Commercial drugs containing flavonoids are active in mice with malaria and in vitro against chloroquine-resistant Plasmodium falciparum

BACKGROUND The main strategy to control human malaria still relies on specific drug treatment, limited now by Plasmodium falciparum-resistant parasites, including that against artemisinin derivatives. Despite the large number of active compounds described in the literature, few of them reached full development against human malaria. Drug repositioning is a fast and less expensive strategy for antimalarial drug discovery, because these compounds are already approved for human use. OBJECTIVES To identify new antimalarial drugs from compounds commercially available and used for other indications. METHODS Accuvit®, Ginkgo® and Soyfit®, rich in flavonoids, and also the standard flavonoids, hesperidin, quercetin, and genistein were tested against blood cultures of chloroquine-resistant P. falciparum, as well as chloroquine, a reference antimalarial. Inhibition of parasite growth was measured in immunoenzymatic assay with monoclonal anti-P. falciparum antibodies, specific to the histidine-rich protein II. Tests in mice with P. berghei malaria were based on percent of parasitaemia reduction. These compounds were also evaluated for in vitro cytotoxicity. FINDINGS The inhibition of parasite growth in vitro showed that Accuvit® was the most active drug (IC50 5 ± 3.9 μg/mL). Soyfit® was partially active (IC50 13.6 ± 7.7 μg/mL), and Ginkgo® (IC50 38.4 ± 14 μg/mL) was inactive. All such compounds were active in vivo at a dose of 50 mg/kg body weight. Accuvit® and quercetin induced the highest reduction of P. berghei parasitaemia (63% and 53%, respectively) on day 5 after parasite inoculation. As expected, the compounds tested were not toxic. MAIN CONCLUSIONS The antimalarial activity of Accuvit® was not related to flavonoids only, and it possibly results from synergisms with other compounds present in this drug product, such as multivitamins. Multivitamins in Accuvit® may explain its effect against the malaria parasites. This work demonstrated for the first time the activity of these drugs, which are already marketed.

Antiplasmodial activity of iron(II) and ruthenium(II) organometallic complexes against Plasmodium falciparum blood parasites

This work reports the in vitro activity against Plasmodium falciparumblood forms (W2 clone, chloroquine-resistant) of tamoxifen-based compounds and their ferrocenyl (ferrocifens) and ruthenocenyl (ruthenocifens) derivatives, as well as their cytotoxicity against HepG2 human hepatoma cells. Surprisingly with these series, results indicate that the biological activity of ruthenocifens is better than that of ferrocifens and other tamoxifen-like compounds. The synthesis of a new metal-based compound is also described. It was shown, for the first time, that ruthenocifens are good antiplasmodial prototypes. Further studies will be conducted aiming at a better understanding of their mechanism of action and at obtaining new compounds with better therapeutic profile.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Several species of Aspidospermaplants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorumextracts, the plant activity against Plasmodium falciparumwas evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL); the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5) displayed high selectivity indexes (SI) (= 56 and 113, respectively), whereas compounds 2 and 3 were toxic (SI < 10). The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Blood shizonticidal activities of phenazines and naphthoquinoidal compounds against Plasmodium falciparum in vitro and in mice malaria studies

Due to the recent advances of atovaquone, a naphthoquinone, through clinical trials as treatment for malarial infection, 19 quinone derivatives with previously reported structures were also evaluated for blood schizonticide activity against the malaria parasite Plasmodium falciparum. These compounds include 2-hydroxy-3-methylamino naphthoquinones (2-9), lapachol (10), nor-lapachol (11), iso-lapachol (12), phthiocol (13) and phenazines (12-20). Their cytotoxicities were also evaluated against human hepatoma and normal monkey kidney cell lines. Compounds 2 and 5 showed the highest activity against P. falciparum chloroquine-resistant blood-stage parasites (clone W2), indicated by their low inhibitory concentration for 50% (IC50) of parasite growth. The therapeutic potential of the active compounds was evaluated according to the selectivity index, which is a ratio of the cytotoxicity minimum lethal dose which eliminates 50% of cells and the in vitro IC50. Naphthoquinones 2 and 5, with activities similar to the reference antimalarial chloroquine, were also active against malaria in mice and suppressed parasitaemia by more than 60% in contrast to compound 11 which was inactive. Based on their in vitro and in vivo activities, compounds 2 and 5 are considered promising molecules for antimalarial treatment and warrant further study.

