The Spontaneous Incidence of Neurological Clinical Signs in Preclinical Species Using Cage-side Observations or High-definition Video Monitoring: A Retrospective Analysis.

When conducting toxicology studies, the interpretation of drug-related neurological clinical signs such as convulsions, myoclonus/myoclonic jerks, tremors, ataxia, and salivation requires an understanding of the spontaneous incidence of those observations in commonly used laboratory animal species. The spontaneous incidence of central nervous system clinical signs in control animals from a single facility using cage-side observations or high definition video monitoring was retrospectively analyzed. Spontaneous convulsions were observed at low incidence in Beagle dogs and Sprague-Dawley rats but were not identified in cynomolgus monkeys and Göttingen minipigs. Spontaneous myoclonic jerks and muscle twitches were observed at low incidence in Beagle dogs, cynomolgus monkeys, and Sprague-Dawley rats but were not seen in Göttingen minipigs. Spontaneous ataxia/incoordination was identified in all species and generally with a higher incidence when using video monitoring. Salivation and tremors were the two most frequent spontaneous clinical signs and both were observed in all species. Data from the current study unveil potential limitations when using control data obtained from a single study for toxicology interpretation related to low incidence neurological clinical signs while providing historical control data from Beagle dogs, cynomolgus monkeys, Sprague-Dawley rats, and Göttingen minipigs.

Drug-Induced Seizures: Considerations for Underlying Molecular Mechanisms.

A broad spectrum of chemical entities have been associated with drug-induced seizure (DIS), emphasizing the importance of this potential liability across various drug classes (e.g., antidepressants, antipsychotics, antibiotics, and analgesics among others). Despite its importance within drug safety testing, an understanding of the molecular mechanisms associated with DIS is often lacking. The etiology of DIS is understood to be a result of either a deficit in inhibitory (e.g., gamma aminobutyric acid) or an elevated excitatory (e.g., glutamate) signaling, leading to synchronous neuronal depolarization affecting various brain regions and impairing normal neurological functions. How this altered neuronal signaling occurs and how these changes interact with other non-brain receptor driven DIS-associated changes such as metabolic disturbances, electrolyte imbalances, altered drug metabolism, and withdrawal effects are poorly understood. Herein, we discuss important molecular mechanisms identified in DIS for several drugs and/or drug classes. With a better understanding of the molecular mechanisms associated with DIS, in vivo or in vitro models may be applied to characterize and mitigate DIS risk during drug development. Susceptibility stratification for DIS presents species differences in the following order beagle dogs > rodents and cynomolgus monkeys > Göttingen minipigs with a more than 2-fold difference between canines and minipigs, which is important to consider during non-clinical species selection. While clinical signs such as myoclonus, severe muscle jerks, or convulsions are often associated with abnormal epileptiform EEG activity, tremors are most of the time physiological and rarely observed with concurrent epileptiform EEG activity which need to be considered during DIS risk evaluation.

Cerebrospinal Fluid Characterization in Cynomolgus Monkeys, Beagle Dogs, and Göttingen Minipigs.

Intrathecal administration is an important route for drug delivery, and in pharmacology and toxicology studies, cerebrospinal fluid (CSF) collection and analysis is required for evaluating blood-brain barrier penetration and central nervous system exposure. The characteristics of CSF in commonly used nonrodent models are lacking. The purpose of this study is to evaluate and provide some insights into normal cellular and biochemical composition of CSF as well as diffusion potential following intrathecal injection across several nonrodent species. Cerebrospinal fluid samples were collected from the cerebellomedullary cistern of beagle dogs, cynomolgus monkeys, and Göttingen minipigs and analyzed for clinical chemistry and cytological evaluation. Diffusion into the intrathecal space following intrathecal injection was assessed following administration of a contrast agent using fluoroscopy. The predominant cell types identified in CSF samples were lymphocytes and monocytoid cells; however, lymphocytes were represented in a higher percentage in dogs and monkeys as opposed to monocytoid cells in minipigs. Clinical chemistry parameters in CSF revealed higher Cl- concentrations than plasma, but lower K+, Ca2+, phosphorus, glucose, creatinine, and total protein levels consistent across all 3 species. Diffusion rates following intrathecal injection of iodixanol showed some variability with dogs, showing the greatest diffusion distance; however, the longest diffusion time through the intervertebral space, followed by monkeys and minipigs. Minimal diffusion was observed in minipigs, which could have been attributed to anatomical spinal constraints that have been previously identified in this species.

