M. M. Caramona

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.

Role of nitric oxide in the activation of NF-kappaB, AP-1 and NOS II expression in articular chondrocytes.

Abstract. Objective and design: Determine the sources of nitric oxide (NO) and evaluate its role in the activation of nuclear Factor-kappaB (NF-κB) and activator protein-1 (AP-1) and in the expression of NO synthase II (NOS II), induced by interleukin-1β (IL-1).¶Material or subjects: Primary cultures of bovine articular chondrocytes.¶Treatment: The cells were treated with IL-1, 5 ng/ml with or without the NO donor S-nitroso-N-acetylpenicillamine (SNAP), in concentrations ranging from 10 to 300 μM.¶Methods: NF-κB and AP-1 activation were evaluated by electrophoretic mobility shift assay. Northern blot was used to detect NOS II mRNA levels and western blot to evaluate IκB-α, NOS I and NOS II protein levels.¶Results: Under basal conditions, chondrocytes expressed NOS I, which was lost upon IL-1 treatment. SNAP inhibited IL-1-induced NF-κB activation and NOS II expression. When added alone, SNAP induced AP-1 activation to approximately the same extent as IL-1.¶Conclusions: These results suggest that, in chondrocytes, NO is a key regulator of the signaling pathways leading from IL-1 to NF-κB and AP-1 activation and to the expression of genes that are involved in the pathophysiology of arthritic diseases.

Hydrogen peroxide mediates interleukin-1beta-induced AP-1 activation in articular chondrocytes: implications for the regulation of iNOS expression.

The pro-inflammatory cytokine interleukin-1β (IL-1) induces articular chondrocytes to produce reactive oxygen species (ROS), including hydrogen peroxide (H2O2), which mediate some IL-1-induced responses. This study aimed at elucidating the role of ROS, particularly H2O2, in mediating IL-1-induced activation of the transcription factor activator protein-1 (AP-1) in primary cultures of articular chondrocytes. AP-1 may function either as an inducer or as a repressor of the inducible nitric oxide synthase (iNOS) gene promoter. Since we observed that AP-1 is not required for iNOS expression in chondrocytes, we also investigated whether it is a repressor of this gene. The results of electrophoretic mobility shift assays showed that both IL-1 and H2O2 activated AP-1 and that inhibition of IL-1-induced ROS production abrogated AP-1 activation. The AP-1 complexes, induced by either IL-1 or H2O2, contained c-Fos/c-Jun and c-Fos/JunD heterodimers, but IL-1 activated AP-1 with a kinetics slower than that observed with H2O2. Pre-activation of AP-1, before stimulation of the cells with IL-1, did not inhibit iNOS mRNA and protein synthesis, relative to cells treated with IL-1 alone. These results indicate that H2O2 is a major mediator of IL-1-induced AP-1 activation in articular chondrocytes and that inhibition of ROS production is an effective strategy to block this IL-1-induced response. This study also identifies c-Fos/c-Jun and c-Fos/JunD heterodimers as the AP-1 transcription factors induced by IL-1, which, although not involved in the transcriptional regulation of the iNOS gene, may be important for the regulation of other genes also relevant in arthritic diseases, namely the collagenase-1 and IL-8 genes.

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.

Catecholamine and MHPG plasma levels, platelet MAO activity, and3H-imipramine binding in heroin and cocaine addicts

AbstractThis work evaluated in a population of heroin and heroin plus cocaine human addicts:1.Norepinephrine (NE), epinephrine (Epi), and 3-methoxy-4-hydroxyphenylglycol (MHPG) (the principal metabolite of brain NE) plasma levels;2.Monoamine oxidase (MAO) activity; and3.3H-imipramine specific binding to the amine carrier in platelets. NE plasma levels were significantly lower in the short-term heroin user groups (1–3 and 4–6 yr), a finding not observed in both the long-term heroin user (>6 yr) and heroin plus cocaine user (>6 yr) groups. Epi levels changed in a similar manner, except that a significant increase was noted in heroin plus cocaine abusers. Conversely, dopamine and MHPG plasma levels increased with the duration of heroin use, and even more with cocaine abuse. Platelet MAO activity increased in all groups. Specific3H-imipramine binding sites showed an increase after 3 yr of heroin abuse and in all heroin plus cocaine addicts. In conclusion, short-term use of heroin decreases NE or Epi release, but with prolonged use, a slow adpatation occurs. In contrast, cocaine inhibits the neuronal Epi uptake, even in a situation of long duration of abuse. Probably the amine levels additionally regulate the amine carrier, resulting in changes that show a different pattern from major depression. These drugs of abuse may also influence directly or indirectly related enzymatic systems.

