Antimalarial drugs : Mode of action and status of resistance

Malaria is a major global health problem, with an estimated 300 to 500 million clinical cases occurring annually. Malaria remains one of the leading causes of disease and death in the tropics, mainly of children under 5 years of age. The most prevalent and dangerous type of malaria is caused by Plasmodium falciparum. P. vivax is a common cause of malaria in Latin America, Asia, and Oceania, but not Africa. P. malariae and P. ovale are much less common. Antimalarials are used in three different ways: prophylaxis, treatment of falciparum malaria, and treatment of non-falciparum malaria. Prophylactic antimalarials are used almost exclusively by travelers from developed countries who are visiting malaria-endemic countries. The antimalarials in common use come from the following classes of compounds: the quinolines (chloroquine, quinine, mefloquine, amodiaquine, primaquine), the antifolates (pyrimethamine, proguanil and sulfadoxine), the artemisinin derivatives (artemisinin, artesunate, artemether, arteether) and hydroxynaphthaquinones (atovaquine).


INTRODUCTION
Malaria is caused by infection with a single-cell parasite, Plasmodium.Four Plasmodium spp.cause malaria in human beings: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae.P. falciparum is the most important because it accounts for the majority of infections and causes the most severe symptoms.Malaria remains one of the leading causes of morbidity and mortality in the tropics.According to the World Malaria Report (2011), there were 106 malaria endemic countries in 2010.
There were 216 million cases of malaria in 2010; 81% of these were in the World Health Organization (WHO) African region.An estimated 3.3 billion people are at risk of malaria.An estimated 655, 000 persons died of malaria in 2010.86% of the victims were children under 5 years of age, and 91% of malaria deaths occurred in the African region.Antimalarials are used in three different ways: prophylaxis, treatment of falciparum malaria, and treatment of non-falciparum malaria.Prophylactic antimalarials are used almost exclusively by travelers from developed countries who are visiting malariaendemic countries.Treatment protocols for falciparum malaria vary, depending on the severity of the disease; fast-acting, parenteral drugs are best for severe, lifethreatening disease.In addition, treatment protocols for falciparum malaria vary geographically and depend on the resistance profiles for strains in particular regions.Non-falciparum malarias, in contrast, rarely are drugresistant.In addition, P. vivax and P. ovale have dormant liver stages that can cause relapses months to years after an infection is cleared, so they need to be treated with an additional agent that can clear this stage.The antimalarials in common use come from following classes of compounds: the quinolines (chloroquine, quinine, mefloquine, amodiaquine, primaquine), the antifolates (pyrimethamine, proguanil and sulfadoxine),the artmisinin chloroquine resistance also is uncertain.Chloroquineresistant parasites accumulate less chloroquine in the food vacuole than do sensitive parasites (Fitch, 1970) and one assumption is that chloroquine resistance is not based on the mode of action of the drug but on the access of the drug to the parasite food vacuole.Early studies indicated that chloroquine resistance was associated with an elevated level of drug efflux.Drugresistant parasites were reported to release preaccumulated chloroquine almost 50 times faster than chloroquine-sensitive isolates (Verdier et al., 1985;Krogstad et al., 1987).Furthermore, verapamil was shown to reduce the apparent rate of drug efflux from chloroquine-resistant parasites (Krogstad et al., 1987).Since verapamil is known to reverse the P-glycoproteinmediated efflux of drugs in multidrug-resistant tumor cells (Martin et al., 1987), this led to the proposal that efflux of chloroquine by a plasmodial P-glycoprotein is responsible for chloroquine resistance.Bray et al. (1999) on the other hand have suggested that chloroquine resistance is caused by reduced affinity of chloroquine for heme, thereby reducing chloroquine uptake.Another proposal is that chloroquine is transported actively through the parasite by the Na + /H + exchanger (NHE) and that resistance to chloroquine is mediated by mutations in the NHE (Wunsch et al., 1998) but this suggestion has been disputed.Wellems et al. (1990Wellems et al. ( , 1991) ) analyzed a cross between a chloroquineresistant and a chloroquine-sensitive strain of P. falciparum, and identified a chloroquine-resistance locus within a 400 kb segment of chromosome 7. Su et al. (1997) mapped the putative chloroquine-resistance locus to a 36 kb region and identified the open reading frames of 8 potential genes within this region.Initially, chloroquine resistance was thought to be caused by cg2, a gene coding for a polymorphic protein located at the parasite periphery.However, recent transformation studies have ruled out cg2 and suggest another gene, pftcr within this region (Fidock et al., 1999).

