Nitric oxide: state of the art in drug design.

Since the great discovery of Furchgott, Ignarro and Murad in the late 90's, nitric oxide (NO) is considered one of the most versatile endogenous molecules, which is involved in important signaling biochemistry pathways of the human body. Thus, it is directly related to pathological processes and its over- or low-production is able to cause damage in systems that are involved. By using certain functional groups present in molecules that already have potential therapeutic value, hybrid compounds, by means of inclusion of NO-donors (e.g., ester nitrates, furoxans, benzofuroxans, NONOates, S-nitrosothiols, metal complexes), can be generated that have a NO release benefit along with maintaining the activity of the native drug. This approach has proved to be useful in many spheres of Medicinal Chemistry, such as cardiovascular, inflammatory, bacterial, fungal, viral, parasitic, ocular diseases and cancer. Potent and selective nitric oxide synthase inhibitors are being designed, mainly through enzyme structure based process, however, due to high homology between the isoforms, these studies have proved to be very difficult. The objective of the research is to achieve a balance between the release of therapeutic amounts of NO, especially in specific site of action, and maintaining the native drug activity. The search for new and effective NO-donor hybrid drugs, as well as selective nitric oxide synthase inhibitors, is an important focus in modern drug design in order to manipulate biochemical pathways involving NO that influence many dysfunctions of the human organism.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs.

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 microM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 æM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Preparação e caracterização inicial de complexo de inclusão entre nitrofurazona e 2-hidroxipropil-Beta-ciclodextrina / Preparation and initial characterization of inclusion complex between nitrofurazone and 2-hydroxypropyl-Beta-cyclodextrin

Nitrofurazona(NF), 5-nitro-2-furaldeído semicarbazona, é um antibiótico de amplo espectro, que apresenta diversos efeitos tóxicos e baixa solubilidade aquosa. A complexação da NF com ciclodextrinas é de grande interesse para o desenvolvimento de uma formulação para este antibiótico que seja mais segura e eficiente. Neste trabalho foi realizada a preparação e caracterização inicial do complexo de inclusão entre NF e hidroxipropil-Beta-ciclodextrina (HP-Beta-CD) através de experimentos para determinação da cinética de complexação, medidas de fotoestabilidade, medidas de constante de afinidade fármaco: ciclodextrina, ensaios de liberação in vitro, estequiometria de formação do complexo e morfologia do complexo por microscopia eletrônica de varredura. Os ensaios de cinética de complexação mostram que para o complexo atingir o equilíbrio são necessárias 17,3h. As isotermas de solubilidade determinadas para a NF em função da temperatura mostraram perfis do tipo A e B indicando que a temperatura é um fator importante na complexação da NF com ciclodextrina. Os experimentos de fotoestabilidade indicam que a inserção da molécula de NF na cavidade interna da ciclodextrina protege o fármaco da fotodecomposição. A cinética de liberação mostra que o perfil de liberação do fármaco é modificado pela presença da ciclodextrina no meio. A estequiometria de complexação entre NF e HP-Beta-CD determinada foi de 1:1 NF:HP-Beta-CD. Os resultados demicroscopia eletrônica de varredura indicam alterações na estrutura cristalina da NF em presença deciclodextrina. Este estudo está baseado na caracterização físico-química da complexação entre NF e HP-Beta-CD podendo ser uma nova potencial opção para utilização terapêutica do NF.

