A histological and immunohistochemical study of beta cells in streptozotocin diabetic rats treated with caffeine

In this study, the histological, immunohistochemical, morphometric, and biochemical changes to pancreatic beta-cells in STZ-induced diabetes were evaluated in rats treated with different doses of caffeine. Fifty adult male Wistar albino rats were divided into five groups: the nondiabetic control group, the diabetic untreated group, and three diabetic groups treated with different doses of caffeine (10, 50, and 100 mg/kg/day). Blood glucose and serum insulin levels were measured. The pancreata were collected and processed into paraffin sections. They were stained using hematoxylin and eosin (H&E) and Masson trichrome stains. The insulin expression in beta-cells was assessed using immunohistochemistry. Morphometrically, the percentage area of anti-insulin antibody reaction, the percentage of beta-cells per total islet cell number, and the average area of the islets were determined. STZ-induced degenerative changes in beta-cells led to decreases in the number of functioning beta-cells and insulin immunoreactivity and to increases in the number of collagen fibers in the islets. In STZ-treated rats, caffeine significantly decreased blood glucose concentration while increasing blood insulin levels at the highest applied dose. It also induced a significant increase in the number of immunoreactive beta-cells. In conclusion, caffeine may have a protective role in the biochemical and microscopic changes in pancreatic beta-cells in diabetes induced in rats through STZ administration. (Folia Histochemica et Cytobiologica 2014, Vol. 52, No. 1, 42–50

Gender differences in biochemical markers and oxidative stress of rats after 28 days oral exposure to a mixture used for weight loss containing p-synephrine, ephedrine, salicin, and caffeine

A associação de p-sinefrina, efedrina, salicina, e cafeína em suplementos alimentares e produtos para perda de peso é muito utilizada em todo o mundo, embora a efedrina tenha sido proibida em muitos países. O objetivo deste estudo foi avaliar o perfil de toxicidade à exposição oral de 28 dias à associação de p-sinefrina, efedrina, salicina e cafeína (na proporção de 10:4:6:80 m/m respectivamente) em ratos Wistar machos e fêmeas. Diariamente, os animais foram observados quanto ao peso corporal, sinais de toxicidade, morbidade e mortalidade. Após 28 dias, os animais foram sacrificados e o sangue coletado para avaliações hematológicas, bioquímicas e de estresse oxidativo. Não se observaram sinais clínicos de toxicidade, tampouco perda significativa de peso, mortes, ou quaisquer alterações significativas nos parâmetros hematológicos. Biomarcadores do estresse oxidativo e bioquímicos mostraram peroxidação lipídica, danos renais e hepáticos (p < 0,05; ANOVA/Bonferroni) em ratos machos (100 e 150 mg/kg) e a redução (p < 0,05; ANOVA/Bonferroni) nos níveis de glutationa reduzida (GSH) em todos os grupos de machos tratados. Nas fêmeas, não houve indícios de estresse oxidativo, nem alterações bioquímicas. O diferente perfil de toxicidade entre os gêneros sugere influência hormonal nos efeitos de mistura administrada. A associação testada pode alterar o estado oxidativo e promover danos renais e hepáticos.The association of p-synephrine, ephedrine, salicin, and caffeine in dietary supplements and weight loss products is very common worldwide, even though ephedrine has been prohibited in many countries. The aim of this study was to evaluate a 28-day oral exposure toxicity profile of p-synephrine, ephedrine, salicin, and caffeine mixture (10:4:6:80 w/w respectively) in male and female Wistar rats. Body weight and signs of toxicity, morbidity, and mortality were observed daily. After 28 days, animals were euthanized and blood collected for hematological, biochemical, and oxidative stress evaluation. No clinical signs of toxicity, significant weight loss or deaths occurred, nor were there any significant alterations in hematological parameters. Biochemical and oxidative stress biomarkers showed lipid peroxidation, and hepatic and renal damage (p < 0.05; ANOVA/Bonferroni) in male rats (100 and 150 mg/kg) and a reduction (p < 0.05; ANOVA/Bonferroni) in glutathione (GSH) levels in all male groups. Female groups displayed no indications of oxidative stress or biochemical alterations. The different toxicity profile displayed by male and female rats suggests a hormonal influence on mixture effects. Results demonstrated that the tested mixture can alter oxidative status and promote renal and hepatic damages

Effects of Paullinia cupana extract on lamotrigine pharmacokinetics in rats: A herb-drug interaction on the gastrointestinal tract with potential clinical impact.

