Memoire Online - Interaction of quinolines and artemisinin based antimalarials drugs with ferriprotoporphyrin IX

Malaria is a major health problem in many countries and according to an estimate of the WHO, more than 500 million infections occur per year. Actually, all over the world, malaria is killing one people after 30 seconds. Because of multidrug resistance of Plasmodium falciparum to current quinoline based drugs, antimalarial drugs are investigated to understand their mode of action and to provide a ratioanal basis to design new drugs. Artemisinin, a sesquiterpene obtained from Chinese herbal drug «qinghaosu «has received considerable as a promising and potent antimalarial in terms of efficacy and cost.

It has been established that hemin is primarily involved in the antimalarial activity of antimalarias. Thus, the interaction of these drugs with hemin may represent a crucial screening test to define their efficacy. In this study, the interaction of hemin with chloroquine, quinine and quinidine in 50% water-propylene glycol at pH 9, 8.1, 7.4, and 6.8 using using a spectrophotometric method. In addition, the results indicated that hemin complexed more strongly with quinidine than with chloroquine and quinine and the binding constants were pH-dependent. Moreover, it was proved that the water-propylene glycol mixture is well suitable to the study of the systems containing hemin and quinoline-based drugs.

Artemisinin and derivates were investigated by UV-Visible at pH 9 and 7.4 and by HPLC/DAD/MS analysis for their reactivity with hemin. It has been showed that artesunate and dihydroartemisinin interacted more strongly with Fe (III) PPIX that artemisinin did. Aqueous DMSO solution is well suitable studying hemin-artemisnin interaction. Both quinoline and artemisinin drugs- hemin complexes exhibited 1:1 stoechiometry.

The reported results showed too that hemin and endoperoxide lactone derived antimalarials slowly react to give rise to several stereoisomers supramolecular adducts (three for artesunate, seven for artemisinin and eight isomers for dihydroartemisinin) while in contrast, it has been reported that only heme (Fe^II) did react with artesiminin based drugs.

4.3 Binding reaction of hemin with chloroquine, quinine and quinidine in water-propylene glycol mixture

4.4.1 Binding reaction of hemin with artemisinin, artesunate and dihydroartemisinin in water-DMSO mixture

4.4.2 Binding reaction of hemin with artesunate in water-propylene glycol mixture

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).

Chapter 2 Literature Survey

In humans, malaria is caused by four species of the genus Plasmodium, namely Plasmonium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae (Wernsdorfer and McGregor, 1988).

Of these, P. falciparum is the most important as it causes almost all malaria-associated deaths. There is, however, significant morbidity associated with P. vivax (Trigg and Kondrachine, 1998). The biology of P. falciparum is fortunately the best understood of the four species. The life cycle of P. falciparum is complex and divided into three overall stages: mosquito, liver and blood stages.

Sexual reproduction of gametocytes occurs in the gut of the female vector mosquito (Anophele genus) and leads to the formation of zygotes that bury themselves in the gut lining of the mosquito. These then develop into oocysts and after some time form sporozoites that migrate to the salivary glands of the mosquito. When an infected mosquito bites a human host, these sporozoites enter the blood stream and rapidly make their way to the liver, invading hepatocytes. During a period of development in the liver of about a week as tissue schizonts, the parasites multiply asexually, finally simultaneously rupturing the host cells and entering the blood stream as merozoites. These merozoites invade red blood cells, entering into the blood cycle consisting of ring, trophozoite and blood schizont stages as shown in Figure 1-1.

Asexual reproduction in the blood cell leads to further merozoites and hence to ever increasing parasitaemia. Some of the merozoites develop into gametocytes. Upon entering the red cell the gametocytes may be taken up by mosquitoes to complete the life cycle. Symptoms of the disease (high fever, headache, malaise, muscle aches...) are entirely associated with the blood stage and so any curative drug must be specifically active against this part of the life cycle (Wernsdorfer and McGregor, 1988).

By knowing what is happening in the blood stage of parasite life cycle, we can understand why, generally speaking, individuals with abnormal hemoglobin S are resistant to malaria? Normally, about 2% of the erythrocytes of individuals with sickle -cell anemia are observed to sickle under low-oxygen concentration conditions found in the capillaries. However, the lower pH of infected erythrocytes increases the proportion of sickling in the capillaries up to 40%. Thus during the early stages of malarial infection, parasite-enhanced sickling probably causes the preferential removal of infected erythrocytes from the circulation. In the latter stages of infection, when the parasitized erythrocytes are attached to the capillary walls, the sickling induced by the low oxygen environment may mechanically and /or metabolically disrupt the parasite. Consequently, bearers of the sickle cell trait in a malarial region have an adaptive advantage (Voet and Voet, 1995).

Figure 1-1 A representation of the life cycle of Plasmodium falciparum. Ring forms, trophozoites and blood schizonts are collectively referred to as the blood stages of the cycle and are the specific targets of chloroquine and related antimalarial drugs. After invading red cells, most merozoites form ring stages and then trophozoites, but a small fraction instead develop into sexual forms called gametocytes which then reproduce in the gut of a mosquito when the insect feeds on the infected host. (Egan et al., 1999).

During its blood stage, P. falciparum utilises host's hemoglobin as a food source. This stage occurs in an acidic compartment within the parasite called a food vacuole that has a pH in the range 5.0-5.6 (Spiller et al., 2002). Plasmodia degrade hemoglobin and use the amino acids derived from proteolytic digestion for their biosynthetic requirements. Hemoglobin degradation is a highly ordered process involving several proteases (Eggleson, 1999; Banerjee, 2002; Rosenthal et al., 2002). Denatured globin formed by the action of plasmepsins is further degraded into small peptides by other proteases. A cysteine protease, falcipain, has been characterized from P. falciparum, which degrades denatured globin (Eggleson, 1999).

Large amounts of free nontoxic heme is released as a product of hemoglobin degradation (Mavakala and Gushimana, 1991). Released heme from hemoglobin is autoxidized into ferric form (hematin, hemin or aquaferriprotoporphyrin IX or H₂O-Fe(III)PPIX) that is highly toxic, inhibiting vacuolar proteases and damaging parasite membranes [Berman and Adams, 1997]. Detoxification of heme is therefore necessary for the survival and growth of malaria parasite (Meshnick, 2002).

In the host, detoxification of heme is achieved by an enzyme called heme oxygenase, which breaks heme to form biliverdin. Another enzyme, biliverdin reductase, converts biliverdin into bilirubin, which is converted into a water-soluble conjugate and excreted through urine. Malaria parasite does not seem to use this pathway for the heme catabolism. Inside the food vacuole of malaria parasite, heme is converted into hemozoin, popularly known as malaria pigment. This hemozoin pigment is a dimer of heme units linked through an iron-carboxylate bond (Pagola et al., 2000).

Pagola et al. have revealed that hemozoin is a hemin dimer with hydrogen bonding between the dimer units in the crystal as shown in Figure1-2. In the light of this, the continued use of the word polymer to describe malaria pigment or -hematin, or the word polymerization to describe its formation is inappropriate and inaccurate (Pagola et al., 2000; Egan, 2002).

Figure 1-2 Chemical structures are shown for (a) hematin (aqua or hydroxyferriprotoporphyrin IX), (b) heme. The dimeric structure for beta-hematin is also indicated (c). The OH-, H2O group is represented by X in (a) and the histidine is represented by Y in (b) (Egan, 2002).

In the dimer, a bond is formed by the linking of central ferric iron of one heme unit with the propionate side chain of another heme. This pigment is inert in parasite and released into the host blood supply after infected erythrocytes burst open at the end of parasite life cycle (Pandey and Tekwani, 1996). Hemozoin is insoluble in organic solvents (methanol, ethanol, and acetone) and mildly soluble in alkaline bicarbonate buffer (100 mM, pH 9.0), whereas free heme is soluble in these solvents.

Numerous conflicting theories have been put forward over the past five decades to describe the mechanism of action of antimalarial drugs. Hypothesis for the mode of action of chloroquine essentially fall into two broad categories: those in which the drug exerts its action outside the food vacuole of the parasite and those in which the activity is located inside the food vacuole.

Chloroquine and related drugs exhibit antibacterial activity, blocking both DNA and RNA synthesis but the required chloroquine concentration is about one thousand times as much as that needed in curative treatment of malaria. No binding of mefloquine to DNA has been observed (Slater, 1993; Egan and Marques, 1999).

Intravacuolar mechanisms seem more plausible because of substantial accumulation of the drugs in the vacuole. Most workers in the field currently favour a hypothesis in which quinoline antimalarial drugs inhibit formation of hemozoin. There is, however disagreement over how this occurs and there are essentially three variations of the hypothesis:

1. Slater and Cerami (1992) originally suggested that these drugs inhibit the putative heme polymerase enzyme.

2. Fitch and Chou (1996) have extended this hypothesis by suggesting that these drugs are potential regulators of the putative heme polymerase enzyme.

