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Neste trabalho foram estudadas, por espectroscopia UV-Vis, as interações do fármaco anti-malária cloroquina com uma série de porfirinas meso fenilas substituídas aniônicas, catiônicas e neutras, e seus complexos Fe(III), em tampão aquoso e em solução metanólica. Com base nos resultados de interação, as ferro(III) porfirinas meso fenilas substiuídas e a FePPIX foram utilizadas como catalisadores para a oxidação de cloroquina por iodosilbenzeno (PhIO) e peróxidos (H2O2 e ácido meta-cloroperbenzóico, CPBA), em ambos os meios. Dos estudos de interação foi observado, em meio aquoso, pH 6,4, a formação de um complexo p-p, resultante do efeito cooperativo das interações eletrostáticas e da interação p-p entre as espécies aniônicas e a cloroquina. Em metanol nenhuma complexação p-p foi observada. As porfirinas e ferro(III) porfirinas neutras e catiônicas não formaram complexos com a cloroquina. As constantes de formação do complexo p-p entre as porfirinas ou ferro(III) porfirinas e cloroquina foram comparáveis áquelas relatadas na literatura para a cloroquina e outras ferro(III) porfirinas naturais. As reações de oxidação foram analisadas por cromatografia líquida de alta eficiência (CLAE). O principal produto obtido em todos os sitemas estudados foi a monodesetilcloroquina. Este é também o principal produto ?in vivo? da oxidação deste fármaco catalisada por CYP-450. A identificação do produto que é gerado em segunda maior quantidade, o derivado desetilamino da cloroquina, foi realizada por CLAE acoplada a espectrometria de massas. Dos resultados das reações de oxidação com PhIO e H2O2 foi possível constatar que: as ferro(III) porfirinas aniônicas são os catalisadores mais eficientes, com rendimentos de até 30 % de monodesetilcloroquina; em solução aquosa elas são seletivas para a deetilação oxidativa da cloroquina, enquanto que em metanol até cinco produtos de oxidação podem ser observados. As ferro(III) porfirinas neutras apresentam baixa atividade catalítica na oxidação da cloroquina por PhIO ou H2O2 pois não interage eletrostaticamente com o substrato. As ferro(III) porfirinas catiônicas não apresentam atividade catalítica devido à repulsão eletrostática com o substrato. As reações de oxidação por CPBA apresentaram baixos rendimentos devido à rápida destruição dos catalisadores quando se utilizou este oxidante. Foram ainda realizados estudos catalíticos utlizando ferro(III) porfirinas suportadas em membrana de polidimetilssiloxano (PDMS) e poli-(1?4)-b-d-glucosamina (quitosana) na oxidação de substratos padrões (ciclocteno, cicloexano e estireno). Destes, apenas a quitosana se mostrou um suporte adequado, resultando em um catalisador eficiente. As ferro(III) porfirinas imobilizadas neste suporte foram utlizadas na oxidação da cloroquina por PhIO, levando aos mesmos rendimentos que estas ferro(III) porfirinas apresentaram em meio homogêneo, mostrando também a dependência interação(FeP:substrato)/atividade catalítica previamente observada para as ferro(III) porfirinas em solução. Os resultados obtidos neste trabalho mostram que as ferro(III) porfirinas aniônicas são eficientes catalisadores biomiméticos da oxidação da cloroquina por PhIO e H2O2, tanto em solução como imobilizados. Além disso, pode-se concluir que a habilidade do catalisador em interagir com o substrato apresenta forte influência na atividade catalítica e deve ser levada em consideração nos estudos de outros sistemas modelos do CYP-450. / In this work, the interaction between the antimalarial drug chloroquine with a series of anionic, cationic, and neutral meso phenyl substitued porphyrins; and their iron(III) complexes, was investigated by UV-Vis spectroscopy, in aqueous buffer and methanolic solution. On the basis of the interaction results, the meso phenyl substitued iron(III) porphyrins and FePPIX were used as catalyst in the oxidation of chloroquine by iodosilbenzene (PhIO) or peroxides (H2O2 e meta-chloroperbenzoic acid, CPBA), in both media. From the interaction studies, it was possible to observe the formation of a p-p complex generated from the cooperative effect of the electrostatic and p-p interactions between the anionic species and chloroquine. In methanol, no complexation was observed. The neutral and cationic porphyrins and iron(III) porphyrins did not form complexes with chloroquine. The association constants of the p-p complex formed between the meso phenyl substitued porphyrins and iron(III) porphyrins with chloroquine were comparable with those reported in the literature for the interaction of chloroquine with others natural iron(III) porphyrins. The oxidation reaction