Neurogenic-derived 6-nitrodopamine is the most potent endogenous modulator of the mouse urinary bladder relaxation.

6-Nitrodopamine (6-ND) modulates vas deferens, seminal vesicles, and corpus cavernosum contractility; however, its role on the lower urinary tract organs has not been evaluated. Investigations of isolated urinary bladders from wild-type (WT) mice revealed 6-ND release was comparable to that of dopamine and adrenaline, whereas noradrenaline was hardly detected, as assessed by liquid chromatography coupled to tandem mass spectrometry. In vitro, 6-ND induced concentration-dependent relaxations in carbachol pre-contracted bladders with high potency (pEC50: 8.04 ± 0.86), independently of eNOS/sGC activity. Co-incubation of 6-ND (1-10 µM) antagonizes the contractile effects of acetylcholine (p < 0.05). Experiments using nitric oxide synthase (NOS) knockout mice demonstrated that 6-ND release from isolated urinary bladder was significantly reduced by neuronal NOS (nNOS-/-) deletion and abolished by triple NOSs deletion (n/i/eNOS-/-), while no significant changes were observed in endothelial (eNOS-/-) or inducible (iNOS-/-) knockout mice. Incubation with tetrodotoxin resulted in a significant decrease in 6-ND release in bladders obtained from WT, but not in nNOS-/- mice. The bladders from nNOS-/- and n/i/eNOS-/- mice exhibited significantly higher contractile responses to electric field stimulation (EFS), compared to eNOS-/-, iNOS-/-, or WT bladders. The hyperreactivity observed in triple NOS knockouts was reversed by the incubation with bladder mucosal layer obtained from a donor WT mice, but not with the muscular layer. These findings clearly demonstrate 6-ND is the most potent endogenous relaxing agent of urinary bladder, and inhibition of its release is associated with bladder hyperreactivity.

Comparative bioavailability study with two sodium valproate tablet formulations administered under fasting conditions in healthy subjects.

AIM: To assess the bioequivalence of two sodium valproate formulations in healthy subjects of both sexes. MATERIALS AND METHODS: The study was conducted using an open, randomized, two-period crossover design with a 2-week washout interval. Plasma samples were obtained over a 96-hour period. Plasma concentrations of valproate were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC/MS) with negative ion electrospray ionization. From the sodium valproate plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained Cmax, AUC, tmax, Ke, and T1/2. RESULTS: The geometric mean with corresponding 90% confidence interval for test/reference percent ratios were 104.43% (90% CI 100.42 - 108.61%) for Cmax, 98.11% (90% CI = 94.66 - 101.70%) for AUClast, and 96.71% (90% CI = 92.97 - 100.60%) for AUC0-inf. CONCLUSION: Since the 90% CI for Cmax and AUClast ratios were all inside the 80 - 125% interval proposed by the US Food and Drug Administration Agency (FDA), it was concluded that the new sodium valproate formulation (epilenil 500-mg coated tablet) without food elaborated by Biolab Sanus Farmaceutica Ltda is bioequivalent to depakene formulation for both the rate and the extent of absorption.

Determinação de captopril por HPLC acoplado a espectômetro de massa: aplicação em estudo de bioequivalência / Determination of captopril by hplctandem mass spectrometry: application in a bioequivalence study

Objetivo: Avaliar a bioequivalência de três diferentes formulações de captopril 25mg (Capoten® como formulação de referência e Captopril produzido pela FURP e Farmanguinhos como formulações testes) em 24 voluntários saudáveis de ambos os sexos. Métodos: Os voluntários selecionados eram livres de doenças, como confirmado pelo exame físico, psiquiátrico, ECG e exames laboratoriais. O estudo foi do tipo aberto, cruzado, em três períodos com 5 dias de intervalo entre eles. As amostras plasmáticas foram obtidas num intervalo de 24 horas e as concentrações de Captopril foram determinadas por cromatografia líquida de fase reversa acoplada à espectrometria de massa (LC-MS-MS). Resultados: A média geométrica para Capoten®/Captopril-FURP 25mg foi 96.9% para AUC0-24, 95.58 % para AUC0-?, e 98.17% for Cmax. O intervalo de confiança (IC) de 90% foi de 84.8-100.65%, 88.5-109.42% e 82.52 116.8%, respectivamente. A média geométrica para Capoten®/ Captopril-Farmanguinhos 25mg foi 99.63 % para AUClast, 98.52% para AUC0-?, e 95.52 para Cmax. O IC de 90% foi de 87.23-113.8%, 86.06-112.79% e 80.29-113.64%, respectivamente. Portanto, os IC de 90% para Cmax, AUClast, AUC0-? estavam dentro da variação de 80-125% proposta pelo Food and Drug Administration. Conclusão: Os comprimidos de 25mg Captopril-FURP e Captopril Farmanguinhos foram bioequivalentes ao Capoten® 25mg em sua taxa e extensão de absorção.

