Protective effects of exogenous and endogenous hydrogen sulfide in mast cell-mediated pruritus and cutaneous acute inflammation in mice.

The recently described 'gasomediator' hydrogen sulfide (H2S) has been involved in pain mechanisms, but its effect on pruritus, a sensory modality that similarly to pain acts as a protective mechanism, is poorly known and controversial. The effects of the slow-releasing (GYY4137) and spontaneous H2S donors (Na2S and Lawesson's reagent, LR) were evaluated in histamine and compound 48/80 (C48/80)-dependent dorsal skin pruritus and inflammation in male BALB/c mice. Animals were intradermally (i.d.) injected with C48/80 (3µg/site) or histamine (1µmol/site) alone or co-injected with Na2S, LR or GYY4137 (within the 0.3-100nmol range). The involvement of endogenous H2S and KATP channel-dependent mechanism were also evaluated. Pruritus was assessed by the number of scratching bouts, whilst skin inflammation was evaluated by the extravascular accumulation of intravenously injected 125I-albumin (plasma extravasation) and myeloperoxidase (MPO) activity (neutrophil recruitment). Histamine or C48/80 significantly evoked itching behavior paralleled by plasma extravasation and increased MPO activity. Na2S and LR significantly ameliorated histamine or C48/80-induced pruritus and inflammation, although these effects were less pronounced or absent with GYY4137. Inhibition of endogenous H2S synthesis increased both Tyrode and C48/80-induced responses in the skin, whereas the blockade of KATP channels by glibenclamide did not. H2S-releasing donors significantly attenuate C48/80-induced mast cell degranulation either in vivo or in vitro. We provide first evidences that H2S donors confer protective effect against histamine-mediated acute pruritus and cutaneous inflammation. These effects can be mediated, at least in part, by stabilizing mast cells, known to contain multiple mediators and to be primary initiators of allergic processes, thus making of H2S donors a potential alternative/complementary therapy for treating inflammatory allergic skin diseases and related pruritus.

Relaxant effect of a metal-based drug in human corpora cavernosa and its mechanism of action.

We studied the mechanisms involved in the human corpora cavernosa (HCC) relaxation induced by a new metal-based nitric oxide (NO) donor, the ruthenium complex cis-[Ru(bpy)2Imn(NO)](+3) (FOR0811). FOR0811 produced relaxation in phenylephrine (PE)-precontracted HCC with a maximal response that achieved 112.9 ± 10.6%. There was no difference between the maximal relaxation induced by FOR0811 when compared with sodium nitroprusside (SNP) (106.8 ± 7.3%), BAY41-2272 (107.6 ± 4.1%) or vardenafil (103.4 ± 3.8%), however, FOR0811 was less potent than SNP and vardenafil. L-N(G)-nitroarginine methyl ester (L-NAME), a NO synthase inhibitor, had no effect in the concentration-response curve elicited by FOR0811. 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a heme-site inhibitor of soluble guanylyl cyclase (sGC) was able to either block or reverse the relaxation induced by FOR0811. On the other hand, the relaxation induced by FOR0811 was not affected by glibenclamide, a blocker of ATP-sensitive potassium channels. FOR0811 (10 µM) was able to increase cyclic guanosine monophosphate (cGMP) levels in corpora cavernosa strips. FOR0811 completely relaxes HCC by a sGC-cGMP-dependent mechanism and can be a lead compound in the development of new stable NO donors.

Phosphodiesterase-9 (PDE9) inhibition with BAY 73-6691 increases corpus cavernosum relaxations mediated by nitric oxide-cyclic GMP pathway in mice.

