Modification of CYP2B6-mediated site of metabolism on Artemisinin molecule as a way to prolong its maximal antimalarial efficacy
Tsyrlov Ib, Chi-Yuan Woo, V.P. Martin, I.N. Shur · International Journal of Infectious Diseases · 2014
Background: A sesquiterpene endoperoxide lactone, artemisinin (AM), has become first-line natural antimalarial drug highly efficient in areas of multidrug resistance. The AM has a short half-life, and is being used with long acting blood schizontocidal drugs with different targets in the parasite. However, a combination therapy was linked to adverse drug interactions, and attributed to lower efficacy of AM. A unique 1,2,4-trioxane ring is essential for activity of the parent substrate, as all known AM metabolites were inactive due to the endoperoxide bridge was reduced to an epoxide. It was reaffirmed, both in vivo and in human liver microsomes that AM to be principally metabolized by CYP2B6 isozyme. Methods & Materials: Previously, under similar circumstances of highly potent parent drugs, and the CYP2B6 demethylasing them into non-active products, we had developed site-directed modification of a substrate molecule by substituting its methyl groups with isopropyl groups. The latters create steric hindrance at CYP2B6 active site thus affecting ligand binding and lowering catalysis. Such strategy was applied in this study. While AM is biosynthesized in Artemisia annual plant extracts from mevalonic acid via dimethylallyl and isopentenyl pyrophosphates, here C-2 dimethylallyl groups at pyrophosphate were substituted with isopropylallyl groups. Isopropylartemisidin (IPAM) molecule appears to retain an intact 1,2,4-trioxane ring incorporating an endoperoxide bridge determined by a specific LC-MS/MS assay. Results: The IPAM was incubated with human liver microsomes from eight different healthy donors, and concentration-time data were assessed by a first-order depletion model. With correlation to the metabolic rate constants for CYP2B6 probe substrates, efavirenz and bupropion, our data revealed that IPAM was metabolized by CYP2B6 at least 12-13 times slower than AM. Using procedure described by Robert et al. (2005), the hydroxylated and glucuronyl-conjugated derivatives of covalent heme adducts were determined in urine of Plasmodium infected mice by means of LC-MS. Namely, the amount of covalent heme-drug adducts was identically high in infected mice treated with 40 nM AM or 40 nM IPAM, compared to mice treated with 40 nM AM metabolites lacking trioxane ring endoperoxide bridge. Conclusion: IPAM appears to represent a new potent antimalarial drug that can target multiple stages of the parasite's life cycle.