Simon Authier

Principles of Safety Pharmacology

Safety Pharmacology is a rapidly developing discipline that uses the basic principles of pharmacology in a regulatory‐driven process to generate data to inform risk/benefit assessment. The aim of Safety Pharmacology is to characterize the pharmacodynamic/pharmacokinetic (PK/PD) relationship of a drugs adverse effects using continuously evolving methodology. Unlike toxicology, Safety Pharmacology includes within its remit a regulatory requirement to predict the risk of rare lethal events. This gives Safety Pharmacology its unique character. The key issues for Safety Pharmacology are detection of an adverse effect liability, projection of the data into safety margin calculation and finally clinical safety monitoring. This article sets out to explain the drivers for Safety Pharmacology so that the wider pharmacology community is better placed to understand the discipline. It concludes with a summary of principles that may help inform future resolution of unmet needs (especially establishing model validation for accurate risk assessment). Subsequent articles in this issue of the journal address specific aspects of Safety Pharmacology to explore the issues of model choice, the burden of proof and to highlight areas of intensive activity (such as testing for drug‐induced rare event liability, and the challenge of testing the safety of so‐called biologics (antibodies, gene therapy and so on.).

Validation of Orthopedic Postoperative Pain Assessment Methods for Dogs: A Prospective, Blinded, Randomized, Placebo-Controlled Study

In the context of translational research, there is growing interest in studying surgical orthopedic pain management approaches that are common to humans and dogs. The validity of postoperative pain assessment methods is uncertain with regards to responsiveness and the potential interference of analgesia. The hypothesis was that video analysis (as a reference), electrodermal activity, and two subjective pain scales (VAS and 4A-VET) would detect different levels of pain intensity in dogs after a standardized trochleoplasty procedure. In this prospective, blinded, randomized study, postoperative pain was assessed in 25 healthy dogs during a 48-hour time frame (T). Pain was managed with placebo (Group 1, n = 10), preemptive and multimodal analgesia (Group 2, n = 5), or preemptive analgesia consisting in oral tramadol (Group 3, n = 10). Changes over time among groups were analyzed using generalized estimating equations. Multivariate regression tested the significance of relationships between pain scales and video analysis. Video analysis identified that one orthopedic behavior, namely ‘Walking with full weight bearing’ of the operated leg, decreased more in Group 1 at T24 (indicative of pain), whereas three behaviors indicative of sedation decreased in Group 2 at T24 (all p<0.004). Electrodermal activity was higher in Group 1 than in Groups 2 and 3 until T1 (p<0.0003). The VAS was not responsive. 4A-VET showed divergent results as its orthopedic component (4A-VETleg) detected lower pain in Group 2 until T12 (p<0.0009), but its interactive component (4A-VETbeh) was increased in Group 2 from T12 to T48 (p<0.001). Concurrent validity established that 4A-VETleg scores the painful orthopedic condition accurately and that pain assessment through 4A-VETbeh and VAS was severely biased by the sedative side-effect of the analgesics. Finally, the video analysis offered a concise template for assessment in dogs with acute orthopedic pain. However, subjective pain quantification methods and electrodermal activity need further investigation.

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.

Video-electroencephalography in conscious non human primate using radiotelemetry and computerized analysis: refinement of a safety pharmacology model.

INTRODUCTION Electroencephalography (EEG) investigations are occasionally required as follow-up studies for safety pharmacology core battery (S7A). Video-EEG monitoring is a standard diagnostic tool in humans but limited data is available on its use in telemetered freely moving macaque monkeys for safety pharmacology investigations. While proconvulsant risk evaluations are routinely conducted in rodents, pharmacological or pharmacokinetic considerations lead to the use of non human primates in toxicology and safety pharmacology in some cases. METHODS Cynomolgus monkeys were instrumented with telemetry implants. Placement of EEG electrode was based on the 10-20 system using three derivations (C3-O1, Cz-Oz and C4-O2). EEG trace analysis was carried out using NeuroScore software. After 24 h of continuous video-EEG monitoring, animals received pentylenetetrazole (PTZ, 10 mg/kg/15 min) until convulsions were noted. Convulsions were immediately treated with diazepam (1.0 mg/kg). A seizure detection protocol with a dynamic spike train threshold was used for the entire EEG monitoring period (total of 44 h) including periods when PTZ was administered. Spectral analysis was done to quantify the absolute and relative amplitude of EEG frequency bands (delta, theta, alpha, sigma and beta waves). Sleep stages were quantified and EEGs during seizures were analyzed using fast Fourier transformation (FFT) to assess dominant frequencies. RESULTS Spike trains were detected by computerized analysis in all animals presenting PTZ-induced seizures while paroxysmal activities were systematically predictive (at least 4-min prior to generalized seizures). Beta activity increased with visual stimulation using monkey treats. Characteristics of EEG for all sleep stages (I, II, III and IV) were present in all animals. Delta activity was predominant in normal awake EEG as well as in all sleep stages. Seizure peak frequency was 3-6 Hz on FFT, corresponding to the discharge of the underlying generator. DISCUSSION EEG-video monitoring can be useful when using non human primates to characterize neurological adverse effects with unpredictable onset. Computerized video-EEG analysis was a valuable tool for safety pharmacology investigations including proconvulsant risk assessment, spectral analysis of frequency bands and sleep stage determination.