3. Egan and coworkers (Egan et al., 1994) have shown that chloroquine, amodiaquine and quinine can directly inhibit formation of synthetic -hematin and suggested that activity of these drugs in vivo involves inhibition of hemozoin formation by direct interaction with Fe(III)PPIX. This hypothesis has also been supported by Dorn and co-workers (Dorn et al., 1995; Dorn et al., 1998) and further support for this type of mechanism has been presented by several other laboratories (Sullivan et al., 1996; Basilico et al., 1997; Hawley et al., 1998) although there are some differences in detail.

These findings motivated a number of studies on antimalarial-hematin interactions in both aqueous and non-aqueous solution, as well as on their interactions with other iron-porphyrins. Many of the earlier studies concentrated on obtaining visible, Mössbauer and NMR spectroscopic evidences for hematin-drug interactions and some association constants were determined. For example, Log K values for the bonding of hemin-drug are 5.52 (chloroquine), 5.39 (amodiaquine), 4.10 (quinine), 4.04 (9-epiquinine) and 3.09 (mefloquine), in 40% aqueous DMSO solution, at an apparent pH of 7.5 and 25^oC (Egan et al., 1997; Adams et al., 1999). It is clear from the recent investigation of Egan and coworkers that only 2- and 4-aminoquinolines and their derivatives form strong complexes with Fe(III)PPIX. Under the conditions of their studies, quinoline, 3-, 5-, 6-, and 8-aminoquinoline, and 4,7-dichloroquinoline exhibited no evidence of complexation with Fe(III)PPIX. Then, there is a simple correlation between hemin binding and -hematin inhibitory activity because those of compounds, which do not form measurable complexes, fail to inhibit -hematin. Surprisingly, however, not all quinolines, which do form strong complexes with Fe(III)PPIX, inhibit -hematin formation.(Egan, 2000). Perhaps, they are capable of inhibiting -hematin formation at high concentration. Egan et al. proposed a detailed model of the structure-function relationships in chloroquine as follows:

1. The 4-aminoquinoline nucleus alone provides an hemin complexing template but is not sufficient for inhibiting the formation of hemozoin;

2. Introduction of the 7-chloro group is responsible for inhibition of hemozoin formation but probably has little influence on the strength of association with hemin;

3. The aminoalkyl side chain is a requirement for strong antiplasmodial activity. It probably assists in drug accumulation in the food vacuole. It also appears to enhance the strength of association with hemin in some cases, but this effect does not appear to be essential for its activity.

Figure 1-3 Proposed structure-function relationships in chloroquine based on findings of Egan and coworkers (Egan et al., 2000).

It has been reported that several enzymes like aspartic proteases, cysteine proteases and metalloproteases (Rosenthal, 1999) are thought to be involved in the degradation of hemoglobin. Many of these enzymes are optimally active at pH 4.5-5.0 and it is argued that the food vacuole would probably need to maintain a similar pH to permit the efficient proteolysis of hemoglobin (Francis et al., 1997). The work of Homewood and coworkers (Homewood et al., 1972) in the early 1970s outlined the potential importance of the pH of the digestive vacuole (pH_DV) in the mode of action of chloroquine (CQ) and similar drugs. CQ is a lipophilic weak base that will pass through biological membranes in the uncharged form. Once, inside acidic compartments, CQ is protonated and trapped because the protonated base is relatively impermeable. If we assume that the digestive vacuole (DV) has a pH of ~5.0, then this mechanism would permit concentrative uptake of the drug (Geary et al., 1986). Homewood suggested that CQ might kill parasites by increasing pH_DV, so that the acid proteases of the parasite could no longer function effectively (Spiller et al., 2002).

Artemisinin was developed from an ancient Chinese herbal remedy. Artemisia annua (sweet wormwood or`qinghao') was used by Chinese herbal medicine practitioners for at least 2000 years. In 1596, Li Shi zhen, a famous herbalist, recommended it to patients with fever. In 1967, Chinese scientists screened a series of traditional remedies for drug activities, and found that extracts of qinghao had potent antimalarial activity. In 1972, the active ingredient was purified and first named qinghaosu (essence of qinghao), and then later renamed artemisinin. Western interest in Artemisinin derivatives (artesunate, artemether, dihydroartemisinin, arteether) began to grow as multidrug resistant Plasmodium falciparum strains began to spread. Hundreds of synthetic second generation artemisinin derivatives and other natural peroxide compounds with good antimalarial activity have been reported like yingzhaosu, arteflene (Lian et al., 1988; Hofneiz et al., 1994) as shown in scheme 2-1. Due to their potent antimalarial activity, fast action, and low toxicity, artemisinin and its derivatives have distinguished themselves as a new generation of antimalarial drugs. Actually, it has been established that the dihydroartemisinin combined to the holotransferrin would be a promising drug against cancer (Singh and Lai, 2001).

The unusual structure of artemisinin molecules might be indicative of a different mode of action from those of other antimalarial drugs and hence the high potency against the resistant strains. Although the mechanism of its antimalarial activity is not clear and still under debate, there is general agreement that the endoperoxide bridge is essential for the antimalarial activity of artemisinin since deoxyartemisinin compounds which lack the endoperoxide moiety are inactive (China cooperative group on qinghaosu, 1982).

Meshnick et al. proposed a two-step mechanism for the antimalarial action of endoperoxide:

In the first step, artemisinin is activated by intraparasitic heme or free Fe (II) ion to produce free toxic carbon-centred radicals, confirmed by electron paramagnetic resonance (EPR) studies (Meshnick et al., 1993; Taranto et al., 2002].

In the second step, once formed, the artemisinin-derived free radicals appear to damage specific intracellular targets, possibly via alkylation (Berman and Adams, 1997).

But Pandey et al. proposed three possible ways for the effect of endoperoxide drugs on malaria (Pandey et al., 1999; Kannan et al., 2002):

-Interaction of artemisinin with hemozoin leading to the breakdown of the hemozoin pigment which could then form a complex with the heme unity.

These mechanisms are supported by the characterization of a covalent adduct between artemisinin and heme (Robert and Meunier, 1997) and by protein alkylation (Meshnick et al., 1991; Yang et al., 1994). Artemisinin also forms covalent adducts with protein but not with DNA (Yang et al., 1994). Thus, heme is both an activator and target of the artemisinin derivatives (Posner et al., 1995).

About the free radicals generated by artemisinin, there are some controversial discussions on the mechanism of their production. There is much stronger evidence that carbon-centred free radicals are involved. In fact, monoelectronic transfer from iron (II) to peroxide resulted in the cleavage of endoperoxide bond with primary formation of an unstable oxygen-centred radical, rearrangement and creation of toxic C4-centred free radicals. It has been proposed that heme attacks the endoperoxide linkage of artemisinin either at the O1 [Shukla et al., 1995] or O2 position [Tonmumphean, 2001] as shown in scheme 2-2. In pathway A, heme iron attacks the compound at the O2 position. Later, it rearranges to form C4 free radical. In pathway B, heme iron attacks the compound at the O1 position after that C3-C4 bond is cleaved to give carbon radical at C4 as shown in scheme 2.

Thus the presence of heme is necessary for the activation of artemisinin into an alkylating agent, which preferentially attacks proteins.

The fact that artemisinin becomes cytotoxic in the presence of ferrous, have triggered some researchers to study its effect on the therapy of cancer. Since iron influx is high in cancer cells, artemisinin and its analogous, after incubation with holotransferrin which increase the concentrations of ferrous iron in cancer cells, selectively kill cancer cells (Singh and Lai, 2001). In addition to the more largely accepted mechanisms summarized above, other mechanisms of action have also been proposed. For example, Jefford proposed that peroxides could interrupt the detoxification process of heme by transferring an O atom to heme, creating iron-oxene or oxyheme intermediates, which subsequently disable parasite (Jefford et al., 1995).

Scheme 2-2 Proposed mechanism of action of artemisinin (Tonmumphean , 200)].

Haynes and co-workers (Haynes et al., 1999) pointed out that activity is due to the trioxane unity acting as a source of hydroperoxide, which provides electrophilic oxygenating species, hydroxyl or alkoxyl radicals via reductive cleavage with Fe (II) or other reducing agents. These species would be able to hydroxylate biomolecules.

In summary, a schematic diagram of hemoglobin degradation and related pathways is given in Figure 2-4 (Pandey et al., 1999; Egan, 2002).

Hemoglobin

Heme

Small peptides

Hemin

Small peptides

Plasmepsin I, II and IV (aspartic proteases),HAP

Peptide transporter

HRP-2

Parasite membrane

Red cell membrane

Drug uptake through biological membrane by passive diffusion phenomena

Blood schizontocidal antimalarials

Serine protease

Figure 2-4 A schematic diagram of hemoglobin degradation and related pathways

DIGESTIVE VACUOLE OF PLASMODIA

2.3 Mechanisms of resistance of parasite to current drugs

Despite the almost universal occurrence of chloroquine resistance, available evidence suggests that resistance arises as a result of a decrease in drug concentration at the site of action, rather than any change in the target of the drug itself. Chloroquine accumulation in the acidic food vacuole of the malaria parasite might occur by passive diffusion down the pH gradient (ion trapping), by import via an ATP-dependent transporter (active uptake) or by binding to FP (CQ receptor). The CQ in the food vacuole will be almost exclusively diprotonated. The plasmodial P-glycoproteinhomolog-1 (Pgh-1) or the Plasmodium falciparum resistance transporter (PfCRT) are implicated in CQ resistance and might modulate quinoline uptake directly, by transporting drugs in and out of the food vacuole, or indirectly, by contributing to the generation of a pH or electrochemical gradient as shown in Figure 2-5. It has long been acknowledged that the pH_DV could be important in the mechanism of CQ resistance: CQ-resistant (CQR) parasites accumulate much less CQ than do their CQ-sensitive (CQS) counterparts (Bray et al., 1998). Following from the weak-base theory, small increases in pH_DV (in the order of 0.2-0.5 pH unit) would substantially reduce the vacuolar concentration of CQ (Yayon et al., 1985; Foley and Tilley, 1998; Dzekunov et al., 2000; Fidock et al., 2000; Hyde, 2002].

