Development and validation of a LC-MS/MS method with electrospray ionization for determination of LASSBio-579 in rat plasma.

A simple and sensitive LC-MS/MS analytical method was developed and validated for the determination of LASSBio-579 in plasma rat, using fluconazole as internal standard. Analyses were performed on a Shimadzu HPLC system using a Shimadzu C18 column and isocratic elution with acetonitrile-water (80:20, v/v), containing 0.4mM ammonium hydroxide and 0.2 mM acetic acid at a flow rate of 1.0 ml/min (split ratio 1:5). A Micromass triple quadrupole mass spectrometer, equipped with an electrospray ionization interface, operated in the positive mode. Plasma samples were deproteinized with acetonitrile (1:2) and 50 microl of the supernatant were injected into the system. The retention times of LASSBio-579 and IS were approximately 4.7 and 2.4 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 30-2000 ng/ml with determination coefficient >0.98. The lower limit of quantification was 30 ng/ml. The accuracy of method was within 15%. Intra- and inter-day relative standard deviations were less or equal to 13.5% and 6.4%, respectively. The applicability of the LC-MS/MS method for pharmacokinetic studies was tested using plasma samples obtained after intraperitoneal administration of LASSBio-579 to male Wistar rats. No interference from endogenous substances was observed, showing the specificity of the method developed. The reported method can provide the necessary sensitivity, linearity, precision, accuracy, and specificity to allow the determination of LASSBio-579 in pre-clinical pharmacokinetic studies.

Antifungal activity of some Brazilian Hypericum species.

Crude methanolic extracts and fractions from the aerial parts of seven species of Hypericum (H. caprifoliatum Cham. and Schltdl., H. carinatum Griseb., H. connatum Lam., H. ternum A. St.-Hil., H. myrianthum Cham. and Schltdl., H. piriai Arechav. and H. polyanthemum Klotzsch ex Reichardt) growing in southern Brazil were analyzed for their in vitro antifungal activity against a panel of standardized and clinical opportunistic pathogenic yeasts and filamentous fungi, including dermatophytes, by the agar dilution method. Chloroform and hexane extracts of H. ternum showed the greatest activity among extracts tested.

Validated HPLC method for determination of LASSBio-581, a new heterocyclic N-phenylpiperazine derivative, in rat plasma.

A rapid, simple and accurate high performance liquid chromatography (HPLC) method was developed and validated for the determination of LASSBio-581 (1-[1-(4-chloro-phenyl)-1H-[1,2,3]triazol-4-ylmethyl]-4-phenyl-piperazine) in rat plasma using ketoconazole as internal standard. Plasma samples were deproteinized with methanol. A good chromatographic separation was achieved using a reversed phase C18 column. Mobile phase consisting of sodium dihydrogen phosphate monohydrate (pH 4.5, 0.02 M) and methanol mixture (35:65, v/v) was used at a flow rate of 1.0 ml/min. The eluate was monitored using a UV detector at 248 nm. The retention times of LASSBio-581 and the internal standard were approximately 3.8 and 5.6 min, respectively. The calibration curves were linear over the concentration range of 0.25-8.0 microg/ml with correlation coefficients >0.99. The limit of quantitation was 0.25 microg/ml. The accuracy of the method was >90%. The intra-day relative standard deviation (R.S.D.) ranged from 6.15 to 10.52% at 0.4 microg/ml, 7.44 to 13.81% at 1.5 microg/ml and 6.10 to 13.94% at 6.0 microg/ml. The inter-day R.S.D. were 9.54, 8.42 and 8.25% at 0.4, 1.5 and 6.0 microg/ml, respectively. No interference from endogenous substances or metabolites were observed. The method has been used to measure plasma concentrations of LASSBio-581 in pharmacokinetic studies in rats.