Diffuse meningeal oligodendrogliomatosis characterized by spinal intra-parenchymal nodules on magnetic resonance imaging in a dog.

Meningeal oligodendrogliomatosis is a relatively rare neoplasm in dogs. Ante-mortem diagnosis is difficult due to nonspecific neurologic signs overlapping other conditions. The only reported consistent feature is a high level of protein in the cerebrospinal fluid. Veterinary literature offers only 1 case report with magnetic resonance imaging (MRI) of canine spinal meningeal oligodendrogliomatosis in a single dog. In contrast to the predominant diffuse meningeal enhancement shown in that report, we present the case of a young female cane corso dog with marked nodular invasion of the spinal cord on MRI, confirmed by histopathology to be consistent with diffuse meningeal oligodendrogliomatosis. Key clinical message: Meningeal oligodendrogliomatosis should be a differential diagnosis when marked nodular invasion of the spinal cord is seen on MRI, both with and without meningeal enhancement.

Oligodendrogliomatose méningée diffuse caractérisée par des nodules spinaux intraparenchymateux lors d'un examen d'imagerie par résonance magnétique chez un chien. L'oligodendrogliomatose méningée est un néoplasme relativement rare chez le chien. Son diagnostic ante-mortem est difficile en raison de signes neurologiques non spécifiques chevauchant d'autres conditions. La seule caractéristique fréquemment signalée est un niveau élevé de protéines dans le liquide céphalo-rachidien. La littérature vétérinaire ne propose qu'un seul rapport de cas illustrant des images obtenues suite à un examen d'imagerie par résonance magnétique (IRM) chez un seul chien diagnostiqué avec une oligodendrogliomatose méningée rachidienne. Contrairement au rehaussement méningé diffus prédominant montré chez ce chien, nous présentons le cas d'une jeune femelle Cane Corso avec une oligodendrogliomatose méningée diffuse confirmée à l'histopathologie, s'étant manifestée à l'IRM par une invasion nodulaire marquée de la moelle épinière.Message clinique clé:L'oligodendrogliomatose méningée doit être un diagnostic différentiel lorsqu'une invasion nodulaire marquée de la moelle épinière est observée à l'IRM, avec ou sans rehaussement méningé.(Traduit par Dre Isabelle Masseau).

A L2HGDH initiator methionine codon mutation in a Yorkshire terrier with L-2-hydroxyglutaric aciduria.

BACKGROUND: L-2-hydroxyglutaric aciduria is a metabolic repair deficiency characterized by elevated levels of L-2-hydroxyglutaric acid in urine, blood and cerebrospinal fluid. Neurological signs associated with the disease in humans and dogs include seizures, ataxia and dementia. CASE PRESENTATION: Here we describe an 8 month old Yorkshire terrier that presented with episodes of hyperactivity and aggressive behavior. Between episodes, the dog's behavior and neurologic examinations were normal. A T2 weighted MRI of the brain showed diffuse grey matter hyperintensity and a urine metabolite screen showed elevated 2-hydroxyglutaric acid. We sequenced all 10 exons and intron-exon borders of L2HGDH from the affected dog and identified a homozygous A to G transition in the initiator methionine codon. The first inframe methionine is at p.M183 which is past the mitochondrial targeting domain of the protein. Initiation of translation at p.M183 would encode an N-terminal truncated protein unlikely to be functional. CONCLUSIONS: We have identified a mutation in the initiation codon of L2HGDH that is likely to result in a non-functional gene. The Yorkshire terrier could serve as an animal model to understand the pathogenesis of L-2-hydroxyglutaric aciduria and to evaluate potential therapies.

EEG: Characteristics of drug-induced seizures in rats, dogs and non-human primates.