There is currently no evidence for clinically relevant artemisinin resistance (Meshnick, 2002), but Kamchonwongpaison and co-workers have reported that P. falciparum parasites in - thalassemic red cells are more resistant to artemisinin and artesunate than parasites in genetically normal cell (Kamchonwongpaison et al., 1994). Because P. falciparum infected thalassemic red cells have a decreased capacity to accumulate drug. In fact, the thalassemics are a group of genetic diseases with defects in globin chain synthesis in -thalassemic, there is decrease in -globin chain whereas in -thalassemia, there is a decrease in -globin synthesis. This result suggests that special precaution should be made for using artemisinin compounds in the treatment of malaria in any area where abnormal globin-gene frequency is high since this will facilitate the development of artemisinin resistance. Paradoxically, thalassemia appears to confer resistance to malaria infection in vitro and in vivo (Weatherall, 1987; Yuthavong and Wilairat, 1993).

Figure 2-5 Possible mechanisms for chloroquine (CQ) uptake and resistance. [Macreadie et al., 2000]

The positive development over the last decade has been the considerable increase in the understanding of processes occurring within the parasite that are relevant to the mode of action of current antimalarials. It also provides knowledge on targets or potential targets for new antimalarial compounds.

It has been established that proteolysis of hemoglobin is considered to be carried out by four aspartic proteases, namely plasmepsins I, II, and IV, and histo-aspartic protease (HAP) (Banerjee, 2002), three cysteine proteases (falcipains) (Rosenthal et al., 2002), and zinc protease (falcilysin) (Eggleson, 1999). All of these represent potential targets for antimalarials (Figure 2-4) and are currently the subject of intense investigation.

Digestion of hemoglobin releases heme into the food vacuole, where it is oxidized to hematin. Heme is another possible drug target and has been implicated in the mode of action of endoperoxide antimalarials, such as artemisinin and its derivatives (Robert et al., 2002).

Hematin is believed to be the target of chloroquine and other quinoline antimalarials (Figure 2-4). It has been demonstrated that histidine-rich protein 2 (HRP-2) has been implicated as an enzyme or, more likely an initiator in the process of formation of hemozoin. Drugs such as chloroquine have been proposed to inhibit hemozoin formation via direct interaction with hematin (Kaschula et al., 2002), by displacing hematin from HRP-2 (Pandey et al., 2001), or by preventing its binding to this protein. HRP-2 represents a hitherto unexplored target for new antimalarials (Figure 2-4). In this issue of chemistry and biology, the development of a new rapid-throughput screening method for investigating the ability of compounds to prevent hematin binding to HRP-2 is reported recently (Choi et al., 2002; Kannan et al., 2002).

These discoveries can be elaborated on in the future to yield new drugs for treatment of this devastating disease (Egan, 2002).

As recommended by the WHO, the combination of at least two drugs having different modes of action must be used to prevent the development of drug resistance. Dechy et al. therefore adopted a covalent bitherapy strategy by preparing new molecules named trioxaquines by covalently attaching a trioxane, the moiety responsible for the activity of artemisinin, to a 4-aminoquinoline entity, a key constituent of chloroquine (Dechy- Cabaret, 2000; Rodriguez et al., 2003). DU-1102, the first trioxaquine proved very active in vitro on chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum, both on laboratory strains (Dechy-Cabaret, 2000) and on human isolates. DU-1102 is a modular molecule that can be prepared through a convergent synthesis from the cis-bicyclo [3.3.0] octane-3, 7-dione in place of the 1,4-cyclohexanedione used to prepare DU-1102 (Dechy-Cabaret et al., 2002) as shown in scheme 2-3.

Scheme 2-3. Convergent synthesis of the trioxaquine, represented by the molecule 4.

Over the years, researchers confronting the extraordinarily complex parasite have suffered a string of disappointments interspersed with some high-profile setbacks, as promising candidate vaccines have failed to perform up to expectations. The scientific obstacles are enormous: Compared to a virus, with its dozen or so genes and relatively monomaniacal approach to evading the human immune system, the malaria parasite has 14 chromosomes, perhaps 7000 genes, and a four-stage life cycle as it passes from humans to mosquitoes and back again. The existing different species of parasites and their multistage life are obstacles for efficiency elaboration of vaccine. Moreover, dozens of new vaccines are in the works, employing a host of technologies that promise to attack the parasite at every vulnerable point of its multistage life. Researchers now predict that within 5 or 10 years they will have a successful vaccine that will actually save lives (Taubes, 2000).

With the actual human, related plasmodium species and mosquito genome sequences, researchers now have in hand the genetic blueprints for the parasite, its vector, and its victim. This will provide the ability to take a holistic approach in understanding how the parasite interacts with the human host. With that approach, new antimalarial strategies should be possible (Pennisi, 2000). Transgenic mosquitoes could be one product of these studies.

The goal would be to replace the natural mosquito populations ravaging developing countries by "designer mosquitoes," genetically modified so that they are unable to transmit malaria parasite. The development of this technique is crucial for scientists studying the biology of the mosquito and its interactions with the malaria-causing parasite. Genetic modification of mosquitoes offers exciting possibilities for controlling malaria, but success will depend on how transformationaffects the fitness of modified insects (Enserink, 2002; Flaminia et al., 2003).

Chapter 3 Experimental materials and methods

Product Name	Molecular Formula	Molecular Weight	Manufacturer
Hemin chloride	C₃₄H₃₂ClFeN₄O	652.0	Sigma
Artemisinin	C₁₅H₂₂O₅	282.34	Guilin Pharmaceutical Factory
Dihydroartemisinin	C₁₅H₂₄O₅	284.35	Beijing Cotexcin New Technology Corporation
Artesunate	C₁₉H₂₈0₈	384.43	Guilin Pharmaceutical Factory
Chloroquine diphosphate	C₁₈H₂₆N₃Cl.2H₃PO₄	515.9	Sigma
Quinine	C₂₀H₂₄N₂O₂	324.4	Merck
Quinidine sulfate	(C₂₀H₂₄N₂O₂)₂.H₂SO₄	746.9	Merck
Sodium hydroxide A.R.	NaOH	40	Beijing Chemical Factory
Hydrochloric acid A.R, 36-38%, d=1.18	HCl	36.46	Beijing Chemical Factory
Sodium bicarbonate	NaHCO₃	84.1	Beijing Chemical Reagent Company
Dimethylsulfoxide	(CH₃)₂SO	78.13	Beijing Xing Jin Chemical Factory
Ethanol	CH₃CH₂OH	46.07	Beijing Yili Fine Chemical reagents Company
Propylene glycol	CH₃CHOHCH₂OH	76.10	Beijing Chemical Reagent Company
Methanol (HPLC grade)	CH₃OH	32.04	Fisher Scientific
Acetic acid	CH₃COOH	47.1	Beijing Chemical Reagent Company
Ethylene glycol	HOCH₂CH₂OH	62.07	Beijing Chemical Reagent Company
Sodium hydrogen phosphate A.R.	Na₂HPO₄.12H₂O	358.14	Beijing Yili Fine Chemical Reagents Company
Sodium dihydrogen phosphate A.R.	NaH₂PO₄.2H₂0	156.01	Beijing Yili Fine Chemical Reagents Company
Tris (hydroxymethyl)-methylamine	C₄H₁₁NO₃	121.09	Beijing Yili Fine Chemical Reagents Company

Appliance	Method of Analysis	Laboratory
Shimadzu UV -Visible 21OOS Spectrophotometer Coupled to CPS-260 thermostat, Japan	Measurements of absorbance at the Soret band of hemin (interaction hemin-artemisinin compounds)	Tsinghua Analysis Center, Beijing
Perkin-Elmer Lambda 2 UV-Visible Spectrophotometer, Germany	Measurements of absorbance at the Soret band of hemin (interaction hemin-quinoline compounds )	Biomacromolecules Laboratory, Kinshasa University (Congo)
LS/MS/MS API 300 Column :SB-C18 Zorbax 4.6x250nm(5m), USA	Measurements of the mass of the hemin-artemisinin compounds complex	Tsinghua Analysis Center, Beijing
Agilent 1100 series LC/MSD Trap/ Column :SB-C18 Zorbax 4.6x250nm(5m), USA	Measurements of the mass of the hemin-artemisinin compounds complex (Analytical HPLC/DAD/MS)	Tsinghua Analysis Center, Beijing
Magnetic Stirrer 90-3	Mixture of work solutions	Chemistry Department, Tsinghua University
Balance Adventurer, USA	Preparation of stock solutions	Chemistry Department, Tsinghua University
Temperature Controller KWS-FIE2-B, China	Incubation at 37^oC	Tsinghua Analysis Center, Beijing
pHmeter PHS-2C(Shanghai Dapu Instruments Limited compagny) equipped with Aurora Electrode(kept soakep in 4 M KCl)	pH measurements of stock and work solutions	Chemistry Department, Tsinghua University

Some physical and chemical properties of solvents which are used to study hemin-antimalarial drugs interactions are summarized on the Table 3-1.

