Vincent Castagné

Behavioral Indices in Antipsychotic Drug Discovery

Schizophrenia is characterized by three major symptom classes: positive symptoms, negative symptoms, and cognitive deficits. Classical antipsychotics (phenothiazines, thioxanthenes, and butyrophenones) are effective against positive symptoms but induce major side effects, in particular, extrapyramidal symptoms (EPS). The discovery of clozapine, which does not induce EPS and is thought effective against all three classes of symptom, has driven research for novel antipsychotics with a wider activity spectrum and lower EPS liability. To increase predictiveness, current efforts aim to develop translational models where direct parallels can be drawn between the processes studied in animals and in humans. The present article reviews existing procedures in animals for their ability to predict compound efficacy and EPS liability in relation to their translational validity. Rodent models of positive symptoms include procedures related to dysfunction in central dopamine and glutamatergic (N-methyl-d-aspartate) and serotonin (5-hydroxytryptamine) neurotransmission. Procedures for evaluating negative symptoms include rodent models of anhedonia, affective flattening, and diminished social interaction. Cognitive deficits can be assessed in rodent models of attention (prepulse inhibition) and of learning/memory (object and social recognition, Morris water maze and operant-delayed alternation). The relevance of the conditioned avoidance response is also discussed. A final section reviews procedures for assessing EPS liability, in particular, parkinsonism (catalepsy in rodents), acute dystonia (purposeless chewing in rodents, dystonia in monkeys), akathisia (defecation in rodents), and tardive dyskinesia (long-term antipsychotic treatment in rodents and monkeys). It is concluded that, with notable exceptions (attention, learning/memory, EPS liability), current predictive models for antipsychotics fall short of clear translational validity.

Use of latency to immobility improves detection of antidepressant-like activity in the behavioral despair test in the mouse

The behavioral despair test (BDT), also called the forced swim test, is an economic, reliable and sensitive test for the detection of potential antidepressant-like activity of new test substances. The vast majority of clinically active antidepressants are active in the BDT, although substances specifically acting on serotonin transmission are generally reported to be less easily detected. Substances active in the BDT decrease the duration of immobility at doses considered as relatively high. In contrast, some psychostimulants are considered as potential false positives since they are also active in the BDT although they are not recognized as clinically active antidepressants. In the present study we have evaluated the usefulness of latency to the first immobility period as an additional parameter in the BDT to further evaluate the effects of antidepressants and psychostimulants administered intraperitoneally in the mouse. The results show that this measure increases the sensitivity of the test for detecting the effects of tricyclic antidepressants (imipramine, desipramine) and selective serotonin/norepinephrine reuptake inhibitors (duloxetine and venlafaxine) but not of serotonin reuptake inhibitors (fluoxetine and escitalopram). In contrast with previous reports, psychostimulants (amphetamine and modafinil) did not affect the duration or the latency to immobility in the BDT. The mouse strain used in the BDT seems to be an important parameter to discriminate between antidepressants and psychostimulants. These results suggest that the measure of the latency to the first immobility improves the predictive validity of the BDT.

Preclinical Behavioral Models for Predicting Antipsychotic Activity

Schizophrenia is a major psychiatric disease that is characterized by three distinct symptom domains: positive symptoms, negative symptoms, and cognitive impairment. Additionally, treatment with classical antipsychotic medication can be accompanied by important side effects that involve extrapyramidal symptoms (EPS). The discovery of clozapine in the 1970s, which is efficacious in all three symptom domains and has a reduced propensity to induce EPS, has driven research for new antipsychotic agents with a wider spectrum of activity and a lower propensity to induce EPS. The following chapter reviews existing behavioral procedures in animals for their ability to predict compound efficacy against schizophrenia symptoms and liability to induce EPS. Rodent models of positive symptoms include procedures related to hyperfunction in central dopamine and serotonin (5-hydroxytryptamine) systems and hypofunction of central glutamatergic (N-methyl-d-aspartate) neurotransmission. Procedures for evaluating negative symptoms include rodent models of anhedonia, affective flattening, and diminished social interaction. Cognitive deficits can be assessed in rodent models of attention (prepulse inhibition (PPI), latent inhibition) and of learning and memory (passive avoidance, object and social recognition, Morris water maze, and operant-delayed alternation). The relevance of the conditioned avoidance response (CAR) is also discussed. A final section reviews animal procedures for assessing EPS liability, in particular parkinsonism (catalepsy), acute dystonia (purposeless chewing in rodents, dystonia in monkeys), akathisia (defecation in rodents), and tardive dyskinesia (long-term antipsychotic treatment in rodents and monkeys).