produtcts were analized by high perfomance liquid chromatography (HPLC). The major product obtained in all the studied systems was monodesethylchloroquine. This compound is also the main ?in vivo? product of chloroquine metabolism catalised by CYP-450. The identification of the second most abundant product, the desethylamine derived from chloroquine, was performed by HPLC coupled with mass spectrometry. The results obtained from the oxidation reactions of chloroquine by PhIO and H2O2 indicated that: (i) the anionic iron(III) porphyrins are efficient catalysts, with yields close to 30 % for monodesethylchloroquine; (ii) in aqueous buffer they are selective toward the oxidative N-deethylation of chloroquine, while in methanol up to five oxidation products are obtained. The neutral iron(III) porphyrins presented low catalytic activity for the chloroquine oxidation by PhIO or H2O2 due to the lack of electrostatic interaction between the catalyst and the substrate. (iii) The cationic iron(III) porphyrins did not exhibit any catalytic activity because of electrostatic repulsion tha tales place between the catalyst and the substrate. Catalyst degradation occurred when CPBA was used as oxidant, leading to very low yields of oxidation products. Catalytic studies using iron(III) porphyrins supported in polydimethylsiloxane (PDMS) membranes and in poly-(1?4)-b-d-glucosamine (chitosan) were also performed for the oxidation of standard substrates (cicloctene, ciclohexane and styrene). Only chitosan was considered to be an adequate support, resulting in efficient catalysis. The iron(III) porphyrins immobilized on this support were used for the oxidation of chloroquine by PhIO, leading to the same yields as those obtained with the same iron(III) porphyrins in homogeneous medium. It is noteworthy that the relations between the interaction FeP:substrate and the FeP catalytic activity previously observed for the iron(III) porphyrins in solution was also observed in these heterogeneous catalysis. The results obtained in this work show that anionic iron(III) porphyrins are efficient biomimetic catalysts for the oxidation of chloroquine by PhIO and H2O2, both when supported and in solution. Also, the catalyst ability to interact with the substrate has a strong influence on its activity and must be considered when studying other CYP-450 model systems.

Neste trabalho foram estudadas, por espectroscopia UV-Vis, as interações do fármaco anti-malária cloroquina com uma série de porfirinas meso fenilas substituídas aniônicas, catiônicas e neutras, e seus complexos Fe(III), em tampão aquoso e em solução metanólica. Com base nos resultados de interação, as ferro(III) porfirinas meso fenilas substiuídas e a FePPIX foram utilizadas como catalisadores para a oxidação de cloroquina por iodosilbenzeno (PhIO) e peróxidos (H2O2 e ácido meta-cloroperbenzóico, CPBA), em ambos os meios. Dos estudos de interação foi observado, em meio aquoso, pH 6,4, a formação de um complexo p-p, resultante do efeito cooperativo das interações eletrostáticas e da interação p-p entre as espécies aniônicas e a cloroquina. Em metanol nenhuma complexação p-p foi observada. As porfirinas e ferro(III) porfirinas neutras e catiônicas não formaram complexos com a cloroquina. As constantes de formação do complexo p-p entre as porfirinas ou ferro(III) porfirinas e cloroquina foram comparáveis áquelas relatadas na literatura para a cloroquina e outras ferro(III) porfirinas naturais. As reações de oxidação foram analisadas por cromatografia líquida de alta eficiência (CLAE). O principal produto obtido em todos os sitemas estudados foi a monodesetilcloroquina. Este é também o principal produto ?in vivo? da oxidação deste fármaco catalisada por CYP-450. A identificação do produto que é gerado em segunda maior quantidade, o derivado desetilamino da cloroquina, foi realizada por CLAE acoplada a espectrometria de massas. Dos resultados das reações de oxidação com PhIO e H2O2 foi possível constatar que: as ferro(III) porfirinas aniônicas são os catalisadores mais eficientes, com rendimentos de até 30 % de monodesetilcloroquina; em solução aquosa elas são seletivas para a deetilação oxidativa da cloroquina, enquanto que em metanol até cinco produtos de oxidação podem ser observados. As ferro(III) porfirinas neutras apresentam baixa atividade catalítica na oxidação da cloroquina por PhIO ou H2O2 pois não interage eletrostaticamente com o substrato. As ferro(III) porfirinas catiônicas não apresentam atividade catalítica devido à repulsão eletrostática com o substrato. As reações de oxidação por CPBA apresentaram baixos rendimentos devido à rápida destruição dos catalisadores