Isabel Vitória Figueiredo

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.

Pharmacological effect of orally delivered insulin facilitated by multilayered stable nanoparticles

Intestinal uptake, insulinemia and hypoglycemic effect of orally delivered insulin encapsulated in polyelectrolytically stable nanoparticles were evaluated in streptozotocin-induced Wistar diabetic rats. Nanoparticles with 396nm mean diameter were formed by alginate and dextran sulfate nucleating around calcium and binding to poloxamer, stabilized by chitosan, and subsequently coated with albumin. The resulting negatively charged nanoparticles retained insulin bioactivity and enhanced pharmacological availability by shielding insulin from enzymatic degradation and through chemical and physical facilitation of permeation through the intestinal membrane. Insulin nanoencapsulated through a simplified method avoiding harsh conditions and organic solvents, reduced plasma glucose levels to 40% of the basal values with a sustained hypoglycemic effect over 24h. Pharmacodynamic and pharmacokinetic parameters were evaluated at a dose of 50IU/kg nanoencapsulated insulin, and 13% oral bioavailability showed a threefold increase in comparison to free insulin. Confocal microscopy showed internalization of nanoencapsulated insulin in the small intestinal mucosa using independently labeled insulin-FITC and alginate-RBITC. Therefore the nanoformulation facilitated the oral delivery of insulin, and potentially that of other therapeutic proteins.

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.

Polymeric nanoparticles modified with fatty acids encapsulating betamethasone for anti-inflammatory treatment.

Topical glucocorticosteroids were incorporated into nanocarrier-based formulations, to overcome side effects of conventional formulations and to achieve maximum skin deposition. Nanoparticulate carriers have the potential to prolong the anti-inflammatory effect and provide higher local concentration of drugs, offering a better solution for treating dermatological conditions and improving patient compliance. Nanoparticles were formulated with poly-ϵ-caprolactone as the polymeric core along with stearic acid as the fatty acid, for incorporation of betamethasone-21-acetate. Oleic acid was applied as the coating fatty acid. Improvement of the drug efficacy, and reduction in drug degradation with time in the encapsulated form was examined, while administering it locally through controlled release. Nanoparticles were spherical with mean size of 300 nm and negatively charged surface. Encapsulation efficiency was 90%. Physicochemical stability in aqueous media of the empty and loaded nanoparticles was evaluated for six months. Drug degradation was reduced compared to free drug, after encapsulation into nanoparticles, avoiding the potency decline and promoting a controlled drug release over one month. Fourier transform infrared spectroscopy and thermal analysis confirmed drug entrapment, while cytotoxicity studies performed in vitro on human keratinocytes, Saccharomyces cerevisiae models and Artemia salina, showed a dose-response relationship for nanoparticles and free drug. In all models, drug loaded nanoparticles had a greater inhibitory effect. Nanoparticles increased drug permeation into lipid membranes in vitro. Preliminary safety and permeation studies conducted on rats, showed betamethasone-21-acetate in serum after 48 h application of a gel containing nanoparticles. No skin reactions were observed. In conclusion, the developed nanoparticles may be applied as topical treatment, after encapsulation of betamethasone-21-acetate, as nanoparticles promote prolonged drug release, increase drug stability in aqueous media, reducing drug degradation, and increase drug permeability through lipid membranes.

Toxicological assessment of orally delivered nanoparticulate insulin

Subacute toxicological assessment on diabetic rats was conducted after 15 days of daily oral administration of nanoparticulate insulin. Haematological and biochemical analyses were conducted on blood and urine, biopsies performed on organs and tissues, and histology analysed by optical microscopy. Insulin-loaded nanoparticles alone did not change liver or kidney functions. The increase of some hepatic parameters was attributed to diabetes physiopathology and to chemical inducement of diabetes and not to the nanoparticle composition since diabetic controls showed the same variations. In terms of kidney function, parameters such as urea nitrogen and creatinine, were also similar to normal rats with the exception of glycosuria. This single effect was due to diabetes physiopathology and the method of induction, and not to the nanoparticle composition, since non-dosed diabetic rats showed the same alteration. Even so, glycosuria levels of animals dosed with insulin-loaded nanoparticles were lower than control diabetic rats which may indicate an effective hypoglycaemic response. Nanoparticles did not exhibit toxicity in haematological parameters. Finally, organ histology was similar between dosed animals and normal rats with the exception of pancreas histology.

