8

Drug Discovery and Development

WHERE WE WANT TO BE IN THE YEAR 2010

The ready availability of relatively low-cost prophylactic and therapeutic antimalarial drugs will dramatically reverse rising malaria morbidity and mortality evident throughout much of the world. Cooperative efforts among academic, government, and industry researchers will lead to the discovery and development of these new drugs, which will be available in several formulations, effective against all developmental stages of the four human malaria parasites, stable without refrigeration for extended periods, and have few unacceptable side effects. These drugs will include compounds that reverse resistance to chloroquine (thereby restoring its utility), others that act quickly (and thus will be of great benefit in treating severe falciparum malaria), and still others that can be administered to severely ill patients by methods other than injection (reducing the risk of acquiring other infections through contaminated needles). Finally, an enhanced understanding of basic host-parasite differences will lead to the creation of sophisticated new drug discovery strategies that will provide a continual stream of novel antimalarial drugs for human testing.



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MALARIA: Obstacles and Opportunities 8 Drug Discovery and Development WHERE WE WANT TO BE IN THE YEAR 2010 The ready availability of relatively low-cost prophylactic and therapeutic antimalarial drugs will dramatically reverse rising malaria morbidity and mortality evident throughout much of the world. Cooperative efforts among academic, government, and industry researchers will lead to the discovery and development of these new drugs, which will be available in several formulations, effective against all developmental stages of the four human malaria parasites, stable without refrigeration for extended periods, and have few unacceptable side effects. These drugs will include compounds that reverse resistance to chloroquine (thereby restoring its utility), others that act quickly (and thus will be of great benefit in treating severe falciparum malaria), and still others that can be administered to severely ill patients by methods other than injection (reducing the risk of acquiring other infections through contaminated needles). Finally, an enhanced understanding of basic host-parasite differences will lead to the creation of sophisticated new drug discovery strategies that will provide a continual stream of novel antimalarial drugs for human testing.

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MALARIA: Obstacles and Opportunities WHERE WE ARE TODAY Principles of Prophylaxis and Treatment There is ample evidence documenting the increasing incidence of malaria worldwide, due in large part to the spread of Plasmodium falciparum. The deteriorating efficacy of existing antimalarial drugs, because of increasing numbers of drug-resistant parasite strains, makes routine prophylaxis and treatment of the disease a therapeutic challenge. Deciding which drug to use depends on a number of factors, including the patient's age and his or her clinical and immune status. Other important considerations are the type of malaria (vivax, falciparum, ovale, or malariae), outcome desired, drug availability and costs, side effects and degree of compliance expected with the prescribed regimen, drug sensitivity of the parasite strain in the area in which the infection was acquired, and the most appropriate route of administration. Antimalarial drugs are used for five basic purposes (Webster, 1990): to prevent infection from establishing itself in the body (causal prophylaxis); to prevent an established infection from manifesting itself clinically (suppressive prophylaxis); to treat an acute attack of malaria in order to relieve symptoms, eliminate asexual stages of the parasite, or completely eliminate malaria parasites from the body (treatment therapy); to eliminate parasites, whether or not they are causing symptoms (curative therapy); and to eliminate persisting liver forms of the parasite (antirelapse treatment). A sixth use of antimalarial drugs, not now employed, relies on mass distribution of compounds that eliminate gametocytes in infected individuals to reduce parasite transmission in human populations. Since causal prophylactic agents are few, logistically difficult to administer (daily doses are required), and often toxic, this approach to preventing malaria is seldom practical. Additionally, there is parasite resistance to one of the major causal prophylactics, pyrimethamine. Most prophylactic drugs suppress parasitemia and clinical disease. Antirelapse or radical curative treatment may be given either after clinical treatment of a relapsing malaria (caused by P. vivax or P. ovale) or following suppressive prophylaxis when exposure to either of these parasites has occurred. The goals of treating an established infection can also vary. In people who have no natural immunity and are only temporarily exposed to the parasite (e.g., migrants, travelers, military personnel, and temporary laborers), infections must be treated vigorously to eliminate all malaria parasites from the body, since parasitemia may reach life-threatening levels in a short period of time. Children up to the age of four living in endemic regions are at serious risk of severe and even fatal infection. Prompt treat-