Phosphodiesterase-9 (PDE9) specifically hydrolyzes cyclic GMP, and was detected in human corpus cavernosum. However, no previous studies explored the selective PDE9 inhibition with BAY 73-6691 in corpus cavernosum relaxations. Therefore, this study aimed to characterize the PDE9 mRNA expression in mice corpus cavernosum, and investigate the effects of BAY 73-6691 in endothelium-dependent and -independent relaxations, along with the nitrergic corpus cavernosum relaxations. Male mice received daily gavage of BAY 73-6691 (or dimethylsulfoxide) at 3 mg kg(-1) per day for 21 days. Relaxant responses to acetylcholine (ACh), nitric oxide (NO) (as acidified sodium nitrite; NaNO2 solution), sildenafil and electrical-field stimulation (EFS) were obtained in corpus cavernosum in control and BAY 73-6691-treated mice. BAY 73-6691 was also added in vitro 30 min before construction of concentration-responses and frequency curves. PDE9A and PDE5 mRNA expression was detected in the mice corpus cavernosum in a similar manner. In vitro addition of BAY 73-6691 neither itself relaxed mice corpus cavernosum nor changed the NaNO2, sildenafil and EFS-induced relaxations. However, in mice treated chronically with BAY 73-6691, the potency (pEC50) values for ACh, NaNO2 and sildenafil were significantly greater compared with control group. The maximal responses (Emax) to NaNO2 and sildenafil were also significantly greater in BAY 73-6691-treated mice. BAY 73-6691 treatment also significantly increased the magnitude and duration of the nitrergic corpus cavernosum relaxations (8-32 Hz). In conclusion, murine corpus cavernosum expresses PDE9 mRNA. Prolonged PDE9 inhibition with BAY 73-6691 amplifies the NO-cGMP-mediated cavernosal responses, and may be of therapeutic value for erectile dysfunction.

Simultaneous determination of dextromethorphan, dextrorphan and doxylamine in human plasma by HPLC coupled to electrospray ionization tandem mass spectrometry: application to a pharmacokinetic study.

In the present study, a fast, sensitive and robust method to quantify dextromethorphan, dextrorphan and doxylamine in human plasma using deuterated internal standards (IS) is described. The analytes and the IS were extracted from plasma by a liquid-liquid extraction (LLE) using diethyl-ether/hexane (80/20, v/v). Extracted samples were analyzed by high performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Chromatographic separation was performed by pumping the mobile phase (acetonitrile/water/formic acid (90/9/1, v/v/v) during 4.0min at a flow-rate of 1.5 mL min⁻¹ into a Phenomenex Gemini® C18, 5 µm analytical column (150 × 4.6 mm i.d.). The calibration curve was linear over the range from 0.2 to 200 ng mL⁻¹ for dextromethorphan and doxylamine and 0.05 to 10 ng mL⁻¹ for dextrorphan. The intra-batch precision and accuracy (%CV) of the method ranged from 2.5 to 9.5%, and 88.9 to 105.1%, respectively. Method inter-batch precision (%CV) and accuracy ranged from 6.7 to 10.3%, and 92.2 to 107.1%, respectively. The run-time was for 4 min. The analytical procedure herein described was used to assess the pharmacokinetics of dextromethorphan, dextrorphan and doxylamine in healthy volunteers after a single oral dose of a formulation containing 30 mg of dextromethorphan hydrobromide and 12.5mg of doxylamine succinate. The method has high sensitivity, specificity and allows high throughput analysis required for a pharmacokinetic study.

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels.

BACKGROUND AND PURPOSE: Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin-induced diabetic mice, focusing on the contribution of Ca(2+) influx through L-type voltage-operated Ca(2+) channels (L-VOCC). EXPERIMENTAL APPROACH: Male C57BL/6 mice were injected with streptozotocin (125 mg·kg(-1) ). Four weeks later, contractile responses to carbachol, α,ß-methylene ATP, KCl, extracellular Ca(2+) and electrical-field stimulation were measured in urothelium-intact DSM strips. Cystometry and histomorphometry were performed, and mRNA expression for muscarinic M(2) /M(3) receptors, purine P2X1 receptors and L-VOCC in the bladder was determined. KEY RESULTS: Diabetic mice exhibited higher bladder capacity, frequency, non-void contractions and post-void pressure. Increased bladder weight, wall thickness, bladder volume and neural tissue were observed in diabetic bladders. Carbachol, α,ß-methylene ATP, KCl, extracellular Ca(2+) and electrical-field stimulation all produced greater DSM contractions in diabetic mice. The L-VOCC blocker nifedipine almost completely reversed the enhanced DSM contractions in bladders from diabetic animals. The Rho-kinase inhibitor Y27632 had no effect on the enhanced carbachol contractions in the diabetic group. Expression of mRNA for muscarinic M(3) receptors and L-VOCC were greater in the bladders of diabetic mice, whereas levels of M(2) and P2X1 receptors remained unchanged. CONCLUSIONS AND IMPLICATIONS: Diabetic mice exhibit features of urinary bladder dysfunction, as characterized by overactive DSM and decreased voiding efficiency. Functional and molecular data suggest that overactive DSM in diabetes is the result of enhanced extracellular Ca(2+) influx through L-VOCC.

