Interaction of quinolines and artemisinin based antimalarials drugs with ferriprotoporphyrin IX

Chapter 1 Introduction

More than 40% of the world's population live in areas where malaria is endemic and each year 300-400 millions cases of infections are recorded. Actually, all over the world, malaria is killing one people after 30 seconds. In Africa, official estimations of annual mortality indicate that 1-3 millions cases of death are due to malaria. Most of the victims are children under 5 years of age (Hoffman, 1996; World Health Organization, 2002). Surprisingly, the tropical African region where malaria is the major cause of death coincides closely with the areas where the sickle-cell disease is prevalent, which is a genetic disorder characterized by mutation of glutamic acid to valine residue in position 6 of the -chain of hemoglobin. Generally, people with sickle-cell gene are resistant to malaria (Voet and Voet, 1995). Malaria, tuberculosis, AIDS, and malnutrition remain the important health problems of the developing countries and contributing significantly to their poverty.

With fast spreading multidrug resistance to commonly used quinoline-based antimalarial drugs (alkaloidal drugs), especially chloroquine by Plasmodium falciparum (human malaria parasite) (Wyler, 1983; Hien and White, 1993; Berman and Adams, 1997), antimalarial drugs are intensively investigated to understand their mode of action and the factors responsible of the resistance. A better understanding of the mode of action may provide a rational basis to improve the antimalarial activity of the existing drugs and to design new drugs like artemisinin derivatives, trioxaquines (China Cooperative Research Group on Qinghaosu and Its Derivatives as Antimalarials, 1982; Klayman, 1985; Krogstad et al., 1985; Jiang et al., 1995; Dechy et al., 2002; Gong et al., 2001; Dechy et al., 2003; Cointeaux et al., 2003).

Historically, since 1940, chloroquine was the most effective drug for treatment and prophylaxis because of its strong therapeutic activity, low toxicity and low cost. A perfect substitute for chloroquine should exhibit the qualities of chloroquine, and if possible, be even better. Among the few new drugs against malaria, the promising, from considerations of potency, low toxicity, resistance, cost, rapidity of action and first order pharmacokinetics, is artemisinin (Artemisia annua), a non-alkaloidal drug from an ancient Chinese herbal drug Qinghao (sweet wormwood). Artemisinin is a saturated endoperoxide lactone molecule and has been used by the Chinese for 2 milleniums as a folk remedy against fever. Lack of chemical resemblance to the quinoline antimalarial might have been the reason of the success of this drug and its congeners including dihydroartemisinin, artesunate, artemether and arteether in treatment of multidrug resistant malaria (Meshnick, 2002). Unfortunately, though more than hundred years have elapsed since the discovery of malaria parasite and its transmission, detailed biochemistry of Plasmodia remains poorly understood (Trigg and Kondrachine, 1998). Despite the proven efficacy of artemisinin against severe malaria, the precise mode of action of it and its derivatives, as well as those quinoline drugs, remains uncertain. Frequently, controversial discussions in the literature are seen (Homewood et al., 1972; Chou et al., 1980; Yayon et al.; 1985; Constantinidis and Satterlee, 1988; Egan et al., 1996; Robert et al., 1997; Cazelles, 2001). The mechanism of action of any drug is important in drug development. Generally, the drug compound binds with a specific target, a receptor to mediate its effects. Strong drug-receptor interactions are required for high drug activity. The most interesting and developed theory suggests the binding of intraparasitic heme or hemin (ferro- or ferriprotoporphyrin IX) with these drugs as the prerequisite for their antimalarial action, leading to the formation of a complex, which is probably toxic to the parasite (Chou et al., 1980; Yayon et al., 1985; Egan et al., 1996; Meshnick et al., 1996; Berman and Adams, 1997; Chen et al., 2001; Robert et al., 2002). It has been established that hemin is present in high amounts in malaria parasites as a result of hemoglobin digestion (Sherman, 1984). From this, hemin is thought of as having critical role in the mechanism of action of artemisinin and its derivatives.

Several approaches can be undertaken to study the interaction of antimalarial drugs with hemin, including thermodynamic and theoretical studies of the binding process. Chou et al. investigated the interaction between hemin and quinoline drug in aqueous medium. This medium, however, presents some limitations in regard to the solubility of the reacting partners. Hemin is soluble in alkaline aqueous solution, whereas quinoline compounds are soluble in acidic aqueous solution. As reported ( Yang et al., 1998; Bilia et al., 2002; Kannan et al., 2002), artemisinin compounds are insoluble in aqueous solutions but they are soluble in ethanol, in dimethyl sulfoxide (DMSO), methanol, acetone, and dimethylacetamide mediums.

