Utilization of mechanistic modelling and simulation to analyse fenspiride proarrhythmic potency - Role of physiological and other non-drug related parameters.

WHAT IS KNOWN AND OBJECTIVE: Fenspiride, a drug that had been used for decades for the treatment of respiratory diseases, was recently withdrawn from the market due to the potential risk of QT prolongation and proarrhythmia. This is the first such withdrawal for many years and hence poses a question whether such risk could have been predicted and to what degree non-drug-specific parameters play a role in the reported QT prolongation and cases of TdP. The study aim was to test various 'what-if' scenarios to assess the influence of age, gender, heart rate, and plasma potassium concentration on QT interval prolongation due to various doses of fenspiride with the use of mechanistic mathematical modelling. METHODS: Concentration-time profiles were simulated with the use of a PBPK model developed based on published physico-chemical data, data from in vitro ADME experiments, and in vivo PK study results. Pharmacodynamic effect, that is, drug-triggered pseudoECG signal modification was simulated using a biophysically detailed model of human cardiac myocytes. Analysis of the qNet metric was also performed to classify proarrhythmic risk related to fenspiride. RESULTS: In the simulation study, arrhythmia was not observed even in the 'what-if' scenarios with extreme exposure, age, heart rate, and plasma potassium concentration. The qNet metric value positioned fenspiride in the intermediate risk class. WHAT IS NEW AND CONCLUSION: It can be hypothesized that the clinically observed arrhythmia cases were not directly caused by fenspiride alone but a combination of multiple factors, including comedications.

Opportunities for use of one species for longer-term toxicology testing during drug development: A cross-industry evaluation.

An international expert working group representing 37 organisations (pharmaceutical/biotechnology companies, contract research organisations, academic institutions and regulatory bodies) collaborated in a data sharing exercise to evaluate the utility of two species within regulatory general toxicology studies. Anonymised data on 172 drug candidates (92 small molecules, 46 monoclonal antibodies, 15 recombinant proteins, 13 synthetic peptides and 6 antibody-drug conjugates) were submitted by 18 organisations. The use of one or two species across molecule types, the frequency for reduction to a single species within the package of general toxicology studies, and a comparison of target organ toxicities identified in each species in both short and longer-term studies were determined. Reduction to a single species for longer-term toxicity studies, as used for the development of biologicals (ICHS6(R1) guideline) was only applied for 8/133 drug candidates, but might have been possible for more, regardless of drug modality, as similar target organ toxicity profiles were identified in the short-term studies. However, definition and harmonisation around the criteria for similarity of toxicity profiles is needed to enable wider consideration of these principles. Analysis of a more robust dataset would be required to provide clear, evidence-based recommendations for expansion of these principles to small molecules or other modalities where two species toxicity testing is currently recommended.

Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor agonists: design, synthesis, and evaluation of PF-3246799 as a treatment for urinary incontinence.

New pyrimido[4,5-d]azepines 7 are disclosed as potent 5-HT(2C) receptor agonists. A preferred example, 7b had minimal activation at either the 5-HT(2A) or 5-HT(2B) receptors combined with robust efficacy in a preclinical canine model of stress urinary incontinence (SUI) and attractive pharmacokinetic and safety properties. Based on this profile, 7b (PF-3246799) was identified as a candidate for clinical development for the treatment of SUI. In addition, it proved to be critical to build an understanding of the translation between recombinant cell-based systems, native tissue preparations and in vivo preclinical models. This was a significant undertaking and proved to be crucial in compound selection.

Strategies to encourage the adoption of social housing during cardiovascular telemetry recordings in non-rodents.