Seizures are amongst the most frequent neurological issues encountered in pre-clinical safety testing. The objective was to characterize EEG morphologies and premonitory signs in drug-induced seizures in preclinical species. A comparative (inter-species) retrospective analysis for drug-induced seizures recorded by video-telemetry was conducted in rats (n = 53), dogs (n = 195), and non-human primates (n = 234). The most frequent premonitory signs were, in rats, myoclonus (100%), tremors (93%), salivation (75%), partial ptosis (58%) and chewing/bruxism (58%); in dogs, tremors (77%), ataxia/uncoordination (60%), myoclonus (45%), salivation (43%), excessive licking (38%), high vocalization (38%) and decreased activity (34%); in non-human primates, tremors (79%), decreased activity (70%), myoclonus (57%), retching/emesis (37%), hunched posture (30%) and ataxia/uncoordination (27%). Seizure duration ranged from 3 s to 14 min with an average of 46 ±â€¯21 s, comparable across species. At seizure onset, spike frequency averaged 9.4 Hz for the three species compared to 4.3 Hz at seizure end. Peak average amplitudes were attained at mid-seizure and amplitudes at seizure end decreased from peak but remained higher than onset amplitudes. Spike duration was inversely correlated with frequency and presented a crescendo pattern. Morphological characteristics can serve to refine automated EEG analysis. From a regulatory perspective, the most common paradigm is to use the most sensitive species in seizure liability studies but translational potential and clinical relevance may be under represented in the decision making process in some cases. EEG morphologies during drug-induced seizures presented remarkable similarities between species and tremors were identified as a predominant premonitory clinical sign in all species.

Reprint of "EEG: Characteristics of drug-induced seizures in rats, dogs and non-human primates".

Seizures are amongst the most frequent neurological issues encountered in pre-clinical safety testing. The objective was to characterize EEG morphologies and premonitory signs in drug-induced seizures in preclinical species. A comparative (inter-species) retrospective analysis for drug-induced seizures recorded by video-telemetry was conducted in rats (n = 53), dogs (n = 195), and non-human primates (n = 234). The most frequent premonitory signs were, in rats, myoclonus (100%), tremors (93%), salivation (75%), partial ptosis (58%) and chewing/bruxism (58%); in dogs, tremors (77%), ataxia/uncoordination (60%), myoclonus (45%), salivation (43%), excessive licking (38%), high vocalization (38%) and decreased activity (34%); in non-human primates, tremors (79%), decreased activity (70%), myoclonus (57%), retching/emesis (37%), hunched posture (30%) and ataxia/uncoordination (27%). Seizure duration ranged from 3 s to 14 min with an average of 46 ±â€¯21 s, comparable across species. At seizure onset, spike frequency averaged 9.4 Hz for the three species compared to 4.3 Hz at seizure end. Peak average amplitudes were attained at mid-seizure and amplitudes at seizure end decreased from peak but remained higher than onset amplitudes. Spike duration was inversely correlated with frequency and presented a crescendo pattern. Morphological characteristics can serve to refine automated EEG analysis. From a regulatory perspective, the most common paradigm is to use the most sensitive species in seizure liability studies but translational potential and clinical relevance may be under represented in the decision making process in some cases. EEG morphologies during drug-induced seizures presented remarkable similarities between species and tremors were identified as a predominant premonitory clinical sign in all species.

Comparison of susceptibility to antimicrobials of bacterial isolates from companion animals in a veterinary diagnostic laboratory in Canada between 2 time points 10 years apart.

The susceptibility to antimicrobials of bacterial species most frequently isolated from companion animals in a veterinary teaching diagnostic laboratory was evaluated retrospectively. A significant decrease between 1990-1992 and 2002-2003 was noted in the susceptibility of dog isolates to the following antimicrobials: Escherichia coli to cephalothin (86% to 61%, P < 0.001); E. coli to ampicillin (85% to 67%, P < 0.001); Proteus spp. to ampicillin (92% to 71%, P < 0.01); coagulase-positive staphylococci (Staphylococcus aureus and Staphylococcus intermedius) to enrofloxacin (99% to 95%, P < 0.01). Significantly increased susceptibilities were also noted as follows: coagulase-positive staphylococci to erythromycin (78% to 90%, P < 0.001) and tetracycline (61% to 77%, P < 0.001). Despite a limited number of results available for cats, a significant increase in susceptibility was noted for Pseudomonas spp. to gentamicin (40% to 100%, P < 0.05) and for E. coli to tetracycline (59% to 80%, P < 0.05). Regular updates on the resistance to antimicrobials used in veterinary medicine are required.