Comparative bioavailability of two escitalopram formulations in healthy human volunteers.

OBJECTIVE: To assess the bioequivalence of two escitalopram formulations (Test formulation: escitalopram (10 mg tablet) manufactured by Apsen Farmacêutica S.A.) Reference formulation: escitalopram (Lexapro; 10 mg tablet) from Lundbeck Brasil Ltda) in healthy volunteers of both sexes. METHODS: The study was conducted using an open, randomized, two-period crossover design with at least a 21-day washout interval. Plasma samples were obtained over a 168 h period. Plasma escitalopram concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). The following pharmacokinetic parameters were obtained from the escitalopram plasma concentration vs. time curves: AUC(last), AUC(inf) and C(max). RESULTS: The limit of quantification for escitalopram was 0.2 ng x ml(-1). The geometric mean with corresponding 90% confidence interval (CI) for Test/Reference percent ratios were 97.35% (90% CI = 90.28-104.96%) for C(max), 99.60% (90% CI = 92.93-106.74%) for AUC(last) and 99.92% (90% CI = 93.34-106.97%) for AUC(inf). CONCLUSION: Since the 90% CI for AUClast, AUCinf and Cmax ratios were within the 80-125% interval proposed by the US FDA, it was concluded that escitalopram formulation manufactured by Apsen Farmacêutica S.A. is bioequivalent to the Lexapro formulation in regard to both the rate and the extent of absorption.

Vardenafil, but not sildenafil or tadalafil, has calcium-channel blocking activity in rabbit isolated pulmonary artery and human washed platelets.

BACKGROUND AND PURPOSE: Phosphodiesterase type-5 (PDE5) inhibitors constitute a novel and important therapeutic option for the treatment of pulmonary hypertension. The effects of the PDE5 inhibitors sildenafil, tadalafil and vardenafil on rabbit isolated pulmonary artery ring preparations and on intracellular Ca2+ concentration of thrombin-stimulated human platelets were investigated. EXPERIMENTAL APPROACH: Rabbit pulmonary artery rings were mounted in 10 mL organ bath containing Krebs solution. Tissues were connected to force-displacement transducers, and changes in isometric force were recorded. Ca2+ flux in human washed platelets was measured. KEY RESULTS: Sildenafil, tadalafil and vardenafil (0.0001-10 microM) concentration-dependently relaxed endothelium-intact and endothelium-denuded pulmonary artery rings. Endothelium denudation caused rightward shifts in the concentration-response curves to sildenafil, tadalafil and vardenafil (9-, 12- and 123-fold, respectively). Incubation with N(omega)-nitro-L-arginine methyl ester (100 microM) or ODQ (1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one) (10 microM) caused similar reductions of PDE5-induced vasorelaxations in intact rings. Sildenafil and tadalafil did not affect the phenylephrine-induced contractions, whereas vardenafil reduced the maximal responses, and shifted the phenylephrine-induced contraction curves to the right in endothelium-denuded rings (5- and 19-fold for 1 and 10 microM, respectively). Vardenafil (but neither sildenafil nor tadalafil) caused a marked rightward shift and a decrease of maximal contractile response to CaCl2. Vardenafil, but neither sildenafil nor tadalafil, significantly reduced the Ca2+ mobilization and Ca2+ influx in thrombin-stimulated washed platelets. CONCLUSIONS AND IMPLICATIONS: Our results indicate that vardenafil, in contrast to sildenafil or tadalafil, also blocked Ca2+ fluxes, thus enhancing its vasorelaxation of the pulmonary artery.

Long-term nitric oxide deficiency causes muscarinic supersensitivity and reduces beta(3)-adrenoceptor-mediated relaxation, causing rat detrusor overactivity.

