Artesunate Activation by Heme in an Aqueous Medium

Abstract

The reaction between the antimalarial drug artesunate (ATS) and ferriprotoporphyrin_(IX) (FPIX) in the presence of glutathione (GSH) has been monitored by nuclear magnetic resonance (NMR) spectroscopy. By following the disappearance of resonances of protons near the endoperoxide group in ATS, the rate at which the drug is activated can be directly measured. In an aqueous medium, the rate of ATS activation is limited by the rate of reduction of the FPIX Fe(III) center by GSH. The reaction is observed to slow dramatically in the presence of other heme binding antimalarial drugs. These findings explain the long observed antagonism between artemisinin derivatives and quinoline-based drugs. This discovery suggests that combination therapy that involves artemisinin or any of its derivatives and a quinoline-based drug may be compromised.

Figure 1.

Structures of antimalarial drugs used in this study and their common abbreviations. ATM, ATS and DHA are derivatives of the parent drug ART that are commonly used in the malaria clinic. LUM, PPQ, and AQ are commonly used “partner” drugs in artemisinin combination therapies (ACTs).

Figure 2.

Artesunate (ATS) and the numbering scheme employed in this work.

Figure 3.

The 3-CH3 resonance of ATS before (a) and after (b) reaction with Fe(II)FPIX heme formed in situ from Fe(III)FPIX and GSH. Peaks marked with “*” are tentatively assigned to acetate and acetyl proton.

Figure 4.

(a) NMR resonance signal of 3-CH3 of ATS obtained in 30 minute intervals from an aqueous sample containing FPIX, GSH and ATS, (b) as in (a), but with overnight incubation of FPIX with GSH before adding ATS.

Figure 5.

Conversion of ATS to C4 centered radical ATS vs time with (top, ×) and without (bottom, ◆) prior complete reduction of FPIX by GSH (see text).

Figure 6.

Conversion of ATS to C4 centered radical ATS vs time (top, ◆) and oxidation of GSH into GSSG vs. time (bottom, ×)

Figure 7.

Proposed mechanism for alkylation of FPIX by ATS (see [23])

Figure 8.

The amount of unreacted ATS as a function of time in the absence (●) vs presence of other FPIX - binding antimalarial drugs: CQ; ◇, PPQ; ◆, AQ; ◻, PY; ▴, LUM; +, QN; ▵, PQ; ∎, and 9-epi-quinine (EPI; ◯)

Figure 9.

The amount of unreacted ATS as a function of time in the presence of CQ, at the following CQ:FPIX ratios: 1:1 (●), 1:2 (◯), 1:4 (◻), and 0:1 (∎).

Figure 10.

Proposed structures for FPIX - ATM adducts formed − (854) and + (1092) the presence of EPI (see text).

Figure 11.

ESI - MS peaks observed for FPIX - ATM adduct formation in the presence of EPI (note peaks at 854 and 1092 corresponding to left, right Fig. 10)