Status of resistance
Chloroquine, soon after introduction in the 1950s, quickly became the main drug of choice globally to treat uncomplicated P. falciparum infections, for instance, as part of the Global Malaria Eradication Campaign launched by the (WHO) in 1955.However, P. falciparum did eventually develop resistance to chloroquine, and has spread to almost all the endemic countries today (Wellems and Plowe, 2001).Chloroquine resistant parasites in Africa were thought by some to share the same origin as the Indo-China strains, but by others to have developed locally as a result of mass drug administration plus intrinsic entomological, epidemiological, and parasitological factors that promoted local resistance selection (Diribe and Warhurst, 1985).
Current molecular studies suggest the Asian origin of African isolates, but at least four different foci of chloroquine resistance have so far been identified (Warhurst, 1995).Resistance to chloroquine has spread to almost all the countries thus limiting the effective use of this low cost antimalarial.

QUININE
Quinine is derived from the bark of the cinchona tree and was used for treating fevers as early as the 17th century, although not until 1820, it was the active ingredient of the bark, isolated and used in its purified form.Quinine is used as a treatment for uncomplicated and severe malaria in many different therapeutic regimens.Quinimax, which is a combination of quinine, quinidine, and cinchonine (all derived from cinchona bark), is also used (Deloron et al., 1985).Quinine must be administered for at least 7 days to non-immune populations (Bunnag and Harinasuta, 1986;Krishna and White, 1996;Bjorkman et al., 1991) but it is effective in immune populations (such as in sub-Saharan Africa) when given for 3 to 5 days because it appears to be potentiated by the host immune system (Miller et al., 1989).In the United States where quinine is not available commercially, quinidine (its D-isomer) is used.Quinine is also used in combination with antibiotics (tetracycline or doxycycline).

Mode of action
Quinine acts in a manner similar to that of chloroquine but with some differences; chloroquine causes clumping of the malaria pigment, whereas quinine antagonizes this process (Peters, 1987).In addition, quinine is a weaker base than chloroquine and has less affinity for heme, implying that mechanisms other than ion transport into the food vacuole and heme-drug interactions are required for the action of these drugs (Foley and Tilly, 1998).Quinine also interacts rather weakly with heme (Kd = 2.63 × 10 -6 M) (Chou et al., 1980), but has been shown to inhibit heme polymerization (Slater 1992;Chou and Fitch, 1993) and heme catalase activity (Ribeiro et al., 1997).In the absence of a specific transporter, quinine is likely to be accumulated less efficiently in the food vacuole than chloroquine.Further work is required to determine whether the mechanism of action of quinine is more closely aligned to that of chloroquine.

Status of resistance
Although quinine treatment failure has been reported, many of these instances can be attributed to inadequate treatment.Reports of clinical quinine resistance have been sporadic, with the highest incidence occurring in Southeast Asia, where it was first reported in 1967 (WHO, 1967).In Thailand, treatment failure rates increased from 6% in 1978 to 14% in 1979 to 1980, and they were up to 38.5% in 1981 (Chongsuphajaisiddhi et al., 1983).A more recent report shows 23% recrudescence in pregnant women after quinine treatment (McGready et al., 1998), which may be an indication that quinine resistance in Thailand is stabilizing, perhaps because of the widespread use of quinine combinations and alternative drugs.In Africa, quinine resistance remains at very low levels, and even in Southeast Asia, cure rates with quinine combinations (for example, quinine-tetracycline) remain high (Watt et al., 1992;Looareesuwan et al., 1994;Bunnag et al., 1996).