Aspidosperma (Apocynaceae) plant cytotoxicity and activity towards malaria parasites. Part I: Aspidosperma nitidum (Benth) used as a remedy to treat fever and malaria in the Amazon

Infusions of Aspidosperma nitidum (Apocynaceae) wood bark are used to treat fever and malaria in the Amazon Region. Several species of this family are known to possess indole alkaloids and other classes of secondary metabolites, whereas terpenoids, an inositol and the indole alkaloids harmane-3 acid and braznitidumine have been described in A. nitidum . In the present study, extracts from the wood bark, leaves and branches of this species were prepared for assays against malaria parasites and cytotoxicity testing using human hepatoma and normal monkey kidney cells. The wood bark extracts were active against Plasmodium falciparum and showed a low cytotoxicity in vitro, whereas the leaf and branch extracts and the pure alkaloid braznitidumine were inactive. A crude methanol extract was subjected to acid-base fractionation aimed at obtaining alkaloid-rich fractions, which were active at low concentrations against P. falciparum and in mice infected with and sensitive Plasmodium berghei parasites. Our data validate the antimalarial usefulness of A. nitidum wood bark, a remedy that can most likely help to control malaria. However, the molecules responsible for this antimalarial activity have not yet been identified. Considering their high selectivity index, the alkaloid-rich fractions from the plant bark might be useful in the development of new antimalarials.

The utility of anti-trypomastigote lytic antibodies for determining cure of Trypanosoma cruzi infections in treated patients: an overview and perspectives

In previous work, we proposed alternative protocols for following patients with treated Chagas disease and these are reviewed herein. Evidence was provided to support the following: (i) functional anti-trypomastigote antibodies are indicative of ongoing chronic Trypanosoma cruzi infections; (ii) specific antibodies detected by conventional serology (CS) with epimastigote extracts, fixed trypomastigotes or other parasite antigens may circulate years after parasite elimination; (iii) functional antibodies are evidenced by complement-mediated lysis of freshly isolated trypomastigotes, a test which is 100 percent specific, highly sensitive, and the first to revert after T. cruzi elimination and (iv) the parasite target for the lytic antibodies is a glycoprotein of high molecular weight (gp160) anchored at the parasite surface. The complement regulatory protein has been cloned, sequenced and produced as a recombinant protein by other groups and is useful for identifying functional anti-T. cruzi antibodies in ELISA tests, thus dispensing with the need for live trypomastigotes to manage treated patients. If used instead of CS to define cures for Chagas patients, ELISA will avoid unnecessary delays in finding anti-T. cruzi drugs. Other highly sensitive techniques for parasite DNA detection, such as PCR, need to be standardized and included in future protocols for the management of patients with drug-treated Chagas disease.

The search for new antimalarial drugs from plants used to treat fever and malaria or plants ramdomly selected: a review

In this review we discuss the ongoing situation of human malaria in the Brazilian Amazon, where it is endemic causing over 610,000 new acute cases yearly, a number which is on the increase. This is partly a result of drug resistant parasites and new antimalarial drugs are urgently needed. The approaches we have used in the search of new drugs during decades are now reviewed and include ethnopharmocology, plants randomly selected, extracts or isolated substances from plants shown to be active against the blood stage parasites in our previous studies. Emphasis is given on the medicinal plant Bidens pilosa, proven to be active against the parasite blood stages in tests using freshly prepared plant extracts. The anti-sporozoite activity of one plant used in the Brazilian endemic area to prevent malaria is also described, the so called "Indian beer" (Ampelozizyphus amazonicus, Rhamnaceae). Freshly prepared extracts from the roots of this plant were totally inactive against blood stage parasites, but active against sporozoites of Plasmodium gallinaceum or the primary exoerythrocytic stages reducing tissue parasitism in inoculated chickens. This result will be of practical importance if confirmed in mammalian malaria. Problems and perspectives in the search for antimalarial drugs are discussed as well as the toxicological and clinical trials to validate some of the active plants for public health use in Brazil

New antimalarial drugs: a search based on plants used in popular medicine to treat fever and malaria

