Pierina Sueli Bonato

Endophytic Fungi: Natural Products, Enzymes and Biotransformation Reactions

Endophytes exhibit a complex web of interactions with host plants and with other endophytic microorganisms and therefore they have been intensively studied over the last several years as prolific sources of new and bioactive natural products. In fact, an impressive number of natural products have been produced by endophytic microorganisms. In addi- tion, some studies have shown endophytes to be good producers of useful enzymes to improve industrial processes. More recently, endophytes have also received attention as biocatalysts in the chemical transformation of natural products and drugs. Results have shown their ability to modify chemical structures with a high degree of stereospecificity. Some reac- tions are similar to those catalyzed by mammal phase I metabolism; therefore endophytes could be used as models for drug metabolism studies. This paper summarizes recent data on endophytes research as a source of novel and bioactive natural products, as producers of enzymes and their use on biotransformation processes.

Recent advances in the determination of enantiomeric drugs and their metabolites in biological fluids by capillary electrophoresis-mediated microanalysis.

This review summarizes the recent developments in the application of electromigration techniques for the enantioselective analysis of drugs and metabolites in biological fluids. During the period covered by this review, it has been observed an increase in the use of negatively charged chiral selectors, particularly sulfobutyl ether‐β‐cyclodextrin and sulfated‐β‐cyclodextrin, and the combination of two different chiral selectors in the running buffer to obtain the resolution of drugs and their metabolites. Low detection limits as required for pharmacokinetic studies were obtained by using concentration techniques, including sample stacking procedures, and more sensitive detection systems, such as laser‐induced fluorescence and mass spectrometry. Finally, the major points are discussed that can be considered to obtain reliable methods for enantioselective bioanalysis.

Pharmacokinetics of combined treatment with praziquantel and albendazole in neurocysticercosis

AIMS Neurocysticercosis is the most common cause of acquired epilepsy in the world. Antiparasitic treatment of viable brain cysts is of clinical benefit, but current antiparasitic regimes provide incomplete parasiticidal efficacy. Combined use of two antiparasitic drugs may improve clearance of brain parasites. Albendazole (ABZ) has been used together with praziquantel (PZQ) before for geohelminths, echinococcosis and cysticercosis, but their combined use is not yet formally recommended and only scarce, discrepant data exist on their pharmacokinetics when given together. We assessed the pharmacokinetics of their combined use for the treatment of neurocysticercosis. METHODS A randomized, double-blind, placebo-controlled phase II evaluation of the pharmacokinetics of ABZ and PZQ in 32 patients with neurocysticercosis was carried out. Patients received their usual concomitant medications including an antiepileptic drug, dexamethasone, and ranitidine. Randomization was stratified by antiepileptic drug (phenytoin or carbamazepine). Subjects had sequential blood samples taken after the first dose of antiparasitic drugs and again after 9 days of treatment, and were followed for 3 months after dosing. RESULTS Twenty-one men and 11 women, aged 16 to 55 (mean age 28) years were included. Albendazole sulfoxide concentrations were increased in the combination group compared with the ABZ alone group, both in patients taking phenytoin and patients taking carbamazepine. PZQ concentrations were also increased by the end of therapy. There were no significant side effects in this study group. CONCLUSIONS Combined ABZ + PZQ is associated with increased albendazole sulfoxide plasma concentrations. These increased concentrations could independently contribute to increased cysticidal efficacy by themselves or in addition to a possible synergistic effect.

Enantioselective analysis of metoprolol in plasma using high-performance liquid chromatographic direct and indirect separations: applications in pharmacokinetics

Direct enantioselective separation on chiral stationary phases and indirect separation based on the formation of diastereomeric derivatives were developed and compared for the HPLC analysis of R(+) and S(-)-metoprolol in human plasma. Plasma samples prepared using solid-phase extraction columns or liquid-liquid extraction were directly analyzed on a Chiralpack AD or on a Chiralcel OD-H columns, respectively. S-(-)-menthyl choroformate was also used to yield diastereomeric derivatives resolved on a RP-8 column. The methods were employed to determine plasma concentrations of metoprolol enantiomers in a pharmacokinetic study of single dose administration of racemic metoprolol to a healthy Caucasian volunteer phenotyped as extensive metabolizer of debrisoquine. The correlation coefficients among enantioselective metoprolol plasma concentrations (5-223 ng/ml) obtained by the three methods were equal or higher than 0.99. The direct method that employed the chiral column Chiralpak AD may be considered the most sensitive, although the three methods demonstrated interchangeable use in the pharmacokinetic investigation.

Simultaneous determination of albendazole sulfoxide enantiomers and albendazole sulfone in plasma

A high-performance liquid chromatographic method has been developed for the simultaneous determination of albendazole sulfoxide (ABZSO) enantiomers and albendazole sulfone (ABZSO2) in human plasma. The resolution of ABZSO enantiomers and ABZSO2 was obtained on a Chiralpak AD column using hexane-isopropanol-ethanol (81:14.25:4.75, v/v/v) as the mobile phase. The drugs were detected by fluorescence (lambda(exc) = 280 nm, lambda(em) = 320 nm). The drugs were extracted from 500 microl plasma with ethyl acetate, and after solvent evaporation, the residues were dissolved in the mobile phase and chromatographed. The method was precise and accurate for the three compounds, as judged by the coefficients of variation and relative errors observed. Linear standard curves were obtained in the concentration range of 5-2500 ng/ml for ABZSO enantiomers and 1-500 ng/ml for ABZSO2. A typical plasma concentration-time profile is presented for one patient under treatment for neurocysticercosis.