Paullinia cupana-containing preparations are being consumed worldwide for weight reduction. As obesity and epilepsy are common comorbidities and lamotrigine (LTG) is a broad-spectrum antiepileptic drug, it is likely to find epilepsy patients taking P. cupana and LTG simultaneously. Thus, this work aimed to investigate the potential interaction between P. cupana extract and LTG in rats. In a study, rats were orally co-administered with a single-dose of P. cupana extract (821 mg/kg) and LTG (10 mg/kg). In another study, rats were orally pre-treated for 14 days with P. cupana extract (821 mg/kg/day) receiving LTG (10 mg/kg, p.o.) on the 15th day. Rats of the respective control groups received the corresponding volume of the extract vehicle. LTG concentrations were determined at several post-dose time-points and submitted to a non-compartmental pharmacokinetic analysis. The co-administration of P. cupana and LTG induced a significant reduction of LTG Cmax and AUC0-24 and prolonged the mean residence time. However, no significant effects were observed on LTG pharmacokinetics following a 14-day pre-treatment period with the extract. In this study changes in the body weight of rats and in some biochemical parameters were also evaluated. Overall, the results revealed a pharmacokinetic-based herb-drug interaction between P. cupana extract and LTG, mainly after their co-administration

Effect of Solvent Interaction & Soluble Microneedles on Skin Permeability to Drug Molecules

Skin forms a formidable barrier protecting the human body from external environmental rigors and excessive loss of water; maintaining equilibrium. The barrier properties of the skin can be attributed to its unique macromolecular organization and morphology. As a route for drug administration, skin presents a large surface area and can be used for both systemic and localized targeted drug delivery applications offering several advantages over conventional drug therapy; avoidance of first pass metabolism, patient compliance, sustained or controlled delivery for an extended period, to name a few. However, the organized structure of the skin, since intended to prevent entry of adverse chemicals, poses a formidable challenge to molecular transport. From a drug delivery perspective, skin is different from GIT in anatomy and functionality, the former being more permeable to drug molecules. Through various peer revieresearch on the drug transport kinetics through skin, it has been realized that the primary barrier to cutaneous drug transport resides in the Stratum Corneum (SC), the uppermost layer of the skin. The 15-20 μm thick lipophilic, torturous morphology of the SC resembles a brick and mortar structure and imposes a limitation on percutaneous drug transport with only a few molecules having the prerequisite physicochemical characteristics to permeate the intact SC. Thus, drug penetration and subsequent diffusion across the SC is a passive process leading to constraints on the amount of drug that is deliverable to achieve the desired therapeutic effect. To increase the number of candidates for cutaneous delivery and to attain appropriate dose levels requires application of certain enhancement strategies. These approaches employ different mechanisms; (i) an external driving force by iontophoresis (ii) reversible modulation of the SC barrier function by chemical penetration enhancers (iii) creating “easy access” transport channels by microneedles. Nevertheless, a thorough understanding of the molecular transport process across the skin is requisite before formulation strategies could be employed to deliver drugs across the skin in a therapeutically pertinent time-frame. The research presented in this dissertation addresses the knowledge gap that pertains to percutaneous drug absorption by investigating the transport of drug molecules into the skin after a short-term exposure (5 minutes) to aqueous and ethanolic drug solution. Further, the research demonstrates the effect of chemical & physical enhancement approaches: chemical penetration enhancers and microneedles on skin permeability to drug molecules

Nonclinical assessment of the potential for herb-drug interactions between herbal extracts present in weight loss supplements and lamotrigine