Dopaminergic profile of new heterocyclic N-phenylpiperazine derivatives

Dopamine constitutes about 80 percent of the content of central catecholamines and has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to report the structural design of two N-phenylpiperazine derivatives, compound 4: 1-[1-(4-chlorophenyl)-1H-4-pyrazolylmethyl]-4-phenylhexahydropyrazine and compound 5: 1-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-ylmethyl]-4-phenylhexahydropyrazine, planned to be dopamine ligands, and their dopaminergic action profile. The two compounds were assayed (dose range of 15-40 mg/kg) in three experimental models: 1) blockade of amphetamine (30 mg/kg, ip)-induced stereotypy in rats; 2) the catalepsy test in mice, and 3) apomorphine (1 mg/kg, ip)-induced hypothermia in mice. Both derivatives induced cataleptic behavior (40 mg/kg, ip) and a hypothermic response (30 mg/kg, ip) which was not prevented by haloperidol (0.5 mg/kg, ip). Compound 5 (30 mg/kg, ip) also presented a synergistic hypothermic effect with apomorphine (1 mg/kg, ip). Only compound 4 (30 mg/kg, ip) significantly blocked the amphetamine-induced stereotypy in rats. The N-phenylpiperazine derivatives 4 and 5 seem to have a peculiar profile of action on dopaminergic functions. On the basis of the results of catalepsy and amphetamine-induced stereotypy, the compounds demonstrated an inhibitory effect on dopaminergic behaviors. However, their hypothermic effect is compatible with the stimulation of dopaminergic function which seems not to be mediated by D2/D3 receptors

Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae)

The aim of the present study was to assess the analgesic activity of the aerial parts of two Hypericum species native to Southern Brazil, H. caprifoliatum and H. polyanthemum. The antinociceptive effect of the H. polyanthemum cyclohexane extract (POL; 180 mg/kg) and of the H. caprifoliatum methanol (MET) and cyclohexane (CH) extracts (90 mg/kg) was evaluated in the hot-plate (ip and po) and writhing (po) tests using male Swiss CF1 mice weighing 22-27 g (N = 10 per group). All extracts displayed antinociceptive effects in the hot-plate test (MET ip = 48 percent, MET po = 39 percent, CH ip = 27 percent, CH po = 50 percent, POL ip = 74 percent, and POL po = 49 percent compared to control). Pretreatment with naloxone (2.5 mg/kg, sc) abolished the effects of CH and POL, and partially prevented the analgesia induced by MET administered by the ip (but not by the po) route. POL and CH (po) significantly reduced the number of writhes induced by acetic acid, while MET was ineffective in this regard. We conclude that the antinociceptive effects of the H. caprifoliatum (CH) and H. polyanthemum (POL) hexane extracts seem to be mediated by the opioid system. Moreover, the antinociceptive activity of the H. caprifoliatum MET extract seems to depend on at least two chemical substances (or groups of substances) with distinct pharmacokinetic profiles and mechanisms of action. Only the naloxone-insensitive component of MET activity showed good bioavailability following oral administration

Dopaminergic profile of new heterocyclic N-phenylpiperazine derivatives.

Dopamine constitutes about 80% of the content of central catecholamines and has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to report the structural design of two N-phenylpiperazine derivatives, compound 4: 1-[1-(4-chlorophenyl)-1H-4-pyrazolylmethyl]-4-phenylhexahydropyrazine and compound 5: 1-[1-(4-chlorophenyl)-1H-1,2,3-triazol-4-ylmethyl]-4-phenylhexahydropyrazine, planned to be dopamine ligands, and their dopaminergic action profile. The two compounds were assayed (dose range of 15-40 mg/kg) in three experimental models: 1) blockade of amphetamine (30 mg/kg, ip)-induced stereotypy in rats; 2) the catalepsy test in mice, and 3) apomorphine (1 mg/kg, ip)-induced hypothermia in mice. Both derivatives induced cataleptic behavior (40 mg/kg, ip) and a hypothermic response (30 mg/kg, ip) which was not prevented by haloperidol (0.5 mg/kg, ip). Compound 5 (30 mg/kg, ip) also presented a synergistic hypothermic effect with apomorphine (1 mg/kg, ip). Only compound 4 (30 mg/kg, ip) significantly blocked the amphetamine-induced stereotypy in rats. The N-phenylpiperazine derivatives 4 and 5 seem to have a peculiar profile of action on dopaminergic functions. On the basis of the results of catalepsy and amphetamine-induced stereotypy, the compounds demonstrated an inhibitory effect on dopaminergic behaviors. However, their hypothermic effect is compatible with the stimulation of dopaminergic function which seems not to be mediated by D2/D3 receptors.

Antinociceptive activity of Hypericum caprifoliatum and Hypericum polyanthemum (Guttiferae).

