Interaction of quinolines and artemisinin based antimalarials drugs with ferriprotoporphyrin IX

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4.4.3 HPLC/MS analysis of hemin-artemisinin based drugs interaction

As already reported by some researchers, artemisinin reacts with heme (Fe^II) but do not react with metal (III) porphyrins (like hemin (Fe^III), (Mn^III) tetraphenylporphyrin ( Robert et al., 1997, Cazelles, 2001; Robert et al., 2002).

Based on the findings of Bilia and co-workers (Bilia et al., 2002) who reported formation of two isomers artemisinin-hemin covalent adduct in DMSO solutions and based on the fact that, so far most of the reported information center on artemisinin, studies on artesunate and dihydroartemisinin are few. Then, we have proposed to investigate the formation of supramolecular adducts between artemisinin derivatives like artesunate and dihydroartemisinin) by HPLC/MS and HPLC/DAD/MS spectrometry using DMSO solutions incubated at 37^oC over 12 h period. The structures of hemin and related artemisinin drugs are represented in Scheme 4-2. For the HPLC/MS analysis, 0.002 M DMSO solutions of drugs and 0.001 M DMSO solutions of hemin were used. The reported results showed that hemin and drug slowly react to give rise to supramolecular adducts. Hemin no mixed to drug showed a peak with a retention time (Rt) of 38.95 min. His MS spectra is characterized by the molecular mass of 616.5 m/z (M⁺-35.5) corresponding to fragment with loss of chloride atom as shown in figure 4-10. After drug addition, we found a chromatographically similar product with an apparent molecular mass close to the theoretical value of 898 (for ART), 900.5 (for DHA), 1000.5 (for AST), indicating a 1:1 heme-artemisinin compounds adduct. In the case of artesunate, three more peaks with Rt 41.26, 49.37, and 53.23 min. Their MS spectra were similar and characterized by four fragments at 1000.5, 940.5, 882.5 and 822.4 m/z as shown in figure 4-11. The first corresponding exactly to a supramolecular complex, result of addition of artesunate (m/z=384.4) with hemin (m/z=616.3). The three latter corresponding to the covalent adducts between a modified artemisinin and hemin without extensive degradation of both partners. [M⁺-60= 940.5] corresponds to an adduct with likely loss of CH₃COOH, [M⁺-118=882.5] to an adduct with loss of HO₂C(CH₂)₂CO₂H (succinic acid) as expected from succinate containing molecule (case of artesunate). [M⁺-178= 882.5] corresponds to an adduct with both loss of CH₃COOH and HO₂C(CH₂)₂CO₂H. These data evidenced the presence of three supramolecular complexes represented by three isomers.

Artesunate Dihydroartemisinin Artemisinin

Scheme 4-2 Structures of hemin and related artemisinin based drugs.

Hemin

Figure 4-10 HPLC-MS spectra of hemin.

Figure 4-11 HPLC-MS spectra of artesunate-hemin complex

We have the similar results with dihydroartemisinin. The HPLC/DAD/MS spectra analysis of DHA showed two kind of peaks: one with Rt 33.5 (m/z=616.5) corresponding to hemin spectra and another peaks with Rt 42.1; 46.6; 51.2; 54.8; 61.1; 64.1; 69.8; 75.7 min. These eight latter have all similar MS spectra and exhibited one intense peak at m/z = 840 m/z. These peaks are attributed to dihydroartemisinin-hemin adduct, resulting from addition of DHA (m/z=284.4) with hemin (m/z=616.3) followed with loss of a fragment of 60 m/z most likely due to loss of CH₃COOH as expected for an acetate containing molecule. These data evidenced the presence of eight supramolecular complexes represented by eight isomers (appendix 2).

Paradoxically to the findings of Bilia et co-workers, we identified seven isomeric hemin-artemisinin covalent adduct with retention time at 37.9; 39.4; 41.2; 42.5; 45.6; 47and 48.6 min as shown at figure. The molecular peak was detected at m/z=838.8 (M⁺), corresponding to the expected mass for an adduct hemin (m/z=616.5)/artemisinin (m/z=283.3) which, lost a fragment of acetic acid molecule within the mass spectrometer (appendix 3).

Consequently, we supported that, both hemin and heme can react with endoperoxide lactone based drugs. Based on literature, it seems heme reacts more quickly with artemisinin compounds than hemin does. But, ours findings suggest that it is useful in the understanding of mechanism of action artemisinin drug to always take account of that hemin which, is metal (III) porphyrin can form with those drugs adduct products.