Complexos de inclusão de indometacina com hidroxipropil-beta-ciclodextrina: estudos de dissolução e coeficiente de partição

Indomethacin, a non steroidal anti-inflamatory, is practically water insoluble. Hydroxypropyl-b-cyclodextrin grants better solubility characteristics to included drugs. Indomethacins complexation protects from hydrolysis, enhancing solubility and dissolution. The aim is to study the influence of complexation methods, freeze and spray-dryer, on indomethacin dissolution and partition coefficient. Dissolution results show that freeze-dried inclusion complex has a better time needed to dissolve 50% and 90% - 1 and 1.8 minutes then other products. The same happens with percentage of indomethacin dissolved at 5, 30 and 60 minutes - 92.6±1.8%; 98.9±1.2% e 100.01±0.1% and also with dissolution efficiency and dissolution profile. Partition coefficient results with the complexes obtained by both methods corroborate the theory that there are several intervenient strengths on this process and not only drugs free fraction that regulates transport to organic phase, reinforcing the environmental pH significance. With phosphate buffer pH 7.0, variations on transport grade by cyclodextrin addition are very small, showing no significant changes on log P*. With phosphate buffer pH 5.5, although not significant, variations are slightly higher. We can conclude that complexation enhances dissolution capacities of lipophylic drugs without changing the characteristics that give them a good ability for membrane diffusion.

Polyphenols from Cymbopogon citratus leaves as topical anti-inflammatory agents.

ETHNOPHARMACOLOGICAL RELEVANCE A variety of plant polyphenols have been reported to have anti-inflammatory, frequently associated with erythema, edema, hyperplasia, skin photoaging and photocarcinogenesis. Cymbopogon citratus (DC). Stapf (Poaceae) is a worldwide known medicinal plant, used in traditional medicine in inflammation-related conditions. AIM OF THE STUDY In this work, the anti-inflammatory potential of C. citratus infusion (CcI) and its polyphenols as topical agents was evaluated in vivo. MATERIALS AND METHODS The plant extract was prepared and its fractioning led two polyphenol-rich fractions: flavonoids fraction (CcF) and tannins fraction (CcT). An oil/water emulsion was developed with each active (CcI, CcF+CcT and diclofenac), pH and texture having been evaluated. Release tests were further performed using static Franz diffusion cells and all collected samples were monitored by HPLC-PDA. In vivo topical anti-inflammatory activity evaluation was performed by the carrageenan-induced rat paw edema model. RESULTS The texture analysis revealed statistically significant differences for all tested parameters to CcF+CcT, supporting its topical application. Release experiments lead to the detection of the phenolic compounds from each sample in the receptor medium and the six major flavonoids were quantified, by HPLC-PDA: carlinoside, isoorientin, cynaroside, luteolin 7-O-neohesperidoside, kurilesin A and cassiaoccidentalin B. The CcF+CcT formulation prompted to the higher release rate for all these flavonoids. CcI4%, CcI1% and CcF+CcT exhibited an edema reduction of 43.18, 29.55 and 59.09%, respectively. CONCLUSIONS Our findings highlight that CcI, containing luteolin 7-O-neohesperidoside, cassiaoccidentalin B, carlinoside, cynaroside and tannins have a potential anti-inflammatory topical activity, suggesting their promising application in the treatment of skin inflammatory pathologies.

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.

Evaluation of hepatic glucose metabolism via gluconeogenesis and glycogenolysis after oral administration of insulin nanoparticles

Nanoparticles were designed to promote insulin intestinal absorption via the oral route, to increase portal insulin levels to better mimic the physiological pathway, providing enhanced glucose control through glycogenolysis and gluconeogenesis. Nanoparticles were formulated with insulin encapsulated in the core material consisting of alginate and dextran sulfate, associated with poloxamer and subsequently coated with chitosan then albumin. A spherical and slightly rough core was observed in electron micrographs with the appearance of a concentration gradient of the polysaccharide structure toward the periphery of the nanoparticle. Atomic force microscopy showed that the fully formed nanoparticles are about 200 nm in diameter with smooth and spherical morphology. Histopathological analysis of organs and tissues of diabetic rats dosed daily for 15 days with insulin nanoparticles was used to evaluate toxicological issues. No morphological or pathological alterations were observed in rat liver, spleen, pancreas, kidney or intestinal sections. Following, the effect of nanoencapsulated insulin on inhibiting hepatic gluconeogenesis was evaluated after a single insulin administration and oral glucose tolerance test, which represents a significant metabolic challenge to the liver. Alterations in the hepatic glucose metabolism of fasted streptozotocin-diabetic rats were determined by the percent contribution of glycogenolysis and gluconeogenesis, measured by using metabolic tracers, however similar gluconeogenesis contribution to the hepatic metabolism was observed between diabetic rats receiving nanoencapsulated insulin or insulin solution. The metabolic results may be explained by the inability of a single dose in shifting the gluconeogenesis/glycogenolysis contributions, sampling time, fasting period or by influence of the kidney enzymes and impairment in insulin signaling observed in stz-diabetic rats.

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.