Functional neurotoxicity evaluation of noribogaine using video-EEG in cynomolgus monkeys.

INTRODUCTION: Continuous video-electroencephalographic (EEG) monitoring remains the gold standard for seizure liability assessments in preclinical drug safety assessments. EEG monitored by telemetry was used to assess the behavioral and EEG effects of noribogaine hydrochloride (noribogaine) in cynomolgus monkeys. Noribogaine is an iboga alkaloid being studied for the treatment of opioid dependence. METHODS: Six cynomolgus monkeys (3 per gender) were instrumented with EEG telemetry transmitters. Noribogaine was administered to each monkey at both doses (i.e., 160 and 320mg/kg, PO) with an interval between dosing of at least 6days, and the resulting behavioral and EEG effects were evaluated. IV pentylenetetrazol (PTZ), served as a positive control for induced seizures. RESULTS: The administration of noribogaine at either of the doses evaluated was not associated with EEG evidence of seizure or with EEG signals known to be premonitory signs of increased seizure risk (e.g., sharp waves, unusual synchrony, shifts to high-frequency patterns). Noribogaine was associated with a mild reduction in activity levels, increased scratching, licking and chewing, and some degree of poor coordination and related clinical signs. A single monkey exhibited brief myoclonic movements that increased in frequency at the high dose, but which did not appear to generalize, cluster or to be linked with EEG abnormalities. Noribogaine was also associated with emesis and partial anorexia. In contrast, PTZ was associated with substantial pre-ictal EEG patterns including large amplitude, repetitive sharp waves leading to generalized seizures and to typical post-ictal EEG frequency attenuation. INTERPRETATION: EEG patterns were within normal limits following administration of noribogaine at doses up to 320mg/kg with concurrent clinical signs that correlated with plasma exposures and resolved by the end of the monitoring period. PTZ was invariably associated with EEG paroxysmal activity leading to ictal EEG. In the current study, a noribogaine dose of 320mg/kg was considered to be the EEG no observed adverse effect level (NOAEL) in conscious freely moving cynomolgus monkeys.

Safety pharmacology investigations on the nervous system: An industry survey.

The Safety Pharmacology Society (SPS) conducted an industry survey in 2015 to identify industry practices as they relate to central, peripheral and autonomic nervous system ('CNS') drug safety testing. One hundred fifty-eight (158) participants from Asia (16%), Europe (20%) and North America (56%) responded to the survey. 52% of participants were from pharmaceutical companies (>1000 employees). Oncology (67%) and neurology/psychiatry (66%) were the most frequent target indications pursued by companies followed by inflammation (48%), cardiovascular (43%), metabolic (39%), infectious (37%), orphan (32%) and respiratory (29%) diseases. Seizures (67% of participants), gait abnormalities (67%), tremors (65%), emesis (56%), sedation (52%) and salivation (47%) were the most commonly encountered CNS issues in pre-clinical drug development while headache (65%), emesis/nausea (60%), fatigue (51%) and dizziness (49%) were the most frequent issues encountered in Phase I clinical trials. 54% of respondents reported that a standard battery of tests applied to screen drug candidates was the approach most commonly used to address non-clinical CNS safety testing. A minority (14% of all participants) reported using electroencephalography (EEG) screening prior to animal inclusion on toxicology studies. The most frequent group size was n=8 for functional observation battery (FOB), polysomnography and seizure liability studies. FOB evaluations were conducted in a dedicated room (78%) by blinded personnel (66%) with control for circadian cycle (55%) effects (e.g., dosing at a standardized time; balancing time of day across treatment groups). The rat was reported as the most common species used for seizure liability, nerve conduction and drug-abuse liability testing.