The National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) is working with industry to promote social housing during cardiovascular telemetry recordings within non-rodent safety pharmacology and toxicology studies. Following surveys to capture current practice, benefits and concerns to adoption of this refinement (2015 and 2017), a 2018 European workshop shared experience and practical advice to address common barriers such as sensitivity of different study designs and the potential for cross-contamination with test article in socially-housed conditions. A similar number of responses were received to each survey (38 in 2015; 36 in 2017), from biopharmaceutical companies and CROs that perform or outsource non-rodent telemetry studies. Each dataset had different respondents, but 19 facilities provided answers regarding dogs and non-human primates (NHPs) for both surveys. More respondents socially-housed their non-rodents in 2017; increases were apparent for both the non-recording/acclimatisation periods and the telemetry recording periods compared with 2015. However, on recording days only 60, 75 and 89% of respondents from Europe and 25, 14 and 36% of respondents from outside of Europe socially-housed their dogs, minipigs or NHPs respectively. The potential for contamination with test article between animals housed together is considered by some facilities as justification for individual housing during recordings, however, survey data did not support this rationale. Nine organisations provided data on prevalence of vomiting during telemetry studies, showing the risk was moderate for dogs and very low for minipig and NHP. Further, if vomiting did occur, this could be managed effectively with little impact on study outcomes or validity and with careful dose selection, the risk is further diminished. A recent increase in published papers and posters on this topic would suggest many more companies are planning, or have recently implemented, this refinement. The continued willingness of the community to share practical experience and publish validation data may lead to this approach becoming the 'new standard' across the industry in the near future, representing a core component of 'best-practice' recommendations to increase animal welfare whilst maintaining quality data provision for investigational and regulatory purposes.

Applying the CiPA approach to evaluate cardiac proarrhythmia risk of some antimalarials used off-label in the first wave of COVID-19.

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH-CLQ)-mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID-19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high-risk cell populations, and in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. In each assay, concentration-response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH-CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH-CLQ inhibited IKr (half-maximal inhibitory concentration [IC50 ]: 1 µM and 3-7 µM, respectively) and IK1 currents (IC50 : 5 and 44 µM, respectively). When combining OH-CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50  > 300-1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH-CLQ. Effects were more pronounced in the high-risk cell population. In hiPSC-derived cardiomyocytes, all drugs showed early after-depolarizations, except AZI. Combining CLQ or OH-CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off-label use in COVID-19, CLQ and OH-CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.

General Principles for the Validation of Proarrhythmia Risk Prediction Models: An Extension of the CiPA In Silico Strategy.

This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration-sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model-based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm.

Better prediction of the local concentration-effect relationship: the role of physiologically based pharmacokinetics and quantitative systems pharmacology and toxicology in the evolution of model-informed drug discovery and development.

Model-informed drug discovery and development (MID3) is an umbrella term under which sit several computational approaches: quantitative systems pharmacology (QSP), quantitative systems toxicology (QST) and physiologically based pharmacokinetics (PBPK). QSP models are built using mechanistic knowledge of the pharmacological pathway focusing on the putative mechanism of drug efficacy; whereas QST models focus on safety and toxicity issues and the molecular pathways and networks that drive these adverse effects. These can be mediated through exaggerated on-target or off-target pharmacology, immunogenicity or the physiochemical nature of the compound. PBPK models provide a mechanistic description of individual organs and tissues to allow the prediction of the intra- and extra-cellular concentration of the parent drug and metabolites under different conditions. Information on biophase concentration enables the prediction of a drug effect in different organs and assessment of the potential for drug-drug interactions. Together, these modelling approaches can inform the exposure-response relationship and hence support hypothesis generation and testing, compound selection, hazard identification and risk assessment through to clinical proof of concept (POC) and beyond to the market.

The molecular cloning and functional expression of the dog CCR5.

Activation of CCR5 by specific chemokines is involved in the regulation of the immunological response of leukocytes at sites of inflammation. In addition, CCR5 serves as a fusion co-factor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). Consequently, several CCR5 antagonists are currently in development for the treatment of HIV-1 infection. The dog CCR5 gene was cloned in order to characterise the chemokine binding site of the dog receptor for comparison across species. The deduced amino acid sequence of the dog CCR5 has close homology to the human receptor (80% identity). A HEK-293 cell line expressing the dog recombinant receptor was generated and immunoblot analysis with an anti-human CCR5 antibody revealed a 58kDa band in the cell lysate. In functional calcium signalling assays, the CCR5 endogenous ligands MIP-1alpha, MIP-1beta and RANTES evoked a robust response in the dog recombinant CCR5 cells. In a CRE-Luc (cAMP response element-luciferase) reporter gene assay, MIP-1beta (0.01-30nM) produced concentration-dependent inhibition of forskolin induced elevation in cAMP levels, and was equipotent in dog, human and macaque recombinant CCR5 cells (EC(50) 0.4, 0.21 and 0.47nM, respectively). These data suggest that chemokine signalling is conserved in the dog CCR5.

