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Antimalarial Drugs from Nature
Eimear Burke, Jane Deasy, Ruairí Hasson, Ruaidhri McCormack, Vikramjit Randhawa and Philip Walsh, 2nd year Medicine
Humans have always coexisted with parasites. Even in medieval times, it was believed that using large amounts of herbs and spices in cooking, in addition to perfumery materials (camphor, sandalwood and incense), afforded protection from malaria. Indeed, the constituent terpenes (like menthol, carvone and thujone) or phenols (like eugenol and myristicin) did work against parasitic infection, by either causing paralysis of the worms or disrupting the parasitic life cycle. The infectious disease malaria is caused by the protozoan parasite of the genus Plasmodium, and it is one of the most dangerous diseases infecting human populations. Approximately 300 - 500 million people are infected annually, and 1.5 - 2.7 million lives are lost to malaria each year. Four species of the sporozoa are recognised as etiological agents in human malaria: Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium falciparum. P. falciparum is the most dangerous with the highest morbidity rates, and generally is the only species that may cause death in humans.1 The resistance of the malarial parasite to drugs and the resistance of the mosquitoes to insecticides have resulted in the resurgence of malaria in many parts of the world. There is a pressing need for vaccines and new drugs.
THE LIFE CYCLE OF THE MALARIA PARASITE
Plasmodium parasites have a complex life cycle, which is shared between a vertebrate host and an insect vector. The parasite enters the bloodstream through the bite of an infected female Anopheles mosquito; only 60 out of the 380 species of this mosquito are able to carry the malarial parasite. It has been discovered that resistant species have a higher trypsin-like activity in their midguts. Also, sporogeny within the mosquito are governed by environmental temperature as mosquitoes are poikilotherms. Sporozoites from the mosquito’s salivary gland that are injected into the human bloodstream travel to the liver and infect hepatic cells. They remain in liver cells (safe from an immune response) for 9 -16 days, undergoing multiple asexual fission and producing merozoites. After being released from the liver, they may continue to re-infect other liver cells or may attach to and penetrate erythrocytes. The parasite directs formation of a "knob" on a red blood cell’s (RBC) surface that secures the infected cell to a blood vessel wall. This is vital, for if this did not occur, the erythrocyte would simply be transported to the spleen and destroyed, along with the parasite. Also, the body’s immune system recognises foreign proteins generated and destroys infected cells. The parasite overcomes this obstacle by producing phenotypic, polymorphic variants of particular surface proteins so that if the immune system recognises one variant, others exist to avoid detection and elimination. A build up of attached erythrocytes onto a vessel wall can block circulation, resulting in hypoxia and even coma or death in cerebral malaria.1
The merozoites differentiate into trophozoites, which devour 75% of the RBC’s haemoglobin in order to replicate its nucleus. During this stage the parasite is called a schizont. Cytoplasm and cell membranes are then placed around each nucleus to form 12-28 merozoites. The parasite ingests haemoglobin by the process of pinocytosis via the cytostome. It is then degraded by two aspartic proteases (plasmepsin I and plasmepsin II) and one cysteine protease (falciparin) in the acidic digestive vacuoles of the malaria parasite. This results in the production of the toxic chemical heme, so the parasite polymerises the heme to harmless hemozoin. Certain chemical and biochemical conditions are necessary for this process to occur: an acidic environment (pH 4.5-5.0), depletion of antioxidants and the presence of ferriprotoporphyrin IX in the Fe+3 state.1
Eventually, the erythrocytes lyse (from osmotic fragility and infection), thus releasing even more merozoites. Pyrogens are also released, causing severe cycles of chills and fever every 36 hours with subtertian malaria (P. falciparum), every 48 hours with tertian malaria (P. vivax) and every 72 hours with quartan malaria (P .malariae). These time spans correspond to the infection and breakdown of erythrocytes. Anaemia also results. Merozoites may also differentiate into microgametocytes and macrogametocytes which do not rupture the erythrocyte. When another feeding mosquito ingests these gametocytes, they develop into female and male gametes, which fuse to form a diploid zygote (an ookinete). This then penetrates the mosquito’s gut wall where it develops into an oocyst. Sporogeny within the oocyst produces many sporozoites, and upon oocyst rupture, they migrate to the salivary gland for injection into another host, beginning the cycle again.1
Figure 1. The Life Cycle of the Malaria Parasite