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MALARIA: Obstacles and Opportunities ment and, in some instances, prophylaxis of these children can be life-saving. In other situations, however, completely eradicating parasitemia may not be necessary or even desirable. For example, older children and adults living in areas where exposure to P. falciparum malaria is constant and uncontrolled derive more protection from their own immunity than from antimalarial drugs. In such settings, medications that help control clinical attacks of malaria and prevent an uncontrolled rise in parasitemia are sufficient, since they prevent death and allow for the gradual acquisition of immunity. It is not the intention of this report to provide current treatment guidelines. Those who are interested in this aspect of malaria are urged to consult recent documents that describe treatment options in detail (World Health Organization, 1990). Status of Prophylaxis and Treatment There are three distinct population groups for whom antimalarial drugs are important: indigenous inhabitants of malarious areas, temporary (nonindigenous) inhabitants of malarious areas, and individuals who become ill with malaria or are diagnosed with the disease in nonmalarious regions of the world. The status of drug treatment and prophylaxis for each of the four malaria parasites is presented below. Plasmodium malariae and P. ovale Drugs used for prophylaxis and treatment of infections caused by P. ovale or P. malariae seem satisfactory. There are no reports of resistance in these species. In terms of both disease severity and numbers of cases, these parasites are of less concern than P. vivax and P. falciparum. Plasmodium vivax Drug treatment and prophylaxis of P. vivax malaria is far less than satisfactory. Most significantly, there are serious side effects with primaquine, a causal prophylactic used to eliminate latent liver-stage parasites. Recently, there have also been problems with drug supplies. Without primaquine, curative therapy or prevention of relapses is not currently possible. Although chloroquine has been used to eliminate blood-stage parasites, and is thus used to treat the symptoms of vivax malaria, there are strains of P. vivax resistant to this drug (Rieckmann et al., 1989). Fresh therapeutic approaches must be developed.

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MALARIA: Obstacles and Opportunities Plasmodium falciparum The situation for drug treatment and prophylaxis of P. falciparum malaria is desperate (Payne, 1987; Moran and Bernard, 1989). Given the toxicity and scarcity of primaquine, causal prophylaxis is not a viable option. Daily doses of proguanil provide causal prophylaxis in areas where resistance to this drug is not present, but the compound is not marketed in the United States. Since this species of parasite does not cause relapse, suppressive prophylaxis should be able to prevent the disease. However, strains of P. falciparum resistant to one or more of the available antimalarial drugs have been documented throughout the world. Resistance to the dihydrofolate reductase inhibitors pyrimethamine and proguanil is widespread, and the potential for serious toxicity with the para-aminobenzoic acid (PABA) anti-metabolites sulfadoxine, sulfalene, and dapsone has limited their use in combination with reductase inhibitors for this purpose. The nearly global spread of chloroquine resistance precludes its routine use for suppressive prophylaxis. In the worst situations, such as along the Thailand-Cambodia border, parasite strains resistant to mefloquine and/or quinine are also disturbingly common, and few viable prophylactic regimens exist. Among the antibiotics with antimalarial activity, most attention is currently focused on doxycycline, a tetracycline derivative suitable for once-daily dosing (Pang et al., 1987, 1988). Concerns about the widespread use of doxycycline or other tetracyclines for prophylaxis of malaria include the emergence of drug-resistant strains of P. falciparum, potentially eroding the clinical utility of these drugs in combination with quinine for the treatment of severe malaria (Bruce-Chwatt, 1987). In addition, there are concerns that doxycycline prophylaxis could select drug-resistant strains of pathogenic bacteria (Peters, 1990a,b). Tetracyclines are broadly available in many malarious areas, however, and the impact on resistance patterns from the use of tetracyclines for malaria prophylaxis is unclear. A recent report suggests that the use of doxycycline for malaria prophylaxis in Thailand was not associated with an increased risk of diarrhea from drug-resistant strains of enteric pathogens (Arthur et al., 1990). Furthermore, tetracyclines are often taken as prophylaxis against “travelers' diarrhea” by visitors to less-developed countries. The safety of these drugs is remarkable. Given these considerations, it is appropriate to use doxycycline, at least for travelers in regions where chloroquine resistance is a problem, for malaria prophylaxis. That there are more treatment options for falciparum malaria than prophylactic regimens is not cause for optimism. The problem of drug resis-

