The antimalarial trioxaquine DU 1301 alkylates heme in malaria - infected 1 mice 2 3

18 The in vivo alkylation of heme by the antimalarial trioxaquine DU1301 afforded covalent 19 heme-drug adducts that have been detected in the spleen of malaria-infected mice. This result 20 indicates that the alkylation capacity of trioxaquines in mammals infected by Plasmodium is 21 similar to that of artemisinin, a natural antimalarial trioxane-containing drug. 22 AC CE PT ED Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Antimicrob. Agents Chemother. doi:10.1128/AAC.00165-08 AAC Accepts, published online ahead of print on 16 June 2008 on D ecem er 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom Ms AAC00165-08 Version 3, May 20th, 2008. 2 Artemisinin and its derivatives have emerged as antimalarial drugs over the past three decades. 23 With a pharmacophore based on a 1,2,4-trioxane, these drugs are highly active against 24 multidrug resistant Plasmodium strains, and are non-toxic, even for children and pregnant 25 women. In addition, no clinically relevant parasite resistance has been reported so far (7, 24, 26 25). Many synthetic peroxides have been synthesized as artemisinin models in the last 15-20 27 years (for recent reviews, see 8, 15, 22). Among them, trioxaquines ® have a unique structure, 28 containing both an aminoquinoline moiety (as in chloroquine) and a synthetic 1,2,4-trioxane 29 entity (as an artemisinin mimic) (2, 4). As previously described (4), trioxaquine DU1301 has 30 been obtained by convergent synthesis from 7-chloro-4-(2-aminoethylamino)quinoline and a 31 1,2,4-trioxane derived from ascaridole (20, 10). Trioxaquines are able to cure malaria infected 32 mice treated orally at 15-20 mg/kg/day for four days ("4-day suppressive test", see ref. 16). 33 Trioxaquines are being developed by Palumed, and one of them (PA1103-SAR116242) is 34 currently under regulatory pre-clinical development with Sanofi-Aventis (13 and refs. therein). 35 The mechanism of action of antimalarial 1,2,4-trioxanes has been intensively studied 36 during the last two decades. The crucial role of heme digestion/aggregation processes in 37 infected erythrocytes has led to investigations of the possible interaction of heme with 38 artemisinin. In the initial work, it was reported that heme-catalyzed peroxide cleavage is 39 responsible for the alkylation of heme and specific parasite proteins (1, 6). Such iron(II) 40 mediated-cleavage of the endoperoxide function generates, in particular, an alkyl radical 41 centered at position C4 of artemisinin or synthetic trioxanes (9, 17). The concentration of free 42 iron ions in living cells is close to zero, whereas hemoglobin concentration is 5 mM in red 43 blood cells (corresponding to a heme concentration of 20 mM). In addition, the extensive 44 ingestion of host hemoglobin by Plasmodium within its food vacuole leads to vacuolar heme 45 concentrations of about 400 mM (21). Bearing in mind the important role of free heme in 46 infected erythrocytes, we reported that artemisinin is efficiently activated by heme in vitro and 47 also in vivo, leading to the alkylation of the heme macrocycle (18, 19). Covalent heme-drug 48 adducts resulting from the alkylation by the drug were detected in the spleen and urine of 49 malaria-infected mice (18). Yet, the exact role of heme-artemisinin adducts in the death of the 50 parasite will probably remain a matter of debate. Nevertheless, it is now well-established that 51 artemisinin alkylates heme with its C4-centered radical, and, as underlined in a recent 52 publication, free heme or hemoglobin heme alkylations are "possibly the only malaria-parasite53 relevant fully characterized alkylation reactions reported so far for [artemisinin]" (23). 