Investigation of the predictive validity of laser-EPs in normal, UVB-inflamed and capsaicin-irritated skin with four analgesic compounds in healthy volunteers.

AIMS: The aim of the present study was to assess the predictivity of laser-(radiant-heat)-evoked potentials (LEPs) from the vertex electroencephalogram, using an algesimetric procedure, testing the anti-nociceptive/anti-hyperalgesic effects of single oral doses of four marketed analgesics (of different compound classes) vs. placebo, in healthy volunteers with three skin types. METHODS: This was a randomized, placebo-controlled, single-blind, five-way-crossover trial. Twenty-five healthy male/female Caucasians were included (receiving celecoxib 200 mg, pregabalin 150 mg, duloxetine 60 mg, lacosamide 100 mg or placebo) in a Williams design, with CO2 laser-induced painful stimuli to normal, ultraviolet (UV) B-inflamed and capsaicin-irritated skin. LEPs and visual analogue scale ratings were taken at baseline and hourly for 6 h postdose from all three skin types. RESULTS: In normal skin, the averaged postdose LEP peak-to-peak-(PtP)-amplitudes were reduced by pregabalin (-2.68 µV; 95% confidence interval (CI) -4.16, 1.19) and duloxetine (-1.73 µV; 95% CI -3.21, -0.26) but not by lacosamide and celecoxib vs. placebo. On UVB-irradiated skin, reflecting inflammatory pain, celecoxib induced a pronounced reduction in LEP PtP amplitudes vs. placebo (-6.2 µV; 95% CI -7.88, -4.51), with a smaller reduction by duloxetine (-4.54 µV; 95% CI -6.21, -2.87) and pregabalin (-3.72 µV; 95% CI -5.40, -2.04), whereas lacosamide was inactive. LEP PtP amplitudes on capsaicin-irritated skin, reflecting peripheral/spinal sensitization, as in neuropathic pain, were reduced by pregabalin (-3.78 µV; 95% CI -5.31, -2.25) and duloxetine (-2.32 µV; 95% CI -3.82, -0.82) but not by celecoxib or lacosamide vs. placebo, which was in agreement with known clinical profiles. Overall, PtP amplitude reductions were in agreement with subjective ratings. CONCLUSIONS: LEP algesimetry is sensitive to analgesics with different modes of action and may enable the effects of novel analgesics to be assessed during early clinical development.

Investigation of combined CYP3A4 inductive/inhibitory properties by studying statin interactions: a model study with the renin inhibitor ACT-178882.

PURPOSE: ACT-178882, a direct renin inhibitor, was used as a model compound in an elaborate drug-drug interaction study with atorvastatin and simvastatin to explore complex CYP3A4 inductive and inhibitory properties. METHODS: Thirty-two healthy male subjects received single doses of 20 mg atorvastatin and 20 mg simvastatin on days 1, 9, 31, and 41. On days 6 to 33, 500 mg ACT-178882 was administered once daily. Plasma concentrations of ACT-178882, simvastatin, and atorvastatin were measured by LC-MS/MS. Routine safety assessments were performed throughout the study. RESULTS: Exposure (as based on area under the curve) to simvastatin and 6ß-hydroxyacid simvastatin increased (90 % confidence interval) 4.63-fold (3.90, 5.50) and 3.71-fold (3.19, 4.32), respectively, when comparing day 9 and day 1. On day 9, exposure to atorvastatin was similar but Cmax decreased, while both variables decreased for ortho-hydroxy atorvastatin when compared to day 1. On day 31, after prolonged administration of ACT-178882, exposure to atorvastatin, ortho-hydroxy atorvastatin, simvastatin, and 6ß-hydroxyacid simvastatin decreased by 14, 19, 21, and 27 %, respectively, when compared to day 9. However, on this day, exposure to simvastatin and its metabolite was still markedly higher when compared to day 1. Effects of ACT-178882 had largely dissipated on day 41. CONCLUSIONS: This design enabled the study of complex time-dependent effects on CYP3A4 activity with clinically relevant substrates.

Relative bioavailability of a newly developed pediatric formulation of bosentan vs. the adult formulation.