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MALARIA: Obstacles and Opportunities tance seriously compromises the therapeutic options for malaria infections acquired in many parts of the world. The choice of a drug should be guided at least in part by an understanding of the drug sensitivities of the parasites in the locality in which the infection originated. Typically, quinine, mefloquine, or halofantrine can be used to cure falciparum malaria that is resistant to pyrimethamine and chloroquine. In areas where artemisinin or artemether is available, these drugs generally seem to be effective. As a last line of defense, prolonged courses of tetracyclines can be useful. However, these antibiotics are slower acting than other antimalarial drugs and, when used alone, are not satisfactory for treating severe disease. For the several areas of the world plagued by strains of multidrug-resistant P. falciparum, only very restricted treatment options exist. As these strains spread, the effectiveness of antimalarial drugs will continue to erode. Available Antimalarial Drugs Causal Prophylactics Primaquine The 8-aminoquinoline class of antimalarial drugs, of which primaquine is the most studied, were derived from methylene blue in one of the earliest efforts in medicinal chemistry. The discovery of methylene blue's antimalarial activity by Guttman and Ehrlich in 1891 is sometimes considered to mark the advent of modern chemotherapy (Carson, 1984). In 1926, the world's first synthetic antimalarial agent, pamaquine, was produced in Germany. A close structural analog of primaquine, this compound proved too toxic for clinical use. Primaquine was subsequently developed during a massive screening program for new antimalarial drugs instituted by the U.S. Army during World War II. The use of primaquine was somewhat hampered by its limited supply, but this situation has been resolved. The utility of primaquine therapy is also limited because it has a relatively low therapeutic index. That is, the dose required to produced a cure is only slightly lower than the amount considered toxic in humans. Prominent side effects include gastrointestinal distress, methemoglobinemia, and in patients with a genetic deficiency in the enzyme glucose-6-phosphate dehydrogenase, oxidant stress-induced hemolytic anemia. Changes in the drug's formulation may reduce the incidence or severity of these reactions. The mechanism of action of primaquine against malaria liver-stage parasites and gametocytes has not been determined. Metabolism of primaquine by the host is thought to be necessary for drug activity, and most investigations have focused on the ability of primaquine metabolites to generate active oxygen species that kill the parasite (Bates et al., 1990). Though