Effects of amphetamine, diazepam and caffeine on polysomnography (EEG, EMG, EOG)-derived variables measured using telemetry in Cynomolgus monkeys.

INTRODUCTION: Medication-induced sleep disturbances are a major concern in drug development as a multitude of prescription drugs alter sleep patterns, often negatively. Polysomnography is used in clinical diagnostics but is also applicable to animal models. Rodent sleep architecture (nocturnal) differs from larger diurnal mammals, including humans, increasing the translational potential of non-rodent species to the clinic. This study aimed to characterize the response to pharmacological agents known to affect sleep structure and EEG activity in a non-human primate (Macaca fascicularis) using telemetry-based polysomnography. METHODS: Animals were instrumented with telemetry transmitters for continuous electroencephalogram (EEG), electro-oculogram (EOG) and electromyogram (EMG) monitoring combined with video. EEG, EMG and EOG were monitored for 12 to 24h to establish baseline values, followed by administration of pharmacological agents (saline, d-amphetamine, diazepam or caffeine). RESULTS: Amphetamine (0.3 and 1mg/kg, by oral administration (PO)) significantly reduced total sleep time, including the duration of both non-rapid eye movement [NREM] sleep and REM sleep. It also decreased EEG activity in low frequencies (i.e., 4-6Hz) during wakefulness. Diazepam (2mg/kg, PO) did not significantly alter sleep duration, but importantly reduced EEG activity in low frequencies (approximately 2-12Hz) during wakefulness, NREM and REM sleep. Finally, caffeine (10 and 30mg/kg, PO) decreased both NREM and REM sleep duration. In addition, spectral analysis revealed important decreases in low frequency activity (i.e., 1-8Hz) during wakefulness with a parallel increase in high frequency activity (i.e., 20-50Hz) during NREM sleep. DISCUSSION: As these observations are similar to previously reported pharmacological effects in humans, results support that EEG, EOG and EMG monitoring by telemetry in Cynomolgus monkeys represents a useful non-clinical model to investigate and quantify drug-induced sleep disturbances.

Reprint of "Effects of amphetamine, diazepam and caffeine on polysomnography (EEG, EMG, EOG)-derived variables measured using telemetry in Cynomolgus monkeys".

INTRODUCTION: Medication-induced sleep disturbances are a major concern in drug development as a multitude of prescription drugs alter sleep patterns, often negatively. Polysomnography is used in clinical diagnostics but is also applicable to animal models. Rodent sleep architecture (nocturnal) differs from larger diurnal mammals, including humans, increasing the translational potential of non-rodent species to the clinic. This study aimed to characterize the response to pharmacological agents known to affect sleep structure and EEG activity in a non-human primate (Macaca fascicularis) using telemetry-based polysomnography. METHODS: Animals were instrumented with telemetry transmitters for continuous electroencephalogram (EEG), electro-oculogram (EOG) and electromyogram (EMG) monitoring combined with video. EEG, EMG and EOG were monitored for 12 to 24h to establish baseline values, followed by administration of pharmacological agents (saline, d-amphetamine, diazepam or caffeine). RESULTS: Amphetamine (0.3 and 1mg/kg, by oral administration (PO)) significantly reduced total sleep time, including the duration of both non-rapid eye movement [NREM] sleep and REM sleep. It also decreased EEG activity in low frequencies (i.e., 4-6Hz) during wakefulness. Diazepam (2mg/kg, PO) did not significantly alter sleep duration, but importantly reduced EEG activity in low frequencies (approximately 2-12Hz) during wakefulness, NREM and REM sleep. Finally, caffeine (10 and 30mg/kg, PO) decreased both NREM and REM sleep duration. In addition, spectral analysis revealed important decreases in low frequency activity (i.e., 1-8Hz) during wakefulness with a parallel increase in high frequency activity (i.e., 20-50Hz) during NREM sleep. DISCUSSION: As these observations are similar to previously reported pharmacological effects in humans, results support that EEG, EOG and EMG monitoring by telemetry in Cynomolgus monkeys represents a useful non-clinical model to investigate and quantify drug-induced sleep disturbances.

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.