Plants have been and still continue to be one of the most important sources of active ingredients. Actually, plants are still the backbone of modern pharmacopoeias and remain as a source of new drug candidates. The use of medicinal plants or plant-based medicinal products is also increasing in many developed countries as an alternative and complementary form for the treatment of diseases. Thus, the concomitant use of plants and conventional medications is emerging as a common practice in patients with hypertension, diabetes, epilepsy, depression, and oncological diseases, as well as in people with obesity and being overweight. Recently, obesity and epilepsy have been related as comorbid conditions with a high prevalence, particularly in patients with refractory epilepsy and under polytherapy. Treatment of patients with epilepsy should, therefore, take into account that the presence of comorbid conditions may compromise the efficacy and safety of antiepileptic drugs, which constitute the main therapeutic approach in epilepsy. Lamotrigine (LTG) is a well-tolerated antiepileptic drug widely used in epilepsy; however, it has a narrow therapeutic range and a considerable interindividual variability in its pharmacokinetics. Therefore, the focus of research addressed in this thesis was the nonclinical assessment of the potential for herb-drug interactions between herbal extracts present in weight loss supplements and LTG, using the rat as whole animal model. After optimization and validation of selective, precise and accurate bioanalytical methods for the quantification of LTG in human samples (plasma and saliva) and in rat samples (plasma and brain), the conditions for proceeding with nonclinical studies were met. Therefore, then a number of nonclinical studies were performed in adult male Wistar rats with the main objective of evaluating the effects of standardized extracts of Paullinia cupana (guarana), Garcinia cambogia (malabar tamarind), Citrus aurantium (bitter orange) and Fucus vesiculosus (bladderwrack) on the kinetics of LTG. To this end, at least two independent pharmacokinetic studies were carried out to evaluate the effects of each herbal extract on the pharmacokinetics of LTG; the first study aimed to evaluate the effects after the co-administration of the extract and LTG, and the second one aimed to evaluate the effects of a 14-day pre-treatment period with the extract on the pharmacokinetics of LTG subsequently administered on the 15th day. Globally, the results of the pharmacokinetic studies involving the four herbal extracts pointed out that P. cupana extract is the one that has higher potential to interact with LTG, while G. cambogia, C. aurantium and F. vesiculosus extracts had minor or no effects on LTG pharmacokinetics. The co-administration of P. cupana extract and LTG caused, in particular, a significant decrease in the peak plasma drug concentration (Cmax) and in the extent of systemic exposure to LTG over the first 24 h (AUC0-24). Based on the findings achieved in these nonclinical studies, an important pharmacokinetic interaction between P. cupana extract and LTG was herein described for the first time, which potentially may have clinical impact in patients treated with LTG. Moreover, the repeated administration of the tested herbal extracts during a 14-day period did not have relevant effects on the body weight gain of rats, which raises doubts about their effectiveness in reducing body weight. So, in conclusion, the nonclinical assessment of herb-drug interactions is of utmost importance to anticipate the potential effects of herbal preparations in the pharmacokinetics of narrow therapeutic index drugs like LTG, constituting these data the starting point for further confirmation and investigation of the relevance of these interactions at a clinical level.As plantas têm sido, e continuarão ainda a ser, uma das fontes mais importantes de princípios ativos. Na realidade, as plantas constituem ainda a “espinha dorsal” das farmacopeias modernas e continuam a ser uma fonte de novos candidatos a fármacos. A utilização de plantas medicinais ou de preparações medicinais à base de plantas está também a aumentar em muitos países desenvolvidos como uma forma alternativa e complementar para o tratamento de doenças. Por conseguinte, o uso concomitante de plantas e medicamentos convencionais é uma prática comum em doentes com hipertensão, diabetes, epilepsia, depressão e doenças oncológicas, assim como em pessoas com obesidade e excesso de peso. Recentemente, a obesidade e a epilepsia têm sido consideradas comorbilidades com uma elevada prevalência, particularmente em doentes com epilepsia refratária e polimedicados. O tratamento de doentes com epilepsia deve, portanto, ter em consideração que a presença de comorbilidades pode comprometer a eficácia e a segurança dos fármacos antiepiléticos, os quais constituem a principal estratégia terapêutica na epilepsia. A lamotrigina (LTG) é um fármaco antiepilético bem tolerado e amplamente utilizado na epilepsia, mas que apresenta uma margem terapêutica estreita e uma variabilidade interindividual considerável na sua farmacocinética. Por isso, o foco de investigação considerado nesta tese foi a avaliação não-clínica do potencial de interação entre extratos de plantas presentes em suplementos à base de plantas para emagrecimento e a LTG, usando o rato como modelo animal. Após a otimização e a validação de métodos bioanalíticos seletivos, precisos e exatos para a quantificação da LTG em amostras humanas (plasma e saliva) e em amostras de rato (plasma e cérebro), as condições para prosseguir com os estudos não-clínicos estavam reunidas. Portanto, de seguida, um conjunto de estudos não-clínicos foi realizado em ratos Wistar machos adultos com o objetivo principal de avaliar os efeitos de extratos padronizados de Paullinia cupana (guaraná), de Garcinia cambogia (tamarindo de Malabar), de Citrus aurantium (laranja-amarga) e de Fucus vesiculosus (bodelha) na cinética da LTG. Para tal, pelo menos dois estudos farmacocinéticos independentes foram realizados para avaliar os efeitos de cada extrato na farmacocinética da LTG; o primeiro estudo teve como objetivo avaliar os efeitos após a coadministração do extrato e da LTG, e o segundo estudo foi realizado para avaliar os efeitos de um período de pré-tratamento de 14 dias com cada extrato na farmacocinética da LTG administrada subsequentemente ao 15º dia. Globalmente, os resultados dos estudos farmacocinéticos envolvendo os quatro extratos de plantas revelaram que o extrato de P. cupana é aquele que tem maior potencial para interagir com a LTG, enquanto que os extratos de G. cambogia, C. aurantium e F. vesiculosus tiveram poucos ou nenhuns efeitos na farmacocinética da LTG. A coadministração do extrato de P. cupana e LTG causou, em particular, um decréscimo significativo da concentração plasmática máxima (Cmax) e da extensão de exposição sistémica à LTG nas primeiras 24 h (AUC0-24). Com base nos resultados obtidos nestes estudos não-clínicos, uma interação farmacocinética importante entre o extrato de P. cupana e a LTG foi aqui descrita pela primeira vez, a qual potencialmente pode ter impacto clínico em doentes tratados com a LTG. Além disso, a administração repetida dos extratos testados durante um período de 14 dias não teve efeitos relevantes sobre o ganho de peso corporal dos ratos, o que levanta dúvidas sobre a eficácia deles na redução do peso corporal. Assim, em conclusão, a avaliação não-clínica de interações planta-fármaco é de extrema importância para antecipar os efeitos potenciais de preparações à base de plantas na farmacocinética de fármacos de índice terapêutico estreito como a LTG, constituindo esses dados o ponto de partida para confirmação posterior e investigação da relevância dessas interações a nível clínico.UID/Multi/00709/201