quando se utilizou este oxidante. Foram ainda realizados estudos catalíticos utlizando ferro(III) porfirinas suportadas em membrana de polidimetilssiloxano (PDMS) e poli-(1?4)-b-d-glucosamina (quitosana) na oxidação de substratos padrões (ciclocteno, cicloexano e estireno). Destes, apenas a quitosana se mostrou um suporte adequado, resultando em um catalisador eficiente. As ferro(III) porfirinas imobilizadas neste suporte foram utlizadas na oxidação da cloroquina por PhIO, levando aos mesmos rendimentos que estas ferro(III) porfirinas apresentaram em meio homogêneo, mostrando também a dependência interação(FeP:substrato)/atividade catalítica previamente observada para as ferro(III) porfirinas em solução. Os resultados obtidos neste trabalho mostram que as ferro(III) porfirinas aniônicas são eficientes catalisadores biomiméticos da oxidação da cloroquina por PhIO e H2O2, tanto em solução como imobilizados. Além disso, pode-se concluir que a habilidade do catalisador em interagir com o substrato apresenta forte influência na atividade catalítica e deve ser levada em consideração nos estudos de outros sistemas modelos do CYP-450. / In this work, the interaction between the antimalarial drug chloroquine with a series of anionic, cationic, and neutral meso phenyl substitued porphyrins; and their iron(III) complexes, was investigated by UV-Vis spectroscopy, in aqueous buffer and methanolic solution. On the basis of the interaction results, the meso phenyl substitued iron(III) porphyrins and FePPIX were used as catalyst in the oxidation of chloroquine by iodosilbenzene (PhIO) or peroxides (H2O2 e meta-chloroperbenzoic acid, CPBA), in both media. From the interaction studies, it was possible to observe the formation of a p-p complex generated from the cooperative effect of the electrostatic and p-p interactions between the anionic species and chloroquine. In methanol, no complexation was observed. The neutral and cationic porphyrins and iron(III) porphyrins did not form complexes with chloroquine. The association constants of the p-p complex formed between the meso phenyl substitued porphyrins and iron(III) porphyrins with chloroquine were comparable with those reported in the literature for the interaction of chloroquine with others natural iron(III) porphyrins. The oxidation reaction produtcts were analized by high perfomance liquid chromatography (HPLC). The major product obtained in all the studied systems was monodesethylchloroquine. This compound is also the main ?in vivo? product of chloroquine metabolism catalised by CYP-450. The identification of the second most abundant product, the desethylamine derived from chloroquine, was performed by HPLC coupled with mass spectrometry. The results obtained from the oxidation reactions of chloroquine by PhIO and H2O2 indicated that: (i) the anionic iron(III) porphyrins are efficient catalysts, with yields close to 30 % for monodesethylchloroquine; (ii) in aqueous buffer they are selective toward the oxidative N-deethylation of chloroquine, while in methanol up to five oxidation products are obtained. The neutral iron(III) porphyrins presented low catalytic activity for the chloroquine oxidation by PhIO or H2O2 due to the lack of electrostatic interaction between the catalyst and the substrate. (iii) The cationic iron(III) porphyrins did not exhibit any catalytic activity because of electrostatic repulsion tha tales place between the catalyst and the substrate. Catalyst degradation occurred when CPBA was used as oxidant, leading to very low yields of oxidation products. Catalytic studies using iron(III) porphyrins supported in polydimethylsiloxane (PDMS) membranes and in poly-(1?4)-b-d-glucosamine (chitosan) were also performed for the oxidation of standard substrates (cicloctene, ciclohexane and styrene). Only chitosan was considered to be an adequate support, resulting in efficient catalysis. The iron(III) porphyrins immobilized on this support were used for the oxidation of chloroquine by PhIO, leading to the same yields as those obtained with the same iron(III) porphyrins in homogeneous medium. It is noteworthy that the relations between the interaction FeP:substrate and the FeP catalytic activity previously observed for the iron(III) porphyrins in solution was also observed in these heterogeneous catalysis. The results obtained in this work show that anionic iron(III) porphyrins are efficient biomimetic catalysts for the oxidation of chloroquine by PhIO and H2O2, both when supported and in solution. Also, the catalyst ability to interact with the substrate has a strong influence on its activity and must be considered when studying other CYP-450 model systems.