The aim of the present study was to assess the analgesic activity of the aerial parts of two Hypericum species native to Southern Brazil, H. caprifoliatum and H. polyanthemum. The antinociceptive effect of the H. polyanthemum cyclohexane extract (POL; 180 mg/kg) and of the H. caprifoliatum methanol (MET) and cyclohexane (CH) extracts (90 mg/kg) was evaluated in the hot-plate (ip and po) and writhing (po) tests using male Swiss CF1 mice weighing 22-27 g (N = 10 per group). All extracts displayed antinociceptive effects in the hot-plate test (MET ip = 48%, MET po = 39%, CH ip = 27%, CH po = 50%, POL ip = 74%, and POL po = 49% compared to control). Pretreatment with naloxone (2.5 mg/kg, sc) abolished the effects of CH and POL, and partially prevented the analgesia induced by MET administered by the ip (but not by the po) route. POL and CH (po) significantly reduced the number of writhes induced by acetic acid, while MET was ineffective in this regard. We conclude that the antinociceptive effects of the H. caprifoliatum (CH) and H. polyanthemum (POL) hexane extracts seem to be mediated by the opioid system. Moreover, the antinociceptive activity of the H. caprifoliatum MET extract seems to depend on at least two chemical substances (or groups of substances) with distinct pharmacokinetic profiles and mechanisms of action. Only the naloxone-insensitive component of MET activity showed good bioavailability following oral administration.

Gênero Pfaffia: aspectos químicos, farmacológicos e implicações para o seu emprego terapêutico / Pfaffia: available chemical and pharmacological data and their implications for its therapeutical use

Os relatos encontrados na literatura científica sobre a composição química e atividades farmacológicas do gênero Pfaffia spp. são apresentados. Os estudos sobre esse gênero são ainda bastante escassos, sendo assim, o uso racional de espécies de Pfaffia, com finalidade terapêutica ainda depende de conhecimento aprofundado de suas propriedades farmacológicas e do desenvolvimento de tecnologias de produção e controle de qualidade.

This review summarizes available chemical and pharmacological data about Pfaffia genre. Scientific studies about these species are still lacking, and their therapeutical rational use depends on the availability of more informations about their pharmacological properties and the development of production and control quality technologies.

Monoamine oxidase inhibitory activity of some Hypericum species native to South Brazil.

The total methanol crude extracts and petroleum ether, chloroform, and methanol fractions obtained from Hypericum species, H. caprifoliatum, H. carinatum, H. connatum, H. cordatum, H. myrianthum, H. piriai, H. polyanthemum and H. brasiliense, all native to South Brazil, were assayed for monoamine oxidase A (MAO A) and MAO B inhibitory activity in rat brain mitochondrial preparations at concentrations ranging from 1 to 20microg mL(-1). Three benzopyrans, HP1 (6-isobutyryl-5,7-dimethoxy-2,2-dimethylbenzopyran), HP2 (7-hydroxy-6-isobutyryl-5-methoxy-2,2-dimethylbenzopyran) and HP3 (5-hydroxy-6-isobutyryl-7-methoxy-2,2dimethylbenzopyran) isolated from H. polyanthemum were also tested at maximal concentrations of 150, 150 and 75/microM, respectively. The lipophilic extracts of H. polyanthemum, H. caprifoliatum and H. piriai displayed MAO A inhibitory activity greater than 50%. Among the benzopyrans, only HP3 showed significant activity, with an IC50 value of 22 microM. The total methanol crude extracts of aerial parts from H. carinatum, H. connatum, H. cordatum, H. polyanthemum and H. piriai were evaluated for antidepressant activity in the Porsolt's forced swimming test in Wistar rats (270 mg kg(-1) day(-1); i.p); however, none of them showed activity.

Plants as source of drugs.

This work presents a study of the importance of natural products, especially those derived from higher plants, in terms of drug development. It describes the main strategies for obtaining drugs from natural sources, fields of knowledge involved, difficulties and perspectives. It also includes a brief discussion of the specific situation in Brazil regarding the use of, trade in, and research into therapeutic resources of natural origin and the general lack of awareness of the use of potentially toxic plants, mainly in folk medicine.