MEFLOQUINE AND HALOFANTRINE
Mefloquine was developed in the 1970s by the United States Army in response to the increasingly poor cure rates of chloroquine, with clinical trials beginning in 1972 (Davidson et al., 1975;Trenholme et al., 1975;Rieckmann et al., 1974).Mefloquine has a very long halflife both in patients with malaria (10.3 to 20.5 days) (Karbwang and White, 1990;Na-Bangchang et al., 1995) and in healthy volunteers (13.8 to 27.5 days).Mefloquine is recommended for prophylaxis and therapy in chloroquine-resistant areas.Despite considerable publicity about possible neuropsychiatric side-effects of mefloquine, the same evidence is not conclusive (Choo, 1996).
Halofantrine is a tricyclic compound that was developed at approximately the same time as was mefloquine and has been used as a second-line agent; its use may be limited by its cardiotoxic side-effects and variable pharmacokinetics.

Mode of action
Mefloquine interacts relatively weakly with free heme, with reported Kd values ranging from 3.3 × 10 -7 to 1.63 × 10 -5 M (Chou et al., 1980;Chevli and Fitch, 1982).Mefloquine has been shown to inhibit heme polymerization in vitro with a similar (Slater and Cerami, 1992;Slater, 1993) or lower (Chou and Fitch, 1993;Raynes et al., 1996) efficiency than chloroquine (that is, close to millimolar levels).Given the lower basicity of mefloquine, it seems unlikely that it would reach the intravacuolar concentration required to inhibit heme polymerization.Furthermore, while chloroquine treatment of P. berghei infected mice was found to cause a decrease in hemozoin production, mefloquine and quinine had no effect (Chou and Fitch, 1993).Mefloquine is also a much less potent enhancer of the peroxidase activity of heme than chloroquine (Sugioka and Suzuki, 1991) and has been shown to interfere with the ability of chloroquine to enhance heme-induced cell lysis (Dutta and Fitch, 1983).
The available data suggest therefore that, mefloquine interferes with a different step in the parasite-feeding process than chloroquine (Geary et al., 1986).Desneves et al. (1996) used the technique of photo affinity labeling to identify two high-affinity, mefloquine-binding proteins with apparent molecular masses of 22 to 23 kDa and 36 kDa in P. falciparum infected erythrocytes.The identities of these polypeptides have not been established yet, but they may be involved in mefloquine uptake or action.There is also increasing evidence to suggest a role for the plasmodial P-glycoprotein (P-glycoprotein homolog-1, Pgh-1) in mefloquine resistance.This raises the possibility that Pgh-1 may also be the target of action of mefloquine.

Mechanism of resistance
Mefloquine resistance in field isolates of P. falciparum is associated with amplification of the pfmdr1 gene (Peel et al., 1993;Wilson et al., 1993;Cowman et al., 1994) and over-expression of its protein product Pgh-1 (Cowman et al., 1994).Moreover, selection for mefloquine resistance in vitro leads to amplification and over expression of the pfmdr1 gene (Cowman et al., 1994;Wilson et al., 1989;Peel et al., 1994).This has led to the idea that Pgh-1 is responsible for at least some forms of mefloquine resistance.Resistance to halofantrine and quinine also increased during mefloquine selection, suggesting a similar underlying mechanism (Peel et al., 1993(Peel et al., , 1994;;Cowman et al., 1994).Resistance to mefloquine is not reversed by verapamil or chlorpromazine, but can be reversed by penfluridol (Peters and Robinson, 1991).