Enantioselective kinetic disposition of albendazole sulfoxide in patients with neurocysticercosis

The enantioselectivity of the kinetic disposition of albendazole sulfoxide (ASOX) was investigated in 18 patients with neurocysticercosis treated with a multiple dose regimen of albendazole for 8 days (5 mg/kg every 8 h). Serial blood samples were collected on the eighth day of treatment during the last dose interval, with prorogation up to 12 h. Albendazole sulfone (ASON) and enantiomers of ASOX were analyzed in plasma samples by HPLC using a Chiralpak AD column and detection by fluorescence. The pharmacokinetic parameters showing statistically significant differences between the (+) ASOX and (-) ASOX enantiomers are presented as respective means (95% CI) as follows: maximum plasma concentration, Cmax = 301.6 (179.7-423.5) vs 54.9 (21.9-87.9) ng.ml-1; elimination half-life, t1/2 = 5.2 (4.1-6.3) vs 3.3 (2.8-3.8) h, area under the plasma concentration-time curve, AUCss0-8 = 1719.2 (978.6-2459.8) vs 261.4 (102.9-419.8) ng.h.ml-1 and apparent clearance, Cl/fm = 5.8 (3.8-7.8) vs 54.0 (35.2-72.7) l.h-1.kg-1. The mean value of 9.2 (7.6-10.9) for the AUC0-8(+)-ASOX/AUC0-8(-)-ASOX ratio demonstrated plasma accumulation of the (+) enantiomer. Sulfone formation capacity, expressed by the AUCss0-8 ratio ASON/ASOX + ASON, was 8.0 (7.0-8.9). The present data indicate enantioselectivity in the kinetic disposition of ASOX in patients with neurocysticercosis.

Therapy for neurocysticercosis: pharmacokinetic interaction of albendazole sulfoxide with dexamethasone.

Albendazole is considered the drug of choice for neurocysticercosis. It is frequently used in combination with dexamethasone to prevent the acute inflammatory reaction due to cysticercal death. It has been reported that dexamethasone increases the plasma level of albendazole sulfoxide, the active metabolite of albendazole. The pharmacokinetic interaction of albendazole sulfoxide with dexamethasone, associated or not with cimetidine, was investigated in 24 patients with active intraparenchymal brain cysticercosis. Eight of these patients received albendazole alone, eight received it in combination with dexamethasone, and eight received it in combination with both dexamethasone and cimetidine. The pharmacokinetic parameters maximum plasma concentration, time to maximum plasma concentration, absorption half-life, and absorption rate constant did not differ between groups, suggesting that the formation of albendazole sulfoxide was not altered by the administration of dexamethasone, combined or not with cimetidine. There were significant differences, however, in the parameters plasma concentration-time curve, oral clearance, elimination half-life, and elimination rate constant, suggesting that dexamethasone, combined or not with cimetidine, decreases the rate of elimination of albendazole sulfoxide.

HPLC screening and GC-MS confirmation of triazine herbicides residues in drinking water from sugar cane area in Brazil

The extensive use of chlorotriazines as selectiveherbicides in agriculture and their relatively highpersistence imply that these compounds are now presentin the environment, contaminating surface and groundwater. In European countries, United States andCanada, the drinking water ordinance demands a limitedconcentration of 0.5 μg L-1 for the sum of allpesticides and 0.1 μg L-1 with respect to eachcompound, implying on the necessity of sensitive andselective analytical methods. In the present study wedescribe two methods for the analysis of atrazine,simazine and ametryn residues in surface and groundwater collected from the Espraiado Stream watershed,Ribeirão Preto region, SP, Brazil. The HPLC methodused for sample screening was based on herbicideextraction with dichloromethane:isopropanol (9:1, v/v)followed by reversed-phase chromatography (RP-8) withdetection at 220 nm. The presence of herbicides wasconfirmed by GC-MS after ethyl acetate extraction. Atotal of 250 samples collected at different sites fromOctober 1995 to July 1996 were analyzed. Ametrynresidues were detected in 17 samples but almost alwaysat concentrations below those maximum levels recommended by international agencies of environmental control.

MICROEXTRAÇÃO EM FASE LÍQUIDA (LPME): FUNDAMENTOS DA TÉCNICA E APLICAÇÕES NA ANÁLISE DE FÁRMACOS EM FLUIDOS BIOLÓGICOS

The analysis of drugs and metabolites in biological fluids usually requires extraction procedures to achieve sample clean-up and analyte preconcentration. Commonly, extraction procedures are performed using liquid-liquid extraction or solid-phase extraction. Nevertheless, these extraction techniques are considered to be time-consuming and require a large amount of organic solvents. On this basis, microextraction techniques have been developed. Among them, liquid-phase microextraction has been standing out. This review describes the liquid-phase microextraction technique based on hollow fibers as a novel and promising alternative in sample preparation prior to chromatographic or electrophoretic analysis. The basic concepts related to this technique and its applicability in extraction of drugs are discussed.

Simple and efficient method for enantioselective determination of omeprazole in human plasma

A practical and selective HPLC method for the separation and quantification of omeprazole enantiomers in human plasma is presented. C18 solid phase extraction (SPE) cartridges were used to extract the enantiomers from plasma samples and the chiral separation was carried out on a Chiralpak AD column protected with a CN guard column, using ethanol:hexane (70:30) as the mobile phase, at a flow rate of 0.5 ml/min. The detection was carried out at 302 nm. The method proved to be linear in the range of 10-1000 ng/ml for each enantiomer, with a quantification limit of 5 ng/ml. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 10%.