Malaria afflicts about 500 million people worldwide thus causing significant global economic toll. The drugs available to treat the disease are rapidly losing their efficacy because of widespread prevalence of drug resistant parasites. Thus there is an urgent need to discover novel malaria therapeutics. This research is focused on to study the properties of a novel naturallike synthetic scaffold and analyze its selectivity, and cellular mechanism of action in Plasmodium falciparum. We have identified a novel compound, 3-amino-2-piperidinequinoline (APQ), which we termed UCF401. APQ demonstrated IC[sub50] at submicromolar concentrations against Plasmodium falciparum using the SYBR Green-I fluorescence assay measuring cellular proliferation. This compound also demonstrated low cytotoxicity against the NIH3T3 and HEPG2 cells using MTS assays, showing an IC50 of 174 [micro]M and 125 [micro]M respectively, suggesting of excellent selectivity. We evaluated the compliance of APQ with Lipinski's parameters and determined the in vitro physicochemical profiles of the compound. Our results show that APQ is a Lipinski parameter compliant and has good physicochemical properties. The cellular mechanism of action of APQ was characterized through the assessment of the effects of the compound at different stages of the parasite's intraerythrocytic life cycle. This assay was done by treating a synchronized cell line with the compound at 5X the IC50 value and then imaging the cells at 12-hour intervals. We found that APQ arrests parasite development at the trophozoite stage. In addition we determined that APQ is parasitocidal after a 96 h exposure. These results demonstrate that APQ can be considered as a validated hit and/or early lead.

As malaria, caused by Plasmodium spp., continues to afflict millions of people worldwide, there is a dire need for the discovery of novel, inexpensive antimalarial drugs. Although there are effective drugs on the market, the consistent development of drug resistant species has decreased their efficacy, further emphasizing that novel therapeutic measures are urgently needed. Natural products provide the most diverse reservoir for the discovery of unique chemical scaffolds with the potential to effectively combat malarial infections, but, due to their complex structures, they often pose extreme challenges to medicinal chemists during pharmacokinetic optimization. In our laboratory we have performed unbiased, cell-based assays of numerous synthetic compounds from chemical libraries enriched with nature-like elements. This screening has led to the discovery of many original chemical scaffolds with promising antimalarial properties. In an attempt to further characterize these scaffolds, the most promising compounds were assayed in order to determine their cytotoxic effects on mammalian cells. In addition, the development of a drug resistant parasite line of Plasmodium falciparum to the most promising compound was done in order to determine the relative probability for parasite resistance development.

The purpose of this study was to describe the characteristics and findings of antimalarial drug efficacy studies conducted in Ethiopia and to use the findings to formulate recommendations for antimalarial drug efficacy monitoring and use of evidence to inform antimalarial treatment policy for the Ethiopian setting. This study reviewed 44 antimalarial efficacy studies conducted in Ethiopia from 1974 to 2011. The analysis of results indicated that chloroquine as the first-line antimalarial drug for the treatment of malaria due to Plasmodium falciparum had a 22% therapeutic failure in 1985. Chloroquine was replaced with sulfadoxine-pyrimethamine in 1998, more than 12 years later, when its therapeutic failure had reached 65%. Sulfadoxinepyrimethamine at the time of its introduction had a treatment failure of 7.7%; it was replaced after seven years in 2004 by artemether-lumefantrine; by then its treatment failure had reached 36%. The WHO recommends the replacement of a first-line antimalarial drug when more than 10% of treatment failure is reported. The replacement drug should have a therapeutic efficacy of more than 95%; while the change itself should be completed within two years. The prolonged delay to replace failing antimalarial drugs in Ethiopia seems to have been influenced mainly by the lack of systematic antimalarial drug efficacy data collection and pragmatic use of the data and evidence gathered.Almost eight years after its introduction, isolated studies show that the efficacy of artemether-lumefantrine has decreased from 99% in 2003 to around 96.3% in 2008. Though this decrease is not statistically significant (chi-square 1.5; P=0.22) and has not reached the threshold of 10%, it is plausible that its efficacy may drop further. This is mainly due to regulatory provisions in the country that allow marketing of oral artemisinin mono-therapies that are not recommended for malaria treatment, use of less effective antimalarial combination drugs in the neighboring countries and widespread drug quality problems. The situation calls for and this study recommends the establishment of stringent drug efficacy monitoring and early warning system and alignment of the antimalarial drug regulatory practices with recommendations of the WHO. / Health Studies / D. Litt. et Phil. (Health Studies)