WHAT IS KNOWN: Bosentan is a dual endothelin receptor antagonist approved for the treatment of pulmonary arterial hypertension (PAH). Since bosentan is frequently used to treat pediatric PAH patients, a pediatric formulation was developed. AIM: To evaluate the pharmacokinetic properties of bosentan and its active metabolite, Ro 48-5033, of the quadrisected, dispersible pediatric vs. the adult tablet after single-dose administration to healthy subjects. Secondary objectives of the study were to compare the pharmacokinetics of two inactive metabolites and the safety of both formulations. MATERIALS AND METHODS: In this open-label, two-way crossover study, subjects (20 - 43 years) were randomized to receive single oral doses of 62.5 mg of bosentan as 1 adult tablet and 64 mg as 2 pediatric tablets of 32 mg. Blood samples were drawn over a 48-hour period to measure bosentan and its metabolites. RESULTS: 16 subjects were enrolled and completed the study. Following treatment with the pediatric formulation, values for Cmax and AUC0-∞ of bosentan were lower than with the adult formulation with geometric mean ratios (90% confidence interval) of 0.82 (0.65, 1.04) and 0.87 (0.78, 0.97), respectively. Similar results were obtained for the primary active metabolite Ro 48-5033. Both treatments were well tolerated. WHAT IS NEW AND CONCLUSION: Although the 90% confidence intervals of the geometric mean ratios of Cmax and AUC0-∞ were not entirely within the conventional bioequivalence range (0.80 - 1.25), no clinically relevant effect of formulation on the pharmacokinetics of bosentan and Ro 48-5033 was detected. Both formulations were well tolerated.

Comparative pharmacokinetic, pharmacodynamic, safety, and tolerability profiles of 3 different formulations of epoprostenol sodium for injection in healthy men.

BACKGROUND: Epoprostenol sodium for injection is approved for the treatment of severe cases of primary pulmonary arterial hypertension. Currently, there are 3 approved formulations of this drug containing the same active ingredient (epoprostenol sodium) but differing with regard to excipients. When compared with epoprostenol sodium formulated with glycine-mannitol (epoprostenol GM), 2 new formulations of epoprostenol sodium, one formulated with arginine-mannitol (epoprostenol AM) and one formulated with arginine-sucrose (epoprostenol AS), have improved stability after reconstitution and dilution. The biocomparability of epoprostenol AM and epoprostenol GM, with regard to pharmacokinetic (PK), pharmacodynamic (PD), safety, and tolerability profiles, has been shown previously. OBJECTIVE: This study compared PK, PD, safety, and tolerability profiles of the 3 different formulations of epoprostenol sodium for injection. METHODS: This was a prospective, single-center, open-label, 2-period, 2-treatment, randomized, crossover, ascending dose study in 2 parts. Twenty healthy men in part 1 and 20 different individuals in part 2 received epoprostenol AM and epoprostenol AS and epoprostenol GM and epoprostenol AS, respectively, in a crossover fashion, as sequential IV infusions of 2, 4, 6, and 8 ng/kg/min for 2 hours each. In each part, the PK profile of epoprostenol was characterized via analysis of the concentration-time profiles of its 2 primary metabolites: 6-keto-prostacyclin F1α and 6,15-diketo-13,14-dihydro-prostacyclin F1α. The effect of the formulations was assessed using the 90% CI of the geometric mean ratio calculated for the exposure PK parameters. The PD variables cardiac output, cardiac index, and heart rate were assessed using echocardiography. Adverse events were recorded through the study. RESULTS: The plasma concentration versus time curves of epoprostenol AM and epoprostenol AS in part 1 and epoprostenol GM and epoprostenol AS in part 2 were similar in shape and almost superimposable. For each study part, the 90% CIs of ratios of geometric means for AUC0-∞ of the assessed epoprostenol formulations were within the range for bioequivalence (0.8-1.25). The increases in cardiac output, cardiac index, and heart rate resulting from infusion with epoprostenol sodium were comparable between all formulations, with maximum values attained after 8 hours. Almost all study participants reported at least one treatment-emergent adverse event, the most common being headache, which was reported in 80% to 85% of study participants. CONCLUSIONS: Overall, the PK, PD, safety, and tolerability profiles of the 3 formulations of epoprostenol sodium for injection are comparable and meet the criteria of bioequivalence. Australian New Zealand Clinical Trials Registry identifier: ACTRN12612001086853.

Pharmacokinetics, pharmacodynamics, and tolerability of ACT-077825, a new direct renin inhibitor after multiple-ascending doses in healthy subjects.

