines. StarDrop software, containing P450 metabolic and DEREK modules, was utilized to scan X-376's chemical structure for metabolic lability and hazardous warnings. X-376 and Encorafenib (ENF as internal standard) were resoluted on the Eclipse Plus C18 column utilizing an isocratic mobile phase method. The X-376 calibration curve was linear from 1 to 3000 ng/mL. The precision and accuracy of this study's UPLC-MS/MS approach were tested for intra- and inter-day measurements. Inter-day accuracy was −1.32% to 9.36% while intra-day accuracy was −1.5% to 10.00%. The intrinsic clearance (Cl_int) and in vitro half-life (t_1/2) of X-376 were 59.77 mL/min/kg and 13.56 min. The high extraction ratio of X-376 supports the 50 mg twice-daily dose for ALK-positive NSCLC and CNS metastases patients. In silico software suggests that simple structural changes to the piperazine ring or group substitution in drug design may improve metabolic stability and safety compared to X-376.

This editorial prefaces the annual themed issue on safety pharmacology (SP) methods which has been published since 2004 in the Journal of Pharmacological and Toxicological Methods (JPTM). Here we highlight content derived from the 2023 Safety Pharmacology Society (SPS) meeting held in Brussels, Belgium. The meeting generated 138 abstracts, reproduced in the current volume of JPTM. As in prior years, the manuscripts reflect various areas of innovation in SP including in silico modeling of stroke volume, cardiac output and systemic vascular resistance, computational approaches that compare drug-induced proarrhythmic sensitivity of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), an evaluation of the utility of the corrected J-Tpeak and Tpeak-to-Tend parameters from the ECG as potential proarrhythmia biomarkers, and the applicability of nonclinical concentration-QTc (C-QTc) modeling of data derived from the conduct of the in vivo QTc study as a component of the core battery of safety pharmacology studies.

Acne vulgaris, a chronic inflammatory skin disease with a high prevalence worldwide, necessitates reliable preclinical models for both understanding its pathogenesis and evaluating potential anti-acne therapies. This study aims to establish a robust mouse model using intracutaneous injection of Cutibacterium acnes bacterial suspension. Three hairless mouse strains (SKH-hr1, SKH-hr2 brown, and SKH-hr2 + ApoE) were systematically compared to ascertain the stains most closely resembling acne in humans. Various assessments, including photo documentation, biophysical evaluation, blood analysis, and histopathology, were conducted. Despite all strains exhibiting acne-like lesions, SKH-hr1 mice emerged as the most suitable model, demonstrating the most satisfactory results across multiple criteria. This research underscores the significance of employing hairless mice strains as models in acne studies to enhance and facilitate the development of effective therapeutic interventions.

Introduction

Cardiac safety assessment, such as lethal arrhythmias and contractility dysfunction, is critical during drug development. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been shown to be useful in predicting drug-induced proarrhythmic risk through international validation studies. Although cardiac contractility is another key function, fit-for-purpose hiPSC-CMs in evaluating drug-induced contractile dysfunction remain poorly understood. In this study, we investigated whether alignment of hiPSC-CMs on nanopatterned culture plates can assess drug-induced contractile changes more efficiently than non-aligned monolayer culture.

Methods

Aligned hiPSC-CMs were obtained by culturing on 96-well culture plates with a ridge-groove-ridge nanopattern on the bottom surface, while non-aligned hiPSC-CMs were cultured on regular 96-well plates. Next-generation sequencing and qPCR experiments were performed for gene expression analysis. Contractility of the hiPSC-CMs was assessed using an imaging-based motion analysis system.

Results

When cultured on nanopatterned plates, hiPSC-CMs exhibited an aligned morphology and enhanced expression of genes encoding proteins that regulate contractility, including myosin heavy chain, calcium channel, and ryanodine receptor. Compared to cultures on regular plates, the aligned hiPSC-CMs also showed both enhanced contraction and relaxation velocity. In addition, the aligned hiPSC-CMs showed a more physiological response to positive and negative inotropic agents, such as isoproterenol and verapamil.

Discussion

Taken together, the aligned hiPSC-CMs exhibited enhanced structural and functional properties, leading to an improved capacity for contractility assessment compared to the non-aligned cells. These findings suggest that the aligned hiPSC-CMs can be used to evaluate drug-induced cardiac contractile changes.

Cardiac valvulopathy (Cardiac Valve Disease; CVD) associated with off-target activation of the 5-hydroxytryptamine (5-HT) 2B receptor has been well recognized, but is still poorly predicted during drug development. The regulatory guidance proposes the use of 5-HT2B binding data (i.e., Ki values) and free maximum therapeutic exposure (Cmax) to calculate safety margins as a threshold of detection (>10) for eliminating the risk of drug-induced cardiac valvulopathy. In this paper, we provide additional recommendations for preclinical prediction of CVD risk based on clinical pharmacodynamic and pharmacokinetic data obtained from drugs with or without 5-HT2B receptor activation. Our investigations showed that 5-HT2B agonist affinity of molecules tested in an in vitro 5-HT2B cell-based functional assay, placed in perspective to their sustained plasma exposure (AUCs) and not to their peak plasma exposure, Cmax (i.e., maximum therapeutic exposure) provide a solid basis for interpreting 5-HT2B data, for calculating safety margins and then, accurately differentiate drugs associated with a clinical risk of CVD from those which are not (despite having some agonist 5-HT2B activity). In addition, we discuss the risk of multi-organ fibrosis linked to 5-HT2B receptor activation, often underestimated, however well reported in FAERS for 5-HT2B agonists. We believe that our recommendations have the potential to mitigate the risk for the clinical development of CVD and fibrosis.

Quantification of the unbound portion of platinum (Pt) in human plasma is important for assessing the pharmacokinetics of the chemotherapeutic drug cisplatin. In this study, we sought to compare the recovery of unbound Pt using Nanosep® filters to 1) traditional filters (Centrifree®, Centrisart$\circledR$, Amicon®) or trichloroacetic acid (TCA) protein precipitation, and 2) unbound, bound, and total Pt concentrations in clinical specimens. For the tested filters, the impact of 1) molecular weight cut-offs, 2) centrifugation force, and 3) total Pt concentration on Pt binding in human plasma was evaluated. Pt was quantified using inductively coupled-plasma mass spectrometry. In human plasma spiked with 0.9 μg/mL Pt, the percent of unbound Pt increased at higher centrifugation speeds. By comparison, the percent of unbound Pt was highest (42.1%) following TCA protein precipitation. When total Pt was ≤0.9 μg/mL, unbound Pt (∼20–30%) was consistent across filters. Conversely, when plasma was spiked with Pt exceeding 0.9 μg/mL, the percent of unbound Pt increased from 36.5 to 48% using ultrafiltration, compared to 63.4% to 79% with TCA precipitation. In patients receiving cisplatin-containing chemotherapy, the fraction of unbound Pt at concentrations exceeding 0.9 μg/mL ranged between 35 and 90%. Moreover, the unbound fraction of Pt in plasma correlated with the concentration of unbound (R² = 0.738) and total Pt (R² = 0.335). In summary, this study demonstrates that 1) the percent of unbound Pt is influenced by total and unbound Pt levels in vitro and in clinical specimens, and 2) ultrafiltration with Nanosep® filters is a feasible method for quantifying unbound Pt concentrations in human plasma.