Antimalarial assay

An additional step in the radical mechanism has been suggested namely, that collapse of C-4 radical intermediate 234 to a neutral but highly reactive alkoxy-epoxide 235 occurs (Scheme 5).104 Protein alkylation then presumably occurs via 235 and not radical intermediates such as 234. Unfortunately, epoxide 235 is probably too unstable to be handled or identified. In the case of 258 however, we were granted the opportunity to test the hypothesis that an intermediate epoxide was responsible for the mode of action. Of the series of three tetracycles, 258 retained nearly two-thirds of the activity of artemisinin. The Fe(II)-induced rearrangement product 281, a quite stable epoxide was submitted for antimalarial assay and found to be completely devoid of activity. As 258 is a potent antimalarial but the epoxide 281 is not, it seems reasonable to suggest that the antimalarial activity of 258 is unrelated to epoxy intermediates. 

This is achieved in practice by combining the use of a primary antimalarial assay with a secondary assay for general cytotoxicity (109). The primary assay is expected to expose any and all active test substances without regard to selectivity, so as to minimize the risk of omitting potentially effective agents. As a consequence, the hits in this assay will include false positives, i.e., substances which inhibit the growth of Plasmodium parasites and mammalian cells to the... 

Compared with the plethoric studies on antiplasmodial activity against P. falciparum in vitro, in vivo antimalarial assays dedicated to quassinoids are scarce and hardly comparable. Nevertheless, the excellent antimalarial activities highlighted in those studies should open the way to complementary studies of their pharmacokinetics and toxicity. Association with other antimalarials should also be investigated in order to define new therapeutic schemes and to lower the administered doses, increasing drugs tolerability. 

In vivo assays were performed using the standard Peters 4-day test. Ihe activity refers to oral antimalarial activity and reflects the concentration of drug required to reduce parasitaemia, in mice infected with Plasmodium berghei, by 50% of the control. 

Carbenes, dioxirane preparation, 1132 Carbocations, antimalarial endoperoxides, 1309 Carbohydrate hydroperoxides, Mo-catalyzed olefin epoxidation, 432, 436 Carbohydrates, TBARS assay, 669 Carbonate esters, oxidative ozonolysis, 737, 738... 

In Vivo, In Vitro and Chemical Assays for the Detection of Antimalarial and Heme Aggregation Activity. 

As is often the case in multidisciplinary research, a number of different assays have been developed to monitor the inhibition of hemozoin aggregation (Table 1). Consequently, a great deal of confusion has arisen in the literature concerning the exact efficacy of a particular hemozoin inhibitor. For this reason, it is worthwhile to discuss the principal assays employed in the evaluation of antimalarial compounds and those assays specifically designed to evaluate heme aggregation inhibition. 

Table 1. Comparison of IC50 Values of Some Typical Antimalarial Agents from Typical Assay Systems.

Most of the reported in vivo data presented in the literature involves the infection of donor mice with a strain of P. berghe [61]. After a parasitemia of circa 30% is achieved in the donor mice, a blood sample is taken, diluted, and injected into experimental and control mice. The parasitemia load is regularly monitored for 3 to 7 days after infection by blood smear analysis as a function of drug dose. While in vivo efficacy assays reveal the true scope of a potential antimalarial s efficacy, the methods are very expensive. Consequently, additional assays have been developed to rapidly assess the effectiveness of lead heme aggregation inhibitors. 

An important in vitro assay used to evaluate antimalarials is the inhibition of P. falciparum growth in culture. In this assay, parasite cultures are exposed to serial dilution of the potential drugs over 48-72... 

A rapid semiautomated microdilution method for the microbiological assay of the chloroquine has been developed by Desgardins (26). Antimalarial activity of chloroquine may be studied against cultured Plasmodium falciparum, microplates are used to prepare serial dilution of the drug. Parasites obtained from continuous stock cultures are subcultured in the micro-plates for 42 h. Inhibition of uptake of a radio labeled nucleic acid precursor by parasites serves as the indicator of antimicrobial activity. 

Antimalarial ADME/tox assays in vitro in vivo testing, hit L identification A r Virtual screening- Predict antimalarial actives, ADME/tox t properties A... 

In parallel, extensive studies on P. falciparum field isolates in Gabon [140-142], Senegal [143], Cambodia [118, 119, 144], and the Thailand Burmese border [145] corroborated the efficacy of FQ on the parasite whatever its resistance level to chloroquine or to other commonly used antimalarials mefloquine, quinine, halofantrine, and artemisinin derivatives [146, 147]. The cross reactivity observed in some studies with CQ was limited and it was demonstrated that it was caused by differences in initial parasitemia among isolates at the start of the assays [141]. Independance of susceptibility of P. falciparum with phenotypic variation of pfcrt gene, responsible for CQ resistance, could be suspected from these results, but this was demonstrated at the molecular level on Cambodia isolates [148] and extended further on other genes currently involved in resistance to aminoquinoline antimalarials [89, 90]. 

Flotow, H. et al. 2002. Development of a plasmepsin II fluorescence polarization assay suitable for high-throughput antimalarial drug discovery. J. Biomol. Screen. 7, 367-371. 

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