BACKGROUND AND PURPOSE: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. EXPERIMENTAL APPROACH: Male Wistar rats received N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and beta-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [(3)H]inositol phosphate, NO synthase (NOS) activity, [(3)H]quinuclidinyl benzilate ([(3)H]QNB) binding and bladder morphology were also performed. KEY RESULTS: Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [(3)H]inositol phosphate in bladder tissue from rats treated with L-NAME. [(3)H]QNB was bound with an apparent K(D) twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. CONCLUSIONS AND IMPLICATIONS: Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced K(D) values for muscarinic receptors and [(3)H]inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.

Prediction and biochemical characterization of intrinsic disorder in the structure of proteolysis-inducing factor/dermcidin.

Proteolysis-inducing factor/dermcidin (PIF/DCD) is a novel human gene, located on chromosome 12, locus 12q13.1, that encodes a secreted 110-amino acid protein. Two transcripts for the protein have been identified in normal skin, breast, placenta and brain, and in various primary and metastatic tumor cells. The putative native-state structure of PIF/DCD has not been resolved. Here, we describe some biochemical features of the soluble recombinant 11-kDa protein produced in Escherichia coli. The native 11-kDa polypeptide displayed an anomalous mobility on 1% SDS-PAGE under reduced conditions and appeared as a single approximately 16-kDa band. Under nonreduced conditions, we detected by mass spectrometry, the presence of multiple peaks corresponding to m/z values of 21 kDa, which we confirmed as a dimeric form with a disulfide bridge between cysteine 34 of each 11-kDa monomer. The native protein exhibited an unusually high susceptibility to proteolytic attack by trypsin, and up to 13 peptides derived from its C-terminus were produced after 5 min of incubation. The secondary structure analysis of PIF/DCD native protein in aqueous solution, by circular dichroism spectroscopy, revealed regions with non-well-defined secondary structure but that acquired alpha-helix and beta-sheet secondary structures in the presence of TFE/water mixtures and micellar and non-micellar SDS molecules. By using PONDR, DisEMBL, DisProt, and GlobPlot computational predictors, we identified a long disorder region at the N-terminus of PIF/DCD amino acid sequence. This segment (from 19-50 residues) is critical for some of its biological activities, including neuron survival. This result is coherent with successive failure of crystallization of the protein. Taken together, these data suggest that the disorder and order transition may be relevant for some biological functions of PIF/DCD.

A bioequivalence study of gliclazide based on quantification by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry.

OBJECTIVE: The aim of this study was to evaluate, in human volunteers, the performance of one gliclazide tablet formulation (gliclazide 80 mg tablet from EMS Indústria Farmacêutica Ltda.) against two reference gliclazide tablet formulations (Diamicron 80 mg tablet from Servier do Brazil Ltda. and Diamicron 80 mg tablet from Servier (Ireland) Industries Limited). METHODS: The study had an open, randomized, three-period crossover design with a one-week washout interval between doses. The samples were obtained over a 48-h interval after each oral administration of gliclazide. The samples were extracted from plasma using diethylether : hexane (80 : 20, v/v) and the extracts were analyzed by high-performance liquid chromatography coupled with electrospray tandem mass spectrometry (HPLC-MS/ MS). Chromatography was performed isocratically using a Jones Chromatography Genesis C8 120A 4u. The method had a chromatographic run-time of 2.5 min and a calibration curve of the range of 0.02- 10 microg x ml(-1) (r(2) > 0.9993). The limit of quantification was 0.02 microg x ml(-1). RESULTS: The geometric mean and 90% confidence intervals (CI) for the Gliclazide/Diamicron (Ireland) ratio were 588.68% (90% CI= 491.16, 705.58%) for AUClast, 423.50% (90% CI = 338.25, 530.23%) for AUCinf, and 1395.77% (90% CI= 1116.62, 1744.72%) for Cmax. The geometric mean and 90% confidence intervals (CI) for the Gliclazide/Diamicron (Brazil) ratio were 249.16% (90% CI = 207.96, 298.54%) for AUCiast, 249.16% (90% CI = 207.96 - 298.54%) for AUCinf, and 188.04% (90% CI - 151.72, 233.05%) for Cmax. CONCLUSION: Since the 90% CI for Cmax, AUClast and AUC(0-infinity) ratios were all outside the 125% interval proposed by the US Food and Drug Administration, we concluded that the gliclazide test formulation were not bioequivalent to either reference formulation. Interestingly, the pharmacokinetic parameters such as Cmax, AUClast of both reference formulations are compatible with neither the literature nor the profile of an immediate release formulation. In addition, both reference formulations were not bioequivalent in themselves, indicating significant differences in reference product formulation.