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MALARIA: Obstacles and Opportunities this is an attractive hypothesis, more research is needed to specify the molecular events that underlie the antimalarial activity of primaquine. In addition to its use as a causal prophylactic, primaquine has on occasion been used as a gametocytocidal drug. Alone among available antimalarial drugs, primaquine has significant activity against these sexual stages. Since by destroying the gametocyte stages primaquine can theoretically reduce malaria transmission, mass treatment of infected individuals could be of potential benefit. Given the drug 's toxicity, however, and the fact that such therapy offers no immediate benefit to the patient, this usage is not generally recommended. No efforts are now being devoted to the discovery of compounds that act specifically against gametocytes. Use of gametocytocidal drugs as part of a malaria control strategy would require wide distribution, repeated dosing, and a high rate of compliance. Since no drug is entirely without side effects and the target population would acquire no short-term therapeutic benefits, the development of such a drug, in the absence of activity against liver- or blood-stage malaria parasites, is not attractive. The need for wide drug distribution and a high compliance rate also reduce the likelihood that gametocytocidal therapy will play a role in future malaria control operations. Schizonticidal Drugs Several compounds demonstrate activity against both liver- and blood-stage malaria parasites. These drugs, including pyrimethamine, proguanil, and doxycycline, are described in the next section. Drugs Used for Treatment Quinine The antifever properties of Peruvian cinchona bark were revealed to Europeans in the early 1600s. More than 200 years later, the structure of the cinchona alkaloids was determined. Quinine is just one of a number of active constituents in this botanical preparation. Quinine remains an important therapeutic agent, especially for drug-resistant P. falciparum infections. As a front-line antimalarial drug, however, quinine is restricted by a short half-life, side effects at therapeutic blood concentrations, and the need to reserve its usage for certain situations, such as treatment of drug-resistant falciparum malaria. There is also some disagreement about the proper route of administration for quinine, although the problems associated with intramuscular use of the drug appear to be less severe than originally thought. Quinine 's role in causing hypoglycemia when used to treat severe malaria has been questioned (White et al., 1983a,b; Taylor et al., 1988). The occurrence of the constellation of symptoms termed “cinchonism, ” described during routine antimalarial therapy, requires further evaluation.

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MALARIA: Obstacles and Opportunities The mechanism of action of quinine is not known, nor is it known whether this drug, and others that contain a quinoline nucleus, share a common mechanism. This topic is discussed in more detail for chloroquine, on which most research attention has focused. Strains of P. falciparum with reduced sensitivity to quinine have been reported. Fortunately, increasing the dose seems to overcome this problem, although this increases the risk of side effects. Quinine resistance appears to be an independent phenotype, sometimes but not always associated with mefloquine or chloroquine resistance. Compounds that reverse chloroquine resistance also reverse quinine resistance but not mefloquine resistance (see below). Penfluridol (Peters and Robinson, in press), which reverses resistance to mefloquine, halofantrine, and artemisinin, has no influence on quinine or chloroquine resistance. Although quinine and chloroquine resistance are affected similarly in a pharmacological sense, the two traits occur independently. The molecular basis for resistance to quinine is not well understood. Just as malaria has historically shaped the outcomes of some wars, so too have wars determined some approaches to malaria chemotherapy. For example, attempts by the Dutch to transport cinchona trees from Peru to other sites in the tropics resulted in the establishment of a cinchona plantation in Java. Eventually, this area accounted for 90 percent of the world's supply of quinine. The seizure of Java by the Japanese during World War II interrupted this supply and forced the Allied nations to begin an intensive search for synthetic quinine replacements, which eventually led to the discovery of nearly all currently available antimalarial drugs. Chloroquine This 4-aminoquinoline was initially synthesized in Germany as part of that country's antimalarial medicinal chemistry program. The drug was thought to be too toxic, however, and development was rejected in favor of its 3-methyl analog, sontoquine. Allied interest in this series was stimulated by the capture of supplies of German sontoquine in Tunis during World War II. Chloroquine was identified anew as a highly effective antimalarial compound by the American drug screening program during World War II. Further development resulted in its becoming a very useful antimalarial drug. It is relatively safe, inexpensive, and in the absence of resistance, highly effective. In fact, the major limitation to its usefulness is the emergence of drug-resistant strains of P. falciparum and, recently, P. vivax. The mechanism of action of chloroquine and other quinoline-containing antimalarial drugs, including other 4-aminoquinolines, quinine, and mefloquine, is not known. Various mechanisms have been proposed, including DNA binding, formation of cytolytic complexes with ferriprotoporphyrin IX (a breakdown product of heme, stored in granules by the parasite), and