Direct effects of caffeine on osteoblastic cells metabolism: the possible causal effect of caffeine on the formation of osteoporosis

BACKGROUND: Caffeine consumption has been reported to decrease bone mineral density (BMD), increase the risk of hip fracture, and negatively influence calcium retention. In this study, we investigated the influence of caffeine on the osteoblasts behaviour. METHOD: Osteoblasts derived from newborn Wistar-rat calvaria was used in this study. The effects of various concentrations of caffeine on bone cell activities were evaluated by using MTT assay. Alkaline phosphatase (ALP) staining, von Kossa staining and biochemical parameters including ALP, lactate dehydrogenase (LDH), prostaglandin E(2 )(PGE(2)) and total protein were performed at day 1, 3, and 7. DNA degradation analysis under the caffeine influence was also performed. RESULTS AND DISCUSSION: The results showed that the viability of the osteoblasts, the formation of ALP positive staining colonies and mineralization nodules formation in the osteoblasts cultures decreased significantly in the presence of 10 mM caffeine. The intracellular LDH, ALP and PGE(2 )content decreased significantly, the LDH and PGE(2 )secreted into the medium increased significantly. The activation of an irreversible commitment to cell death by caffeine was clearly demonstrated by DNA ladder staining. CONCLUSION: In summary, our results suggest that caffeine has potential deleterious effect on the osteoblasts viability, which may enhance the rate of osteoblasts apoptosis

Behavioral and biochemical characterization of elevated “I-maze” as animal model of anxiety

AbstractThe elevated I-maze is a modification of the elevated plus-maze model of anxiety in mice. The design of I-maze comprises a straight wooden passage, resembling the English letter “I,” divided equally into three areas; two enclosed areas (close arms) at both ends of the “maze” and an open area in the center of two enclosed areas. The I-maze completely avoids the central platform of elevated plus-maze, removing any ambiguity in time spent on central platform and allowing uninterrupted animal exploration. In this model, diazepam (1 mg/kg) and gabapentin (10 mg/kg) significantly increased the percentage of time spent in the open areas (%TO) and the number of unprotected head dips (uHDIPS), and reduced the number of protected head dips (pHDIPS) and stretch attend postures (SAP) from close to open arm. Similarly, fluoxetine (5 mg/kg) significantly increased %TO and uHDIPS, and significantly decreased SAP from close to open arm, but it did not have any significant effect on pHDIPS. The 5-HT3 receptor antagonist, ondansetron (0.1 mg/kg), did not produce any significant change in all the behaviors, observed, as compared to vehicle-treated control mice. On the other hand, the anxiogenic agent, caffeine (15 mg/kg), did produce a significant decrease in %TO and uHDIPS, and significantly increased pHDIPS and SAP from close to open arm. Mice confined in open area of I-maze bring the relevant biochemical changes associated with anxiety behavior, showing significant increase in the levels of plasma nitrate and plasma corticosterone. These data indicate that a combination of novel design of elevated I-maze and a detailed behavioral analysis provides a sensitive model for the measurement of anxiety