Absence of functional deficits in rats following systemic administration of an AAV9 vector despite moderate peripheral nerve and dorsal root ganglia findings: A clinically silent peripheral neuropathy.

Adeno-associated virus (AAV)-based vectors are commonly used for delivering transgenes in gene therapy studies, but they are also known to cause dorsal root ganglia (DRG) and peripheral nerve toxicities in animals. However, the functional implications of these pathologic findings and their time course remain unclear. At 2, 4, 6, and 8 weeks following a single dose of an AAV9 vector carrying human frataxin transgene in rats, non-standard functional assessments, including von Frey filament, electrophysiology, and Rotarod tests, were conducted longitudinally to measure allodynia, nerve conduction velocity, and coordination, respectively. Additionally, DRGs, peripheral nerves, brain and spinal cord were evaluated histologically and circulating neurofilament light chain (NfL) was quantified at 1, 2, 4, and 8 weeks, respectively. At 2 and 4 weeks after dosing, minimal-to-moderate nerve fiber degeneration and neuronal degeneration were observed in the DRGs in some of the AAV9 vector-dosed animals. At 8 weeks, nerve fiber degeneration was observed in DRGs, with or without neuronal degeneration, and in sciatic nerves of all AAV9 vector-dosed animals. NfL values were higher in AAV9 vector-treated animals at weeks 4 and 8 compared with controls. However, there were no significant differences in the three functional endpoints evaluated between the AAV9 vector- and vehicle-dosed animals, or in a longitudinal comparison between baseline (predose), 4, and 8 week values in the AAV9 vector-dose animals. These findings demonstrate that there is no detectable functional consequence to the minimal-to-moderate neurodegeneration observed with our AAV9 vector treatment in rats, suggesting a functional tolerance or reserve for loss of DRG neurons after systemic administration of AAV9 vector.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Reprint of: Nonclinical species sensitivity to convulsions: An IQ DruSafe consortium working group initiative.

Clinical development of compounds that carry a convulsion liability is typically limited by safety margins based on the most sensitive nonclinical species. To better understand differences in sensitivity to drug-induced convulsion of commonly used preclinical species, a survey was distributed amongst pharmaceutical companies through an IQ consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) resulting in convulsion-related data on 80 unique compounds from 11 companies. The lowest free drug plasma concentration at which convulsions were observed and the no observed effect level for convulsions were compared between species to determine their relative sensitivity. Additionally, data were collected on other endpoints including use of electroencephalography, premonitory signs, convulsion type, the reason why development was stopped, and the highest development phase reached. The key outcomes were: (1) the dog was most often determined to be the most sensitive species by both non-exposure and exposure-based analyses, (2) there was not a clear sensitivity ranking of other species (NHP, rat and mouse), (3) CNS symptoms were frequently present at exposures that were not associated with convulsions, but no single reliable premonitory indicator of convulsion was identified, and (4) the lack of convulsions in the compounds that were tested in humans in this dataset may suggest that convulsion liability is well mitigated via current drug development strategies.

Translation of in vitro cannabinoid 1 receptor agonist activity to in vivo pharmacodynamic endpoints.

INTRODUCTION: Building an understanding of in vivo efficacy based on the evaluation of in vitro affinity or potency is critical in expediting early decision making in drug discovery and can significantly reduce the need for animal studies. The aim of the present study was to understand the translation of in vitro to in vivo endpoints for the cannabinoid receptor 1 (CB1). METHODS: Using a selection of CB1 agonists we describe an evaluation of in vitro to in vivo translation comparing in vitro receptor affinity or functional potency, using both cAMP and ß-arrestin endpoints, to various in vivo CB1 agonist-associated endpoints. RESULTS: We demonstrate that in vitro CB1 agonism significantly correlates with the CB1-induced cue in the drug discrimination model in vivo, but not with other purported CB1 agonist-mediated in vivo endpoints, including hypothermia and sedation. Thus, these data challenge common perceptions regarding CB1 agonist-induced tetrad effects in rodents. DISCUSSION: This work exemplifies how in vitro profiling of receptor affinity or potency can predict in vivo pharmacodynamic effects, using the CB1 as an example system. The translatability of in vitro activity to in vivo efficacy allows for the ability to rapidly contextualize off-target CB1 in vitro findings, allowing clear and rapid definition of the risk posed by such activity without the need for extensive animal studies. This has significant implications in terms of early decision making in drug discovery and reducing the use of animals in research, while also outlining a template for expanding the approach for additional targets.