This presentation will be divided into three parts:1-the ongoing situation of human mataria in the Brazilian Amazon, where it is endemic, causing over 500,000 new cases each year; 2 - approaches our group used to find new drugs to treat mataria through experimental models and using medicinal or randomly selected plants; and 3 - our results with extracts or molecules showing antimalarial activities against the blood stages, as well as with one plant active against sporozoites. The latter, a plant used for prophylaxis, is known as "Indian beer" and grows on the margins of the Amazon River. Totally inactive against the blood-stage parasites in mice and chickens and against the human Plasmodium falciparum malaria in cultures, it is active against the sporozoites in chicken mataria. Purification of the active molecule(s) from fresh plants, now in progress, as well as clinical trials for the validation of its popular use in Brazil, are needed.

Immune response to sporozoite and blood stage antigens among subjects exposed to malaria in Brazil: a review

A proteçäo contra malária adquirida por indivíduos expostos em áreas endêmicas é amplamente modulada pelo padräo de transmissäo. No Brasil, crianças e adultos säo igualmente acometidos e a infecçäo tende a ser seguida de doença clínica com sintomas de intensidade variável podendo ocorrer vários episódios sucessivos de malária por Plasmodium vivax e/ou P. falciparum. Entretanto, as taxas de prevalência variam dentro de uma mesma área endêmica sendo os casos relatados distribuídos de forma näo homogênea. Nesta revisäo säo discutidos os estudos atuais sobre a resposta imune humora e celular dirigida contra esporozoítas e formas sagüíneas de P. falciparum e P. vivax entre brasileiros expostos a diferentes situaçoes de transmissäo, enfatizando nossos estudos prévios conduzidos no Estado de Mato Grosso e em um foco de transmissäo localizado fora da área endêmica. O estudo da resposta imune naturalmente adquirida por populações expostas a baixos níveis de transmissäo poderá fornecer informaçoes importantes para o esclarecimento de aspectos relevantes sobre os mecanismos envolvidos na proteçäo contra a doença. Esses conhecimentos somados aos muitos verificados para as áreas hiperendêmicas poderäo permitir a escolha de possíveis vacinas protetoras e também a avaliaçäo adequada de voluntários vacinados nas áreas endêmicas.

Antígenos solúveis liberados por tripomastigotas de Trypanosoma cruzi utilizados no teste de Elisa para detectar cura em pacientes chagásicos após tratamento específico / Soluble antigens released by Trypanosoma cruzi trypomastigotes used Elisa to detect cure in chagasic patients following specific treatment

Dois antígenos solúveis de tripomastigotas do Trypanosoma cruzi, um obtido de sobrenadantes de culturas celulares (AgSb) e o outro excretado/secretado por essas formas em meio de cultura (AgES), foram avaliados em um teste de ELISA para o diagnóstico da infecçao chagásica e controle de cura de pacientes tratados. Os pacientes tratados apresentavam testes de lise mediada pelo complemento e hemoculturas repetidamente negativos, apesar de permanecerem com a sorologia convencional positiva (pacientes dissociados). O teste de lise negativo indica que estes pacientes eliminaram a infecçao. Entre os controles com infecçao ativa, os AgSb e os AgES detectaram respectivamente 93 e 100 por cento dos casos. No entanto, entre os pacientes dissociados, o teste de ELISA, utilizando os AgSb e AgES, foi positivo com 28 por cento e 5 por cento dos soros, respectivamente. Portanto, este teste com os AgES é indicado para o controle de cura da doença de Chagas, podendo vir a substituir a reaçao de lise mediada pelo complemento no acompanhamento sorológico individual de pacientes tratados.

The immune response to malaria sporozoite antigens in animal models and humans: a retrospective overview and present goals