Rodent Models of Depression: Forced Swim and Tail Suspension Behavioral Despair Tests in Rats and Mice

The development of antidepressants requires simple rodent behavioral tests for initial screening before undertaking more complex preclinical tests and clinical evaluation. Presented in the unit are two widely used screening tests used for antidepressants, the forced swim (also termed behavioral despair) test in the rat and mouse, and the tail suspension test in the mouse. These tests have good predictive validity and allow rapid and economical detection of substances with potential antidepressant‐like activity. The behavioral despair and the tail suspension tests are based on the same principle: measurement of the duration of immobility when rodents are exposed to an inescapable situation. The majority of clinically used antidepressants decrease the duration of immobility. Antidepressants also increase the latency to immobility, and this additional measure can increase the sensitivity of the behavioral despair test in the mouse for certain classes of antidepressant. Testing of new substances in the behavioral despair and tail suspension tests allows a simple assessment of their potential antidepressant activity by the measurement of their effect on immobility. Curr. Protoc. Pharmacol. 49:5.8.1‐5.8.14.

Comparison of methods for the assessment of locomotor activity in rodent safety pharmacology studies

INTRODUCTION General neurobehavioral assays, like a modified Irwin test or a functional observational battery, are necessary for central nervous system (CNS) safety pharmacology testing near the end of the target validation (early discovery) stage of preclinical drug development. However, at earlier stages, when a greater number of test compounds must be screened for potential CNS side effects, locomotor activity assessment may be a better tool for the comparison of compounds. METHODS Spontaneous locomotor activity counts obtained from two automated test systems - an infrared beam-based activity meter (Actimeter) and the mechanical vibration-based LABORAS - were compared in rats dosed with chlorpromazine (2-8mg/kg) or caffeine (3-24mg/kg), p.o. A modified Irwin test was also performed to visually observe the neurobehavioral effects. RESULTS In all three assays, dose-dependent sedation- and excitation-related effects were observed with chlorpromazine and caffeine, respectively. The two automated activity-detection systems exhibited similar sensitivities in determining changes in locomotor activity, but with the LABORAS being more sensitive than the Actimeter in detecting caffeine-induced increases in vertical activity (rearing behavior). DISCUSSION Infrared beam-based activity detection systems and LABORAS provide relatively-comparable quantitative data regarding locomotor activity. Practical considerations, such as relative cost versus degree of versatility, should be considered when deciding which system to use for the screening of test compounds during the earliest stages of preclinical drug development.