Nonclinical species sensitivity to convulsions: An IQ DruSafe consortium working group initiative.

Clinical development of compounds that carry a convulsion liability is typically limited by safety margins based on the most sensitive nonclinical species. To better understand differences in sensitivity to drug-induced convulsion of commonly used nonclinical species, a survey was distributed amongst pharmaceutical companies through an IQ consortium (International Consortium for Innovation and Quality in Pharmaceutical Development) resulting in convulsion-related data on 80 unique compounds from 11 companies. The lowest free drug plasma concentration at which convulsions were observed and the no observed effect level for convulsions were compared between species to determine their relative sensitivity. Additionally, data were collected on other endpoints including use of electroencephalography, premonitory signs, convulsion type, the reason why development was stopped, and the highest development phase reached. The key outcomes were: (1) the dog was most often determined to be the most sensitive species by both non-exposure and exposure-based analyses, (2) there was not a clear sensitivity ranking of other species (NHP, rat and mouse), (3) CNS symptoms were frequently present at exposures that were not associated with convulsions, but no single reliable premonitory indicator of convulsion was identified, and (4) the lack of convulsions when compounds were tested in humans in this dataset may suggest that convulsion liability is well mitigated via current drug development strategies.

Use of Nerve Conduction Assessments to Evaluate Drug-Induced Peripheral Neuropathy in Nonclinical Species-A Brief Review.

The peripheral nervous system (PNS) is subject to a wide range of structural and functional insults including direct damage to axons, loss of myelin, and progressive deficits in saltatory conduction. Drugs that damage the PNS often result in neuropathies that impact the structure and function of targeted nerves. In most cases, both sensory and motor neurons are affected with damage initially evident in the distal extremities. Drug-induced neuropathies are potentially reversible following cessation of treatment, but early stages of neuropathy can be subclinical and asymptomatic making diagnosis difficult. Nerve biopsy is highly validated and provides definitive evidence of nerve injury and corresponding severity; however, it is limited in some respects and electrophysiological measures can complement histopathological assessments and provide a functional measure of potential toxicity. In a drug development setting, nerve conduction assessments are valuable to monitor nerve function longitudinally if nerve damage is suspected or confirmed, and importantly, can be used to monitor progression and/or recovery of a drug-induced neuropathy. This review will summarize the methodology used in nerve conduction assessments as well as discuss data interpretation and considerations for use in nonclinical species. Finally, the use of nerve conduction assessments in nonclinical drug development is discussed.

Social partnering alters sleep in fear-conditioned Wistar rats.

Social support, when provided following a traumatic experience, is associated with a lower incidence of stress-related psychiatric disorders. Our hypothesis was that providing a social interaction period with a naive conspecific would improve sleep architecture in response to cued fear conditioning in Wistar rats. Rats were randomly assigned to either the socially isolated or socially partnered groups. Rats assigned to the socially isolated group were individually housed following electrode implantation and fear conditioning. Rats assigned to the socially partnered group were initially paired-housed, and then one rat from each pair was randomly chosen for sleep electrode implantation and fear conditioning. Rats from both groups were habituated to a recording chamber, and baseline sleep was recorded over 22 hours. One day later (Training Day), they were fear-conditioned to 10 presentations of a tone (800 Hz, 90 dB, 5 sec) co-terminating with a mild electric foot shock (1.0 mA, 0.5 sec), at 30-sec intervals. While rats in the socially isolated group were left undisturbed in their home cage for 30-min, socially partnered rats interacted for 30 minutes with their non-stressed rat partner immediately after fear conditioning and while the auditory tones were presented on Days 1 and 14. The results indicated that social interaction increased sleep efficiency in partnered rats compared to isolated rats following the fear conditioning procedure. This was due to an increase in the amount of rapid eye movement sleep (REMS) during the light phase. Evaluation of REMS microarchitecture revealed that the increase in REMS was due to an increase in the number of single REMS episodes (siREMS), which represented a more consolidated REMS pattern. A surprising finding was that partnered rats had a greater number of sequential REMS episodes (seqREMS) at Baseline, on the Training Day and on Day 1 when compared to isolated rats. The greater number of seqREMS episodes in partnered rats may be due to the partnering procedure and not fear conditioning, as the effect was also seen at Baseline. Thus it appears that while the partnering procedure may have given rise to a fragmented REMS pattern, social partnering promoted a greater consolidation of REMS in response to the fear conditioning procedure.