Promoção do uso racional de fitoterápicos: uma abordagem no ensino de Farmacognosia

Neste trabalho é apresentada uma experiência no ensino de Farmacognosia, cuja base principal é o estudo dos aspectos farmacoterapêuticos de fitofármacos e fitoterápicos, visando o estabelecimento de seu uso racional. São apresentados dados sobre a comercialização e ações institucionais destinadas à normatização, padronização e utilização racional destes produtos no mundo, com propostas e condutas na prática farmacêutica para aquisição e dispensação adequada de fitoterápicos.

In this paper we present a pharmacognosy teaching experience which focuses mainly on the pharmacotherapeutic aspects of phytomedicines and drugs from natural sources aiming their rational use. World commercial data and institutional actions destined to provide normalization, standardization and rational use as well some propositions for the acquisition of these products and patient counseling are presented.

Psychopharmacological screening of Pfaffia glomerata Spreng. (Amarathanceae) in rodents.

The alcoholic extract of Pfaffia glomerata roots (100, 500, 1000 mg/kg, intraperitoneally (i.p.), and 500, 1000, 1500 mg/kg, per os) was studied in several behavioral animal models for the evaluation of central activity: open field, barbiturate sleeping time, pentilenotetrazole (PTZ)-induced convulsions, elevated plus-maze, step-down inhibitory avoidance and forced swimming test. The acute treatment (500 mg/kg, i.p.) interfered with the open-field habituation, decreased sleep latency and increased barbiturate-induced sleeping time, protected partially the animals of PTZ-induced convulsions, decreased the memory retention in step-down inhibitory avoidance, and did not have an important effect in the elevated plus-maze test and forced swimming test. The same extract at 1000 mg/kg per os did not cause any effect in barbiturate sleeping time and pentilenotetrazole-induced convulsions models. Thus, the effect on the memory was deeper evaluated in the step-down inhibitory avoidance task. When administered by intraperitoneal route, the extract showed a dose-dependent effect causing full amnesia at 1000 mg/kg. On the other hand, when it was given by oral route at 500, 1000 and 1500 mg/kg, no influence on the memory retention was observed. These results suggest that the alcoholic extract of P. glomerata roots presents different effects depending on the route of administration: by i.p route, it seems to be a central nervous system depressant agent; by oral route, it seems to be ineffective, at least in the tested doses.

Screening for the antidepressant activity of some species of Hypericum from South Brazil.

The crude methanol extracts of three species of the genus Hypericum (H. brasiliense, H. caprifoliatum and H. myrianthum) native to South Brazil were evaluated for the antidepressant activity according to the forced swimming test, a classical animal model for antidepressant drug screening. Among the species studied, only H. caprifoliatum showed activity. Thus, this plant was fractionated with solvents in increasing polarity (petroleum ether; petroleum ether: chloroform (1:1); chloroform and methanol). The petroleum ether fraction was the only one which demonstrated antidepressant activity at a dose of 270 mg/kg/day (i.p.). The chemical analyses showed that this fraction is rich in phenolic compounds, mainly of the phloroglucinol type.

Inhibition of cytochrome P450-dependent monooxygenases by an alkaloid fraction from Helietta apiculata markedly potentiate the hypnotic action of pentobarbital.

Crude alkaloid fraction (CAF) isolated from the leaves of Helietta apiculata showed the presence of furoquinolines. The extract was investigated to determine if it can enhance the sensitivity of the central nervous system (CNS) to the hypnotic action of pentobarbital. Administration of CAF to mice in a dose range of 300-500 mg/kg prior to an injection of pentobarbital (40 mg/kg, i.p.) was associated with a statistically significant decrease of sleep latency and prolongation of pentobarbital-induced sleeping time. Pretreatment of rats with the same alkaloid extract (150 mg/kg, i.p. for 4 days) prior to administration of pentobarbital (40 mg/kg, i.p.) caused not only significant reduction of the levels of microsomal proteins, total cytochrome P450 enzymes and a decrease of aminopyrin-N-demethylation and 3,4-benz(a)pyrene hydroxylation but also changed the pattern of cytochrome P450. It is concluded that the CAF isolated from H. apiculata can potentiate the CNS depressant effect of pentobarbital due to alteration of barbiturate metabolism through inhibition, mainly, of cytochrome P450-dependent enzymes.