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MALARIA: Obstacles and Opportunities variants of the lysosomotropic hypothesis, which holds that accumulation of the drug in the acidic food vacuole of the parasite somehow disrupts its function. None of these hypotheses is supported by conclusive proof, and all have been questioned on the basis of experimental results and theoretical considerations (Ginsburg and Geary, 1986; Schlesinger et al., 1988; Geary et al., 1990; Meshnick, 1990). It is not known whether these drugs share a common mechanism of action. It is particularly distressing that quinine was the first drug whose structure was determined, 170 years ago, yet very little is known about how it exerts its therapeutic effects. Considerable effort has been spent investigating the mechanism by which malaria parasites develop resistance to chloroquine (Krogstad et al., 1988). Most research favors the hypothesis that in resistant strains, a specific protein that transports the drug out of the parasite is either amplified or altered to increase its activity (Martin et al., 1987). The process is probably similar to the glycoprotein-mediated efflux of antitumor drugs observed in multidrug-resistant tumor cells (Foote et al., 1990). Although this is an attractive theory with a considerable body of supporting evidence, there may be other explanations of the efflux process (Ginsburg and Stein, in press), and some complicating pharmacological questions need to be resolved. Furthermore, recent genetic evidence suggests that the efflux genes are not linked to resistance (Wellems et al., 1990). Other 4-aminoquinolines, including amodiaquine, hydroxychloroquine, amopyroquine, and pyronaridine, are also occasionally used to treat malaria. Of these, amodiaquine has been used the most, but none of these agents has a significant advantage over chloroquine. Although cross-resistance to amodiaquine is not uniformly observed in chloroquine-resistant P. falciparum, it is common enough to prevent routine use of the drug in these cases. Because amodiaquine often causes agranulocytosis and hepatitis —extremely serious side effects—its use is no longer recommended under any circumstances. Pyrimethamine and Pyrimethamine-Antimetabolite Combinations Pyrimethamine is a diaminopyrimidine derivative. Like the biguanides discussed below, it is an inhibitor of the enzyme dihydrofolate reductase. Inhibition of this enzyme disrupts DNA synthesis. Pyrimethamine, which was developed at Burroughs Wellcome in the 1940s, is structurally homologous to the biguanide compound, proguanil. Pyrimethamine has two major disadvantages: it does not work well against some strains of P. vivax; and when used alone, it quickly selects for resistant strains of P. falciparum. Currently, pyrimethamine is used only in combination with one of several PABA antimetabolites, including sulfadoxine or sulfalene (both sulfonamides) or dapsone (a sulfone). The potential antimalarial activity of sulfonamides was demonstrated

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MALARIA: Obstacles and Opportunities when the sulfonamide-containing azodye prontosil was found to be active against P. falciparum and the primate malaria parasite P. knowlesi. Specific antimalarial activity was discovered during the development of the sulfonamide antibiotics. Interestingly, the sulfonamides are not very effective against P. vivax, suggesting that folate synthesis is unnecessary for this parasite. By themselves, the sulfa drugs act slowly against P. falciparum which reduces their clinical utility. Since in combination with a dihydrofolate reductase inhibitor these compounds have such enhanced activity, there is little reason to consider using them as single agents for treating malaria. The choice of dapsone, sulfalene, or sulfadoxine (from among many active compounds) is made on the basis of their half-life in humans. These compounds are relatively long acting, as is pyrimethamine, and drug synergy is most beneficial when drugs with similar pharmacokinetics are combined. The long half-life of the sulfa component of the two-drug combination has, unfortunately, meant the occurrence of a major and serious side effect, skin reactions. In their most severe form, these can progress to a sometimes fatal condition known as Stevens-Johnson syndrome, which may be a type of idiosyncratic or allergotoxic effect. The incidence of the syndrome depends in part on the duration of treatment and is apparently more common with longer-acting drugs, such as the antimalarial agents (Miller et al., 1986). Because of the risk of Stevens-Johnson syndrome, combinations of sulfa drugs and pyrimethamine are no longer recommended for prophylaxis of falciparum malaria. However, combinations still play a role in the treatment of P. falciparum infections, though their utility has been eroded by the widespread development of resistance. Only the pyrimethamine-sulfadoxine combination is marketed in the United States. The mechanism of action of pyrimethamine and its synergism with the sulfa drugs are understood in elegant detail (Vennerstrom et al., 1991). Pyrimethamine binds tightly to dihydrofolate reductase from malaria parasites but poorly to its mammalian counterpart. Inhibition of this enzyme prevents nucleic acid synthesis. Dihydrofolate reductase from P. falciparum has been cloned, sequenced, and modeled on the basis of crystallographic analyses of similar reductases from other organisms. PABA is a component of folic acid; PABA antimetabolites act to decrease the synthesis (and thus intracellular concentrations) of folate. Only organisms that cannot acquire folate from the environment are adversely affected by sulfa drugs. By reducing folate concentrations, sulfa drugs enhance the inhibition of dihydrofolate reductase by pyrimethamine (Milhous et al., 1985). Resistance to pyrimethamine is also well understood. Isolates of P. falciparum resistant to the drug consistently show substitution of a single