Telemetry video-electroencephalography (EEG) in rats, dogs and non-human primates: methods in follow-up safety pharmacology seizure liability assessments.

INTRODUCTION Non-clinical seizure liability studies typically aim to: 1) confirm the nature of EEG activity during abnormal clinical signs, 2) identify premonitory clinical signs, 3) measure plasma levels at seizure onset, 4) demonstrate that drug-induced seizures are self-limiting, 5) confirm that conventional drugs (e.g. diazepam) can treat drug-induced seizures and 6) confirm the no observed adverse effect level (NOAEL) at EEG. Our aim was to originally characterize several of these items in a three species comparative study. METHODS Cynomolgus monkey, Beagle dog and Sprague-Dawley rat with EEG telemetry transmitters were used to obtain EEG using the 10-20 system. Pentylenetetrazol (PTZ) was used to determine seizure threshold or as a positive seizurogenic agent. Clinical signs were recorded and premonitory signs were evaluated. In complement, other pharmacological agents were used to illustrate various safety testing strategies. RESULTS Intravenous PTZ doses required to induce clonic convulsions were 36.1 (3.8), 56.1 (12.7) and 49.4 (11.7) mg/kg, in Beagle dogs, cynomolgus monkeys and Sprague-Dawley rats, respectively. Premonitory clinical signs typically included decreased physical activity, enhanced physiological tremors, hypersalivation, ataxia, emesis (except in rats) and myoclonus. In Sprague-Dawley rats, amphetamine (PO) increased high (approximately 40-120Hz), and decreased low (1-14Hz) frequencies. In cynomolgus monkeys, caffeine (IM) increased power in high (14-127Hz), and attenuated power in low (1-13Hz) frequencies. In the rat PTZ infusion seizure threshold model, yohimbine (SC and IV) and phenobarbital (IP) confirmed to be reliable positive controls as pro- and anticonvulsants, respectively. DISCUSSION Telemetry video-EEG for seizure liability investigations was characterized in three species. Rats represent a first-line model in seizure liability assessments. Beagle dogs are often associated with overt susceptibility to seizure and are typically used in seizure liability studies only if required by regulators. Non-human primates represent an important model in seizure liability assessments given similarities to humans and a high translational potential.

Cardiovascular and respiratory safety pharmacology in Göttingen minipigs: Pharmacological characterization.

INTRODUCTION Similarities between pigs and humans support the relevance of Göttingen minipigs for regulatory safety pharmacology. The minipig is the species of choice for cardiovascular safety pharmacology when pivotal repeat toxicology studies are conducted in this species. METHODS 4 male Göttingen minipigs with cardiovascular telemetry transmitters received intravenous saline, esmolol (0.5, 1, 2, 4 and 8mg/kg), medetomidine (0.04mg/kg), remifentanil (0.5, 1, 2, 4, 8 and 16μg/kg) and dopamine (2, 8, 10, 20, 30 and 50μg/kg/min) and oral sotalol (3 and 10mg/kg). Respiratory monitoring was conducted in 3 male and 3 female Göttingen minipigs receiving intravenous saline and methacholine (0, 3.4, 13.5 and 68μg/kg). RESULTS Heart rate (HR) corrected QT was optimal with a method based on analysis of covariance (QTca) followed by Fridericias standard formula. Esmolol induced a decrease in HR. Medetomidine was associated with an initial hypertension with bradycardia followed by sustained hypotension, bradycadia and prolonged QTc. Remifentanil induced a dose-dependent QTc shortening with an increase in arterial pressures. Sotalol caused a decrease in HR and systolic arterial pressure with an increase in PR and QTc intervals. Dopamine induced an increase in arterial and pulse pressures. Methacholine increased tidal volume, respiratory rate and minute volume. DISCUSSION The results suggest that the minipig is a valid alternative to other non-rodent species for cardiovascular and respiratory safety pharmacology studies when this species is justified.