This study was conducted to characterize the multiple-dose tolerability, pharmacokinetics, and pharmacodynamics of ACT-077825, a new direct renin inhibitor, in healthy male subjects. In this single-center, double-blind, placebo-controlled, active-controlled (20 mg of enalapril), randomized multiple-ascending dose study, ACT-077825 was administered once a day. for 7 days in the 50-1000 mg dose range to sodium- and potassium-restricted subjects. ACT-077825 pharmacokinetics on days 1 and 7 were characterized by dose-proportional increases in Cmax and AUCτ. At steady state, accumulation was modest (1.5- to 1.7-fold). Enalapril caused an increase in plasma active renin concentration and plasma renin activity (PRA). ACT-077825 dose dependently increased active renin on days 1 and 7 and inhibited PRA dose dependently only on day 1. On day 7, the maximal PRA inhibition was attained after 250 mg of ACT-077825. In contrast to enalapril, ACT-077825 did not induce any consistent lowering effect on blood pressure when compared with placebo. Of the reported adverse events, diarrhea, headache, and postural dizziness were more frequent. The incidence of diarrhea was greater in the 1000-mg group and a dose of 500 mg of ACT-077825 was identified as the maximum tolerated dose. Overall, pharmacokinetic, pharmacodynamic, and tolerability profiles warrant the further investigation of ACT-077825 in patients with hypertension.

Effect of Multiple-Dose Diltiazem on the Pharmacokinetics of the Renin Inhibitor ACT-077825.

This open-label, randomized study evaluated the effects of steady-state diltiazem on the pharmacokinetic, safety, and tolerability profile of a single dose of the novel renin inhibitor ACT-077825. Twelve healthy Caucasian male subjects (20-50 years) received in treatment sequence A, a single dose of 100 mg ACT-077825 (Days 1 and 17), and oral diltiazem 300 mg once daily (Days 14-26). In treatment sequence B, subjects received a single dose of 100 mg ACT-077825 (Days 4 and 22) and oral diltiazem 300 mg once daily (Days 1-13). ACT-077825 alone and combined with diltiazem was generally well tolerated. On average, the systemic exposure to ACT-077825 was higher in the presence of diltiazem. For AUC0-∞ and t1/2 , the upper limit of the 90% confidence interval (CI) of the geometric mean ratios was outside the study-specific 0.5-2.0 equivalence boundaries, that is, 1.92 (90% CI: 1.30, 2.83) and 1.58 (90% CI: 1.22, 2.04), respectively. In conclusion, diltiazem markedly affected the pharmacokinetics of ACT-077825, probably via inhibition of CYP3A4 activity, without changing its safety and tolerability profile in healthy male subjects. Whether such an interaction will require for therapeutic dose adjustment of ACT-077825 co-administered with diltiazem has to be assessed once the dose-response relationship of ACT-077825 in hypertensive patients is determined.

Determination of 6-keto prostaglandin F1α and its metabolites in human plasma by LC-MS/MS.

An HPLC-MS/MS method was developed and validated for the quantification of 6-keto prostaglandin F1α, the stable hydrolysis product of prostacyclin, and its metabolites 2,3-dinor-6-keto prostaglandin F1α and 6,15-diketo-13,14-dihydro prostaglandin F1α in human plasma. For sample preparation, a solid phase extraction step was combined with a column switching approach for analytes enrichment and further sample clean-up of the processed sample. The assay was validated in the concentration range 50.0-5000 pg/mL for 6-keto prostaglandin F1α and 6,15-diketo-13,14-dihydro prostaglandin F1α, and 100-10,000 pg/mL for 2,3-dinor-6-keto prostaglandin F1α. The inter-batch precision was better than 12.7%, 9.2%, and 9.4% for 6-keto prostaglandin F1α, 2,3-dinor-6-keto prostaglandin F1α, and 6,15-diketo-13,14-dihydro prostaglandin F1α, respectively. The inter-batch accuracy was between 97.3% and 100.8% for 6-keto prostaglandin F1α, between 97.5% and 103.0% for 2,3-dinor-6-keto prostaglandin F1α, and between 92.0% and 100.0% for 6,15-diketo-13,14-dihydro prostaglandin F1α. Further it has been demonstrated that the analytes were stable in plasma for 20 h at room temperature, during three freeze-and-thaw cycles, for 96 days at -25 °C storage temperature, and 50h in the autosampler tray at room temperature.

Integrated pharmacokinetics and pharmacodynamics of epoprostenol in healthy subjects.