54 AC CE PT ED on D ecem er 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom Ms AAC00165-08 Version 3, May 20th, 2008. 3 Trioxaquine DU1301 was designed as a potential alkylating agent of heme, and such an 55 alkylating ability has been evidenced in vitro (11). To confirm that the formation of heme56 trioxaquine adducts is a biologically relevant process, we examined the in vivo alkylating 57 ability of DU1301, a representative member of this new class of antimalarial drugs in parasite58 infected mice. 59 60 To address the question of in vivo alkylating properties of trioxaquines, Plasmodium 61 vinckei petteri-infected mice were treated orally with DU1301, a trioxaquine prototype (Figure 62 1). A set of three Swiss female albino mice (25-30 g) were i.p. inoculated with erythrocytes 63 parasitized by P. vinckei petteri. When the parasitemia (determined microscopically on Giemsa 64 thin blood smears) was higher than 40% (after 3 days), mice were treated orally with a single 65 dose of trioxaquine DU1301 (2.5 mg diluted in 100 μL of DMSO, corresponding to a dose of 66 100 mg/kg). As control experiments, a set of three healthy mice received the same dose of 67 DU1301 and a set of infected mice received pure DMSO (no treatment). Mice were sacrificed 68 2h after treatment and organs (spleen, liver and kidneys) were collected. 69 The detection of heme-DU1301 adducts in mouse organ extracts first required an 70 efficient extraction of these hydrophobic compounds from biological tissues. Each organ was 71 crushed with sand in a mortar. Heme-DU1301 adducts are poorly soluble in common aqueous 72 and organic solvents, and stacking of heme with quinoline moieties could account for this low 73 solubility (11). Strong π−π interactions between heme and the quinoline ring of antimalarials 74 has been previously evidenced by NMR analysis (14). Because of the low solubility of these 75 heme-DU1301 adducts, pyridine was found to be the best solvent for their extraction (1.0 to 76 1.5 mL, vortex for 20 min). The organ extracts were analyzed by LC-MS without any 77 additional treatment. 78 The characterization of small amounts of hydrophobic adducts having a low solubility 79 in biological extracts, was much more complicated than the “usual” in vitro characterization of 80 adducts prepared on the bench on a 10-100 mg scale (11). Because of the difficulties with the 81 organ extracts, it was essential to optimize the analytical conditions using chemically prepared 82 adducts (11), in order to have reliable, accurate and sensitive detection of the in vivo adducts. 83 Analytical separations were performed on a 5-μm C18 Modulo-Cart QS Uptisphere column 84 (250 x 4.6 mm) equipped with a pre-column with the same packing material. Compounds were 85 eluted in the following conditions: solvent (A) = MeOH/H2O/TFA, 70/30/0.05, solvent (B) = 86 AC CE PT ED on D ecem er 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom Ms AAC00165-08 Version 3, May 20th, 2008. 4 MeOH/TFA, 100/0.05; gradient: from A/B = 100/0 to A/B = 0/100 over 25 min, followed by 87 10 min at A/B = 0/100; elution rate was 0.5 mL/min and UV/visible detection was performed 88 at 340 and 400 nm using a diode-array detector. The injected volume of pyridine extract was 89 100 μL. Positive-ion electrospray mass spectrometry was performed on a Q-Trap AB Sciex 90 quadrupole instrument (Figure 2). 