Selective down-regulation of [(125)I]Y0-alpha-conotoxin MII binding in rat mesostriatal dopamine pathway following continuous infusion of nicotine.

Prolonged exposure to nicotine, as occurs in smokers, results in up-regulation of all the neuronal nicotinic acetylcholine receptor subtypes studied so far, the only differences residing in the extent and time course of the up-regulation. alpha6beta2*-Nicotinic acetylcholine receptors are selectively enriched in the mesostriatal dopaminergic system and may play a crucial role in nicotine dependence. Here we show that chronic nicotine treatment (3mg/kg/day for two weeks, via s.c. osmotic minipumps) caused a significant decrease (36% on average) in the binding of [(125)I]Y(0)-alpha-conotoxin MII (a selective ligand for alpha6beta2*-nicotinic acetylcholine receptors in this system) to all the five regions of the rat dopaminergic pathway analyzed in this study. After one week of withdrawal, binding was still lower than control in striatal terminal regions (namely the caudate putamen and the accumbens shell and core). In somatodendritic regions (the ventral tegmental area and the substantia nigra) the decrease was significant at the end of the treatment and recovered within one day of withdrawal. This effect was not due to displacement of [(125)I]Y(0)-alpha-conotoxin MII binding by residual nicotine. In fact the binding was not changed by 565 ng/g nicotine (obtained with a single injection of nicotine), a concentration much higher than that found in the brain of rats chronically treated with nicotine (240 ng/g). In addition, consistent with previous studies reporting an up-regulation of other subtypes of nicotinic acetylcholine receptors, we found that nicotine exposure significantly increased (40% on average) the binding of [(125)I]epibatidine (a non-selective agonist at most neuronal heteromeric nicotinic acetylcholine receptors) in three up to five regions containing only alpha-conotoxin MII-insensitive [(125)I]epibatidine binding sites, namely the primary motor, somatosensory and auditory cortices. In conclusion, this work is the first to demonstrate that alpha6beta2*-nicotinic acetylcholine receptors, unique within the nicotinic acetylcholine receptor family, are down-regulated following chronic nicotine treatment in rat dopaminergic mesostriatal pathway, a finding that may shed new light in the complex mechanisms of nicotine dependence.

Investigation of the effects of lamotrigine and clozapine in improving reversal-learning impairments induced by acute phencyclidine and D-amphetamine in the rat.

RATIONALE: Phencyclidine (PCP), a glutamate/N-methyl-D-aspartate (NMDA) receptor antagonist, has been shown to induce a range of symptoms similar to those of patients with schizophrenia, while D-amphetamine induces predominantly positive symptoms. Previous studies in our laboratory have shown that PCP can selectively impair the performance of an operant reversal-learning task in the rat. Furthermore, we found that the novel antipsychotic ziprasidone, but not the classical antipsychotic haloperidol, could prevent the PCP-induced deficit. OBJECTIVES: The aim of the present study was to validate the model further using the atypical antipsychotic clozapine and then to investigate the effects of lamotrigine, a broad-spectrum anticonvulsant that is known to reduce glutamate release in vitro and is able to prevent ketamine-induced psychotic symptoms in healthy human volunteers. A further aim was to compare effects of PCP and D-amphetamine in the test and investigate the effects of the typical antipsychotic haloperidol against the latter. METHODS: Female hooded-Lister rats were food deprived and trained to respond for food in a reversal-learning paradigm. RESULTS: PCP at 1.5 mg/kg and 2.0 mg/kg and D-amphetamine at 0.5 mg/kg significantly and selectively impaired performance in the reversal phase of the task. The cognitive deficit induced by 1.5 mg/kg PCP was attenuated by prior administration of lamotrigine (20 mg/kg and 30 mg/kg) or clozapine (5 mg/kg), but not haloperidol (0.05 mg/kg). In direct contrast, haloperidol (0.05 mg/kg), but not lamotrigine (25 mg/kg) or clozapine (5 mg/kg), prevented a similar cognitive impairment produced by D-amphetamine (0.5 mg/kg). CONCLUSIONS: Our findings provide further data to support the use of PCP-induced disruption of reversal learning in rodents to investigate novel antipsychotic drugs. The results also provide evidence for different mechanisms of PCP and D-amphetamine-induced disruption of performance in the test, and their different sensitivities to typical and atypical antipsychotic drugs.

Some considerations about stability study design.

The aim of a stability study is to check whether or not a drug maintains its initial properties (over a given range) in a period of time. There are many parameters considered in a stability study, but in this paper only the active content will be considered. Evaluation of this parameter is performed by measuring the content of the active ingredient in the drug at different times and on expiration to see if it is within specification (1). The problem also involves estimating whether the drug will perform satisfactorily and maintain its initial properties in the future. In this paper, we provide a criterion for the evaluation of experimental design used in a stability study for predicting the content of an active ingredient in a drug, starting from the confidence limit calculated following International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) recommendations. The point of view adopted is akin to hypothesis testing. The main question is this: "If the drug fulfills the requirements at a given time in the future, what is the probability that the data collected for that drug will show its suitability?" For this purpose, two concepts are used-producer gain and consumer loss-which are defined as follows: producer gain is the advantage (for the producer) of recognizing a drug as "good" (provided it is good and within specification); consumer loss is the loss (for the consumer) when the drug is no longer good, but the data collected indicate that it is good. The aim of the experimental design in this study is to increase the probability of producer gain and maximize it on the basis of a given consumer loss.

A comparison of the in vitro Comet assay with the in vitro chromosome aberration assay using whole human blood or Chinese hamster lung cells: validation study using a range of novel pharmaceuticals.

The in vitro Comet assay, a sensitive, quick and relatively cheap test, could become a valid alternative to the commonly used in vitro chromosomal aberration test, in the preliminary evaluation of new chemical entities early in the development of new pharmaceuticals. A validation of the Comet assay procedure using whole human blood or CHL cells was carried out in comparison with a cytogenetic test utilizing the same target cells with the following compounds which demonstrated positive results in standard chromosomal aberration tests: two well-documented clastogens, methyl methanesulphonate and cyclophosphamide, and eight novel drugs in early development. A 3 h exposure time, in both the absence and presence of metabolic activation, was used for the in vitro Comet assay. Agreement between the results of the Comet assay and the chromosomal aberration tests was found to be satisfactory on a qualitative basis, although positive results in the Comet assay were always at higher doses than in the cytogenetic test. This indicates a reduction in sensitivity using the former genotoxicity end-point. In order to try to explain this observation, a range of exposure times (0.25, 0.5, 1, 2 and 3 h) were investigated in two further experiments to determine the optimal time for detecting Comet induction in this assay procedure. Maximum levels of DNA damage (in terms of Comet induction) were recorded at earlier sampling times (0.25-1 h) in whole human blood using the same positive doses observed in HPLT. Further studies need to be performed to confirm these findings. It is possible that strand breaks are too short lived to allow detection after a 3 h treatment period (due to preferential repair), indicating the need for shorter exposure times in some cases to optimize their detection.