AIM: The aim of the study was to report the first thorough characterization of the pharmacokinetics (PK) and pharmacodynamics (PD) of epoprostenol in an integrated manner. METHOD: Twenty healthy male subjects received two formulations of i.v. epoprostenol, in a crossover design, in sequential infusions of 2, 4, 6 and 8 ng kg(-1) min(-1) for 2 h each. A sensitive assay was developed which allowed accurate PK characterization of epoprostenol via analysis of the concentration-time profiles of its two primary metabolites, 6-keto-prostacyclin F(1α) and 6,15-diketo-13,14-dihydro-prostacyclin F(1α) . PD parameters included cardiac output (CO), cardiac index (CIn) and heart rate (HR). RESULTS: The pharmacokinetics of epoprostenol deviated slightly from dose-proportionality, probably due to a food effect. After infusion of the two formulations of epoprostenol, the t(1/2) values expressed as geometric mean (95% confidence interval) were 0.25 h (0.14, 0.46) and 0.22 h (0.13, 0.38) for 6-keto-prostacyclin F(1α) , and 0.32 h (0.22, 0.45) and 0.34 h (0.26, 0.46) for 6,15-diketo-13,14-dihydro-prostacyclin F(1α) . A single compartment infusion model with first order elimination adequately described the PK of 6-keto-prostacyclin F(1α) . This model also characterized the food effect. Stepwise infusions with epoprostenol resulted in a progressive increase in CO, CIn and HR. CONCLUSION: Of the two metabolites analyzed, the appearance of 6-keto-prostacyclin F(1α) in plasma was more closely associated with the haemodynamic effects of i.v. epoprostenol. PK and PD profiles showed that CIn relates proportionally and linearly to the plasma concentrations of 6-keto-prostacyclin F(1α) . These results suggest that 6-keto-prostacyclin F(1α) is a suitable surrogate marker of plasma concentrations of epoprostenol.

Entry-into-humans study with a new direct renin inhibitor.

PURPOSE: To evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of escalating single oral doses of ACT-077825, a novel orally active renin inhibitor, in healthy male subjects. METHODS: In this single-center, double-blind, placebo- and active-controlled (with enalapril) randomized study, 70 subjects received a single dose of ACT-077825 (1-1,000 mg), placebo, or enalapril 20 mg under fasted conditions. The main pharmacokinetic endpoints were area under the plasma ACT-077825 concentration-time curve from time zero to infinity and the terminal half-life (t(1/2)). The pharmacodynamic endpoints included immunoactive active renin (iAR) plasma concentrations and plasma renin activity (PRA). Standard laboratory and safety data were collected. RESULTS: Of the few adverse events reported, diarrhea and headache were the most frequent. The pharmacokinetics of ACT-077825 were dose-proportional in the dose range 100 to 1,000 mg. Terminal t(1/2), best characterized following a dose of 1,000 mg, was 41.6 h and t(max) 4-5 h post-dose. ACT-077825 dose-dependently increased iAR and decreased PRA, effects that were associated with a decrease in blood pressure at 1,000 mg, similar to following treatment with enalapril. CONCLUSION: The results provide evidence that ACT-077825, with a pharmacokinetic profile consistent with a once-a-day dosing regimen, may represent an effective antihypertensive agent and pave the way toward a multiple-ascending dose study.

Imaging trait anxiety in high anxiety F344 rats: Focus on the dorsomedial prefrontal cortex.

Functional magnetic resonance imaging (fMRI) has become an important method in clinical psychiatry research whereas there are still only few comparable preclinical investigations. Herein, we report that fMRI in rats can provide key information regarding brain areas underlying anxiety behavior. Perfusion as surrogate for neuronal activity was measured by means of arterial spin labeling-based fMRI in various brain areas of high anxiety F344 rats and control Sprague-Dawley rats. In one of these areas, the dorsomedial prefrontal cortex (dmPFC), c-Fos labeling was compared between these two strains with immunolabeling. The effects of a neurotoxic ibotenic acid lesion of the dmPFC in F344 rats were examined in a social approach-avoidance anxiety procedure and fMRI. Regional brain activity of high anxiety F344 rats was different in selective cortical and subcortical areas as compared to that of low anxiety Sprague-Dawley rats; the largest difference (i.e. hyperactivity) was measured in the dmPFC. Independently, c-Fos labeling confirmed that F344 rats show increased dmPFC activity. The functional role was confirmed by neurotoxic lesion of the dmPFC that reversed the high anxiety-like behavior and partially normalized the brain activity pattern of F344 rats. The current findings may have translational value as increased activity is reported in an equivalent cortical area in patients with social anxiety, suggesting that pharmacological or functional inhibition of activity in this brain area should be explored to alleviate social anxiety in patients.

Examining face and construct validity of a noninvasive model of panic disorder in Lister-hooded rats.