91 92 The LC-MS analysis of spleen extracts of a malaria-infected mouse treated with 93 trioxaquine DU1301 (100 mg/kg/100 μL of DMSO per os) is reported in Figure 3a-c. Currents 94 of ions with m/z = 1101.0 and 949.2, which correspond to adducts 1 and 2, respectively, were 95 detected with retention times of 18.5 21.7 min, along with heme (tR = 16.5 min). Retention 96 times and mass spectra were consistent with those observed with chemically prepared adducts 97 (11). Furthermore, the isotopic pattern of signals at m/z 1101.1 and 949.2 clearly showed the 98 presence of both one iron atom (M-2 due to 54Fe) and one chlorine atom (M+2 due to 37Cl) 99 (Figure 2). This result is fully consistent with a reductive activation of the peroxide bond of 100 trioxaquine DU1301 by iron(II)-heme occurring within infected erythrocytes. This reaction 101 leads to the formation of an oxygen-centered radical that rapidly rearranges to give an alkyl 102 radical (Figure 1) able to alkylate heme at meso positions, giving rise to the “complete” 103 covalent adduct heme-DU1301, 1. Heme-drug adduct 2, generated by hydrolysis and loss of 104 the terpene moiety, probably in the spectrometer, was also detected. 105 Adducts 1 and 2 were both detected in all infected mice treated with DU1301. In the 106 controls, these heme-trioxaquine adducts were undetectable in all spleen extracts of healthy 107 mice treated under the same conditions (Figure 3d-e), and in all spleen extracts of untreated 108 (only excipient) malaria-infected mice (data not shown). 109 Heme-DU1301 adducts remained below detectable levels in the kidneys and urine of 110 malaria infected mice that had been treated with trioxaquine. However, it should be considered 111 that the spleen, an organ devoted to the elimination of damaged red blood cells, is the first 112 candidate for accumulation of heme-drug adducts. Further metabolism of these adducts is a 113 dynamic process whereas their distribution may be responsible for the fact that they remained 114 below the detection limit in kidney and urine. 115 116 The alkylation of heme by DU1301 occurred only in infected mice treated at 117 pharmacologically relevant doses. The presence of these heme-drug adducts should therefore 118 be considered as evidence of the alkylating capacity of these trioxane-containing drugs 119 AC CE PT ED on D ecem er 4, 2017 by gest httpaac.asm .rg/ D ow nladed fom Ms AAC00165-08 Version 3, May 20th, 2008. 5 triggered by the presence of the parasite in mice. The detection of heme-DU1301 adducts 120 within a malaria-infected mammal indicates that heme alkylation by DU1301 is an efficient 121 reaction that occurs in vivo and might be considered as a key element concerning the 122 mechanism of action of this antimalarial agent. This result underlines the importance of the 123 alkylating ability of trioxane-based antimalarial drugs and also confirms that artemisinin and 124 trioxaquines share the same heme-alkylating properties. In addition, since artemisinin and the 125 citrate salt of DU1301 are both active on the same stages of synchronized P. falciparum 126 parasites (ring, trophozoite and gametocyte) (2), these pharmacological features strongly 127 suggest that this common alkylating reactivity might be one of the key factors of their 128 antimalarial activity. 