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MALARIA: Obstacles and Opportunities amino acid at one location in dihydrofolate reductase. Proguanil resistance is associated with substitution at a different amino acid location, and strains resistant to both drugs show substitutions at both sites (Peterson et al., 1990). A detailed understanding of the inhibitor-enzyme interaction offers an opportunity for rational drug design or discovery. It should be possible to identify compounds that bind to dihydrofolate reductase but are unaffected by the mutations associated with pyrimethamine and proguanil resistance. Drugs derived from such a program would restore the usefulness of this strategy for treating falciparum malaria. Proguanil (Chlorguanil) and Chlorproguanil The discovery of antimalarial activity of the biguanides predates the development of the diaminopyrimidines. These compounds were initially prepared as acyclic analogs of a series of anilino pyrimidines under investigation at Imperial Chemical Industries in the United Kingdom. Like pyrimethamine, proguanil is a causal prophylactic agent for P. falciparum, but it acts slowly against the blood-stage parasite. It is not particularly useful for treating vivax malaria. Unlike pyrimethamine, proguanil has a short half-life, and so prophylactic use requires daily dosing. Strains of P. falciparum resistant to proguanil are common, seriously limiting its use in therapy. Chlorproguanil contains one more chlorine atom than proguanil, which increases the drug's potency and half-life (Peters, 1990b). Neither drug is currently marketed as a single oral dose in combination with a PABA anti-metabolite, however, and clinical trials conducted in Thailand with proguanil in combination with sulfonamide suggest that these drugs may have utility in areas endemic for multidrug-resistant malaria (Karwacki et al., 1990). Neither proguanil nor chlorproguanil is marketed in the United States. The mechanism of action of the biguanides is the same as that of pyrimethamine. Technically, the biguanides are “prodrugs” that must be metabolically converted by the human host to a cyclic compound, cycloguanil, that inhibits dihydrofolate reductase (Webster, 1990). Mefloquine The development of mefloquine was a collaborative achievement of the U.S. Army Medical Research and Development Command, the World Health Organization (WHO), and Hoffman-LaRoche, Inc. The drug was recently licensed in the United States and several other countries. A 4-quinoline methanol, mefloquine may be considered a quinine analog. It was first synthesized by the Army's medicinal chemistry program in the late 1960s. Clinical and field trials during the past 17 years have confirmed the effectiveness of a single dose of mefloquine for rapidly clearing P. falciparum parasitemia and alleviating symptoms. Preliminary studies of mefloquine