Nitrofurazone (NF), 5-nitro-2-furaldehyde semicarbazone, a broad-spectrum antibiotic, has reported toxic effects and low solubility in water. It would be of great interest to form inclusion complexes between NF and a cyclodextrin, to develop more effective and safer antibiotic formulations. This paper focuses on the preparation of inclusion complexes of NF with 2- hydroxypropyl- -cyclodextrin (HP- -CD) and their initial characterization by evaluating rates of complex formation, photostability, solubility isotherms, release rate profiles, stoichiometry of the complexes and their morphology, as revealed by scanning electron microscopy. The kinetic tests of complex formation revealed that 17,3 h is enough for stabilization of the NFcyclodextrin complex. The solubility isotherm studies showed that the isotherm changes from type A to type B, as a function of temperature. The photostability experiments showed that the insertion of the NF in the HP- -CD cavity protects the drug from photodecomposition. The release kinetic tests showed that the profile of NF release from the complex is altered by the presence of HP- -CD in the medium. A Job's plot indicated that the stoichiometry of the complex was 1:1 NF:HP- -CD. The scanning electron micrographs showed changes in the crystal structure of NF in the complex. This study focused on the physicochemical properties of drug-delivery formulations that could potentially be developed into a novel type of therapy with NF.

A duplicated nitrotienyl derivative with antimycobacterial activity: synthesis, X-ray crystallography, biological and mutagenic activity tests.

A duplicated nitrotienyl derivative was obtained as a by-product from the synthesis of a proposed molecular hybrid of a nitrotienyl derivative and isoniazid with an expected dual antimycobacteria mechanism. The structure was shown to be the 5,5'-dinitro-2-(2,3-diaza-4-(2'-tienyl)buta-1,3-dienyl)tiophene by X-ray crystallography. The minimal inhibitory concentration (MIC) determination of this compound proved to be promising against Mycobacterium pathogenic strains such as M. avium and M. kansasii, although it had a high level of mutagenicity, as observed in mutagenic activity tests.

Synthesis and characterization of membranes obtained by graft copolymerization of 2-hydroxyethyl methacrylate and acrylic acid onto chitosan.

Chitosan based membranes to be applied on wound healing as topical drug delivery systems were developed by graft copolymerization of acrylic acid (AA) and 2-hydroxyethyl methacrylate (HEMA) onto chitosan using cerium ammonium nitrate as chemical initiator. Evidence for graft copolymerization of the vinyl monomers onto chitosan was obtained by FTIR and DMTA. Swelling degree, cytotoxicity, thrombogenicity and haemolytic activity of these membranes were evaluated. Chitosan-graft-AA-graft-HEMA showed to be the best matrix for drug delivery systems than chitosan-graft-AA because it retains good swelling properties, but the content in HEMA has improved cytocompatibility, hemocompatibility and thrombogenic character.

Voltammetric behavior of benznidazole at a DNA-electrochemical biosensor.

Benznidazole is a drug used commonly as a therapeutic agent against Chagas' disease in Brazil. To clarify the cytotoxic action of benznidazole the electrochemical reduction of benznidazole has been investigated using a DNA-electrochemical biosensor, prepared by modification of a glassy carbon electrode with DNA, and the results compared with reduction at a bare glassy carbon electrode. The dependence of peak potential with pH follows slopes of 59 and 52 mV per pH unit in acid media, respectively, which corresponds to a mechanism involving the same number of electrons and protons. In neutral and alkaline solution no significant dependence of peak potential with pH was found. During the electrochemical reduction of benznidazole the formation of the hydroxylamine derivative occurs, involving a total of four electrons. The potentials for reduction were less negative when using the DNA-modified glassy carbon electrode than at the bare glassy carbon electrode although the mechanism was the same, and at pH 7.51 the peak current was four times higher than that obtained with the bare electrode. The DNA-biosensor enabled pre-concentration of the drug onto the electrode surface and the in situ damage caused to the DNA on the electrode surface by the product of benznidazole reduction could be detected electrochemically. The results are in agreement with the hypothesis that the hydroxylamine derivative is the reactive species responsible for the cytotoxic action of benznidazole.

An approach for the rational design of new antituberculosis agents.

Tuberculosis (TB) kills more youth and adults than any other infectious disease in the world today. The emergence of new strains of Mycobacterium tuberculosis resistant to some or all current antituberculosis drugs is a serious and crescent problem. The resistance is often a corollary to HIV infection and drug-resistant TB is more difficult and more expensive to treat, besides to be more likely fatal. Thus, it is still necessary to search for new antimycobacterial agents. The identification of novel targets need the identification of biochemical pathways specific to mycobacteria and related organisms. Many unique metabolic processes occur during the biosynthesis of mycobacterial cell wall components. In this report, we examine one of these attractive targets for the rational design of new antituberculosis agents--the mycolic acids.