Objective: To assess three different captopril tablet formulations of 25mg for their bioavailability (Capoten® as the reference formulation and Captopril from FURP and Farmanguinhos as the test formulations) in 24 healthy volunteers of both sexes. Methods: The volunteers were free from serious disease, as assessed by physical and psychiatric examination, EKG, and laboratory tests. The study was open, with a three-period crossover design and a five-day washout period. Plasma samples were obtained over a 24-hour interval. Captopril concentrations were determined by reversed phase liquid chromatography tandem mass spectrometry (LC-MS-MS). Results: The geometric mean for Capoten® /Captopril - FURP 25 mg was 96.9 % for AUC0-24, 95.58 % for AUC0-?, and 98.17% for Cmax. The 90% confidence intervals (CI) were 84.8-100.65%, 88.5-109.42% and 82.52-116.8%, respectively. The geometric mean for Capoten®/Captopril-Farmanguinhos 25 mg was 99.63 % for AUClast, 98.52% for AUC0-?, and 95.52 for Cmax. The 90% CI were 87.23-113.8%, 86.06-112.79% and 80.29-113.64%, respectively. Therefore, the 90% CI for Cmax, AUClast, AUC0-? were within the 80-125% interval proposed by the Food and Drug Administration. Conclusion: Captopril- FURP and Captopril-Farmanguinhos 25 mg tablets were bioequivalent to Capoten® 25 mg, according to both the rate and extent of absorption.

Immunolocalization of nitric oxide synthase isoforms in human archivaland rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50mM Tris–HCl buffer, pH 9.5, for 20 min at 900 W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10mMsodium citrate buffer,pH6.0, for 15 min at 900 W.Inflammatory cells in ahumanlung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli.Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens.

Administration of BAY 41-2272 prevents bladder dysfunction in nitric-oxide deficient rats.

OBJECTIVE: to evaluate the protective effects of BAY 41-2272, a soluble guanylate cyclase activator, on changes in cystometric parameters in rats deficient in nitric oxide (NO). METHODS: Rats were divided into the following groups: (a) control; (b) DMSO; (c) L-NAME; (d) BAY 41-2272 alone; (e) L-NAME + BAY 41-2272. The NO synthase blocker L-NAME (20 mg/rat/day) was given in drinking water concomitantly or not with BAY 41-2272 (10 mg/kg/day, given by gavage). RESULTS: Chronic L-NAME treatment markedly increased the mean arterial blood pressure, and co-treatment with BAY 41-2272 nearly reversed L-NAME-induced rise on mean arterial blood pressure. Non-void contractions were significantly increased in L-NAME group (0.90 ± 0.1 number/minute) compared with either DMSO or control group (0.49 ± 0.1 number/minute), which were prevented by co-treatment with BAY 41-2272 (0.56 ± 025 number/minute; p < 0.05). The threshold and peak pressure increased by 70 and 44%, respectively, after chronic L-NAME treatment, while co-treatment with BAY 41-2272 largely attenuated both effects (27 and 22% increase, respectively). The frequency of micturition cycles decreased by about of 50% in L-NAME-treated rats compared with control animals, and co-treatment with BAY 41-2272 normalized this parameter. CONCLUSIONS: Our data show that long-term oral administration of BAY 41-2272 counteracts the bladder dysfunction seen in NO-deficient rats, indicating that restoration of the NO-cGMP pathway by this compound may be of beneficial value to treat bladder symptoms.

Amlodipine reduces the antimigratory effect of diclofenac in spontaneously hypertensive rats.