Table 3-1: Comparison of physical, chemical and spectroscopic properties of water, DMSO and propylene glycol (PREG) (Marcus, 1999).

	Water	DMSO	PREG
Molecular weight (g.mol^-1) :	18.02	78.13	76.10
Density at 25^oC(g.cm-3) :	0.9974	1.0958	1.0326
Melting point (^oC) :	0.00	18.55	-60.15
Boiling point(^oC) :	100.00	189.05	187.65
Surface tension at 25^oC :	71.8	43.0	36.5
Viscosity at 25^oC( mPa s) :	0.8903	1.991	42.2
Dielectric constant at 25^oC :	78.36	46.45	32
Diameter (nm) :	0.343	0.513	0.533
Dipole momemt (D)	1.85	4.06	2.25
Hydrogen bond donation ability :	1.17	0.00	0.83
Electron -pair donicity :	0.47	0.76	0.78
Polarity / polarizability :	1.09	1.00	0.76
Proton affinity (KJ mol^-1) :	697	834	828
PKa at 25^oC :	-	-	14.80
PKb at 25^oC :	-	15.5	-
Spectroscopic windows, UV(nm) :	190	265	-

Solubility of hemin and antimalarial drugs in some usual solvents are summarized in table 3-2. ART (artemisinin), AST (artesunate), DHA (dihydroartemisinin), EG (ethyleneglycol), PREG (propyleneglycol), ETOH (ethanol); +, a (in alkaline solution); +, b (in bicarbonate solution); +, c (soluble at pH >8).

Table 3-2. Solubility of hemin and antimalaraial drugs in studied medium

Reactants	Solvents
Reactants	H₂O (pH 9)	NaHCO₃ 5 %	EG	EG 50 %	PREG	PREG 50 %	ETOH 80 %	DMSO	DMSO 25 %	DMSO 40 %
Hemin	+	-	-	+, a	-	+, a	+	+	+	+
ART	-	-	-	-	-	-	+	+	-	+, c
AST	-	+	-	+, b	-	+, b	+	+	+	+
DHA	-	-	-	-	-	-	+	+	-	+, c
Chloroquine	-	-	-	+	-	+		+	-	+
Quinine	-	-	-	+	-	+		+	-	+
Quinidine	-	-	-	+	-	+		+	-	+

Tris-HCl buffer solutions were prepared by mixing different amounts of 0.2 M Tris and O.2 M HCl to give required pH.

25 mL of Tris with 20.7mL of HCl and diluting it with 45.7 mL of distillated water (in case of pH 7.4).

25 mL of Tris with 2.5 mL of HCl and diluting it with distillated water to 100 mL (in case of pH 9).

Phospahte buffer solutions were prepared by mixing amounts of 0.2 M disodium phosphate and O.2 M of sodium phosphate to give required pH.

40% aqueous DMSO solutions (v/v) were prepared by mixing 40 mL of DMSO and 60 mL of corresponding buffer so that the final pH of the mixture was 7.4 or 9.

In addition, 25% aqueous DMSO were prepared by mixing 25 mL of DMSO and 75 mL of corresponding buffer in the same conditions.

2 M of hydrochloric acid and sodium hydroxide served to adjust the pH of all solutions.

Both 50% ethylene glycol and propylene glycol water mixture were prepared by mixing 50mL of each of them with equivalent volume of buffer, adjusted to the pH of work solutions..

In the propylene glycol medium, hemin stock solution, 306 in concentration, was prepared by first dissolving 10 mg of in 25 mL of alkaline distilled water, followed by addition of equivalent volume of propylene glycol.

(25 or 40%) aqueous DMSO stock solutions of hemin (306 ) were prepared by first dissolving 10 mg of it in 12.5 or 20 mL of DMSO, followed by addition 37.5 or 30 mL of buffer.

DMSO stock solutions of hemin (1.0 mM) were prepared by dissolving 6.52 mg of it in 10mL of DMSO (for HPLC-MS analysis).

Aqueous ethanol 85% stock solutions of hemin (306 ) were prepared by dissolving 5 mg of it in 21.25 mL of ethanol and 3.75 mL of distillated water.

Aqueous stock solutions of hemin (306 ) were prepared by dissolving 5 mg of it in 25 mL of alkaline distillated water.

All stock solutions of hemin were refrigerated under 4^oC and, stored in the dark and were stable for at least two weeks.

Chloroquine, quinine and quinidine stock solutions (0.02 M) were prepared by dissolving 51.6 mg, 32.4 mg and 78.3 mg of them, respectively, in 25 mL of acidic distilled water, completed with addition of equivalent volume of propylene glycol.

In 40% DMSO aqueous solutions, artesunate, dihydroartemisinin and artemisinin stock solutions (0.002 M) were prepared by dissolving 19.2 mg, 14.2 mg and 14.1mg of them, respectively, in 10 mL of DMSO, completed with addition of 15 mL of buffer.

In DMSO solutions, artesunate, dihydroartemisinin and artemisinin stock solutions (2 mM) were prepared by dissolving 7.69 mg, 5.69 mg and 5.65 mg of them, respectively, in 10 mL of DMSO (for HPLC-MS measurements).

In 50% propylene glycol solutions, artesunate stock solution (0.002 M) was prepared by first dissolving19.2 mg of it in 12.5 mL of 5 % sodium bicarbonate, followed by addition of equivalent volume of propylene glycol.

Various conventional methods thin layer chromatography (TLC) (Pras et al., 1991), Gas chromatography (GC) (Fulzele et al., 1991), GC-MS and tandem mass spectroscopy MS\MS (Dhingra et al., 2000), HPLC with UV detection (Pras et al., 1991) and with electrochemical detection (HPLC-EC) (Acton et al., 1985) have been proposed and assessed to detect and quantify artemisinin. Radio immuno assay (RIA) and Enzyme electrochemical detection (ELISA) comprise the unconventional techniques to detect artemisinin (Dhingra et al., 2000).

In fact, TLC is not reliable technique to quantify artemisinin due to the poor staining characteristics of the intact molecule and interference with other constituents of the plants. TLC is useful as an assay method only after a tedious chromatographic enrichement (Pras et al., 1991). Gas chromatography also has been applied for the analysis of artemisinin. However, ART is thermolabile compound (stable upto 150^oC) and decomposes on the column. High-pressure liquid has been used chromatography with ultra violet detection but the plethora of crude extract constituents that absorb in the low wavelength region required to detect artemisinin effectively its peak. Moreover, artemisinin needs to be derivatized due to its lack of chromophores (Pras et al., 1991). This process can hamper the result by derivatizing the other compounds present in the crude extract. Moreover, ART is sensitive to acid and base treatment. The most sensitive way for detecting an quantifying artemisinin in crude plant extract without any molecular breakdown or interference from other related compounds and which does not require any derivatization or sample purifiction is High pressure liquid chromatography with electrochemical detection (HPLC-EC). HPLC-EC measures ART directly because the peroxide moiety undergoes electrochemical reduction. This method is highly sensitive and can detect nanogram levels of artemisinin. However, the reductive electrochemical detection involves very special precautions as molecular oxygen is reduced at the low cathodic potentiel of -0.8 V (Acton et al., 1985).

The unconventional methods (RIA and ELISA) are sensitive and highly specific than conventional methods to detect in artemisinin levels in small samples of plant tissues from young seedlings and from cell or tissue cultures. Although RIA is more sensitive, the use of radioactive compounds present a series of problems of special acquisition and use requirements, uncertain stability, high cost, health hazards and disposal difficulties (Dhingra et al., 2000). Hence ELISA is as sensitive as RIA, safer and is based on the peroxide bridge for antibody specificity to detect artemisinin and closely related compounds in crude extracts of artemisia annua (Dhingra et al., 2000).

In our study, we used UV-Vis spectroscopic and HPLC-MS with UV detection for investigating the binding of hemin with antimalarial drugs.

Absorption measurements based upon ultraviolet and visible radiation find widespread application for the identification and determination of myriad inorganic and organic species. Molecular ultraviolet/visible absorption methods are perhaps the most widely used of all quantitative analysis techniques in chemical and clinical laboratories throughout the world. Important characteristics of spectrophotometric and photometric methods include: (1) wide applicability to both organic and inorganic systems, (2) typical sensitivities of 10^-4to 10^-6M, (3) moderate to high selectivity, (4) good accuracy, (5) ease and convenience of data acquisition (Skoog et al., 1998).