During the seventeenth century, malaria was a global epidemic causing untold havoc in several countries, claiming a heavy toll of human lives and eluding an effective cure even from eminent physicians of the day. In 1630, a great discovery was made by the Spanish when they found the quina tree on the eastern side of the Andes mountains. The bark of this ‘fever tree’ was found to be a natural remedy of this deadly disease. The name is believed to originate from the Countess of Chinchon, wife of a Spanish Viceroy who in 1638, fell desperately ill with malaria. She was cured using the herbal remedy of ‘quinquina’ bark and in her honour, the tree was named Cinchona. Cinchona is a genus of tropical evergreen trees and shrubs, belonging to the family Rubiaceae. Not all species of cinchona can be used to produce quinine; the most useful species are C. officinalis, C. calisaya and C. pubescens.2
Throughout the 1600’s to mid-1800’s, quinine bark was the most widely used treatment for malaria, proving to be the first chemical compound used successfully to treat an infectious disease. In 1820, Parisians Pierre Peletier and Joseph Coventou isolated from a fresh supply of bark a bitter gum that was soluble in both alcohol and acid. Of the 36 alkaloids found in the cinchona bark, only four possessed antimalarial properties, with Quinine being the most effective. Its molecular formula was found to be C20H24N2O2, enabling it to bind strongly to blood proteins and form complexes that are toxic to the malarial parasite.2
Quinine’s Mode of Action
There are two main derivatives of quinine: chloroquine and mefloquine. Mefloquine is more widely used due to emerging chloroquine resistance. However, chloroquine in combination with proguanil (paludrine) has proven a sensible alternative during pregnancy because of its relatively low toxicity. Although quinine and its derivatives have been used as malaria prophylactics for over 50 years, much confusion surrounds the true mechanism of these drugs. Electron microscope studies on the effect of quinoline-containing drugs on P. falciparum have shown that the first physical changes are swelling of the food vacuole and accumulation of undigested haemoglobin. This vacuole is the site of haemoglobin degradation to provide amino acids for growth. This suggests that these drugs operate by blocking action of the food vacuole.3
Chloroquine is a diprotic weak base which is attracted to the acidic pH of the parasite’s food vacuole. Once in the vacuole, it becomes deprotonated and membrane-impenetrable, and accumulates in the vacuole.
The latest research suggests that the target for drug action is ferriprotoporphyrin IX (FP), a self-toxic protein involved in the polymerisation pathway of haem to haemozoin (malaria pigment). FP is necessary as plasmodia lack haem oxygenase enzymes. The exact mechanism of this polymerisation is still under investigation, and current theories are conflicting. Regardless of the nature of the pathway, chloroquine is capable of blocking the polymerisation process. It has been shown that saturation of chloroquine uptake is mediated by binding to FP. The chloroquine-FP complex may act as a catalytic poison to the polymerisation reaction. Chloroquine operates against asexual forms of pathogenic malaria parasites (called the "hemo-schizontocidal effect"). However, it is inefficient against gametocytes or exo-erythrocytic liver forms. Chloroquine is known to induce many side effects. The most serious reaction is pigmentary retenitis with an irreversible loss of visual field; however, this only occurs after an accumulative dose of 1000 mg or more. Skin, hair and nail alterations may arise, and in very rare cases, chloroquine use can lead to neuropsychiatric problems, impairment of blood production, ringing in the ears and photosensitization. 4,5
Mefloquine, the other main derivative, inhibits the uptake of chloroquine in infected cells by blocking ingestion of haemoglobin. Lack of Hb disrupts generation of FP to which chloroquine would bind. This mechanism explains the antagonistic effect of chloroquine and mefloquine on parasite growth, and the phenomenon that increased resistance of parasites to chloroquine parallels an increased sensitivity to mefloquine. Studies on the mode of action of mefloquine and quinine suggest that inhibition of haemoglobin degradation is not an essential component of their function; they may inhibit haemoglobin ingestion by inhibiting the endocytotic process. Melfloquine interferes with the transport of haemoglobin and other substances from erythrocytes to the food vacuoles of the malaria parasite.6 Mefloquine also affects only the asexual form of the parasite, with no effect on exo-erthrocytic liver forms or on gametocytes. Side effects include: vertigo, nausea, vomiting, abdominal pain and diarrhoea. On very rare occasions neuropsychiatric symptoms may occur.5
QINGHAOSU / ARTEMISININ
Another alleviating compound was first recorded by Li Shizen, who discussed the use of Qing hao as an antimalarial drug in his Compendium of Materia Medica in 1596. He realised that the shivers and fever of malaria could be combated by Qing hao preparations, and his herbal remedy led to the development of one of today’s most effective antimalarial drugs. Another reference to the herb is contained in the Zhou Hou Bei Ji Feng (handbook of prescriptions for emergency treatments) written in 340 A.D. In the book, the following recipe was found: "In order to reduce fevers, soak one handful of Qing hao in one litre of water, strain the liquor, and drink it all." 7 Even without sophisticated knowledge of organic chemistry or complicated experimental research the ancient Chinese herbalists succeeded in harnessing the medicinal properties of the Qing hao plant.
