Maria Margarida Castel-Branco

THE MAXIMAL ELECTROSHOCK SEIZURE (MES) MODEL IN THE PRECLINICAL ASSESSMENT OF POTENTIAL NEW ANTIEPILEPTIC DRUGS

The choice of appropriate animal models for the initial in vivo testing of potential anticonvulsant compounds is one of the most important steps in the successful search for new antiepileptic drugs. The purpose of this paper is to describe the most important aspects to take into account when performing the maximal electroshock seizure (MES) test in the routine laboratory screening of new antiepileptics: the conventional and threshold MES test experimental procedures, the factors affecting experimental data (laboratory conditions, administration vehicles and drug formulations, time after drug administration, and stimulus duration and site of stimulation) and the assessment of anticonvulsant activity are discussed.

Lamotrigine pharmacokinetic/pharmacodynamic modelling in rats

The aim of this study was to perform a pharmacokinetic/pharmacodynamic (PK/PD) modelling of lamotrigine following its acute administration to rats. Adult male Wistar rats were given 10 mg/kg of lamotrigine intraperitoneally. Plasma and brain samples were obtained at predetermined times over 120 h post‐dose and analysed by liquid chromatography. The anticonvulsant profile against maximal electroshock seizure stimulation was determined over 48 h after dosing. As a linear relationship between lamotrigine plasma and brain profiles was observed, only the plasma data set was used to establish the PK/PD relationship. To fit the effect–time course of lamotrigine, the PK/PD simultaneous fitting link model was used: the pharmacokinetic parameters and dosing information were used in the one‐compartment first‐order model to predict concentrations, which were then used to model the pharmacodynamic data with the sigmoid Emax model, in order to estimate all the parameters simultaneously. The following parameters were obtained: Vd = 2.00 L/kg, kabs = 8.50 h−1, kel = 0.025 h−1, ke0 = 3.75 h−1, Emax = 100.0% (fixed), EC50 = 3.44 mg/L and γ = 8.64. From these results, it can be stated that lamotrigine is extensively distributed through the body, its plasma elimination half‐life is around 28 h and a lamotrigine plasma concentration of 3.44 mg/L is enough to protect 50% of the animals. When compared with humans, the plasma concentrations achieved with this dose were within the therapeutic concentration range that had been proposed for epileptic patients. With the present PK/PD modelling it was possible to fit simultaneously the time‐courses of the plasma levels and the anticonvulsant effect of lamotrigine, providing information not only about the pharmacokinetics of lamotrigine in the rat but also about its anticonvulsant response over time. As this approach can be easily applied to other drugs, it becomes a useful tool for an explanatory comparison between lamotrigine and other antiepileptic drugs.

Influence of administration vehicles and drug formulations on the pharmacokinetic profile of lamotrigine in rats

Given that administration vehicles and drug formulations can affect drug bioavailability, their influence on the pharmacokinetic profile of lamotrigine (LTG), a new‐generation anti‐epileptic drug, was studied in rats. Three different formulations administered intraperitoneally at a dose of 10 mg/kg were used: (1) LTG suspended in a 0.25% methylcelulose solution, (2) LTG dissolved in a 50% propylene glycol solution, and (3) LTG isethionate dissolved in distilled water. Plasma and brain homogenate levels were determined in order to evaluate vehicle‐dependent drug absorption. The results demonstrated rapid absorption of LTG when it was administered as an aqueous solution, in contrast to a slower and more erratic absorption after the injection of either the lipophilic solution or the suspension. A plasma peak was achieved 15 min post‐dose with the aqueous solution, with a brain peak being achieved 15 min later, while with the other formulations both plasma and brain homogenate peaks were reached 2 h after LTG administration. This study suggests that LTG isethionate dissolved in distilled water is the most suitable formulation for successful LTG pharmacokinetic studies in rats.