Enormous numbers of inorganic, organic and biochemical species absorb ultraviolet or visible radiation and are thus amenable to direct quantitative determination. Many nonabsorbing species like artemisinin and derivatives can also be determined spectrophotometrically by causing them to react with a chromophoric reagent (i.e. hemin) to yield a product that absorbs in the ultraviolet or visible region.

Absorption spectroscopy is based upon electromagnetic radiation in the wavelength region of 160 to 780nm. Then, molecular absorption spectroscopy is based on the measurement of the transmittance T or the absorbance A of solutions contained in transparent cells having a path length of d cm. Ordinarily, the concentration c of an absorbing analyte is linearly related to absorbance as represented by the equation 3-1.

This equation is a mathematical representation of Beer-Lambert's law, where is the molar extinction coefficient. Absorbance of a solution is often influenced by such variables as the nature of the solvent, pH, temperature, electrolyte concentration, reaction time and presence of interfering substances (Skoog et al., 1998).

Spectrophotometry is a valuable tool for elucidating the composition of complex ions in solution and for determining their formation constants. In this study, we opted for the mole-ratio method, where a series of solutions is prepared in which the analytical concentration of one reactant is held constant while that the other is varied. A plot of absorbance versus mole ratio of the reactants is then prepared.

In our study, the interactions of hemin with quinoline based drugs were investigated in water-propylene glycol mixture at pH 9, 8.1, 7.4, and 6.8 using spectrophotometric method. Temperature was controlled at 25^oC. Although quinolines compounds showed some bands of absorption in UV-Visible domain, the titration was carried out at the hemin characteristic Soret band at 396 nm by mixing a constant volume (0.3 mL) of hemin solution with various volumes of drug solutions, and then diluted to 5 mL. Thereby, hemin remains at constant concentration (19 ) while drugs concentrations are changed in the range of 0-1300 .

Hemin-artemisinin compounds in water-DMSO or in water-propylene glycol mixture were studied too, using spectrophotometric method, at pH 9 and 7.4.

The spectrophotometer was equipped with a thermostatic cell and temperature was controlled at 37^oC. Due to its lack of chromophores group, artemisinin absorb weakly in the low wavelength region and made his quantification more difficult. Then, the titration was carried out at the hemin characteristic Soret band at 398 (in 50 % PREG) and 402nm (in 40 % DMSO) by mixing a constant volume (0.1 mL) of hemin solution with various volumes of drug solutions, and then diluted to 10 mL by addition of Tris-PEG or Tris-DMSO mixtures. Thereby, hemin remains at constant concentration (3 ) while drugs concentrations are changed in the range of 0-200 . Before each measurements of absorbance, the work solutions were incubated at 37^oC. UV-Visible spectra were carried out after 10 or 24 hours of incubation.

Chromatography is a separation, identification, purification and quantification technique that dates from the work of the Russian chemist Mikhail Tswett in 1903. There are a variety of chromatography techniques, in common use, all of which work on a similar principle. The mixture to be separated is dissolved in a solvent, called the mobile phase, and passed over an adsorption material, called the stationary phase, which is fixed in place in a column or on a solid surface. Those components that are strongly retained by the stationary phase move only slowly with the flow of mobile phase. In contrast, components that are weakly held by the stationary phase travel rapidly. As a consequence of these differences in mobility, sample components separate into discrete bands, or zones, that can be analyzed qualitatively and/or quantitatively. From the chromatogram, several parameters like the retention time can be deduced to characterize the separation and the efficiency (Niessen, 1999).

Chromatographic processes can be classified according to the type of equilibration process involved, which is governed by the type of stationary phase. Various bases of equilibration are: adsorption (TLC), partition (HPLC), ion exchange (IEC), Molecular Exclusion Chromatography and affinity chromatography.

High-performance liquid chromatography is the most widely used of all the analytical separation techniques. The reasons for the popularity of the method is its sensitivity, its ready adaptability to accurate quantitative determinations, its suitability for separating volatile species, similar polarties components or thermally fragile ones, and above all, its widespread applicability to substances that are of prime interest to industry, to many fields of science, and to the public.

Discussions about HPLC methods often revolve around the internal diameter (id) or bore of the column to be used. Standard bore columns have an id of 4 or 5 mm while narrow bore are half that or less. Packed with the same materials, the narrow bore column will require less solvent for the same resolving power since the analytes can be eluted at a lower flow rate, under 0.5 ml/min, than the 2 to 3 ml/min used for standard bore. Narrow bore columns are 4 to 6 times more sensitive (b) using the injection volume required for a standard bore column (a). The eluting analytes can be detected by a variety of techniques, the most universal being UV-visible absorbance (1) which, with diode-array (DAD) technology provides spectral confirmation in the third dimension. Particular analytes have specific physical characteristics that enable detection based on fluorescence, phosphorescence or chemiluminescence (2), refractive index or electrochemical HPLC can be coupled with others analytical methods like HPLC-mass spectrometry (HPLC-MS), HPLC-nuclear magnetic resonance (HPLC-NMR).

In its simplest form, MS (mass spectrometry), a technique used to characterize and separate ions by virtue of their mass/charge (m/z) ratios can be helpful in structure determination as the fragmentation can give useful informations about the structure. Mass spectrometry data from HLPC-MS has two dimensions: Time and Mass. Time describes the isolated time of molecule (retention time, Rt) and Mass represents the mass/charge ratios. Mass spectrometry, especially HPLC/MS/MS, is an important and quite useful technique for the detection, identification, quantitation and analysis of small pharmaceutical molecules, peptides, proteins, and oligonucleotides and their metabolites and degradants. There are several common modes of obtaining mass spectra. These include: Time-of-flight (TOF), quadrupole, ion trap, magnetic sector, and combinations of these. Ionization techniques commonly used in biotechnology and pharmaceutical analysis for non-volatile samples include Matrix-Assisted Laser Desorption/Ionization (MALDI), Electrospray Ionization (ESI), Inductively Couple Plasma (ICP), electron capture ionization (ECI), Atmospheric Pressure Chemical Ionization (APCI) and Fast Atom Bombardment (FAB). Each technique has its own set of advantages and disadvantages. That is, no one technique will solve all problems.

An Agilent1100 Series LC/MSD system consists of an ion trap mass spectrometer and a HPLC as shown in Figure 4-. The mass spectrometer is equipped with electrospray (ESI) and atmospheric pressure chemical ionization (APCI) ion sources and is able to operate in positive and negative ion modes. Samples can be analyzed by direct injection into the ion source or following separation using high performance liquid chromatography. Both qualitative and quantitative analyses are available by using full scan, single ion or selected reaction monitoring. A variety of tandem mass spectrometry experiments can be performed with ions produced by ESI and APCI methods (Figure 4-1).

Figure 3-1 Schematic diagramm of Agilent 1100 Series LC/MSD Trap (Agilent Technologies, 2001).

HPLC/MS analysis was performed on LS/MS/MS API 300. The Column was a 4.6 x 250 mm (5m), SB-C18 Zorbax (Hewlett & Packard, USA); the open temperature was 30^oC. The eluents were A: H2O adjusted to pH 3.2 by CH3COOH and B: methanol. The following gradient was applied: from 50 % A and 50 % B to 25 % A and 75 % B within 70 minutes. Flow elution was 1mL min^-1, 20 L of samples were injected.

MS spectra were registered in positive and sometimes in negative ion mode. The positive MS spectra were performed on an LCQ electrospray directly coupled to the HPLC.

HPLC/DAD/MS analysis was performed on Agilent 1100 series LC/MSD Trap, under the same work conditions. Except this appliance is equipped with UV DAD detector and UV-Vis spectra were recorded in the range 200-450nm. Products were detected at 412 nm in order to follow the modification of the porphyrin chromophore.

All work solutions were mixed under magnetic stirring for 5 min and prepared, daily before each experiment or analysis and protected from light. 2 M of hydrochloric acid and sodium hydroxide served to adjust the pH of all solutions. 0.1 M tris (hydroxymethyl)-methylamine was used as buffer for all solutions. For all HPLC-MS analysis, 1mL of 2 mM DMSO solution of artemisinin compounds was mixed with 1 mL of 1 mM DMSO hemin solution and incubated at (37 \u177À1) ^oC over 10h.

It is assumed that the interaction between hemin (H) and antimalarial drug (Q) can be described according to the equilibrium shown below:

In diluted solutions, the association constant of complex K can be written as follows:

where is the concentration of complex and and are the initial concentrations of hemin and drug, respectively

Combining Equations (3-2), (3-3), (3-4) and (3-5), the resulting quadratic equation can be written as:

where A and d are the optical density and the light path, respectively, and are the molar extinction coefficients of hemin and its complex solutions.

Combining Equations (3-4) and (3-7), with Equation (3-8), the following equation is obtained:

Where A₀ is the molar extinction of hemin solution at

=0, and ( = -) is the difference of the molar extinction coefficients between hemin complex and free hemin. The basic data are initial concentrations of hemin (

) and drug (

) and the corresponding optical absorption of hemin (A). With these data, parameters particularly the equilibrium constant K, can be fitted according to Eq. (3-9) with the help of Microsoft Origin 6.1 package.