129 In addition, aminoquinolines (such as chloroquine) are considered to inhibit the 130 formation of hemozoin by π−π stacking (21, 3, 5). It has been recently reported that 131 trioxaquine DU1301 efficiently prevents the in vitro formation of β-hematin at a lower132concentration than chloroquine itself, whereas the synthetic trioxane precursor of DU1301 does133not inhibit the dimerization of hemin (12). On the other hand, heme-artemisinin adducts also134inhibit β-hematin dimerization and are themselves unable to dimerize (12). These results135suggest that, beside the alkylating ability of the peroxide moiety, trioxaquines may prevent136heme aggregation within the parasite either (i) by stacking of their quinoline fragment with137heme, or (ii) by formation of unpolymerizable heme-drug adducts. Thus, trioxaquines should138be considered as hybrid antimalarial molecules with a dual mode of action: heme-alkylation139and inhibition of heme polymerization.140141 References1421. Asawamahasakda, W., I. Ittarat, Y.-M. Pu, H. Ziffer, and S. R. Meshnick. 1994.143 Reaction of antimalarial endoperoxide with specific parasite proteins. Antimicrob. Agents144Chemother. 38:1854-1858.1452. Benoit-Vical, F., J. Lelièvre, A. Berry, C. Deymier, O. Dechy-Cabaret, J. Cazelles, C.146 Loup, A. Robert, J.-F. Magnaval, and B. Meunier. 2007. Trioxaquines are new147antimalarial agents active on all erythrocytic forms, including gametocytes. Antimicrob.148Agents Chemother. 51:1463-1472.149 3. Bray, P. G., O. Janneh, K. J. Raynes, M. Mungthin, H. Ginsburg, and S. A. Ward.1501999. Cellular uptake of chloroquine is dependent on binding to ferriprotoporphyrin IX151and is independent of NHE activity in Plasmodium falciparum. J. Cell. Biol. 145:363-376.152ACCEPTED onDecemer4,2017bygesthttpaac.asm.rg/Downladedfom Ms AAC00165-08 Version 3, May 20th, 2008.6 4. Dechy-Cabaret, O., F. Benoit-Vical, C. Loup, A. Robert, H. Gornitzka, A. Bonhoure,153H. Vial, J.-F. Magnaval, J.-P. Séguéla, and B. Meunier. 2004. Synthesis and154antimalarial activity of trioxaquine derivatives. Chem. Eur. J. 10:1625-1636.155 5. Egan, T. J., and H. M. Marques. 1999. The role of haem in the activity of chloroquine156and related antimalarial drugs. Coord. Chem. Rev. 190-192:493-517.1576. Hong, Y.-L., Y.-Z. Yang, and S. R. Meshnick. 1994. The interaction of artemisinin with158malarial hemozoin. Mol. Biochem. Parasitol. 63:121-128.1597. Ittarat, W., A. L. Pickard, P. Rattanasinganchan, P. Wilairatana, S. Looareesuwan,160K. Emery, J. Low, R. Udomsangpetch, and S. R. Meshnick. 2003. Recrudescence in161 artesunate-treated patients with falciparum malaria is dependent on parasite burden not on162parasite factors. Am. J. Trop. Med. Hyg. 68:147-152.1638. Jefford, C. W. 2007. New developments in synthetic peroxidic drugs as artemisinin164mimics. Drug Discov. Today 12:487-495, and references therein.1659. Jefford, C. W., F. Favarger, V. H. Maria da Graca, and Y. Jacquier. 1995. The166decomposition of cis-fused cyclopenteno-1,2,4-trioxanes by ferrous salts and some167oxophilic reagents. Helv. Chim. Acta. 78:452-458.16810. Jefford, C. W., A. Jaber, and J. Boukouvalas. 1989. The regioand stereo-controlled169synthesis of cis-p-menth-3-ene-1,2-diol by means of a 1,2,4-trioxane intermediate. J.170 Chem. Soc., Chem. Commun. 1916-1917.17111. Laurent, S. A.-L., C. Loup, S. Mourgues, A. Robert, and B. Meunier. 2005. Heme172alkylation by artesunic acid and trioxaquine DU1301, two antimalarial trioxanes.173 ChemBioChem. 6:653-658.17412. Loup, C., J. Lelièvre, F. Benoit-Vical, and B. Meunier. 2007. Trioxaquines and heme-175artemisinin adducts inhibit the in vitro formation of hemozoin better than chloroquine.176 Antimicrob. Agents Chemother. 51:3768-3770.17713. Meunier, B. 2008. Hybrid molecules with a dual mode of action: dream or reality? Acc.178Chem. Res. 41:69-77.179 14. Moreau, S., B. Perly, C. Chachaty, and C. Deleuze. 