Leishmaniasis phytotherapy. Nature's leadership against an ancient disease.

The use of phytotherapy to treat human diseases has its roots in pre-historical times. Despite the modern advances achieved in the field of synthetic chemistry, the most efficient drugs available have their genesis directly or indirectly related with the vegetal kingdom. Indigenous communities have long used plant extracts to treat illnesses. Many of these extracts have shown effective action, with new bioactive compounds being extracted and screened every year. These extracts have also proven to be good sources of therapeutic agents to the treatment of Leishmaniasis. This work highlights some of these agents, while trying to emphasize the importance of plants as a source of new and powerful drugs against this widespread disease.

In vitro evaluation of quinidine sensitivity in Brazilian Plasmodium falciparum isolates: comparative analysis to quinine and chloroquine.

Falciparum malaria represents a serious and an increasing world public health problem due to the acquired parasite's resistance to the most available drugs. In some endemic areas, quinidine, a diastereoisomer of the antimalarial quinine, has been employed for replacing the latter. In order to evaluate the use of quinidine as an alternative to the increasing loss of quinine effectiveness in Brazilian P. falciparum strains, as has been observed in the Amazon area, we have assayed quinidine, quinine and chloroquine. The in vitro microtechnique was employed. All isolates showed to be highly resistant to chloroquine. Resistance to quinine was not noted although high MIC (minimal inhibitory concentration) values have been observed. These data corroborate the decreasing sensitivity to quinine in strains from Brazil. Quinidine showed IC50 from 0.053 to 4.577 micromol/L of blood while IC50 from 0.053 to 8.132 micromol/L of blood was estimated for quinine. Moreover, clearance of the parasitemia was observed in concentrations lower than that used for quinidine in antiarrhythmic therapy, confirming our previous data. The results were similar to African isolate.

Potential tuberculostatic agents: micelle-forming copolymer poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid.

With the objective of obtaining slow-acting isoniazid derivatives, of potential use as chemoprophylactics or chemotherapeutics in tuberculosis, the micelle-forming copolymer of poly(ethylene glycol)-poly(aspartic acid) prodrug with isoniazid was synthesized. The derivative obtained was found to be active in Mycobacterium tuberculosis culture, with a minimal inhibitory concentration (MIC) 5.6 times lower than that of the tuberculostatic drug.

Dissociation and electrooxidation of primaquine diphosphate as an approach to the study of anti-chagas prodrugs mechanism of action.

This paper describes the voltammetric behavior of primaquine as a previous support to the further understanding of the delivery and action mechanisms of its respective synthesized prodrugs. There are few papers describing the drug behavior and most of the time no correlation between oxidation process and pH is done. Our results showed that primaquine oxidation is a one-step reaction involving two electrons with the charge transfer process being strongly pH-dependent in acid medium and pH-independent in a weak basic medium, with the neutral form being easily oxidized. This leads to the conclusion that quinoline nitrogen ring neutralization is a determinant step to the formation of the oxidized primaquine form. The existence of a relationship between the primaquine dissociation equilibrium and its electrooxidation process is shown. This work points the importance of voltammetric methodology as a tool for further studies on quantitative relationship studies between chemical structure and biological activity (QSAR) for electroactive drugs.

In vitro evaluation of erythromycin in chloroquine resistant brazilian P. falciparum freshly isolates: modulating effect and antimalarial activity evidence.

Erythromycin, a reversal agent in multidrug-resistant cancer, was assayed in chloroquine resistance modulation. The in vitro microtechnique for drug susceptibility was employed using two freshly isolates of Plasmodium falciparum from North of Brazil. The antimalarial effect of the drug was confirmed, with an IC50 estimates near the usual antimicrobial therapy concentration, and a significant statistical modulating action was observed for one isolate.