One of the reasons contributed to the conflicting results reported in the literature about interaction hemin-antimalarial drug interactions is the inappropriate choice of the working medium. It has been well shown that the study of Fe(III)PPPPIX in aqueous solution is problematic because of its tendency to aggregate or dimerize. As can be seen from Figure 7, the spectra (b) of hemin in alkaline aqueous solution showed a large band from 350 to 400 nm which is attributed to an oxodimere represented as (H2O)Fe-O-Fe(H2O), whereas hemin was monomeric and exhibited a sharp Soret peak with a maximum at 396-398 nm (50 % PREG or 50 % EG), at 400 nm (25 % DMSO, 80 % ethanol), at 402 nm (40 % DMSO), at 404 nm (DMSO). The slight shift observed towards longer wavelength is due to the change of medium.

Practically, propylene glycol mixture presents the same thermodynamic advantage as ethylene glycol mixture and is much less toxic than the latter. It was used to study both interaction pairs of hemin-quinoline and hemin-artesunate. Since Artemisinin and dihydroartemisinin are insoluble in both propylene glycol and 25 % DMSO, the bonding of hemin with artemisinin compounds was investigated in 40 % DMSO aqueous solutions. Because of their density, polarity, wide temperature range of the liquid state and ability to have bonding hydrogen with water molecules, DMSO and PREG mix easily with water. Particularly, DMSO is an extraordinarily efficient solvent for many kinds of substances including both organic and inorganic compounds. The heat of mixing of DMSO and water indicates there are stronger interactions between DMSO and water than between DMSO molecules (Yu and Quinn, 1994). At high DMSO concentrations, water-structure is disrupted due to the formation of the DMSO-water complexes.

The spectrum range from 300 to 500 nm and 250 to 650 nm were selected to study the interactions of hemin-quinoline and hemin-artemisinin, respectively. This is because the induced spectral modifications in the presence of the drugs are more significant in this range than in the remainder of the UV-visible region.

Tris-HCl buffer was preferred to phosphate buffer because the latter showed some incompatibilty in terms of solubility (formation of precipitate) in 40 % DMSO and was not suitable when pH>8, although it was well used both in 25 % DMSO and water-propylene glycol mixture at pH 7.4.

4.3 Binding reaction of hemin with chloroquine, quinine and quinidine in water-propylene glycol mixture.

More specifically, the wavelength of 396 nm was selected to determine the constants of complexation because of the greatest variation of the optical density observed in the presence of the antimalarial drugs.

Titration of hemin by increasing amount of drugs in mixed water-propylene glycol solutions gives typical spectral changes as exemplified in Figure 4-2. They are similar to those observed on deuterohemin-quinine, hemin-chloroquine and hemin-quinine interactions in other mediums (Constantinidis and Satterlee, 1988; Gushimana et al., 1993, 1996).

The absorption band centered around 332 nm is from the quinoline derivative and that centered at 396 nm is from hemin. As can be seen from Figure 4-2, addition of chloroquine drug modifies markedly the hemin spectrum, but the peak maximums are still at about 396 nm. This indicates that the complexation does not involve significant modifications on the structure of the porphyrin ring of the ferriprotoporphyrin IX.

Another feature that can be seen for all the three drugs is the appearance of an isosbetic point located at around 350 nm on the titration curves. The experimental data were fitted into a 1:1 complex model as described mathematically in Eq. (3-9). What are shown in Figure are selected such results with the total drug concentration as the only changing parameter. It can be seen that the extinction of the hemin solution decreases with increasing total drug concentration.

This trend is consistent with previous results and can be attributed to complex formation between the drug and hemin (Constantinidis and Satterlee, 1988; Gushimana et al., 1993, 1996).

The solid curves in the figure are fitted data with the experimental results according to Eq. (3-9). Correlation coefficients of the nonlinear fittings are better than 0.9, which implies that the titration curves can be well described by the 1:1 complexation scheme. Similar variation in absorbance of hemin at 396 nm as function of total drug concentration has been obtained at other values of pH and the results are also consistent with the formation of 1:1 complex.

Values of binding constants at various pH obtained from these titration curves are summarized in Table 4-1. As highlighted by values of binding constants in Table 4-1, K values are in the same order of magnitude as those obtained in water-ethylene glycol mixture (Gushimana et al., 1993, 1996).

In fact, the complexation of ferriprotoporphyrin IX with the drug is believed to play the role to bring back the hemin into solution in order to prevent it from polymerization. The ability of quinoline drug to complex with hemin will inhibit the formation of hemozoin (-hematin) in vivo. The drug that has a greater affinity with hemin should maintain more hemin in solution and is thus more effective. This means that quinidine should have the highest efficiency, then comes chloroquine, and finally quinine, based on the data in Table 4-1. But in practical applications, an opposite trend is observed, probably due to the emergence of new resistant strains of malaria parasites against the existing and commonly used antimalarial drugs.

As a matter of fact, in some areas (the case in D.R.Congo, for example) quinine appears more effective than chloroquine. This proves that the strength of haematin-quinoline interactions does not directly correlate with antiplasmodial activity. This indicates that haematin binding is a necessary, but not sufficient requirement for antiplasmodial activity (Egan et al., 1994).

Scheme 4-1 Structures of three quinoline-based drugs, quinine (1), quinidine (2) and chloroquine (3).

In regard to the molecular basis of the hemin-drug interactions, rather less is known about the structures of these complexes. In fact, the complexes between 4-aminoquinolines and hemin are almost certainly p-p complexes (Egan et al., 1994).

This means that there is an interaction between the aromatic ring of the quinoline and the porphyrin structure. In addition, hydrophobic interaction, electronic and steric factors also play important roles in influencing the structures of such complexes. Results from the present study show that chloroquine interacts more strongly with ferriprotoporphyrin IX than quinine does, indicating some additional interaction of the side chain of the quinoline with Fe(III)PPIX. This finding rejoins the result of Egan and co-workers which reported the association constants of chloroquine (log K= 5.52) and quinine (log K= 4.10) in 40% aqueous DMSO at pH 7.5 (Egan et al., 2000). It is suggested that the flexible side aliphatic chain of the chloroquine structure, which is less crowded than that of the stiff quiniclidine group of the quinine structure, stabilizes hemin-chloroquine interaction. It is also supposed also that a hydrogen-bonding interaction between the side-chain amine group of chloroquine and the heme propionate group may play a role in the hemin-chloroquine complex stability. More likely, there may be some direct Van der waals interaction between the side chain of quinoline and the porphyrin ring. In addition, the stability of these complexes is supported by computational results. A molecular mechanics study of the interaction between chloroquine and an iron-porphyrin model for N-acetylmicroperoxidase-8 revealed a minimum energy arrangement with coplanar interaction of the quinoline and iron-porphyrin ring, but could not define a preferred conformation for the complex (Marques, 1996).

It is interesting to note that the conformation of drugs affects their affinity with hemin. As can be seen from Scheme 1, quinine differs from quinidine only at positions C-8 and C-9, the former has 8S9R structure and the latter has 8R9S structure (Ribeiro, 1997). The data showed significantly different affinity to hemin of the two chiral isomers.

Further more, it can also be seen that K values are pH-dependent. That dependence is probably due to acido-basic equilibrium influence on electrostatic interactions between hemin and the drugs. Due to their different pKa values, reacting partners have different electric charge at different pH values (Constantinidis and Satterlee, 1988; Gushimana et al., 1993; Kuhn, 1995).

4.4.1 Binding reaction of hemin with artemisinin, dihydroartemisinin and artesunate in water-DMSO mixture.

Figure 4-4 Spectroscopic changes of Fe(III)PPIX at Soret band (402 nm) as a function of increasing concentartions of drug :

0 (a), 14 (b), 18 (c), 70 (d), 100 (e), 160 M of ART (f) after 24 hours with 40 % DMSO aqueous as solvent and 0.1 M Tris-HCl buffer (pH 9).

Typical spectral changes observed upon hemin-sesquiterne endoperoxide lactone-based drugs complexation are shown in figure 4-4. They are similar to those observed on hemin-artesunate and hemin-artemisinin interactions in other mediums (Berman and Adams, 1997, Bilia et al., 2002).

The wavelength of 402 nm was selected to determine the constants of complexation. Complex formation was monitored by the decline in Absorbance (402 nm) whereas absorbance of hemin, incubated with drug, was essentially unchanged after 24 hrs.

The spectral changes resulting from addition of quinoline based drug to Fe(III)PPIX and those resulting from artemisinin drug-hemin interaction are different. The latter shows a substantial hypochromic effect of the hemin Soret band at 402 nm, accompanied with a red shift of 2-5 nm, whereas in bonding of hemin with quinoline, the peak maximums are still at about 396nm. It means that artemisinin and derivatives are auxochromes because their reaction to hemin leads to red shift, which is clearly apparent after 10 hours. The new peak around 407nm may belong to the complex.