Protective effect of prior physical conditioning on relaxing response of corpus cavernosum from rats made hypertensive by nitric oxide inhibition.

The aim of this work was to evaluate the influence of run training on the responsiveness of corpus cavernosum (CC) from rats made hypertensive by treatment with nitric oxide (NO) synthesis inhibitor. Wistar rats were divided into sedentary control (C-SD), exercise training (C-TR), N(omega)-nitro-L-arginine methyl ester (L-NAME) sedentary (LN-SD) and L-NAME trained (LN-TR) groups. The run training program consisted in 8 weeks in a treadmill, 5 days/week, each session lasted 60 min. L-NAME treatment (2 and 10 mg/rat/day) started after 4 weeks of prior physical conditioning and lasted 4 weeks. Concentration-response curves were obtained for acetylcholine (ACh), sodium nitroprusside (SNP), sildenafil and BAY 41-2272. The effect of electrical field stimulation (EFS) on the relaxations responses of CC was evaluated. Run training prevented the arterial hypertension induced by L-NAME treatment (LN-SD: 135+/-2 and 141+/-2 mm Hg for both doses of L-NAME) compared to LN-SD groups (154+/-1 and 175+/-2 mm Hg, for 2 and 10 mg of L-NAME, respectively). Run training produced an increase in the maximal responses (E(max)) of CC for ACh (C-SD: 47+/-3; C-TR: 52+/-1; and LN-TR: 53+/-3%) and SNP (C-SD: 89+/-1; C-TR: 98+/-1; and LN-TR: 95+/-1%). Both potency and E(max) for ACh were reduced in a dose of 10 mg of L-NAME, and run training restored the reduction of E(max) for ACh. No changes were found for BAY 41-2271 and sildenafil. Relaxing responses to EFS was reduced by L-NAME treatment that was restored by prior physical conditioning. In conclusion, our study shows a beneficial effect of prior physical conditioning on the impaired CC relaxing responses in rats made hypertensive by chronic NO blockade.

Comparative bioavailability of two losartan formulations in healthy human volunteers after a single dose administration.

OBJECTIVE: To compare the bioavailability of two potassic losartan immediate release tablet (50 mg) formulations (Losartan from Laboratórios Cristália Ltd., Brazil, as a test formulation and Cozaar from Merck Sharp & Dohme Farmacêutica Ltd., Brazil, as a reference formulation) in 25 volunteers of both sexes. MATERIAL AND METHODS: The study was conducted in an open, randomized, 2-period crossover design and a 1-week washout period. Plasma samples were obtained over a 24-hour interval. The concentrations of losartan and its active metabolite losartan acid were analyzed by combined reversed phase liquid chromatography and tandem mass spectrometry (LC-MS-MS) with negative ion electrospray ionization using a selected ion monitoring method. From the losartan and losartan acid plasma concentrations vs. time curves the following pharmacokinetic parameters were obtained: AUClast, AUC0-inf and Cmax. RESULTS: The geometric mean and respective 90% confidence interval (CI) of Losartan/Cozaar losartan percent ratios were 92.9% (82.2-105.0%) for Cmax, 99.0% (92.5-105.9%) for AUClast, and 99.1% (92.7-105.8%) for AUC0-inf. Furthermore, the geometric mean and respective 90% CI of Losartan/Cozaar losartan acid percent ratios were 98.5% (91.5-106.0%) for Cmax, 97.9% (93.3 102.7%) for AUClast, and 98.1% (93.6-102.9%) for AUC0-inf. CONCLUSION: Since the 90% CI for Cmax, AUClast and AUC0-inf were within the 80-125% interval proposed by the US Food and Drug Administration, it was concluded that the potassic losartan immediate release 50 mg tablet was bioequivalent to the Cozaar immediate release 50 mg tablet, according to both the rate and extent of absorption. While there were no significant differences in the bioequivalence assessed by either losartan or losartan acid, future bioequivalence studies on losartan may be performed by quantifying losartan alone as the parent compounds are more discriminative.

A phase I trial of the bombesin/gastrin-releasing peptide (BN/GRP) antagonist RC3095 in patients with advanced solid malignancies.