Current approaches and issues in non-clinical evaluation of abuse and dependence

Preclinical assessment of drug abuse and dependence has been the subject of several recent regulatory guidelines. Both the European and US authorities recommend a tiered approach and are generally aligned on the methods which should be used. The first tier simply compares the pharmacology of the novel substance to known drugs of abuse. The second tier aims to identify abuse and dependence liability more directly. The most direct approach to assessing reinforcing properties is the i.v. self-administration procedure. Unfortunately there is no standardized procedure for evaluating substances with differing potencies, reinforcement properties or pharmacokinetics (PK). Indeed, the choice of training substance, species and procedural parameters can radically affect the outcome. Apart from the lower cost of the rat, the primate presents several advantages for self-administration studies (potentially greater similarity to humans in behavioral effects, active doses and PK). Although it does not measure abuse liability directly, drug discrimination is a powerful method for assessing the similarity of a test substance to a known drug of abuse. In this procedure an animal uses the interoceptive effects of the substance as the discriminative stimulus to determine which of two responses to make. For certain classes of substance, such as hallucinogens acting via the 5-HT(2A) receptor, discrimination is the only procedure currently able to identify them. Drug dependence is assessed by the occurrence of withdrawal effects on drug discontinuation. Although conceptually simple, many factors (duration and frequency of drug treatment, dose/exposure levels, duration of observation after discontinuation) can complicate interpretation. Telemetry may represent a novel approach which allows continuous observation of somatic and behavioral parameters during drug withdrawal thereby increasing sensitivity. Presently available tools can identify essentially all substances known to cause abuse or dependence with little risk of false positives. It remains unclear how effective these models will be with entirely novel substances. Nonetheless, drug abuse/dependence is an area of safety pharmacology where the predictive value of animal models remains very high.

Continuous evaluation of drug withdrawal in the rat using telemetry: Effects of morphine and chlordiazepoxide

INTRODUCTION The procedures used to assess withdrawal must be sensitive and widely applicable, i.e. not specific to any particular drug class. Furthermore, the measurements should not be affected by repeat testing. METHODS We have used implanted telemetry devices to continuously follow body temperature, locomotor activity (LMA), heart rate (HR) and mean arterial blood pressure (mean ABP) in addition to food intake and body weight gain over 20days of treatment and 8days of withdrawal. The effects of morphine (32 and 64mg/kg p.o., b.i.d.) and chlordiazepoxide (16, 32 and 64mg/kg p.o., b.i.d.) were studied in rats. RESULTS The results show that during the treatment phase chronic morphine reduced food intake and body weight gain, increased body temperature, HR, mean ABP and LMA. These effects continued over the 20days of treatment. In contrast, chlordiazepoxide slightly increased food intake and body weight gain throughout the treatment period. It also decreased body temperature and LMA but increased HR and mean ABP after the first few administrations but these effects disappeared over the 20days of treatment. Following discontinuation, both morphine- and chlordiazepoxide-treated rats showed a dose-related decrease in food intake and loss of weight on days 2 and 3 of discontinuation. Morphine discontinuation also induced a nocturnal hypothermia and a diurnal hypertension (i.e. during the light phase) which lasted for 4-5days and also moderate diurnal increases in locomotor activity and heart rate over the first 3days of discontinuation. Chlordiazepoxide discontinuation induced small increases in telemetry parameters some of which, such as the effect on locomotor activity, lasted for more than 5days. The intensity and duration of effects for both substances were broadly dose-related. DISCUSSION These data show that telemetry can increase the sensitivity of withdrawal experiments to changes that might otherwise be missed and allows a better definition of the time-course of withdrawal effects. This technique is therefore useful as part of safety pharmacology abuse liability evaluation of novel test substances across a broad range of pharmacological and therapeutic classes.

Face-to-face comparison of the predictive validity of two models of neuropathic pain in the rat: analgesic activity of pregabalin, tramadol and duloxetine.

We compared the preclinical analgesic activity of three marketed drugs with different pharmacological properties, pregabalin, tramadol and duloxetine, described as effective against neuropathic pain in the clinic. These drugs were tested against evoked pain in two different neuropathic models in the rat, the Bennett (CCI) and the Chung (SNL) models. The selected endpoints were tactile allodynia, tactile hyperalgesia, heat hyperalgesia and cold allodynia. Although all three drugs displayed analgesic activity, the effects observed varied according to the behavioral evaluation. Pregabalin showed clear analgesic effects against cold allodynia and tactile hyperalgesia in both the CCI and Chung models. Tramadol was active against all four endpoints in the Chung model with similar effects in the CCI model, apart from tactile allodynia. Duloxetine inhibited tactile allodynia and heat hyperalgesia in both neuropathic pain models. It also displayed efficacy against tactile hyperalgesia in the CCI model and against cold allodynia in the Chung model. These data confirm that the CCI and the Chung models of neuropathic pain do not detect the activity of analgesics with the same sensitivity. Furthermore, the mode of stimulation (tactile or thermal) and the type of endpoint (allodynia or hyperalgesia) can further influence the observed efficacy of gold standards as well as novel compounds developed for treating neuropathic pain symptoms.