About the decrease in absorbance of the Soret band of hemin, two possible processes can be envisaged which would lead to these changes. Either addition of micromolar concentrations of drug induces aggregation of hemin or the changes reflect drug association with hemin. While a large decrease in the absorbance of the Soret band is often an indication of aggregation, equally large decreases are caused by formation of p-p (donor-acceptor) complexes (Egan et al., 1997). Generally, spectral changes of iron porphyrins in the visible region vary depending on the conditions of solvents and pH and the nature of interacting species. The decrease of hemin absorbance is dependent on the drug concentration. A dilution experiment showed that Beer's law is strictly adhered to in the presence of 306 M drug and in the concentration range 0.99 to 48 M Fe(III)PPIX thus providing no evidence of hemin aggregation in this concentration range. Thus, the most reasonable explanation for these spectral changes is the presence of drug-hemin association. There is another feature on the titration curves of artemisinin and derivatives, i.e. a quite regular spectral changes over time with a well-defined isosbetic point around 325 nm. This behaviour is suggestive of an equilibrium between two species. These spectral modifications suggest a progressive disruption of delocalised -electron system of the hemin tetrapyrrole ring. In addition, another band at 290 nm with low intensity was also observed, the origin of that is unclear at this time. Without drug, the band of hemin was still unchanged after 24hours.

Typical kinetic analysis of the mixture of hemin plus artesiminin and derivatives are shown in Figure 9. For example, for 24 M of drug (at pH 9), the residual intensity of the soret band, compared to the control (hemin, 100%), is:

Figure 4-5 Variation of absorbance at 402 nm observed when hemin is titrated with artemisinin derivatives (24 M) within 60 hours of observation in 0.1 M Tris buffer (pH 9, 37^oC).

At 37^oC, the curves of absorbance-time relationship for the complexation showed that the time taken to reach absorbance minimun is shorter for DHA. It was found that the reaction rate is faster with dihydroartemsinin, followed by artesunate than artemisinin did.

Figure 4-6 Variation in absorbance of hemin at 402 nm as function of total concentartion of artemisinin -based drugs after 10 and 24 hours of incubation at 37^oC (pH 9).

The solid lines are best fits of the data to a 1:1 association model obtained by nonlinear least-squares analysis. Association constants for all of the compounds are summarized in Table 4-2. It should be pointed out that the conditions under which the association constants have been measured are of course quite different from those in food vacuole of the parasite (pH~ 5.4, acidic aqueous medium). These are only apparent or conditional association constants. It can be seen that the extinction of the hemin solution decreases with increasing in total drug concentration. Generally, at drug concentrations smaller than 50 M (molar ratio hemin / drug =1:17), there is a significantly and progressive decay in Soret absorbance, more perceptible in the case of DHA. At highest concentrations of drug (molar ratio hemin / drug 1: 25), this decrease is less significant, indicating the saturation of drug binding to heme. Correlation coefficients of the non linear fittings are better than 0.9, which implies that the titration curves can be well describes by the 1:1 association between drug and Fe(III)PPIX. Similar variation in absorbance of hemin at 402 nm as function of total drug concentration has been obtained at pH 7.4 and the results are also consistent with the formation of 1:1 complex. Values of binding constants at related pH obtained from these titration curves are summarized in Table 4-2.

Figure 4-7 Variation in absorbance of hemin at 402 nm as function of total concentration of artemisinin-based drugs after 24 hours of incubation at 37^oC (pH 9).

4.4.2 Binding reaction of hemin with artesunate in water-propylene glycol mixture.

Because of insolubility of artesiminin and dihydroartemisinin in water-propylene glycol mixture, we only reported the bonding reaction of hemin with artesunate in this medium. Figure 4-8 shows typical spectral changes observed when hemin is titrated with artesunate. The wavelength of 398nm was selected to determine the constants of complexation. The main spectroscopic changes consist of a red shift of about 3-4 nm and a decrease in intensity of the Soret band. In addition, the spectra of artesunate showed another absorption peak at 290 nm, already observed in DMSO aqueous medium. The titration curves showed, in the range 250-650 nm, a quite regular spectral change over time with a well-defined isosbetic point around 330 nm.

Figure 4-8 Spetroscopic changes of Fe(III)PPIX Soret band (398nm) as a function of increasing concentartions of drug :

AST (10 h): 0 (a), 1.4 (b), 2.4 (c), 70 (d), 120 (e), 160 M of AST (f) after 10 hours,

AST (24 h): 0 (a), 6 (b), 14 (c), 50 (d), 70 (e), 160 M of AST (f) after 24 hours with water-propylene glycol mixture as solvent and 0.1 M Tris-HCl buffer (pH 9).

Similar variation in absorbance of hemin at 402 nm as function of total drug concentration has been obtained at pH 7.4 and the results are also consistent with the formation of 1:1 complex as shown in Figure 4-9.

Figure 4-9 Variation in absorbance of hemin at 398 nm as function of total concentration of artesunate (pH 9) after 10 or 24 hours of incubation at 37^oC.

Values of binding constants at related pH obtained from these titration curves are summarized in Table 4-3.

In the light of above results, we observed that fixing the concentration of hemin at 3 M and varying drug from 0 to 200 M, the Soret band of hemin decreased. Along with the increase in incubation time and drug concentration, it arises a new peak centred at 290 nm, indicating that the peak belongs to artemisinin compounds. The origin of which is unclear at this time but we suggested that it is a decomposed product of artemisinin as a result of reaction between hemin and drugs. The spectral propriety of this product with strong absorbance is similar to that observed by Green and co-workers (Green et al., 2000) caused by alkali decomposition of ART. They called it ARTS 290 and have described this compound as an enolate/carboxylate. Enolates readily couple to electrophilic dyes such as diazonium salts (Zollinger, 1991), therefore it is possible that

Figure 4-10. Spectral changes of the alkali decomposition product of 0.001 M artesunate to 1 M NaOH after 20 min.

the alkali decomposition product of ART may be similar in structure. In order to verify this assertion, we mixed 0.001 M of AST with 1M of NaOH at room temperature for 20 min. We observed exactly a peak at 290 nm, which has the same characteristic of ART 290 as shown in figure 4-10.

It has been postulated that the interaction of artemisininn with the target heme or hemin in vivo proceeds through the complexes, in which the peroxide bridge of trioxanes coordinates with the iron of hemin, leading to the formation of drug-hemin adduct.

This long-lived intermediate has the spectral characteristics of a heme peroxide (Berman and dams, 199)] compound analogous, in which the hemin is covalently bound to artemisinin via a iron-oxygen-carbon bond, formed by attack of the iron center on the endoperoxide group of drug. This metastable species gradually decays over the ensuing (24 hours for drug highest concentration or 48 hours for drug lowest concentration), to final product devoid of Soret absorbance, indicative of disruption of the heme tetrapyrrole ring system.

Based on data in Tables 4-2 and 4-3, AST should have greater affinity with hemin, then DHA and finally ART. In regard to their constants bindings values, AST and DHA should have the highest efficiency than ART. Practically, it has been shown that artemisinin derivates are at least five times higher than artemisinin. In regard to their molecular structure (Scheme 4-2) it is probably that succininic group of artesunate interacts with hemin propionic group via intra-molecular hydrogen bonding and make this bonding stronger than that of artemisinin does. In addition, in vivo metabolism, artemisinin and derivatives (artesunate, arteether, artemether) are hydrolyzed mainly to the active derivative DHA. DHA, a first generation analogous and metabolite of ART, is known with artesunate to be therapeutically more active than artemisinin (China Cooperative Research Group, 1982, Kamchonwongpaisan and Meshnick, 1996). It was demonstrated too by analyzing the reaction products of DHA plus hemoglobin and of DHA plus globin, DHA reacted rapidly with hemoglobin (as shown in spectral changes in Figure 4-4) but not with globin. This may be a major reason for fast action of these drugs compared to quinoline antimalarials. This is supported equally by the binding constants values (calculated in water propylene glycol mixture) of artesunate (0.43) which are higher than chloroquine (0.17) and quinine (0.05) do. In pratical applications, a similar trend is observed because artemisinin compounds are now at the top of list of new antimalarial drugs in terms of efficacy, rapidity of action and first order pharmacokinetic against chloroquine -resistant strains of plasmodium.

Our results showed that the physical property of the medium of reaction affects the rate of interaction between hemin and drug. We supposed that because of the weak dielectric constants of DMSO and PREG compared to that of water, the bonding constant values of the related artemisinin drug would be more greater in aqueous medium.

Water solubility of artesunate may be due to the presence of succinc group, which increases its polarity make it distinct from other derivatives. DMSO which is highly polar aprotic solvent, with negligible hydrogen bond donation compared to water, may be solvate artesunate anion only through ion-dipole interactions. Less an anionic ligand is solvated, the more readily the complex is formed. From literature, it is known aprotic dipolar solvent permit good complexation while allowing also reasonable solubility of the reacting species (Marcus, 1999). Then, artesunate could coordinate strongly with hemin. The structure of artemisinin-hemin complexes are not well known, may be they are transfert-charge complexes.

In water-DMSO mixture, the binding of hemin to artesunate is better than that in water-propylene glycol. In fact, the propylene glycol, with high viscosity, may be decrease significantly the reactivity of artesunate in this medium than water-dimethyl sulfoxide does.