The purpose of this study was to describe the characteristics and findings of antimalarial drug efficacy studies conducted in Ethiopia and to use the findings to formulate recommendations for antimalarial drug efficacy monitoring and use of evidence to inform antimalarial treatment policy for the Ethiopian setting. This study reviewed 44 antimalarial efficacy studies conducted in Ethiopia from 1974 to 2011. The analysis of results indicated that chloroquine as the first-line antimalarial drug for the treatment of malaria due to Plasmodium falciparum had a 22% therapeutic failure in 1985. Chloroquine was replaced with sulfadoxine-pyrimethamine in 1998, more than 12 years later, when its therapeutic failure had reached 65%. Sulfadoxinepyrimethamine at the time of its introduction had a treatment failure of 7.7%; it was replaced after seven years in 2004 by artemether-lumefantrine; by then its treatment failure had reached 36%. The WHO recommends the replacement of a first-line antimalarial drug when more than 10% of treatment failure is reported. The replacement drug should have a therapeutic efficacy of more than 95%; while the change itself should be completed within two years. The prolonged delay to replace failing antimalarial drugs in Ethiopia seems to have been influenced mainly by the lack of systematic antimalarial drug efficacy data collection and pragmatic use of the data and evidence gathered.Almost eight years after its introduction, isolated studies show that the efficacy of artemether-lumefantrine has decreased from 99% in 2003 to around 96.3% in 2008. Though this decrease is not statistically significant (chi-square 1.5; P=0.22) and has not reached the threshold of 10%, it is plausible that its efficacy may drop further. This is mainly due to regulatory provisions in the country that allow marketing of oral artemisinin mono-therapies that are not recommended for malaria treatment, use of less effective antimalarial combination drugs in the neighboring countries and widespread drug quality problems. The situation calls for and this study recommends the establishment of stringent drug efficacy monitoring and early warning system and alignment of the antimalarial drug regulatory practices with recommendations of the WHO. / Health Studies / D. Litt. et Phil. (Health Studies)

No / Background Plasmodium falciparum drug resistance is a major public health challenge in sub-Sahara Africa. Many people are now resorting to the use of herbs in managing malaria due to the increasing treatment failures with the conventional drugs. In this study the ethanolic extract of Polyalthia longifolia (Sonn) Thw. var. pendula, a variety fondly used in folklore medicine in Ghana was investigated for potential antimalarial drug development. Method The ethanolic extract of P. longifolia (Sonn) Thw. var. pendula stem bark was screened against the multidrug resistant, K1 strain of P. falciparum by the parasite lactate dehydrogenase (pLDH) assay and a good antiplasmodial activity (IC50 22.04 ± 4.23 µg/ml) was observed which led to further chromatographic analysis in search for actives. Results Bioassay guided fractionation of the extract yielded; three clerodane diterpenes [16-hydroxycleroda-3,13-dien-16,15-olide (1), 16-oxocleroda-3,13E-dien-15-oic acid (2) and 3,16-dihydroxycleroda-4(18),13(14)Z-dien-15,16-olide (3)], a steroid [beta-stigmasterol (4)] and two alkaloids [darienine (5) and stepholidine (6)]. While compounds 4, 5 and 6 exhibited weak antiplasmodial activity (IC50 22–105 µg/ml), the clerodane diterpenes exhibited significantly potent (p<0.005) blood schizonticidal activity (IC50: 3–6 µg/ml). This is the first report of the antiplasmodial activity of compounds 2 and 3. In combination assay with chloroquine, compounds 1, 2, 3 and 5 antagonized the antiplasmodial activity of chloroquine while 4 and 6 demonstrated a synergistic action. Conclusion The potent antiplasmodial activity of the extract of P. longifolia (Sonn) Thw. var. pendula and compounds therein strongly suggests its usefulness as an antimalarial agent and supports its inclusion or exploitation in formulations of herbal remedies for malaria in Ghana.