Status of resistance
Resistance to mefloquine has been rising inexorably ever since this drug was introduced in the 1970s (Boudreau et al., 1982;Espinal et al., 1985;Draper et al., 1985;Brasseur et al., 1990;Gay et al., 1990;Raccurt et al., 1991;Looareesuwan et al., 1992;White, 1994).In an area of Thailand where mefloquine was used intensively, substantial mefloquine resistance developed within 5 years of its introduction (White, 1994;Mockenhaupt, 1995).Cure rates with mefloquine have now dropped to 41% in some areas of Thailand (Nosten et al., 1991;Fontanet et al., 1993).Combination regimens with either artenusate or artemether have been introduced in an effort to stem the development of resistance to mefloquine (Price et al., 1995).There have been reports of intrinsic mefloquine resistance in regions of Africa Saifi et al. 151 where the drug had not been used (Oduola et al., 1992;White, 1994), although it now appears that this may have been due to pre-existing levels of quinine resistance.
Resistance to mefloquine in the field was first noted in an area of Thailand (Boudreau et al., 1982) where quinine resistance was already widespread.The ease with which mefloquine resistance develops is exemplified by experiments, showing that mefloquine resistance readily can be induced by applying drug pressure during continuous passage of P. berghei through Anopheles gambiae (Fonseca et al., 1995).Mefloquine has been used in higher doses as a solo treatment in much of Southeast Asia, although high levels of resistance to this therapy are now occurring there as well (ter Kuile et al., 1992).Mefloquine is still effective in most African countries and can be used in areas of chloroquine resistance.Mefloquine and halofantrine show a high degree of in vitro cross-resistance (Basco et al., 1995;Gay et al., 1990;Pradines et al., 1998;Rojas-Rivero et al., 1992;Basco and Le Bras, 1993), although evidence of in vivo cross resistance is limited.It indicates that increasing levels of resistance to mefloquine may limit the effective chemotherapy lifetime of both mefloquine and halofantrine.

ANTIFOLATES
Some of the most widely used antimalarial drugs belong to the folate antagonist class, albeit their role in malaria control is hampered by rapid emergence of resistance under drug pressure (Plowe et al., 1998).Inhibition of enzymes of the folate pathway results in decreased pyrimidine synthesis, hence, reduced DNA, serine, and methionine formation.Activity is exerted at all growing stages of the asexual erythrocytic cycle and on young gametocytes.Traditionally, antifolates are classified into two: 1. Type-1 antifolates (sulfonamides and sulfones) mimic p-aminobenzoic acid (PABA).They prevent the formation of dihydropteroate from hydroxymethyldihydropterin catalyzed by dihydropteroate synthase (DHPS) by competing for the active site of DHPS (a bifunctional enzyme in plasmodia coupled with 2-amino-4-hydroxy-6hydroxymethyl-dihydropteridine pyrophosphokinase [PPPK]).2. Type-2 antifolates (pyrimethamine, biguanides and triazine metabolites, quinazolines) inhibit dihydrofolate reductase (DHFR, also a bifunctional enzyme in plasmodia coupled with thymidylate synthase [TS]), thus preventing the NADPH-dependent reduction of H2folate (DHF) to H4folate (THF) by DHFR.

Mode of action
The antifolate drugs inhibit either dihydrofolate reductase (DHFR) (pyrimethamine, cycloguanil) or dihydropteroate synthase (DHPS) (sulfadoxine).These are two key enzymes in de novo folate biosynthesis; inhibition of this metabolic pathway leads to the inhibition of the biosynthesis of pyrimidines, purines, and some amino acids.Antifolate antimalarial drugs interfere with folate metabolism, a pathway essential to malaria parasite survival.

Mechanism of resistance
This class of drugs includes effective causal prophylactic and therapeutic agents, some of which act synergistically when used in combination.Unfortunately, the antifolates have proven susceptible to resistance in the malaria parasites.Resistance is caused by point mutations in DHFR and DHPS, the two key enzymes in the folate biosynthetic pathway which are targeted by antifolates.Resistance to these drugs arises relatively rapidly in response to drug pressure and is now common worldwide.Resistance to DHFR and DHPS inhibitors is conferred by single mutations of the gene encoding for the respective enzyme, resulting in substitutions in the amino acid chain.There are areas of the DHFR and DHPS genes with identified mutations that are found in isolates that fail to respond to pyrimethamine/sulfa treatment.These occur principally at codons 108, 51, 59, 164, and also occasionally 50, 140, and the ``Bolivian repeat'' of the DHFR gene and codons 436, 437, 540, 581, and 613 of the DHPS gene (Plowe et al., 1998).There is a broad correlation between increased frequency of such mutations and resistance to pyrimethamine/sulfa drugs across the world (Wang et al., 1997).