Water Soluble Cyclophosphamide Adducts of Rhodium(II) Keto-Gluconate and Glucuronate. Synthesis, Characterization and In Vitro Cytostatic Assays.

The synthesis, characterization and biological assays of two new rhodium carboxylate sugar derivatives and respective cyclosphosphamide adducts are described. The compounds, characterized by (13)C and (1)H NMR, infrared and UV-visible spectra, presented high water solubility and hydration grades were confirmed given the concordance between thermal and CHN analyses. The adducts were active in vitro against K-562 cells.

Synthesis and in vitro evaluation of potential antichagasic dipeptide prodrugs of primaquine.

American trypanosomiasis (Chagas' disease) is an endemic parasitic disease afflicting more than 20 million people in Latin America. Currently, therapy is unsatisfactory and only two drugs are available. Primaquine, an antimalarial drug, has trypanocidal activity. Dipeptide derivatives of primaquine, Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ, were synthesized. The choice of the peptides was based on the primary specificity of cruzipain, the major cysteine proteinase from T cruzi. The prodrugs obtained were tested on the LLC-MK2 cell culture infected with trypomastigotes forms of T. cruzi. Phe-Arg-PQ, Lys-Arg-PQ, and Phe-Ala-PQ were active in all stages.

Antimalarial effect in vitro and lack of modulating effect of desipramine and imipramine.

Based on previous studies in vitro of the modulating effect of desipramine on chloroquine-resistance of Plasmodium falciparum, the effect of desipramine and imipramine on freshly isolated resistant Brazilian strains of the parasite was investigated. Both drugs in therapeutic doses showed an unexpected antimalarial effect in vitro in duplicate tests (IC50 = 44.26 and 46.53 micrograms/L for desipramine, and 83.93 and 41.26 micrograms/L for imipramine), but no reversal of resistance when added to cultures together with chloroquine.

Doseamento da cimetidina pelo metodo de Kjeldahl. / Determination of cimetidine by Kjeldahl's method

O nitrogenio contido na molecula da cimetidina foi determinado quantitativamente pelo metodo de Kjeldahl usando quatro condicoes diferentes. A variante que deu melhor resultado consiste em hidrolisar previamente o grupo ciano do farmaco com acido antes de submeter a cimetidina a acao oxidante do acido sulfurico concentrado

Latenciacao de quimioterapicos: preparacao de polimeros sacaridicos de agentes esquistossomicidas e antimalaricos / Latentiation of chemotherapeutical agents: preparation of saccharide polymers of schistosomicidal and antimalaria agents

Visando a obtencao de pro-farmacos de acao prolongada e de menor toxicidade que os prototipos, foram preparados polimeros sacaridicos de agentes esquistossomicidas (anfotalida e pararrosanilina) e de antimalaricos (dapsona, pirimetamina, trimetoprina e sulfadimetoxina). Os produtos foram obtidos mediante reacao do cloroformiato de amido a temperatura ambiente e a 60 graus C com os agentes biologicamente ativos. Dos produtos obtidos, apenas o polimero sacaridico da dapsona preparado a temperatura de 60 graus C (CFA-lt) e o polimero sacaridico da pirimetamina preparado a temperatura ambiente (CFA-3) mostraram-se ativos - o primeiro, parcialmente - quando ensaiados, respectivamente, em malária e esquistossomíase experimentais

Antimalarial activity of saccharidic polymers of dapsone and sulfadimethoxine.

With the purpose of obtaining pro-drugs of dapsone and sulfadimethoxine, those chemotherapeutic agents were attached through covalent bonding to starch polymeric dialdehyde (Sumstar-190). The antimalarial activity of the two resulting compounds - the dapsone saccharidic polymer (PS6) and the sulfadimethoxine saccharidic polymer (PS7) - in mice experimentally inoculated with Plasmodium berghei was significantly increased with this molecular modification. Mice infected with malaria and kept without treatment together with others which received different doses of PS6 and PS7 were also partially or totally cured, possibly due to the ingestion of excrements containing the parent chemotherapeutic agents.