The interaction of hemin and artemisinin compounds is supported too by molecular mechanic calculations. The molecular electrostatic potentiel (MEP) of artemisinin showed that it has two main region with negative potential which may interact with the positively charged iron ion of the heme, one of these regions covers endoperoxide oxygen O1 and O2 whereas the other encompasses the carboxyl oxygen atom 011 and O14 (Scheme 2-2). This suggests that electrostatic interaction between artemisinin and heme can involve large part of the artemisinin structure. However, the carboxyl and peroxide oxygen atoms, which been the most electronegative part of molecule, will probably be more competitive for complexation (Shukla et al., 1995). In addition, Tonmunphen and co-workers reported automated molecular docking of artemisinin to heme. The docking between artemisinin and heme indicated that artemisinin approaches heme by pointing O1 at the peroxide linkage toward the iron center, a mechanism that is controlled by steric hindrance whith a lowest binding energy of -33.13 kcal.mol^-1(Tonmunphen et al., 2001, Taranto et al., 2001). This is probably due to pyramidal geometry of hemin (where iron is out of the main plan formed by the four pyrrole N atoms) which facilitates the approach of Fe to the endoperoxide moiety.

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.

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.

Hemin, product of hemoglobin degradation in vacuole food of parasite, interacted both with quinoline and artemsinin compounds. The major advantage of water-propylene glycol and water-DMSO mixture is that hemin still monomeric and, under these conditions, interpretation of results is not complicated. Both quinoline and artemisinin drugs- hemin complexes exhibited 1:1 stoechiometry. It was found that water-propylene glycol is suitable for hemin-quinolines interactions whereas aqueous DMSO solution is suitable studying hemin-artemisnin interaction. For quinoline based drugs, the results indicated that hemin complexed more strongly with quinidine than with chloroquine and quinine, and the binding constants were pH-dependent. Because of lack of quantitative data, about the bonding of hemin with endoperoxide lactone based antimalarial drugs, we focused our investigation in it. Artemisinin and derivates were studied by UV and HPLC/DAD/MS analysis for their reactivity with hemin. The reported results showed that hemin and endoperoxide lactone derived antimalarials slowly react to give rise to several stereoisomers supramolecular adducts (three for artesunate, seven for artemisinin and eight isomers for dihydroartemisinin) while many studies certified that metal (III) porphyrins react poorly with those drugs. Generally, hemin (Fe^III) is firstly reduced to heme (Fe^II) in the presence of reducing agent like glutathione (in high concentration in erythrocytes)[Robert et al., 2002], secondly the resulting heme will react with artemisinin drug. Thermodynamic data supported too our results and showed that artesunate and dihydroartemisinin interacted more strongly with Fe (III) PPIX that artemisinin did. Then, it must be considered too that Fe (III) PPIX should be a potentiel target of artemisinin derived drugs.

It is already known that active endoperoxides react with porphyrins while inactive ones do not, suggesting that this reaction may be important in parasite. In the light of preview researches, we suggest that in vivo, artemisinin and derivatives diffuses into the food vacuole, where its reacts with newly formed monomere heme (released from digested hemoglobin) or hemin to generate whether a covalent heme-artemisinin or whether a covalent hemin adduct. This long-lived intermediate may ultimately participe, via C4-centred alkylating radical, in the oxidative damage of membrane, which is lethal for the malaria parasite. The full characterization of a covalent artemisinin-hemin adduct is a key in the understanding of the mode of action of this antimalarial drug, the lead molecule for the rational design of cheap and highly efficient endoperoxide-containing molecules against the chloroquine-resistant strains. Thus due to the high sensitivity, simplicity and feasibility of the two analytical methods should be considered a rapid and inexpensive approach in the search for new lead compounds having an antimalarial activity similar to that of artemisinin. These methods can be considered as widespread analysis techniques in the search of false artemisinin, artesunate and dihydroartemisinin drugs on the market.

Since DMSO solutions are a suitable medium of hemin-artemisinin adduct formation, we recommend to produce this adduct in high yield under optimal experimental conditions. Then, the artemisinin adduct products will be separated by reverse HPLC, isolated and his different isomers characterized for future parasitologic essay.

The structure of those isomers can be elucidated by R-X diffraction, or by NMR spectroscopy. But because of paramagnetic character of artemisinin-hemin adduct with Fe (III), some precautions will be taken, like the demetallisation of those adducts.

As physiological medium is aqueous medium, it is suitable to find the experimental optimal conditions in aqueous medium will permit both to control the dimerization of hemin and take account of solubility of antimalarial drugs. In the future, we suggest also:

-To study the artemisinin-drug interaction at food vacuole pH of plasmodium in the used medium.

-To investigate the bonding of hemin with artemisinin based drugs in presence of glutathione (reducing agent present in high concentration in erythrocytes).

-To investigate by two-dimensional infrared correlation spectroscopy the nature of interaction between artemisinin of drug and hemin.

I am very grateful to Prof. Dr Yu Zhi Wu for the supervision of this research. His helpful suggestions, comments, and scientific instructions were very benefit for the planning and the conduct of this study.

I would like to thank professors Xing Zhi, Yang Cheng Dui, Li and Guo An of Tsinghua Analysis Center for their wonderful cooperation.

I thank also Prof. Dr Yav Gushimana, Dr Mpiana and Nlandu of Kinshasa Univerity for their fruitful collaboration.

I am thankful for our group meeting members four their friendly assistance in my working and living.

I acknowledge all the Chemistry Department members and the foreign students office for their hospitality.

I wish to express acknowledge to the Chinese Scholarship Council for the financial support of this research.

I am pleased to thank my wife Agnes Tshombo K. and my daughter Ketsia Mavakala for their love and affection and to them, I dedicate this work.

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Table A1.1 Absorbance values of hemin Soret band as a function of artemisinin concentartion, at various pH , in water-DMSO mixture, in 0.1M Tris-HCl buffer at 37^oC.

Conc.(M)	E (420nm), pH 9						E(402nm), pH 7.4
	DHA		ART		AST		AST
	10h	24h	10h	24h	10h	24h	10h	24h
0	0.255	0.255	0.255	0.255	0.255	0.255	0.291	0.291
3	0.230	0.214	0.240	0.202	0.235	0.216	0.261	0.253
6	0.212	0.183	0.229	0.190	0.229	0.200	0.253	0.240
10	0.200	0.162	0.225	0.188	0.225	0.169	0.242	0.199
14	0.182	0.133	0.222	0.184	0.216	0.151	0.227	0.185
18	0.170	0.116	0.215	0.167	0.214	0.143	0.209	0.167
24	0.150	0.096	0.210	0.162	0.210	0.132	0.141	0.128
50	0.094	0.058	0.190	0.116	0.200	0.121	0.138	0.112
70	0.078	0.050	0.166	0.083	0.100	0.103	0.132	0.108
100	0.049	0.038	0.147	0.065	0.188	0.094	0.124	0.102
120	0.047	0.037	0.127	0.055	0.189	0.086	0.108	0.092
160	0.040	0.035	0.109	0.045	0.186	0.078	0.107	0.090
200	0.038	0.035	0.096	0.039	0.183	0.078	0.105	0.087

Table A1.2 Absorbance values of hemin Soret band as a function of artesunate concentartion, at various pH in water-propylene glycol mixture, in 0.1M Tris-HCl buffer at 37^oC.

Conc. ( M)	AST
	E (398 nm), pH 9		E (398 nm), pH 7.4
	10 h	24 h	10 h	24 h
0	0.294	0.294	0.200	0.200
3	0.285	0.258	0.185	0.175
6	0.262	0.233	0.176	0.161
10	0.251	0.208	0.17	0.156
14	0.244	0.196	0.158	0.147
18	0.225	0.169	0.146	0.134
24	0.183	0.143	0.131	0.123
50	0.124	0.116	0.129	0.112
70	0.0865	0.082	0.114	0.108
100	0.051	0.058	0.107	0.094
120	0.045	0.051	0.093	0.092
160	0.045	0.056	0.09	0.090
200	0.042	0.045	0.087	0.086

Mavakala K, Nlandu B B, Mpiana P T, Gushimana Z Y and Yu Zhi-Wu (\u23561Î¾Ö¾Îä), Binding Reaction of Hemin with Chloroquine, Quinine and Quinidine in Water-propylene Glycol Mixture. Chinese Journal of Chemistry, 2003, volume 21.

Interaction of quinolines and artemisinin based antimalarials drugs with ferriprotoporphyrin IX

Abstract

Chapter 1 Introduction

Chapter 2 Literature Survey

Hemoglobin

Heme

Small peptides

Hemin

Small peptides

Plasmepsin I, II and IV (aspartic proteases),HAP

Peptide transporter

HRP-2

Parasite membrane

Red cell membrane

Drug uptake through biological membrane by passive diffusion phenomena

Blood schizontocidal antimalarials

Serine protease

DIGESTIVE VACUOLE OF PLASMODIA

2.3 Mechanisms of resistance of parasite to current drugs

Chapter 3 Experimental materials and methods

Hemin-chloroquine

Hemin-quinine

Hemin-quinidine