Molecular cloning and radioligand binding characterization of the chemokine receptor CCR5 from rhesus macaque and human.

The aim of this study was to determine if macaque represents a suitable species for the pre-clinical evaluation of novel CCR5 antagonists, such as maraviroc (UK-427,857). To do this we cloned and expressed CCR5 from rhesus macaque and compared the binding properties of [125I]-MIP-1beta and [3H]-maraviroc with human recombinant CCR5. [125I]-MIP-1beta bound with similar high affinity to CCR5 from macaque (K(d) = 0.24 +/- 0.05 nM) and human (K(d) = 0.23 +/- 0.05 nM) and with similar kinetic properties. In competition binding studies the affinity of a range of human chemokines for macaque CCR5 was also similar to human CCR5. Maraviroc inhibited binding of [125I]-MIP-1beta to CCR5 from macaque and human with similar potency (IC50 = 17.50 +/- 1.24 nM and 7.18 +/- 0.93 nM, respectively) and antagonised MIP-1beta induced intracellular calcium release mediated through CCR5 from macaque and human with similar potency (IC50 = 17.50 +/- 3.30 nM and 12.07 +/- 1.89, respectively). [3H]-maraviroc bound with high affinity to CCR5 from macaque (K(d) = 1.36+/-0.07 nM) and human (K(d) = 0.86 +/- 0.08 nM), but was found to dissociate approximately 10-fold more quickly from macaque CCR5. However, as with the human receptor, maraviroc was shown to be a high affinity, potent functional antagonist of macaque CCR5 thereby indicating that the macaque should be a suitable species in which to evaluate the pharmacology, safety and potential mechanism-related toxicology of novel CCR5 antagonists.

Erythromycin slows aging of Saccharomyces cerevisiae.

Life span was measured by counting budding cycles in cohorts of yeast cells treated with erythromycin, paraquat, or geneticin. Paraquat treatment increases oxidative stress; geneticin treatment increases errors during cytoplasmic protein synthesis. Treating with either or both compounds resulted in shorter life spans. Saccharomyces cerevisiae strain K65-3D grown in 16 microg/ml erythromycin, a treatment that results in more accurate protein synthesis by bacteria, had a mean life span that was significantly longer (27%) than that of untreated yeast cells. The life spans of petite variants with no detectable respiratory activity or extranuclear DNA were not affected by this dose of erythromycin, which appeared, therefore, to exert its effect on aging by means of mitochondria. Fitting the data to Weibull and Gompertz distributions allowed calculation of an accelerated life model that relates life span to dose of erythromycin.

Measurement of hyper- and hypotension during repeated dose toxicity studies in either freely moving or physically restrained cynomolgus monkeys.

INTRODUCTION: The measurement of cardiovascular endpoints in standard toxicology studies remains a challenge as the routinely used non-invasive methods require physical restraint, causing an increase of sympathetic neural activity, leading to excitement and potentially hypertension in the experimental animals. In this study, a miniature telemetry blood pressure transmitter was used to evaluate if the acute hyper- and hypotension could be detected in free moving cynomolgus monkeys as well as physically restrained animals using positive control drugs. Furthermore, as a comparator, routine high definition oscillometry (HDO) was performed in restrained animals. METHODS: Hemodynamic parameters were monitored continuously from conscious, freely moving animals following oral administration of vehicle (water) or 1 and 10mg/kg of etilefrine, and 1 and 4mg/kg of dihydralazine as positive control articles. A second dose session was performed to confirm the reproducibility of results and a third dose session combined with physical restraint procedures for blood collection and HDO measurements. RESULTS: There was a dose-dependent, statistically significant increase in the systolic blood pressure following oral doses of etilefrine at all 3 dose sessions. This effect was less apparent during session 3, probably due to the physical restraint applied for the blood sampling and HDO measurement. No differences in the blood pressure were measured using HDO. On all three dose sessions following oral doses of dihydralazine the expected statistically significant decrease in the diastolic pressure could be clearly measured even when the telemetric data recordings were combined with physical restraint. DISCUSSION: Due to the advantages of the minimally invasive telemetry technique compared to HDO and the possibility of prolonged measurement periods, it is an invaluable tool for blood pressure measurement in freely moving animals in toxicology studies.