Evaluation of gastric toxicity of indomethacin acid, salt form and complexed forms with hydroxypropyl-β-cyclodextrin on Wistar rats: histopathologic analysis

Indomethacin (IM) is a non‐steroidal anti‐inflammatory drug which inhibits prostaglandin biosynthesis. It is practically insoluble in water and has the capacity to induce gastric injury. Hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) is an alkylated derivative of β‐CD with the capacity to form inclusion complexes with suitable molecules. IM is considered to form partial inclusion complexes with HP‐β‐CD by enclosure of the p‐chlorobenzoic part of the molecule in the cyclodextrin channel, reducing the adverse effects. The aim of this paper is to evaluate the gastric damage induced by the IM inclusion complex prepared by freeze‐drying and spray‐drying. A total of 135 Wistar rats weighing 224.4 ± 62.5 g were put into 10 groups. They were allowed free access to water but were maintained fasted for 18 h before the first administration until the end of the experiment. IM acid‐form, IM trihydrated‐sodium‐salt and IM‐HP‐β‐CD spray and freeze‐dried, at normal and toxic doses, were administered through gastric cannula once/day for 3 days. Seventy‐two hours after the first administration, the animals were sacrificed and the stomachs collected and prepared for morphological study by using the haematoxylin‐eosin technique. Lesion indexes (rated 0/4) were developed and the type of injury was scored according to the severity of damage and the incidence of microscopic evidence of harm. Microscopic assessment demonstrated levels of injury with index one on 10–25%. The type of complexation method had different incidence but the same degree. The results show that IM inclusion complexation protects against gastric injury, reducing the incidence and the maximum degree of severity from 4 to 1, with a better performance of the spray‐dried complex.

Gastroprotective effect of Cymbopogon citratus infusion on acute ethanol-induced gastric lesions in rats.

ETHNOPHARMACOLOGICAL RELEVANCE Treatment of gastric ulcers with medicinal plants is quite common in traditional medicine worldwide. Cymbopogon citratus (DC) Stapf. leaves infusion has been used in folk medicine of many tropical and subtropical regions to treat gastric disturbances. The aim of this study was to assess the potential gastroprotective activity of an essential oil-free infusion from C. citratus leaves in acute gastric lesions induced by ethanol in rat. MATERIALS AND METHODS The study was performed on adult male Wistar rats (234.0±22.7g) fasted for 24h but with free access to water. The extract was given orally before (prevention) or after (treatment) intragastric administration of absolute ethanol. Effects of dose (28 or 56mg/kg of body weight) and time of contact of the extract with gastric mucosa (1 or 2h) were also assessed. Animals were sacrificed, being the stomachs removed and the lesions were assessed by macroscopic observation and histopathology. RESULTS C. citratus extract, given orally before or after ethanol, significantly (P<0.01) reduced gastric mucosal injury compared with control group (vehicle+ethanol). The effect does not appear to be dose-dependent. Results also suggested that the extract is more effective when the time of contact with gastric mucosa increases. CONCLUSIONS The results of this assay confirm the gastroprotective activity of C. citratus extract on experimental gastric lesions induced by ethanol, contributing for the pharmacological validation of its traditional use.

Hepatic and renal toxicities of indomethacin acid, salt form and complexed forms with hydroxypropyl-β-cyclodextrin on Wistar rats after oral administration

Indomethacin (IM), a non‐steroidal anti‐inflammatory drug, has the capacity to induce hepatic and renal injuries when administrated systemically. The aim of this study is to assess the IM absorption from complexed forms when orally administered to rats, by means of a comparative evaluation of its capacity to induce hepatic and renal injury in different forms, namely IM acid, IM sodium salt or IM complexed with hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD), using freeze‐ and spray‐drying methods.

Lamotrigine kidney distribution in male rats following a single intraperitoneal dose

As it has been previously shown that lamotrigine (LTG) accumulates in the kidney of male rats, the purpose of the present investigation was to characterize the kidney profiles of LTG and its kidney distribution pattern in male rats, in order to confirm if a preferential distribution exists and to analyse if it does or does not affect the LTG systemic pharmacokinetics. Adult male Wistar rats were intraperitoneally injected with 5, 10 and 20 mg/kg of LTG. The concentration–time profiles of LTG in plasma and whole kidney were determined over 120 h postdose. The distribution of LTG in the rat kidney was investigated in another group of rats by measuring LTG levels in the renal cortex and medulla. The LTG plasma concentration–time profiles revealed a linear relationship with dose. However, a slight increase in the LTG elimination half‐life with dose was observed. In contrast, a nonlinear relationship was established between LTG kidney levels and the dose administered. Consequently, nonparallel patterns were observed between LTG plasma and kidney profiles. The LTG kidney distribution pattern revealed an accumulation of LTG in the renal cortex. The present study demonstrated that LTG distributes preferentially to the kidneys of the male rat in a dose‐dependent manner and suggests that such distribution may slightly affect the systemic kinetics of the drug.