Global Metabolomic Identification of Long-Term Dose-Dependent Urinary Biomarkers in Nonhuman Primates Exposed to Ionizing Radiation

Due to concerns surrounding potential large-scale radiological events, there is a need to determine robust radiation signatures for the rapid identification of exposed individuals, which can then be used to guide the development of compact field deployable instruments to assess individual dose. Metabolomics provides a technology to process easily accessible biofluids and determine rigorous quantitative radiation biomarkers with mass spectrometry (MS) platforms. While multiple studies have utilized murine models to determine radiation biomarkers, limited studies have profiled nonhuman primate (NHP) metabolic radiation signatures. In addition, these studies have concentrated on short-term biomarkers (i.e., <72 h). The current study addresses the need for biomarkers beyond 72 h using a NHP model. Urine samples were collected at 7 days postirradiation (2, 4, 6, 7 and 10 Gy) and processed with ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight (QTOF) MS, acquiring global metabolomic radiation signatures. Multivariate data analysis revealed clear separation between control and irradiated groups. Thirteen biomarkers exhibiting a dose response were validated with tandem MS. There was significantly higher excretion of l-carnitine, l-acetylcarnitine, xanthine and xanthosine in males versus females. Metabolites validated in this study suggest perturbation of several pathways including fatty acid β oxidation, tryptophan metabolism, purine catabolism, taurine metabolism and steroid hormone biosynthesis. In this novel study we detected long-term biomarkers in a NHP model after exposure to radiation and demonstrate differences between sexes using UPLC-QTOF-MS-based metabolomics technology.

Combined cardiopulmonary assessments with implantable telemetry device in conscious freely moving cynomolgus monkeys.

Female cynomolgus monkeys were surgically implanted with telemetry transmitters recording ECG (DII), arterial pressure, physical activity, body temperature, and tidal volume. Respiratory rate (RR) and tidal volume (TV) were monitored simultaneously with the telemetry transmitter using impedance. Impedance-based monitoring of RR and TV by telemetry correlated with controlled TV and with pneumotachometer (>98%) in restrained animals. Control drugs with cardiovascular and respiratory effects, including saline, medetomidine (0.01, 0.02 and 0.04mg/kg) and cocaine (0.5, 1.0 and 1.5mg/kg) were administered intravenously. An averaging epoch of 5min was used for analysis of respiratory data. Medetomidine induced significant respiratory depression with decrease in RR and TV in freely moving animals while cocaine increased TV, RR and minute ventilation (MV) with concomitant increase in heart rate when compared with time matched values from saline-treated animals. The onset, duration and magnitude of cardiovascular and respiratory changes were correlated. This highlights the dependency of the cardiovascular and respiratory systems. The use of cardiopulmonary monitoring can allow continuous monitoring including during night time when variability of respiratory parameters is lower. Monitoring of cardiovascular and respiratory parameters in the same animals could also help to decrease the number of animals used in research.

Innovation in safety pharmacology testing

This issue of the Journal of Pharmacological and Toxicological Methods (JPTM) is themed. It is the eighth in a series, arising from the Annual Safety Pharmacology Society (SPS) meeting. The SPS is now in its 10th year as an independent branch of biological sciences (distinct from pharmacology and toxicology) and is the primary forum for driving advances in safety pharmacology. The theme of the meeting and this journal issue is innovation, and the focus is non-clinical safety assessment of new chemical entity (NCEs). The content is informed by regulatory guidance documents (S7A and S7B) prior to first in human (FIH) studies. The manuscripts cover a broad spectrum of safety pharmacology topics from theory to practice, with interrogation of state-of-the-art techniques, and profiling of methods that are in development for safety assessment. Philosophical and strategic issues are addressed, with consideration of the use of novel methods for population pharmacokinetic (PK) analysis, abuse liability, electrocardiogram (ECG) analysis algorithms, in vitro cardiac slice preparations, human pluripotent stem cells, and a brief discussion regarding the assessment of changes in the QRS complex of the ECG indicative of drug-induced blockade of cardiac sodium channels. Safety pharmacology methods continue to evolve.

Non-clinical models: Validation, study design and statistical consideration in safety pharmacology

The current issue of the Journal of Pharmacological and Toxicological Methods (JPTM) focuses exclusively on safety pharmacology methods. This is the 7th year the Journal has published on this topic. Methods and models that specifically relate to methods relating to the assessment of the safety profile of a new chemical entity (NCE) prior to first in human (FIH) studies are described. Since the Journal started publishing on this topic there has been a major effort by safety pharmacologists, toxicologists and regulatory scientists within Industry (both large and small Pharma as well as Biotechnology companies) and also from Contract Research Organizations (CRO) to publish the surgical details of the non-clinical methods utilized but also provide important details related to standard and non-standard (or integrated) study models and designs. These details from core battery and secondary (or ancillary) drug safety assessment methods used in drug development programs have been the focus of these special issues and have been an attempt to provide validation of methods. Similarly, the safety pharmacology issues of the Journal provide the most relevant forum for scientists to present novel and modified methods with direct applicability to determination of drug safety-directly to the safety pharmacology scientific community. The content of the manuscripts in this issue includes the introduction of additional important surgical methods, novel data capture and data analysis methods, improved study design and effects of positive control compounds with known activity in the model.