1985. A nuclear magnetic resonance180study of the interactions of antimalarial drugs with porphyrins. Biochim. Biophys. Acta181840:107-116.182 15. O’Neill, P. M. and G. H. Posner. 2004. A medicinal perspective on artemisinin and183related endoperoxides. J. Med. Chem. 47:2945-2964.184ACEPTED onDecemer4,2017bygesthttpaac.asm.rg/Downladedfom Ms AAC00165-08 Version 3, May 20th, 2008.7 16. Peters, W., J. H. Portus, and B. L. Robinson. 1975. Chemotherapy of rodent malaria.185XXII. Value of drug-resistant strains of Plasmodium berghei in screening for blood186schizontocidal activity. Ann. Trop. Med. Parasitol. 69:155-171.187 17. Posner, G. H., C. H. Ho, D. Wang, L. Gerena, W. K. Milhous, S. R. Meshnick, and W.188Asawamahasakda. 1994. Mechanism-based design, synthesis, and in vitro antimalarial189testing of new 4-methylated trioxanes structurally related to artemisinin: the importance of190a carbon-centered radical for antimalarial activity. J. Med. Chem. 37:1256-1258.19118. Robert, A., F. Benoit-Vical, C. Claparols, and B. Meunier. 2005. The antimalarial drug192artemisinin alkylates heme in infected mice. PNAS 102: 13676-13680; erratum: PNAS1932006, 103: 3943.19419. Robert, A., and B. Meunier. 1997. Characterization of the first covalent adduct between195artemisinin and a heme model. J. Am. Chem. Soc. 119:5968-5969.19620. Schenck, G. O., and K. Ziegler. 1944. Die Synthese des Ascaridols. Naturwiss. 157-157.19721. Sullivan, D. J., Jr., I. Y. Gluzman, D. G. Russel, and D. E. Goldberg. 1996. On the198molecular mechanism of chloroquine's antimalarial action. PNAS 93:11865-11870.19922. Tang, Y., Y. Dong, and J. L. Vennerstrom. 2004. Synthetic peroxides as antimalarials.200Med. Res. Rev. 24:425-448.20123. Tang, Y., Y. Dong, X. Wang, K. Sriraghavan, J. K. Wood, and J. L. Vennerstrom.202 2005. Dispiro-1,2,4-trioxane analogues of a prototype dispiro-1,2,4-trioxolane:203mechanistic comparators for artemisinin in the context of reaction pathways with iron(II).204J. Org. Chem. 70, 5103-5110.205 24. White, N. J. 2008. Qinghaosu (Artemisinin): The price of success. Science 320:330-334.20625. Wongsrichanalai, C., A. L. Pickard, W. H. Wernsdorfer, and S. R. Meshnick. 2002.207Epidemiology of drug resistant malaria. Lancet Infect. Dis. 2:209-218.208 209210F. B. E. G. is indebted to the EU-AntiMal program for PhD fellowship. CNRS, INSERM and211 ANR are acknowledged for financial support. This study was approved by the French212Institutional Animal Experimentation Ethic Committee (approval # MP/R/06/33/11/07). Dr.213Guy Lavigne (LCC-CNRS, Toulouse) is acknowledged for English editing.214 215216ACCEPTED onDecemer4,2017bygesthttpaac.asm.rg/Downladedfom Ms AAC00165-08 Version 3, May 20th, 2008.8 Figure legends217Figure 1. Reductive activation of trioxaquine DU1301 by iron(II)-heme, leading to the218covalent heme-drug adducts 1 and 2. “Quin” stands for the aminoquinoline fragment.219 Figure 2. (a) LC-MS of chemically prepared heme-trioxaquine adducts; UV-visible trace at220400 nm. (b) Ionic current trace (XIC) for m/z = 1101.1, (c) XIC trace for m/z =221949.2.222 Figure 3. (a-c) LC-MS of the spleen extract of a mouse infected by P. vinckei petteri and223orally treated with DU1301 (single dose 100 mg/kg): (a) UV-visible trace at 400 nm,224(b) XIC trace for m/z = 1101.1, (c) XIC trace for m/z = 949.2. (d-e) LC-MS of the225 spleen extract of a healthy mouse orally treated with DU1301 (single dose 100226mg/kg): (d) XIC trace for m/z = 1101.1, (e) XIC trace for m/z = 949.2.227228 ACCEPTED onDecemer4,2017bygesthttpaac.asm.rg/Downladedfom ACCEPTEDonDecemer4,2017bygesthttpaac.asm.rg/Downladedfom