Bombesin/gastrin-releasing peptides (BN/GRP) were shown to bind selectively to cell surface receptors, stimulating the growth of various types of malignancies in murine and human models. The novel BN/GRP synthetic receptor antagonist, RC-3095, was able to produce long-lasting tumor regressions in murine and human tumor models in vitro and in vivo. Animal toxicology studies showed no detectable organ toxicity apart from local irritation at the injection site. The purpose of this study was to determine the safety and feasibility of the administration of RC-3095 by daily subcutaneous injections in patients with advanced and refractory solid malignancies. Twenty-five patients received RC-3095 once or twice-daily at doses ranging from 8 to 96 ug/kg. Dose was escalated in groups of 3-5 patients per dose level. The only toxicity observed was local discomfort in the injection site at the highest doses. A single dose administration of RC-3095 at the highest dose level (96 ug/kg) was tested in a clearly hypergastrinemic individual with the Zollingen-Ellison syndrome and produced a decrease in plasma gastrin down to 50% of basal levels in 6 h. There was no objective tumor responses in patients included in the study. A short-lasting minor tumor response was observed in a patient with a GRP-expressing progressive medullary carcinoma of the thyroid. Due to problems with the analytical method, plasma pharmacokinetic data was obtained only from two patients included at the highest dose level. In these patients, RC-3095 reached plasma concentrations >100 ng/mL for about 8 h, which were within therapeutic levels on the basis of prior data obtained in mice and rats. The plasma elimination half-life was between 8.6-10.9 h. Due to the occurrence of local toxicity at the injection site, the dose escalation procedure could not be fully evaluated up to a maximum tolerated dose. Thus, a recommended dose of RC-3095 for Phase II trials could not be clearly established. Considering the novelty of its mechanism of action and impressive preclinical anti-tumor activity, further studies exploiting new formulations of RC-3095 for human use, such as slow-release preparations, and analogues with a more favorable pharmacokinetics are warranted.

Comparative bioavailability of two ramipril formulations after single-dose administration in healthy volunteers.

OBJECTIVE: To assess the bioequivalence of a ramipril 5 mg tablet formulation (ramipril test formulation from Laboratórios Biosintética Ltda (Sao Paulo, Brazil) and Triatec from Aventis Pharma (Sueano, Brazil) standard reference formulation) in 26 healthy volunteers of both sexes. METHODS: The study was conducted using an open, randomized, 2-period crossover design with a 2-week washout interval. Plasma samples were obtained over a 36-hour period. Plasma ramipril and ramiprilat concentrations were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) with positive ion electrospray ionization using multiple reaction monitoring (MRM). From the ramipril and ramiprilat plasma concentration vs. time curves, the following pharmacokinetic parameters were obtained: AUClast, AUCinf and Cmax. RESULTS: The limit of quantification was 0.2 ng x ml(-1) and 1.0 ng x ml(-1) for ramipril and ramiprilat, respectively. The geometric means and 90% confidence intervals (CI) for Ramipril/Triatec and Ramiprilat/Triatec percent ratios were: 104.69% (90% CI = 93.21-117.59%) for Cmax, 102.49% (90% CI = 92.76-113.24%) for AUClast, 103.60% (90% CI = 93.56 114.73%) for AUCinf, 108.48% (90% CI = 98.86-119.03%) for Cmax, 105.88% (90% CI = 101.55-110.39%) for AUClast, 97.30% (90% CI = 90.17-104.99%) for AUCinf, respectively. CONCLUSION: Since the 90% CI for AUClast, AUCinf and Cmax ratios were within the 80-125% interval proposed by the U.S. FDA, it was concluded that the ramipril formulation produced by Laboratórios Biosintética Ltda is bioequivalent to the Triatec formulation in both rate and extent of absorption.

In vitro mutagenicity of anti-inflammatory parsalmide analogues PA7, PA10, and PA31 triggered by biotransformation into hydroxy derivatives.

In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino-N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino-N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino-N-butyl-2-phenoxy-benzamide] and PA31 [5-amino-N-butyl-2-(p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/beta-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2-19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2-14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.

A bioequivalence study of citalopram based on quantification by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry.