Safety pharmacology investigations in toxicology studies: an industry survey.

INTRODUCTION: The Safety Pharmacology (SP) Society (SPS) conducted an industry survey in 2012 in an attempt to define current industry practices as they relate to inclusion of safety pharmacology (SP) endpoints into Toxicology studies. METHODS: A total of 361 participants from Asia (9.1%), Europe (19.4%) and North America (71.4%) responded to the survey. The preponderance of respondents were toxicologists (53.2%) followed by safety pharmacologists (27.2%) and scientists involved in the conduct of both disciplines (19.6%). Most participants (58.6%) were from pharmaceutical companies employing more than 500 employees. RESULTS: A majority (68.2%) reported having experience in designing, performing or interpreting the SP component of a study when performed as part of a toxicology study. Some participants (42.0%) had submitted data to a regulatory agency where ICHS7 studies were performed as part of a toxicology study rather than as a standalone study. When comparing species that were used in studies in which SP was added to toxicology studies, canines were the most frequently reported animals used for new chemical entities (NCE) whereas non-human (NH) primates were the most frequent for the assessment of biological agents. The most frequent primary motivator for adding ICHS7 SP endpoints to regulatory toxicology studies was to generate additional data to allow for determination of an integrated risk assessment thereby testing Confidence in Safety (CIS) to better manage and/or mitigate risk. The current ability to add safety pharmacology endpoints into regulatory toxicology studies was used to address a specific concern (by 42.1% of respondents) to allow management of risk more effectively (36.8%) or to generate data that contributes to cessation of the progression of a compound (21.1%). For an NCE, SP measurements in toxicology studies were conducted in addition to standalone SP studies (by 40.6% of respondents) or in addition/instead of standalone safety pharmacology studies (by 39.8% of respondents). For biological agents, a majority (74.3%) indicated SP measurements in toxicology were conducted instead of standalone studies as outlined in the ICHS6 guideline while inclusion of SP endpoints in toxicology studies for biological agents in addition to standalone studies was reported by only 25.7% of the respondents. DISCUSSION: The survey highlights that obtaining regulatory agreement for the proposed combined SP/Tox study designs may be useful before study conduct in some cases. Respondents suggest that such discussion could occur at the pre-IND meeting before the IND/CTA enabling program.

Pharmacokinetic-pharmacodynamic modelling of the effect of Moxifloxacin on QTc prolongation in telemetered cynomolgus monkeys.

INTRODUCTION: Delayed ventricular repolarisation is manifested electrocardiographically in a prolongation of the QT interval. Such prolongation can lead to potentially fatal Torsades de Pointes. Moxifloxacin is a fluoroquinolone antibiotic which has been associated with QT prolongation and, as a result, is recommended by the regulatory authorities as a positive control in thorough QT studies performed to evaluate the potential of new chemical entities to induce QT prolongation in humans. The sensitivity of the cynomolgus monkey as a quantitative preclinical predictor of the PK-QTc relationship is discussed. METHODS: Cardiovascular monitoring was performed in the telemetered cynomolgus monkey for 22 h following oral administration of Moxifloxacin (10, 30 and 90 mg/kg) or placebo. QTc was derived using an individual animal correction factor (ICAF): RR-I = QT-I--(RR-550)* (IACF). A PKPD analysis was performed to quantify the increase in placebo-adjusted QTc) elicited by administration of Moxifloxacin. In addition, the rate of onset of hERG channel blockade of Moxifloxacin was compared to Dofetilide by whole cell patch clamp technique in HEK-293 cells stably expressing the hERG channels. RESULTS: Moxifloxacin induced a dose dependent increase in QTc). A maximum increase of 28 ms was observed following administration of 90 mg/kg Moxifloxacin. The corresponding maximum free systemic exposure was 18µM. Interrogation of the PK-QTc relationship indicated a direct relationship between the systemic exposure of Moxifloxacin and increased QTc. A linear PKPD model was found to describe this relationship whereby a 1.5 ms increase in QTc was observed for every 1 µM increase in free systemic exposure. DISCUSSION: The exposure dependent increases in QTc observed following oral administration of Moxifloxacin to the cynomolgus monkey are in close agreement with those previously reported in human subjects. A direct effect linear relationship was found to be conserved in both species. As a result of the quantitative agreement in both species, the utility of the telemetered cynomolgus monkey as a preclinical predictor of QTc) prolongation is exemplified. Furthermore, the rate of onset of hERG channel blockade observed in patch clamp offers a mechanistic insight into the relative rates of channel blockade observed in vivo with both Moxifloxacin and Dofetilide.