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MALARIA: Obstacles and Opportunities in pregnant women (beyond the first trimester) with falciparum malaria indicate tolerance comparable to that with quinine (World Health Organization, 1990). The triple-drug combination mefloquine-sulfadoxine-pyrimethamine is registered in at least 11 countries. WHO no longer recommends its use, however, since its effectiveness in areas of pyrimethamine-sulfadoxine resistance is unproven. Mefloquine itself is reserved for use in regions where drug resistance is a serious problem (World Health Organization, 1990). Recently, problems have arisen with mefloquine use. The cure rate for mefloquine-sulfadoxine-pyrimethamine treatment of P. falciparum in Thailand fell from 96 percent in 1985 to as low as 50 percent in 1990 in some areas of the country (W. Rooney, Ministry of Health, Thailand, personal communication). Indications that the cure rate was dropping came from follow-up data of patients treated in clinics at the Cambodia-Myanmar border. Field studies are under way. Mefloquine can produce adverse neurological and psychiatric reactions. Data from clinical trials testing the drug's therapeutic potential and surveys of travelers taking mefloquine for malaria prophylaxis suggests that its use is associated with a wide range of side effects, including ataxia, depression, stupor, and seizures. The risk of such adverse reactions is on the order of 1 percent following a treatment dose of 1 gram or more, and is 1 in 5,000 following a prophylactic dose. It is not known whether these complications can be reduced by altering the drug's formulation (Lobel et al., 1991). The mechanism of action of mefloquine, like that of quinine, remains a matter of conjecture. Strains of P. falciparum resistant to mefloquine may export the drug more efficiently than sensitive strains. However, mefloquine resistance is pharmacologically distinguishable from resistance to the structurally similar drug quinine. Halofantrine Halofantrine, an aminoalcohol, is a member of the 9-phenanthrenemethanol class of drugs. It was first identified as a potential antimalarial agent during World War II. In the 1960s, when it became clear that chloroquine would have a limited life span, further work was done on this compound by the Walter Reed Army Institute of Research (WRAIR) (Horton, 1988). Commercial development of halofantrine began in 1984. Clinical trials have confirmed its efficacy in both P. falciparum and P. vivax infections in semi-immune patients in Malawi, the Solomon Islands, Thailand, Pakistan, France, French Guyana, Gabon, and parts of East and West Africa (Horton and Parr, 1989). The drug is effective against chloroquine-resistant falciparum malaria, although it shares a few structural features with quinine and mefloquine, the mechanism of action is thought to differ and may be unique. Data that

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MALARIA: Obstacles and Opportunities vitro drug screening using radioisotopes have been developed to complement animal testing in the drug discovery process. Well-characterized clones of falciparum malaria parasites have been derived by a specialized process of direct visualization and single cell micromanipulation. A stable and well-defined pattern of drug susceptibility makes these clones extremely useful for evaluating candidate drugs of diverse chemical classes (Milhous et al., 1989). World Health Organization The Special Programme for Research and Training in Tropical Diseases (TDR), a joint project of the United Nations Development Programme, the World Bank, and WHO, administers an extensive antimalarial drug development program. TDR efforts include the further development of mefloquine, particularly for use in pregnant women and children; studies of the arteether and artemether derivatives of artemisinin; development and evaluation of halofantrine, with an emphasis on improving bioavailability; research on the blood schizonticides, such as the biguanides already on the market; and the development of novel blood and tissue schizonticides, such as the 1,2,4-trioxanes and hydroxynaphthoquinones. Researchers supported by TDR also are working to develop in vitro drug susceptibility tests and tests for detecting the presence of antimalarial drugs in blood and urine. The mechanisms of antimalarial drug resistance, the efficacy of drugs that may reverse resistance, and drug combinations that might delay the development of resistance are under investigation. Other TDR-supported scientists are attempting to synthesize antimalarial drugs for use in preclinical studies, investigate alternatives to microscopy for diagnosing malaria, identify potential drug targets in the malaria parasite's metabolic pathways, and develop cultivation methods for vivax and malariae bloodstage parasites. Role of Drug Companies Unfortunately, antimalarial drug discovery and development is of little interest to pharmaceutical firms. Because the vast majority of malaria cases occur in poor individuals living in less developed countries with nonconvertible currencies, even if a new and highly effective antimalarial compound were developed, a drug company likely would suffer a poor return on the money it invested in research and development. The ease with which novel antimalarial drugs are developed depends on the structural diversity and number of compounds available for screening and the strategies used to identify them. Although WHO supports research related to malaria chemotherapy, the only centralized facility de-