Status of resistance
For all the antifolate drugs, a high number of mutations may be an indicator of clinical resistance, but as the number of mutations becomes smaller, the accuracy in predicting clinical resistance decreases.Resistance to the fansidar combination is widespread, especially in Southeast Asia.
Fansidar has been used as a second-line drug in areas of chloroquine resistance; the loss of fansidar effectiveness caused by increasing resistance has important implications for Africa, where the number of inexpensive alternatives is limited.The development of resistance to sulfadoxine-pyrimethamine by Plasmodium parasites is a major problem for the effective treatment of malaria, especially P. falciparum.Although the molecular basis for parasite resistance is known, the factors providing the development and transmission of these resistant parasites are less clear.In Tanzania, increasing rate of chloroquine resistance led to change in its first line treatment of uncomplicated malaria to sulfadoxinepyrimethamine.This antifolate combination seemed to be an effective and reasonable alternative but resistance to sulfadoxine-pyrimethamine was rapidly gaining ground, facilitated by the slow elimination from the body.High level (45%) of sulfadoxine-pyrimethamine treatment failures were recorded in Muheza, North East Tanzania by Mutabingwa et al. (2001).Nazila et al. (2000) observed that when a combination of pyrimethamine and sulfadoxine was used in Kenya, drug resistant parasites were selected rapidly.A study of pyrimethaminesulfadoxine effectiveness was carried out between 1997 and 1999 at Kilify on the Kenyan Coast, and it concluded that prevalence of triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of pyrimethaminesulfadoxine as a new malaria treatment.Gatton et al. (2004) worked on evolution of resistance to pyrimethamine-sulfadoxine in P. falciparum.Their findings indicate three stages in the development of drug resistance.
The first is the collection of existing parasites with genetic mutations in the DHFR and DHPS gene and it was driven by long half-life of pyrimethamine-sulfadoxine combinations.The second stage involves the selection of parasites with allelic types of higher resistance within the host during an infection.And in the third stage, clinical treatment failure becomes prevalent as the parasites develop sufficient resistance mutation to survive therapeutic doses of the drug combinations.They emphasized on importance of correct treatment of confirmed malaria cases to avoid development of parasite resistance to pyrimethamine-sulfadoxine.

ARTEMISININ DERIVATIVES
For nearly 2,000 years, a cold-water extract of sweet wormwood (Artemisia annua, ''qinghao'') has been used in China for the treatment of fevers.The active ingredient of this plant was isolated in 1970 by Chinese scientists.Artemisinin (or qinghaosu) and its derivatives (artesunate, artemether, and arteether) have been used extensively in China and Southeast Asia, where there are high levels of resistance to the majority of the quinolinecontaining drugs and to all the antifolate drugs (Meshnick et al., 1996).The artemisinin-type compounds in current use are either the natural extract artemisinin itself or the semi-synthetic derivatives (dihydroartemisinin, artesunate, artemether).They achieve higher reduction rates of parasitaemia per cycle than any other drug known to date (White, 1997).

Mode of action
Artemisinin and its derivatives are sequiterpene lactones.
Once administered, the artemisinin derivatives are hydrolyzed rapidly to the biologically active metabolite dihydroartemisinin.The mode of action of the artemisinin drugs has not been completely elucidated.The present knowledge is reviewed by Meshnick et al. (1996) and Cumming et al. (1997).The structure of artemisinin is unusual, and its activity is thought to depend on the presence of the endoperoxide bond, as molecules without it have no antimalarial activity (Brossi et al., 1988).The endoperoxide bond may interact with iron or heme, decomposing into free radicals (Meshnick et al., 1993(Meshnick et al., , 1996;;Paitayatat et al., 1997).Unlike many redox reactions, this process is not reversible, so a single drug molecule will produce only one free radical.The effect of free radicals on the malaria parasite is still not fully understood.Because the concentration of free radicals is insufficient to cause general membrane damage, one theory is that a ''specific free radical target'' exists (Meshnick, 1994).The artemisinin free radical can form a covalent bond with either heme or other parasite proteins (Yang et al., 1993(Yang et al., , 1994) ) and an initial hypothesis was that a heme-artemisinin compound might inhibit the production of hemozoin.No evidence, however, of reduced quantities of hemozoin in artemisinin-treated P. falciparum cultures has been found (Asawamahasakda et al., 1994).Artemisinin also has been shown to bind to 6 specific P. falciparum proteins, one of which is a member of the translationally controlled tumor protein family but the precise effect of this protein alkylation on the parasite is still to be determined.