Drug induced shortening of the QT/QTc interval: an emerging safety issue warranting further modelling and evaluation in drug research and development?

INTRODUCTION: A session dedicated to the issue of drug-induced QT and/or QTc interval (QT/QTc) shortening of the electrocardiogram (ECG) was held at the 2007 Safety Pharmacology Society (SPS) meeting in Edinburgh. METHODS: The session included a presentation on the results of a cross company survey on QT/QTc-shortening, a podium debate with speakers arguing "for" and "against" QT/QTc shortening being a safety issue and a panel discussion with the audience. RESULTS: Compared to QT/QTc prolongation, relatively little is known about the relevance to safety of drug-induced QT/QTc shortening. As with QT/QTc prolongation, there are genetic syndromes and pharmaceutical agents which cause shortening of QT/QTc. The potential safety issue of QT/QTc shortening and its suitability as a biomarker of drug-induced cardiac arrhythmias, are unclear, however, the type of arrhythmia associated with prolongation and shortening are thought to differ. Prolongation is associated with torsades de pointes, whereas, shortening of QT/QTc is proposed to be associated with the more severe arrhythmia, ventricular fibrillation (VF). The industry-wide survey (53 total responses representing 45 different companies) indicates that the number of compounds that induce QT/QTc shortening has increased over the past 5 years with 51% of responses reporting QT/QTc shortening in pre-clinical studies and 22% reporting a corresponding clinical experience. The reason for the increase is not clear but there is a clear business impact with 13% (7/56) of these compounds being discontinued in the pre-clinical phase due to QT/QTc shortening. The majority of companies with clinical experience of QT/QTc shortening have engaged with the regulatory agencies and these experiences will be valuable in shaping how the pharmaceutical industry and the agencies view drug-induced QT/QTc shortening in the future. DISCUSSION: Currently it is not clear how much shortening of QT/QTc is required before it might be considered a safety issue and indeed, whether QT/QTc shortening is a suitable biomarker for cardiac arrhythmias. It is clear, however, that with our current understanding, compounds which shorten QT/QTc will attract close regulatory scrutiny and carry a business risk. The need to better understand this potential cardiac safety issue points to further research including; model development to determine the mechanism(s) of action of drug-induced QT/QTc shortening and the translation between the non-clinical and clinical situation.

Zebrafish assays as early safety pharmacology screens: paradigm shift or red herring?

The recent flurry of interest in the potential use of the zebrafish (Danio rerio) in Drug Discovery has also led to the development of a range of assays purported to be useful as early screens in safety pharmacology. The purpose of this commentary is to take stock of the available zebrafish assays in the context of alternative mammalian cell-based assays, and of the validation outcomes to date. In addition, we report the results of a recent survey of the membership of the Safety Pharmacology Society regarding their views on zebrafish assays. The survey data indicate that the preferred way forward would be a collaborative effort between the pharmaceutical/biotechnology industry (as potential/eventual customers), and the zebrafish contract research companies (as suppliers), alongside expert input from academia and regulatory authorities.

The discovery of indole-derived long acting beta2-adrenoceptor agonists for the treatment of asthma and COPD.

The design and profile of a series of indole containing long acting beta(2)-adrenoceptor agonists is described. Evaluation of these analogues using an in vitro guinea pig trachea tissue model demonstrates that analogues within this series have salmeterol-like duration of action with potential for long duration of action in humans.