Amlodipine, an antihypertensive drug, and diclofenac, an antiinflammatory drug, may generally be combined, particularly in elderly patients; therefore, the potential for their interaction is high. We aim to determine if amlodipine interferes with the antimigratory effect of diclofenac. For this, male spontaneously hypertensive rats (SHRs) were treated with either diclofenac (1 mg.kg.d, 15 d) alone or combined with amlodipine (10 mg.kg.d, 15 d). Leukocyte rolling, adherence, and migration were studied by intravital microscopy. Diclofenac did not change (180.0 +/- 2.3), whereas amlodipine combined (163.4 +/- 5.1) or not (156.3 +/- 4.3) with diclofenac reduced the blood pressure (BP) levels in SHR (183.1 +/- 4.4). Diclofenac and amlodipine reduced leukocyte adherence, migration, and ICAM-1 expression, whereas only diclofenac reduced rolling leukocytes as well. Combined with amlodipine, the effect of the diclofenac was reduced. Neither treatment tested increased the venular shear rate or modified the venular diameters, number of circulating leukocytes, P-selectin, PECAM-1, L-selectin, or CD-18 expressions. No difference could be found in plasma concentrations of both drugs given alone or in association. In conclusion, amlodipine reduces leukocyte migration in SHR, reducing endothelial cell ICAM-1 expression. Amlodipine reduces the effect of the diclofenac, possibly by the same mechanism. A pharmacokinetic interaction as well as an effect on the other adhesion molecules tested could be discarded.

Ticlopidine quantification in human plasma by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Application to bioequivalence study.

A rapid, sensitive and specific method to quantify ticlopidine in human plasma using clopidogrel as the internal standard (IS) is described. The analyte and the IS were extracted from acidified plasma by liquid-liquid extraction using diethyl ether-hexane (80 : 20, v/v). The extracts were analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry (HPLC/MS/MS). Chromatography was performed isocratically on a Jones Genesis C(8) 4 microm analytical column (150 x 4.1 mm i.d.). The method had a chromatographic run time of 3.0 min and a linear calibration curve over the range 1.0-1000 ng ml(-1) (r(2) > 0.999427). The limit of quantification was 1.0 ng ml(-1). This HPLC/MS/MS procedure was used to assess the bioequivalence of two ticlopidine 250 mg tablet formulations (ticlopidine test formulation from Apotex do Brasil, Brazil, and Ticlid from Sanofi-Synthelabo, standard reference formulation). A single 250 mg dose of each formulation was administered to healthy volunteers. The study was conducted using an open, randomized, two-period crossover design with a 2 week washout interval. Since the 90% confidence interval for C(max) and area under the curve ratios were all inside the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that ticlopidine formulation from Apotex do Brasil is bioequivalent to Ticlid formulation with respect to both the rate and the extent of absorption.

Contribution of C-fibers to leucocyte recruitment in bronchoalveolarlavage fluid and pleural cavity in the rat

The effect of neonatal capsaicin (8 methyl-N-vanillyl-6-nonenamide) treatment on the leucocyte infiltration into the airways and pleural cavity was investigated in rats actively sensitized with ovalbumin. The animals were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or vehicle (10% ethanol and 10% Tween 80). At adult ages, the animals were actively sensitized with ovalbumin (200 ìg, s.c.) and 14 days later they were intratracheally (or intrapleurally) challenged with ovalbumin. The substance P level in bronchoalveolar lavage fluid of the capsaicin group was reduced by >90% compared to control group (vehicle), confirming the efficacy of capsaicin treatment. In the capsaicin group, the number of neutrophils (but not of eosinophils and mononuclear cells) in bronchoalveolar lavage fluid of sensitized animals was significantly higher than the control group. Intrapleural injection of ovalbumin in sensitized rats caused a significant neutrophil influx at 6 h that was markedly increased in the capsaicin-pretreated animals compared to control group. The counts of eosinophils and mononuclear cells in the pleural exudates did not differ significantly between capsaicin and control groups. The increased levels of immunoglobulin (Ig)E, IgG1 and IgG2a anti-ovalbumin antibodies in serum of sensitized rats did not differ between capsaicin and control groups. In conclusion, the exacerbated pulmonary neutrophil recruitment caused by the capsaicin neonatal treatment is unrelated to increase in serum immunoglobulin antibodies, and suggests a protective role for C-fibers in attenuating the allergic neutrophil infiltration.