OBJECTIVE: The aim of this study was to compare the bioavailability of two citalopram formulations (citalopram from Eurofarma Laboratórios Ltda., as the test formulation, and cipramil from Schering-Plough, Brazil, as the reference formulation) in healthy volunteers. METHODS: The study had an open, randomized, two-period crossover design with a two-week washout interval between doses. The samples were obtained over a 168-hour interval after each oral administration of citalopram (one 20 mg tablet of each formulation). The analyte and the internal standard were extracted from plasma using diethylether: dichloromethane (70 : 30, v/v) and the extracts were analyzed by high-performance liquid chromatography coupled to electrospray tandem mass spectrometry. Chromatography was done isocratically using a Genesis C8 analytical column (4 microm, 2.1 mm i.d. x 100 mm). The method had a chromatographic run time of three minutes and a linear calibration curve over the range of 0.5 - 200 ng x ml(-1) (r2 > 0.999887). The limit of quantification was 0.5 ng x ml(-1). RESULTS: The geometric mean and 90% confidence intervals (CI) for the citalopram/cipramil ratio were 98.28% (94.24 - 102.49%) for AUClast, 96.44% (90.20 - 103.11%) for AUCinf, and 98.54% (94.70 - 102.54%) for Cmax. CONCLUSION: Since the 90% CI for Cmax, AUClast and AUC(0-infinity) ratios were all within the 80 - 125% interval proposed by the US Food and Drug Administration, we concluded that the citalopram formulation elaborated by Eurofarma Laboratórios Ltda. was bioequivalent to the cipramil formulation in its rate and extent of absorption.

Purification and initial characterization of a novel protein with factor Xa activity from Lonomia obliqua caterpillar spicules.

A novel protein with factor Xa-like activity was isolated from Lonomia obliqua caterpillar spicules by gel filtration chromatography and reversed-phase high-performance liquid chromatography. The protein had a mass of 20745.7 Da, as determined by mass spectrometry, and contained four Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of the protein and the cysteine residues linked by disulfide bridges. The positions of 24 sequenced tryptic peptides, including the N-terminal, were deduced by comparison with a homologous protein from the superfamily Bombycoidea. Approximately 90% of the primary structure of the active protein was determined.

Phentolamine relaxes human corpus cavernosum by a nonadrenergic mechanism activating ATP-sensitive K+ channel.

To investigate the pharmacodynamics of phentolamine in human corpus cavernosum (HCC) with special attention to the role of the K+ channels. Strips of HCC precontracted with nonadrenergic stimuli and kept in isometric organ bath immersed in a modified Krebs-Henseleit solution enriched with guanethidine and indomethacine were used in order to study the mechanism of the phentolamine-induced relaxation. Phentolamine caused relaxation (approximately 50%) in HCC strips precontracted with K+ 40 mM. This effect was not blocked by tetrodotoxin (1 microM) (54.6+/-4.6 vs 48.9+/-6.4%) or (atropine (10 microM) (52.7+/-6.5 vs 58.6+/-5.6%). However, this relaxation was significantly attenuated by L-NAME (100 microM) (59.7+/-5.8 vs 27.8+/-7.1%; P<0.05; n = 8) and ODQ (100 microM) (62.7+/-5.1 vs 26.8+/-3.9%; P<0.05; n = 8). Charybdotoxin and apamin (K(Ca)-channel blockers) did not affect the phentolamine relaxations (54.6+/-4.6 vs 59.3+/-5.2%). Glibenclamide (100 microM), an inhibitor of K(ATP)-channel, caused a significant inhibition (56.7+/-6.3 vs 11.3+/-2.3%; P<0.05; n = 8) of the phentolamine-induced relaxation. In addition, the association of glibenclamide and L-NAME almost abolished the phentolamine-mediated relaxation (54.6+/-5.6 vs 5.7+/-1.4%; P<0.05; n = 8). The results suggest that phentolamine relaxes HCC by a nonadrenergic-noncholinergic mechanism dependent on nitric oxide synthase activity and activation of K(ATP)-channel.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay.

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ss-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251. Streptase was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase and Solustrep formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251 activity per vial, Streptase (75.7 +/- 5.0 units) and Streptonase (94.7 +/- 4.6 units) had the highest activity, while Unitinase (31.0 +/- 2.4 units) and Strek (32.9 +/- 3.3 units) had the weakest activity. Solustrep (53.3 +/- 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.