RATIONALE: Increasing evidence suggests that defensive escape behavior in Lister-hooded (LH) rats induced by ultrasound application may be an animal model of panic disorder. OBJECTIVE: The objectives of this study were to further explore the face and construct validity of ultrasound-induced escape behavior by characterizing the autonomic and neuroendocrine response to ultrasound, and to examine the underlying neuronal structures by comparing the effects of the anxiolytic with panicolytic properties, diazepam, with a preclinical anxiolytic without panicolytic-like activity, the NOP agonist Ro 64-6198. MATERIALS AND METHODS: LH rats were implanted with telemetry transmitters to monitor heart rate and core body temperature before, during, and after ultrasound application. Blood samples were taken after ultrasound application for corticosterone analysis. Ultrasound-induced c-Fos expression was measured in different periaqueductal gray (PAG) and amygdala subregions after treatment with diazepam or Ro 64-6198. RESULTS: Ultrasound application increased heart rate and body temperature, but did not alter plasma corticosterone levels. Ultrasound application increased c-Fos expression in the dorsal and dorsolateral PAG (dPAG, dlPAG) and amygdaloid subregions. Diazepam, but not Ro 64-6198, reduced c-Fos expression in the dPAG/dlPAG, while Ro 64-6198, but not diazepam, reduced c-Fos expression in the central amygdala. CONCLUSIONS: Similar to human panic attacks, ultrasound application to LH rats activated the autonomic, but not the neuroendocrine, stress system. Also, like in humans, the current data confirm and extend that the dPAG/dlPAG plays a key role in ultrasound-induced escape behavior. These observations suggest that ultrasound-induced escape behaviors in LH rats have face and construct validity for panic disorders.

The steroid sulfatase inhibitor COUMATE attenuates rather than enhances access of dehydroepiandrosterone sulfate to the brain in the mouse.

Intraperitoneal injection of adult male mice with the neuroactive steroid dehydroepiandrosterone sulfate (DHEAS) at 1 and 40 mg/kg caused dose-dependent increases in the concentration of both this compound and its corresponding free steroid DHEA in brain within 1 h of injection. Pretreatment of these animals for 24 h with the steroid sulfatase inhibitor COUMATE at a dose (10 mg/kg, p.o.) shown previously to cause almost complete inhibition of this enzyme in liver and brain was expected to increase the amount of the DHEAS dose reaching the brain. Surprisingly however, the increases in brain concentrations of DHEAS and DHEA after injection of DHEAS i.p. were attenuated by pretreatment with COUMATE. The results suggest that the arylsulfamate based steroid sulfatase inhibitors such as COUMATE interfere with the influx of the DHEAS anion into the brain.

Defensive-like behaviors induced by ultrasound: further pharmacological characterization in Lister-hooded rats.

RATIONALE: In rats, dorsal periaqueductal gray (dPAG) stimulation elicits escape behavior that is thought to be related to fear and panic. A noninvasive technique--exposure to ultrasound-has been reported to stimulate the dPAG and induce escape followed by freezing in Lister-hooded (LH) rats. OBJECTIVE: Further characterize pharmacologically the ultrasound--induced defensive behaviors test with anxiolytics acting via different mechanisms. MATERIALS AND METHODS: LH rats, treated with clinically validated anxiolytics, putative anxiolytics, or compounds devoid of anxiolytic properties, were exposed to ultrasound. Baseline locomotion before and duration of escape and freezing behaviors during ultrasound were measured. RESULTS: The low-potency benzodiazepine receptor agonists, diazepam and chlordiazepoxide, selectively reduced escape compared to baseline locomotor activity. The high-potency agonist alprazolam, the mGlu2/3 receptor agonist LY 354740, and the mGlu5 receptor antagonist MTEP reduced escape but did not show such a separation. The voltage-dependent calcium channel inhibitors, pregabalin and gabapentin, selectively reduced escape. The nociceptin OFQ peptide receptor agonist Ro 64-6198 did not affect escape but reduced freezing, an effect that was not produced by any of the other compounds. Buspirone and morphine did not affect escape. As expected, haloperidol reduced escape in a nonselective manner. CONCLUSIONS: The present data demonstrate that ultrasound-induced defensive behaviors in LH rats can be independently modulated by anxiolytics of different classes. In particular, ultrasound-induced escape shows sensitivity to the majority of acute therapeutics effective in panic disorder, although sensitivity to compounds with slow onset of action (e.g., antidepressants) remains to be demonstrated.