A stable state of hemin in neutral or weakly acid aqueous solution cannot be achieved because of its tendency to dimerize. Studies of hemin-antimalarial drugs interactions under these conditions render results unreliable and difficult to interpret. As a result, nonconsistent stoichiometries of hemin-quinoline complexes in aqueous solution, such as 2:1, 4:1, and 7:1 complex were reported (Moreau et al., 1982; Dorn et al., 1998; Egan et al., 2000). Therefore, it is important to take into account both the influence of hemin dimerization and the prerequisite that reacting partners must be soluble in the medium when studying hemin-drug interactions. According to literatures, two methods can be used to circumvent this problem. One is to use nonaqueous, mixed solvent, or detergent solutions. The other is to use heme-peptides or iron porphyrins, which exhibit a lower tendency to aggregate, to model the behaviour of hemin (Marques et al., 1996). Several studies on the interaction with antimalarial drugs have been performed using the latter strategy. Constantinides and Satterlee have investigated the bonding of quinine and chloroquine to Fe(III)uro-porphyrin and 1 :2 stoichiometry (drug :hemin) was reported (Constantinidis and Satterlee, 1988), in contrast the bonding of heme-peptide N-acetylmicroperoxidase-8 (N-AcMP-8) with quinine and 9-epiquine exhibited 1:1 complexation and 2:1 stoichiometry with chloroquine. These conflicting results suggest the importance of performing studies with hemin itself, while at the same time taking care to ensure that it is maintained in a strictly monomeric state.

Unfortunately, quantitative data in mixed solvent systems using hemin, are lacking. Effort has been made to solve the problem by using 40% aqueous DMSO to study the complexes between several quinoline antimalarials and monomeric hemin and 1:1 stoichiometry was proposed (Egan et al., 2002).

In our previous studies, the interactions of hemin with chloroquine and quinine and that of deuterohemin (hemin without vinyl groups) with quinine were investigated in 50% water-ethyleneglycol mixture (Gushimana et al., 1993; Gushimana et al., 1996).

In this medium, which is thermodynamically close to water, (Gushimana et al., 1993) the dimerization process of hemin could be well controlled. A good correlation between thermodynamic and kinetic data had been determined, leading to the conclusion of a 1:1 stoichiometry of the complex. Although this complex can be used for parasitologic assays in vitro, ethylene glycol is toxic (Haley and Berndt, 1987). It is thus important to separate the complex from the solvent before it is used for parasitologic assays in vivo. Another way to solve the problem is to find an alternative medium, which presents the same thermodynamic advantage as ethylene glycol-water mixture and, at the same time, is nontoxic.

Morever, the interactions of hemin with artemisinin compounds were qualitatively investigated in the past using mediums such as water-acetonitrile, -dimethyl sulfoxide, -ethanol mixtures and dimethylacetamide medium. It was found that artemisinin reacted with heme (Fe^II) but do not react directly with metal (III) porphyrins like hemin (Fe^III) or (Mn^III) tetraphenylporphyrin (Berman and Adams, 1997; Cazelles et al., 2001; Robert et al., 2002). Unfortunately no consistent quantitative thermodynamic and kinetic data have been reported ( Yang et al., 1998; Bilia et al., 2002; Meshnick, 2002; Kannan et al., 2002).

In this MSc work, the interaction of hemin with quinoline-based drugs, i.e. chloroquine, quinine, and quinidine, was firstly examined in 50% water-propylene glycol mixture at various pH values using a spectrophotometric titration method. Water-propylene glycol mixture is interesting because propylene glycol is much less toxic and commonly used as pharmaceutical adjuvant. Interactions of endoperoxide lactone based antimalarias, i.e. artemininin, dihydroartemininin and artesunate, with hemin were then investigated using UV-Vis spectroscopic and HPLC-MS methods. Because of weak solubility of artemisinin and dihydroartemisinin in 40% aqueous DMSO at pH 7.4, we report the interaction of hemin with artemisinin, artesunate and dihydroartemisinin in this medium, at pH 9 at 37^oC. The relatively nontoxicity property and miscibility with water of DMSO allow it to have wide applications in cell biology as solvent for drugs (Yu and Quinn, 1994).