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INTRODUCTION: Pupillary function is a sensitive pharmacological readout in humans, but drug effects are not well characterized in rodents. Hence improved methods are needed for quantifying pupillary responses in conscious albino rodents. METHODS: A novel camera-based pupillary imaging method was developed for conscious rats under red-light settings to investigate the effect of various reference compounds on pupil and iris diameter and light reflex. The chosen compounds (pilocarpine, tropicamide, phenylephrine, clonidine, cocaine, morphine) possess well characterized effects in humans. In rats, the compounds were administered topically as eye drops and/or systemically by intraperitoneal injection. RESULTS: Red-light was utilized in order to induce intermediate pupillary width at baseline conditions in conscious albino and pigmented rats, thereby enabling detection of light-induced constriction (miosis) and dilation (mydriasis) of the pupil. Pupil diameter and light reflex were affected by both topical and systemic administration of pilocarpine, tropicamide, phenylephrine, clonidine, cocaine, morphine. However only pharmacologically induced mydriasis was found in albino rats, whereas pigmented rats displayed both mydriasis and miosis albeit with differential responses. The pupillary imaging system is suitable for stand-alone studies as well as for pupil function assessment within the frame of a typical CNS study evaluating behavior (modified Irwin test), locomotor activity and body temperature. DISCUSSION: Strain-specific pharmacological responses were detected for pupillary function, with slightly more human-like responses in pigmented rats than albino rats, illustrating that effects on the rat pupil/iris cannot be directly translated to humans. Nevertheless, changes in pupillary diameter and/or pupillary light reflex in rats is a functional endpoint that can be quantified during early testing, and may trigger more detailed mechanistic characterization beyond the safety pharmacology core CNS battery.
RESUMO
INTRODUCTION: Irwin/FOB testing is routinely conducted to investigate the neurofunctional integrity of laboratory animals during preclinical development of new drugs, however, the study design frequently varies to meet specific needs. Representatives of several European-based pharmaceutical companies performed a "state-of-the-art" assessment of how they conduct their CNS safety evaluation using Irwin/FOB tests. METHODS: This assessment consisted of (1) a survey of current/historical practice, (2) an evaluation of historical studies with reference compounds (amphetamine, chlorpromazine) to determine intercompany reproducibility of results, and (3) an interlaboratory test using reference compounds (MK-801, chlorpromazine) to determine whether partially standardized conditions (animals, sex, doses, vehicles, administration route, observation time points, systemic exposure) might reduce variability of results. RESULTS: Our survey revealed several similarities, e.g., main endpoints of home cage and openfield observations, species, and positive control substances, but also a high level of heterogeneity between different companies with regard to behavioral endpoints during handling and reflex testing, scoring, group size, and timing of studies. Analysis of heterogeneously designed historical studies with amphetamine and chlorpromazine showed the anticipated behavioral changes, albeit with quantitative variability, and identified more robust (e.g., activity, posture, muscle tone, startle reflex, body temperature) and less robust (piloerection, stereotypical behavior, palpebral closure, respiration) Irwin/FOB parameters. A partially standardized interlaboratory test with MK-801 and chlorpromazine showed the expected behavioral changes and principally confirmed the historically-based more/less robust Irwin/FOB parameters, however, it also showed exposure variability and did not show a markedly reduced quantitative variability of behavioral results. DISCUSSION: Our survey and intercompany test results demonstrate certain heterogeneity in design and conduct of Irwin/FOB tests by pharmaceutical companies. Although the general behavioral profiles for the reference compounds were consistently found, quantitative variability of results remained even under partially standardized conditions. This suggests the importance of a high level of standardization with regard to the Irwin/FOB test modification used, scoring system, and observer training, in order to achieve an improved intercompany comparability of Irwin/FOB results.