The effects of serotonin reuptake inhibitors on locomotor activity in gerbils.

In the current study we examined the effects of serotonin reuptake inhibitors on the locomotor activity of gerbils, and undertook experiments to understand the mechanisms involved in their effects. The selective serotonin reuptake inhibitors (SSRIs) fluoxetine (1-30 mg/kg, i.p.) and escitalopram (0.03-10 mg/kg, i.p.) dose-dependently increased locomotor activity, whereas the serotonin and noradrenaline reuptake inhibitor duloxetine (0.3-30 mg/kg, i.p.) did not. The noradrenaline reuptake inhibitor (NRI) reboxetine, which alone did not significantly affect locomotion (1-30 mg/kg, i.p.), markedly reduced the effects of escitalopram. The locomotor effects of fluoxetine and escitalopram were dependent on novelty since both compounds showed rapid habituation in novel cages and were inactive when tested in home cages. Both diazepam (0.3-10 mg/kg, i.p.) and d-amphetamine (0.3-10 mg/kg, s.c.) increased locomotor activity but only the effects of diazepam were novelty-dependent. The finding that SSRIs increased locomotion, with a negative modulatory role for NRI, in a novelty-dependent manner, similar to diazepam, suggests that anxiety plays an important role in the present paradigm. The increase in locomotion as observed in our test conditions can be readily used as a selective and sensitive in vivo assay for serotonin transport inhibition in gerbils.

A combined marble burying-locomotor activity test in mice: a practical screening test with sensitivity to different classes of anxiolytics and antidepressants.

Over the last decades, the inhibition of spontaneous burying of glass marbles by mice has been used as an index of anxiolytic drug action in the so-called marble burying test. Indeed, acute administration of rapid-onset (e.g. diazepam) and slow-onset (e.g. fluoxetine) anxiolytics inhibit marble burying. However, non-anxiolytic compounds such as classical antipsychotics also reduce marble burying thus suggesting that the predictive validity of this procedure for anxiety may be limited. In the present study, after having selected a strain of mice (C57BL/6J) that showed spontaneous avoidance of glass marbles, we tried to improve the predictive validity of the marble burying test for anxiety by measuring locomotor activity during the marble burying test and--if needed--in control experiments by using a videotracking system. Twenty-four reference compounds were tested including anxiolytics, anxiogenics, antidepressants, antipsychotics and other classes. By comparing marble burying scores with locomotor measures, we found that, based on our criteria, most of the anxiolytics and antidepressants selectively inhibited marble burying in contrast to most of the other compounds (e.g. haloperidol, morphine). Two putative anxiolytics, i.e. the nociceptin orphanin FQ peptide receptor agonist Ro 64-6198 and the metabotropic glutamate 5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, also showed a selective profile. We propose this modified procedure, requiring only a limited number of animals, as a valuable screening test for the detection of compounds having anxiolytic effects.

Social approach-avoidance behavior of a high-anxiety strain of rats: effects of benzodiazepine receptor ligands.

RATIONALE: To better understand anxiety disorders with social impairments as well as to identify new treatments, it is important to develop preclinical procedures involving social components of anxiety. Recently, a novel procedure was reported: the rat Social Approach-Avoidance (SAA) test. In this test, the time spent by a test rat in a large social compartment containing an unfamiliar stimulus rat reflects the anxiety state of the animal. It was shown that pre-stressing the test rat increased the avoidance of the social compartment as characterized by an increase in the time spent in the nonsocial compartment. OBJECTIVE: (1) To use a high-anxiety strain, F-344 rats, instead of pre-stressed animals, (2) to automate the test by using video tracking to measure time spent in both compartments and their subdivisions, and (3) to validate this modified test with known benzodiazepine receptor ligands. MATERIALS AND METHODS: F-344 rats were treated with either chlordiazepoxide or diazepam (benzodiazepine receptor agonists), or RO 19-4603 or FG 7142 (benzodiazepine receptor inverse agonists). RESULTS: The agonists produced anxiolytic-like effects, whereas the inverse agonists produced anxiogenic-like effects. These effects were most marked in the two extreme zones in terms of distance to the stimulus rat. CONCLUSIONS: F-344 rats display spontaneous avoidance behavior in the modified SAA procedure, and approach-avoidance behavior in these rats is sensitive to benzodiazepine agonists and inverse agonists in a bidirectional manner. The finding that the assessment of time spent into two virtual zones in each of the compartments markedly increased the sensitivity to both anxiolytics and anxiogenics will be discussed using the concept of physical defensive distance.