Reduced γ range activity at REM sleep onset and termination in fear-conditioned Wistar-Kyoto rats.

Recent investigations of rapid eye movement sleep (REMS) continuity have emphasized the importance of transitions both into and out of REMS. We have previously reported that, compared to Wistar rats (WIS), Wistar-Kyoto rats (WKY) responded to fear conditioning (FC) with more fragmented REMS. Gamma oscillations in the electroencephalogram (EEG) are synchronized throughout the brain in periods of focused attention, and such synchronization of cell assemblies in the brain may represent a temporal binding mechanism. Therefore, we examined the effects of FC on EEG gamma range activity (30-50Hz) at REMS transitions in WKY compared to WIS. Relative power in the gamma range (measured as a percent of total power) at Baseline and upon re-exposure to the fear-inducing conditioning stimulus was measured 35s before REMS onset to 105s after REMS onset (ARO) and 85s before REMS termination (BRT) to 35s after REMS termination. After baseline recording, rats received 10 tones, each co-terminating with an electric foot shock. On Days 1 and 14 post-conditioning, rats were re-exposed to three tones. Fast-Fourier transforms created power spectral data in the gamma frequency domain. Relative power was extracted from an average of 4-5 REMS transitions. Relative gamma power was always higher in WIS. On Day 14, at 15s and 25s ARO, WKY had significant increases in relative gamma power from Baseline. WIS had a significant increase on Day 1 at 25s ARO. Despite the increases in relative gamma power, WKY never achieved levels attained by WIS. Moreover, at 5s BRT, only WKY had a significant decrease in relative gamma power from Baseline to Day 14. Gamma range activity may indicate neural activity underlying maintenance of REMS continuity. Low relative gamma power at REMS transitions may be associated with increased REMS fragmentation in WKY after FC.

Fear conditioning fragments REM sleep in stress-sensitive Wistar-Kyoto, but not Wistar, rats.

Pavlovian conditioning is commonly used to investigate the mechanisms of fear learning. Because the Wistar-Kyoto (WKY) rat strain is particularly stress-sensitive, we investigated the effects of a psychological stressor on sleep in WKY compared to Wistar (WIS) rats. Male WKY and WIS rats were either fear-conditioned to tone cues or received electric foot shocks alone. In the fear-conditioning procedure, animals were exposed to 10 tones (800 Hz, 90 dB, 5s), each co-terminating with a foot shock (1.0 mA, 0.5s), at 30-s intervals. In the shock stress procedure, animals received 10 foot shocks at 30-s intervals, without tones. All subjects underwent a tone-only test both 24h (Day 1) and again two weeks (Day 14) later. Rapid eye movement sleep (REMS) continuity was investigated by partitioning REMS episodes into single (inter-REMS episode interval >3 min) and sequential (interval ≤ 3 min) episodes. In the fear-conditioned group, freezing increased from baseline in both strains, but the increase was maintained on Day 14 in WKY rats only. In fear-conditioned WKY rats, total REMS amount increased on Day 1, sequential REMS amount increased on Day 1 and Day 14, and single REMS amount decreased on Day 14. Alterations were due to changes in the number of sequential and single REMS episodes. Shock stress had no significant effect on REMS microarchitecture in either strain. The shift toward sequential REMS in fear-conditioned WKY rats may represent REMS fragmentation, and may provide a model for investigating the neurobiological mechanisms of sleep disturbances reported in posttraumatic stress disorder.