Mechanism and status of resistance
Artemisinin-resistant strains have been developed both in P. falciparum cultures (Inselburg, 1985) and in P. yoelii mouse models (Chawira et al., 1986).There also have been some indications of increasing in vitro resistance in field isolates (Gay et al., 1994).P. falciparum resistance to artemisinins, which was confirmed on the Cambodia-Thailand border in 2009, is now suspected in parts of Myanmar and Viet Nam.However, Artemisinin-based combination therapies (ACTs) remain highly effective in almost all settings, so long as the partner drug in the combination is locally effective.Artemisinin derivatives have a gametocytocidal activity (Peters, 1993), a feature that, in combination with their pharmacokinetic and pharmacodynamic properties, may well delay the development of drug resistance in the field.Resistance is difficult to induce experimentally, and is labile (low levels of resistance are achieved after sustained drug pressure) but not retained once drug pressure is removed (Peters and Robinson, 1999).Available data suggest that resistance to this class of compounds would be multigenic and share similarities with the quinoline family, as demonstrated in vitro on a series of parasite isolates (Meshnick, 1999).

ATOVAQUONE
The antimalarial activities of hydroxynaphthoquinones were discovered during World War II.Atovaquone is the first effective compound in this class.Currently, it is being marketed as Malarone, which contains a fixed combination of atovaquone and proguanil.

Mode of action
Atovaquone {2-[trans-4-(40-chlorophenyl) cyclohexyl]-3hydroxy-1,4-naphthoquinone}, hydroxynaphthoquinone is used for both the treatment and prevention of malaria in a fixed combination with proguanil.Whilst known to act primarily on mitochondrial functions, its mode of action and synergy with proguanil are not completely understood.This matter is further complicated by the diverse functions of mitochondria in various organisms and by technical difficulties with experiments.It is generally agreed that atovaquone acts on the mitochondrial electron transfer chain, although more recently, its activity and synergy with proguanil has been ascribed to its interference with mitochondrial membrane potential.Atovaquone inhibits cytochrome c reductase activity in P. falciparum (Fry and Pudney, 1992).
Atovaquone is a ubiquinone analogue that binds to the cytochrome bc1 complex of the parasite mitochondrial electron transport chain.The malaria mitochondria electron transport chain disposes of electrons generated by dihydroororate dehydrogenase during the synthesis of pyrimidines (Gutteridge et al., 1979) and the inhibition of this process by atovaquone may kill the parasite (Hammond et al., 1985).More recently, it has been shown in P. yoelli that atovaquone also dissipates the mitochondrial membrane potential of the parasite which may kill the parasite by initiating a process similar to apoptosis.

Mechanism and status of resistance
When atovaquone was first used in clinical trials in Thailand, the treatment failure rate was 33%, regardless of duration of therapy (Looareesuwan et al., 1999).This high level of treatment failure suggests that either a natural background of resistant mutants exists or resistance arises rapidly by the acquisition of point mutations in the cytochrome b gene.Mutations in cytochrome b have been found in atovaquone-resistant Pneumocystis carini and P. yoelii strains, indicating that they may be the cause of atovaquone resistance (Walker et al., 1998;Srivastava et al., 1999).Because of the high rate of treatment failure, atovaquone has been combined with other drugs, including proguanil, doxycycline, and tetracycline.All of these combinations yielded high cure rates (Looareesuwan et al., 1999;Redloff et al., 1996), and the atovaquone-proguanil combination (Malarone)