In 1972, a crystalline compound was extracted from the qinghaosu plant, known in western countries as "artemisinin." In 1979, chemists successfully determined the structure of artemisinin using X-ray crystallographic analysis. They discovered that artemisinin is a sesquiterpene with five oxygen atoms, two of them in a peroxide bridge system over a seven-member ring and two others in a lactone ring structure.8
Figure 2: The chemical structure of artemsinin
Its empirical formula is C15H22O5. Artemisinin is highly insoluble in oil and water and therefore can only be administered orally. No major side effects have been reported, but high parenteral doses of artemisinin derivatives in rats led to selective brain stem neuropathy, which has fuelled debate over safety. 9
Artemisinin’s Mode of Action
Artemisinin-based compounds in current use are either extracted from the parent compound found in the Artemisia annua plant, or are semi-synthetic derivatives like dihydroartemisinin, artemether, arteether and artesunate. These compounds contain a trioxane pharmacophore (Figure 3) but their mode of action is still not fully understood.
The peroxide bridge in the trioxane pharmacophore is essential for the expression of antimalarial activity. Peroxides undergo reductive scission by low-valency transition metals to generate oxygen-centred radicals. These radicals, due to affinity for hydrogen, might generate carbon-centred radicals and produce an FP=O, leading to an epoxide which is a highly active alkylating agent. The carbon-centred radicals may alkylate either haem itself or other proteins, such as Translationally Controlled Tumor Protein (TCTP). It remains to be seen whether the alkylation is specific for the TCTP-haem complex or whether any protein that binds haem can be similarly alkylated. It is also unclear whether haem-bound artemisinin can react with proteins which do not bind haem.5
Figure 3: The chemical structure of the trioxane pharmacophore
This "carbon-centred radical theory" is not universally accepted. Arguments against such a theory are that the formation of carbon-centred radicals is common in reductive cleavage of endoperoxides, but not necessarily linked with appreciable antimalarial activity. Also, the radical-protein complexes are unlikely to survive long enough to reach the target biomolecules. Instead, it is proposed that the trioxane pharmacophore within artemisinin acts as a source of hydroperoxide, through generation of an oxo-stabilised cation upon heterolysis of the C3-O2 bond. This would provide electrophilic oxygenating species or hydroxyl and alkoxyl radicals via reductive cleavage with exogenous iron(II) or other reducing agents. These species would be capable of hydroxylating biomolecules or abstracting hydrogen atoms.8
A consideration of structure-activity relationships involving semi-synthetic and fully synthetic artemisinin derivatives lends support to a bioactivation process that does not involve reductive scission of the intact peroxide. Certain rearranged dihydroartemisinin derivatives are relatively active in vitro against chloroquine-resistant and sensitive clones of P. falciparum, but the formation of ‘carbon-centred radicals’ via intramolecular H-atom abstraction with these compounds is not apparent.8
There is no unequivocal evidence that either reductive scission or hydroperoxide ring-opening mediated by ferrous haem, exogenous ferrous ions or any other agent actually occurs in the highly oxidizing, acidic environment of the food vacuole in the parasite. Each bioactivation pathway will lead to different products upon reaction with target molecules, which will prove invaluable in identifying the true mode of action of these compounds in the future.8
Activity of Artemisin Derivatives
Artemisinin and its derivatives act as blood schizonticides. Derivatives prove useful because artemisinin itself has poor aqueous solubility and decomposes in other protic solvents, resulting in poor bioavailability. Its two main derivatives are artemether and artesunate. Artemether is a rapidly-acting antimalarial drug. The presence of its endoperoxide bridge is essential for its function. This generates a single oxygen molecule and leads to the formulation of free radicals. While it does not last very long in the body, it is metabolised in the liver to a demethylated derivative – dihydroartemisinin - which has a half-life surviving 10 hours more than artemether. As a result of free radicals, dihydroartemisinin morphologically changes the parasites’ membranes. Artemether has no clinical neurotoxicity and no major side effects. However, monotherapy often results in relapse and coma resolution time is significantly delayed. On rare occasions, there can be a decrease in reticulocyte count and changes in ECG patterns.9