The comparative sensitivity of three in vitro safety pharmacology models for the detection of lidocaine-induced cardiac effects

INTRODUCTION In the current ICH S7B guideline, in vitro evaluation of proarrhythmic liability is limited to the risk of QT interval prolongation, whilst the effect of new chemical entities on cardiac conductivity is often overlooked. The aim of this work was to compare the effects of the sodium channel blocker, lidocaine in three in vitro safety pharmacology models: hNa(v)1.5 channel test, atrial action potential (AP) and Purkinje fiber AP and to identify the most sensitive model for detecting cardiac conduction slowing. METHODS Whole-cell patch-clamp methods were used to record the sodium current (I(Na)) encoded by hNa(v)1.5 in stably transfected HEK293 cells at ambient temperature. Transmembrane APs were recorded in rabbit Purkinje fibers and rabbit and guinea-pig left stimulated atria at physiological temperature. Parameters involved in depolarization or repolarization were reported. RESULTS Lidocaine (from 10 to 1000 μM) decreased the amplitude of I(Na) (IC(50): 256±37 μM) in a concentration-dependent manner. In the Purkinje fiber assay, lidocaine (10, 30 and 100 μM) had no effects on maximal upstroke velocity (Vmax), but shortened AP duration at 90% repolarization (APD(90)). At 30 and 100 μM, lidocaine also increased AP triangulation. In guinea-pig atria, lidocaine decreased Vmax starting from 30 μM and conduction velocity (CV) at 100 μM, but had no effects on other parameters. In rabbit atria, lidocaine decreased Vmax and CV at 100 μM without affecting APD(90). The effects of 100 μM lidocaine on Vmax and CV were more marked in rabbit than in guinea-pig atria. CONCLUSION Rabbit atria are more sensitive than rabbit Purkinje fibers or guinea-pig atria for detecting lidocaine-induced cardiac conduction slowing. These data suggest that isolated rabbit atria in addition to the hNa(v)1.5 channel assay could be relevant models to predict drug-induced conduction slowing.

240 A RAPID AND SENSITIVE METHOD TO EVALUATE TACTILE HYPERALGESIA IN THE RAT

possible involvement of glial cells in spinal sensitization in an acute myositis model. Methods: Myositis was induced by the administration of 400mg of carrageenan (Cg) in the right gastrocnemius (GS) muscle of rats, with saline as a control. The nociceptive threshold was evaluated using the electronic von Frey hairs. Behavioral measures were assessed before and 2 h after induction of myositis, and a decrease of the withdrawal threshold (grams) was used as a nociceptive parameter. Immunohistochemical assays to evaluate astrocyte and microglia activation (GFAP and OX-42, respectively) were performed in the lumbar dorsal horn of spinal cord. Intrathecal injection of fluorocitrate, a disruptor of glial function, was used to examine potential spinal glial mediation. Results: The administration of Cg into the GS muscle induced mechanical hyperalgesia. Immunohistochemical data showed that glial cells are activated after acute myositis, as immunostaining for GFAP and OX-42 was significantly higher in Cg-treated animals. Fluorocitrate was able to decrease Cg-induced pain sensitivity and the astrocyte and microglia activation. Conclusions: Our results suggest that glial cells are involved in the development of the nociceptive response induced by acute muscle inflammation. Understanding the role of glial cells during miositis-induced nociception may lead to new strategies for the management of muscle pain, one of the most frequent types of clinically relevant pain.