Prejunctional α2A-autoreceptors in the human gastric and ileocolic arteries

This study was undertaken to determine the subtype of prejunctional α2-autoreceptors in human blood vessels. Segments of gastric and ileocolic arteries were incubated with [3H]noradrenaline and subsequently perifused with modified Krebs-Henseleit solution containing cocaine (12 µM). Five periods of electrical stimulation (S1–S5) were applied (1 Hz, 1 ms, 50 V for 1 min). Concentration-response curves for the facilitatory effect of eight α-adrenoceptor antagonists [rauwolscine, 2-[2-(2-methoxy-1,4-benzodioxanyl)] imidazoline (RX821002), yohimbine, phentolamine, idazoxan, 2-(2’,6’-dimethoxyphenoxyethyl)-aminomethyl-1,4-benzodioxan (WB4101), spiroxatrine and prazosin] were determined. All antagonists enhanced the stimulation-evoked overflow of tritium, indicating the existence of α2-autoreceptors. The EC30% values of the antagonists (concentrations that increased the evoked overflow of tritium by 30%) were taken as a measure of affinity to the autoreceptors. Correlations between the pEC30% values obtained in the present study and the pKi values of the same antagonists at cloned human α2A-, α2B-, α2C-adrenoceptors expressed in Chinese hamster lung cells and at α2D-adrenoceptors in the rat submaxillary gland or the bovine pineal gland showed that the α2-autoreceptors in the human gastric and ileocolic arteries resemble most closely the α2A-subtype.

Semicarbazide-sensitive amine oxidase activity and total nitrite and nitrate concentrations in serum: novel biochemical markers for type 2 diabetes?

The aim of this study was to evaluate the activity of semicarbazide-sensitive amine oxidase (SSAO) and the total nitrite and nitrate (NOx) concentrations in serum from type 2 diabetic patients and control subjects in order to evaluate if they could be used as novel diabetic markers. We studied 38 type 2 diabetic patients and 35 control subjects. Serum samples from those subjects were evaluated by radiochemical methods for SSAO activity using 14C-benzylamine. Serum NOx concentrations were obtained as an index of nitric oxide production by the Griess reaction. Serum SSAO activity was higher in type 2 diabetic patients than in control group and serum SSAO in type 2 diabetic correlated with age, serum creatinine and total cholesterol. Serum NOx levels in type 2 diabetic patients were also significantly higher than those in the control group. Serum NOx levels in control group correlated with serum SSAO activity. In conclusion, the increase in the SSAO activity and NOx levels observed in type 2 diabetic patients could be parameters to take in account and play relevant role in diabetes development. SSAO and NOx are suggested as markers for prognostic of diabetes.

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.

Lamotrigine pharmacokinetic evaluation in epileptic patients submitted to VEEG monitoring

ObjectiveThe aim of the present study was to evaluate the pharmacokinetic profile of lamotrigine (LTG) in epileptic patients submitted to video-electroencephalography (VEEG) monitoring and, in addition, to investigate the influence of concomitant antiepileptic drugs (AEDs) on the kinetics of LTG.MethodsThe analysis assumed a one-compartment open model with first-order absorption and elimination. The kinetic estimates obtained in this population were validated by using the Prediction-Error approach. The influence of medication was also assessed by the calculation of the LTG concentration-to-dose ratio. Patients (n=135) were divided into four groups according to the co-medication: Group 1, patients taking LTG with enzyme-inducer agents; Group 2, patients receiving LTG with valproic acid; Group 3, patients receiving both inducers and inhibitors of LTG metabolism; Group 4, patients under AEDs not known to alter LTG metabolism.ResultsThe obtained estimates for clearance (CL) (L/h/kg) [0.075±0.029 (Group 1), 0.014±0.005 (Group 2), 0.025±0.008 (Group 3) and 0.044±0.011 (Group 4)] appear to be the most appropriate set to be implemented in clinical practice as prior information, as demonstrated by the accuracy and precision of the measurements. In addition, the influence of co-medication on the LTG profile was further confirmed by the basal LTG concentration-to-dose ratio.ConclusionThe results of the present investigation may contribute to achieving the goal of optimizing patients’ clinical outcomes by managing their medication regimen through measured drug concentrations. Patients submitted to VEEG monitoring may benefit from this study, as the results may be used to provide better drug management in this medical setting.

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.