Artesunate is a water-soluble semi-synthetic hemisuccinate derivative of artemisinin. It is the most rapidly effective of all the antimalarial drugs because of its instantaneous bioavailability. It is synthesized by reacting dihydroartemisinin and succinic acid anhydride in an alkaline medium. This type of reaction yields an ester linkage in alpha configuration. A combination of mefloquine and artesunate is highly effective even against multi-drug resistant malaria.9
Drug resistance occurs selectively in the species P. falciparum. The other three species have no documented resistance apart from the regionalized choroquine resistance observed in P. vivax, concentrated largely in Papua New Guinea and Irian Jaya (Indonesia). The reasons for the development and spread of drug resistance involve the interaction of drug-use patterns, characteristics of the drug itself, human host factors, parasite characteristics, and vector and environmental factors.9,10 However, only gene mutations confer resistance to the parasites in nature. A summary on the determinants of drug resistance is shown in the Table 1.
Table 1. Determinants of Antimalarial Drug Resistance
The gene pfmdr1, encoding P-glycoprotein homologue 1 (Pgh1), is linked to chloroquine resistance through mutation.11 In multi-drug-resistant mammalian cancer cells, the P-glycoprotein is an ATP–dependent pump that expels chemotherapeutic agents from the cell. In P. falciparum, the P-glycoprotein is located mainly in the membrane of the digestive vacuole of the parasite and evidence suggests it is involved in nucleotide-dependent transport across the membrane.12 Mutations in other (unidentified) genes are also required to confer complete resistance to the parasites. Changes in Pgh1 can modulate resistance to quinine, mefloquine and halofantrine. Artemisinin also showed decreased sensitivity against various strains of P. falciparum due to this mutation.13 Another gene, pfcrt, coding for a vacuolar membrane transporter protein (PfCRT ) is also associated with chloroquine resistance.11
Resistance to chloroquine arises due to the ability of the P. falciparum to release chloroquine 40-50 times more rapidly than a normal susceptible parasite. Calcium channel blockers like verapamil, vinblastine and daunomycin enhanced the accumulation of chloroquine in a resistant parasite and also inhibited the release of chloroquine. These changes were not found in normal susceptible parasites.13 Calcium channel antagonists are thought to interact with the P-glycoprotein transport system in the membrane of the parasite.15
It has been observed that a mutation which results in the over-expression of the pfmdr1 gene causes increased resistance to mefloquine, paralleled with quinine, but decreased resistance to chloroquine.12 Although increased resistance to mefloquine may occur in the future, it remains an effective drug in most countries. The combination of mefloquine with the qinghaosu derivative artesunate appears promising in combating malaria.14
There is no solid evidence for resistance to artemisinin, although recurrence is associated with the monotherapy of artemisinin and its derivatives at a high rate. In order to prevent this return, artemisinins are used with longer-acting antimalarial medications in combined treatments.12
Quinine and artemisinin are two plant-based antimalarials which were discovered centuries ago. However, certain aspects of their mechanisms are still not yet fully understood. In the absence of vaccines, these compounds and their derivatives have been crucial in the control of malaria. The complexity of the parasite mechanism coupled with progressive resistance to malarial drugs presents researchers with numerous difficulties in the development of both effective vaccines and more powerful pharmaceuticals. One of the biggest obstacles in the battle against malaria is poverty in areas where the disease is most prevalent. Thus, measures must be put in place to ensure that these drugs are affordable and accessible to those most in need.
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Fig. 1. is adapted from: Ridley RG. Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 2002 Feb 7; 415 (6872): 674.
Figures 2 & 3 were drawn by Philip Walsh.