Research and ultimate goals of our sdutides in mamaria immunity is reviewed. A new experimental vector for Plasmodium gallinaceum, an avian malaria, the mosquito Aedes fluviatilis is described. Sporozoites recovered from oocyst (OoS), before they enter salivary glands, are infective for chickens although somewhat less so than the mature salivary gland sporozoites (SGS). The repetitive portion of the circumsporozoite (CS) membrane protein is not responsible for migration/maturation of the OoS into the salivary glands or for infectivity for the vertebrate although present in both populations. The older sporozoites, recovered from mosquitos 4-5 weeks after infection, gradually lose infectivity for chickens. An age-dependent response of chickens to a sporozoite-induced malaria is described, young animals being more susceptible. Monoclonal antibodies (MoAb) have been produced against surface antigens of P. gallinaceum sporozoites, epecific for OoS and SGS antigens, as judged by indirect immunofluorescence and circumsporozoite precipitation tests. By Western blot or by competitive ELISA, these MoAbs recognize the repeat epitope of the CS protein, two polypeptides of molecular weight 64 an 76 kDa, as do the sera from mice immunized with sporozoites. Tested against live sporozoites, these MoAb showed variable protective activity, some of them totally inhibiting parasite infectivity in in vivo and in vitro tests. Cross-reactivity occurred between these MoAb and sporozoites of Plasmodium berghei as well as with their CS protein repeats but no cross-protection was demonstrated against this rodent malaria. Our studies in human malaria in the Amazon area are restricted to the last two years, and to a small sample (180) of individuals recently exposed to transmission of Plasmodium falciparum and P. vivax. A variable but low number (9-30 percent) had anti-CS antibodies and a positive cellular immune response (30-40 percent) in vitro to recombinant CS proteins (of P. falciparum and P. vivax). We found no correlation between these two parameters; between antigen responsiveness and time of exposure to malaria transmission (up to ten years); or between the number of previous malaria infections. In previous studies we showed that individulas who had been exposed but did not acquire malaria, in a focal area of transmission in Minas Gerais, had antisporozoite antibodies...

Hemocultura: técnica sensível na detecçäo do Trypanosoma cruzi em pacientes chagásicos na fase crônica da doença de chagas / Hemoculture: sensitive technique in the detection of trypanosoma cruzi in chagasic patients in the chronic phase of Chagas disease

A sensibilidade de hemoculturas, realizadas uma ou três vezes, foi estudada em 52 pacientes na fase crônica da doença de Chagas. Modificaçoes foram introduzidas na técnica tais como, diminuiçao do período de processamento do sangue, homogeneizaçao suave e exame até 120 dias do cultivo. Os resultados mostram alto percentual de positividade, ou seja, 79 por cento e 94 por cento dos pacientes foram positivos, respectivamente, com um ou três testes. A julgar pelo número de tubos positivos, em cada paciente, a parasitemia foi baixa em 59 por cento deles, média em 16 por cento e alta em 25 por cento. Nao houve diferenças significativas nos resultados positivos em funçao da idade dos pacientes, que variou de 14 a 82 anos. Nossos resultados demonstram que a hemocultura é uma metodologia sensível para o diagnóstico parasitológico da doença de Chagas e ideal para monitorar cura em pacientes submetidos a tratamento.

Antimalarial chemotherapy with natural products and chemically defined molecules

In the present work we have described the in vivo antimalarial actrivity of six different plants. Two of them (Verninia brasiliana and Eupatorium squalidum) were tested in a randomic approach among 273 crude extracts from plants; four (Acanhospermum australe, Esenbeckia febrifuga, Lisianthus specious and Tachia guianensis) were selected after screening 22 crude extracts from different medicinal and some of them showed antimalarial activity in vitro. Some aspects of recent research with natural products aiming to produce drugs are discussed.

Effects of levamisole on experimental infections by Plasmodium berghei in mice

Levamisol (fenilimidotiazol), considerado um potente imunoestimulante, quando administrado a camundongos suíços näo causou aumento significante nos pesos do timo, figado, ou baço, apesar de a droga ter sido usada em diferentes tempos antes da remoçäo desses órgäos. Doses elevadas da droga usadas no esquema profilático de 4 dias näo tiveram efeito antimalárico. Entretanto quando dada a camundongos com malária, 24 horas antes, ao mesmo tempo ou 24 horas após inoculaçäo de uma cepa de Plasmodium berghei cloroquina-sensível ou uma cepa cloroquina resistente o levamisol reduziu, ainda que discretamente, a parasitemia nos graus tratados, sendo a dose de 1 mg/kg o melhor esquema. Foi observado também atraso na mortalidade por malária nos grupos tratados com o levamisol. No entanto, todos os animais morreram. Os dados sugerem que o levamisol tem efeito imunoestimulante, ainda que discreto, na resposta imune de animais, deprimida pela malária