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Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

Mixed tetraoxanes 5a and 13 synthesized from cholic acid and 4-oxocyclohexanecarboxylic acid were as active as artemisinin against chloroquine-susceptible, chloroquine-resistant, and multidrug-resistant Plasmodium falciparum strains (IC50, IC90). Most active 13 is metabolically stable in in vitro metabolism studies. In vivo studies on tetraoxanes with a C(4‘ ‘) methyl group afforded compound 15, which cured 4/5 mice at 600 and 200 mg·kg-1·day-1, and 2/5 mice at 50 mg·kg-1·day-1, showing no toxic effects. Tetraoxane 19 was an extremely active antiproliferative with LC50 of 17 nM and maximum tolerated dose of 400 mg/kg. In Fe(II)-induced scission of tetraoxane antimalarials only RO• radicals were detected by EPR experiments. This finding and the indication of Fe(IV)&dbd;O species led us to propose that RO• radicals are probably capable of inducing the parasite's death. Our results suggest that C radicals are possibly not the only lethal species derived from peroxide prodrug antimalarials, as currently believed.

The high level of attrition of drugs in clinical development has led pharmaceutical companies to increase the efficiency of their lead identification and development through techniques such as combinatorial chemistry and high-throughput (HTP) screening. Since the major reasons for clinical drug candidate failure other than efficacy are pharmacokinetics and toxicity, attention has been focused on assessing properties such as metabolic stability, drug-drug interactions (DDI), and absorption earlier in the drug discovery process. Animal studies are simply too labor-intensive and expensive to use for evaluating every hit, so it has been necessary to develop and implement higher throughput in vitro ADME screens to manage the large number of compounds of interest. The antimalarial drug development program at the Walter Reed Army Institute of Research, Division of Experimental Therapeutics (WRAIR / ET) has adopted this paradigm in its search for a long-term prophylactic for the prevention of malaria. The overarching goal of this program is to develop new, long half-life, orally bioavailable compounds with potent intrinsic activity against liver- and blood-stage parasites. From the WRAIR HTP antimalarial screen, numerous compounds are regularly identified with potent activity. These hits are now immediately evaluated using a panel of in vitro ADME screens to identify and predict compounds that will meet our specific treatment criteria. In this review, the WRAIR ADME screening program for antimalarial drugs is described as well as how we have implemented it to predict the ADME properties of small molecule for the identification of promising drug candidates.