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MALARIA: Obstacles and Opportunities voted to antimalarial drug screening is WRAIR. Pharmaceutical companies possess collections of compounds which, for the most part, have never been screened for antimalarial activity. A number of pharmaceutical companies are investigating drugs with antiprotozoal activity, including activity against Eimeria species, Toxoplasma gondii, and Cryptosporidium species. Although these organisms are related to Plasmodium species, the drugs under study have not been tested for antimalarial qualities. Other Participants A variety of academic, national, and industrial laboratories are or have previously been involved in antimalarial drug development. Several are mentioned in the next section. Drugs Under Development When a candidate antimalarial drug moves from being evaluated in animals to being assessed in humans, the first step of the process —phase I testing—uses incrementally increasing doses given to healthy volunteers in an effort to determine the drug's safety and patients' tolerance for it. Once the drug's safety is assured, phase II testing is conducted to judge its efficacy in volunteers or naturally infected patients with low-level parasitemias and mild clinical illness. In phase III testing, the drug is initially given only to patients hospitalized with moderately severe disease; later, wide-scale use of the drug in a limited area, with careful monitoring to evaluate efficacy and detect low frequency side effects, takes place. Following phase III, an application may be submitted to FDA for licensure of the drug. Phase IV, post-marketing surveillance, involves careful scrutiny for low-prevalence side effects (Fernex, 1984a,b). Although there are several promising leads, few antimalarial drug candidates have reached phase I testing. Arteether This ethyl ether derivative of artemisinin has been selected by WHO and the U.S. Army for collaborative development as an intramuscular formulation to treat malaria. It may have some advantages over artemether, another artemisinin analog, in terms of toxicity. WR 238605 Extensive primate studies suggest that this 8-aminoquinoline, a product of WRAIR drug development efforts, will have better efficacy, less toxic-

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MALARIA: Obstacles and Opportunities ity, better oral bioavailability, and a longer half-life than primaquine. Human testing awaits the filing of an investigational new drug application with FDA. It is of considerable interest that WR 238605 has also shown activity against the opportunistic pathogen Pneumocystis carinii in rodents (Bartlett et al., 1991). Pneumocystis carinii pneumonia is a common and serious infection in AIDS patients. A partner for development of the drug has not yet been selected. BW566c This compound is a member of the hydroxynaphthoquinone class of compounds. The antimalarial activity of these compounds was discovered in the 1940s, when they were synthesized and tested as part of an effort to develop a synthetic replacement for quinine. Several drugs in this class, including menoctone, have gone on to human testing, but further development proved unwarranted. BW566c was identified as an antimalarial candidate at the British drug company Burroughs Wellcome (Gutteridge, 1989). Interest intensified when, like WR 238605, it was found to possess anti-P. carinii activity. It is now in human trials for the treatment of P. carinii pneumonia. Development of BW566c as an antimalarial agent depends on the results of these studies; it is not likely to undergo further research and development if it has only antimalarial potential, since Burroughs Wellcome, like most other pharmaceutical companies, has terminated its antimalarial drug discovery program. There is some evidence that the hydroxynaphthoquinones act by inhibiting electron transport at ubiquinone-sensitive sites in the parasite mitochondria. This inhibition is coupled to inhibition of the enzyme dihydroorotate dehydrogenase, which is necessary for pyrimidine (and thus nucleic acid) synthesis. Antibiotics Antimalarial activity has been found in various fluoroquinoline antibiotics and newer erythromycin derivatives, including azithromycin and roxithromycin. Most research has focused on the quinolones, which are already approved by the FDA for human use. One of these, ciprofloxacin, is now in human trials for malaria. Whether or not these drugs will have advantages over the tetracyclines and clindamycin remains to be seen. Agents That Reverse Chloroquine Resistance A wide variety of drugs have been found to restore the potency of chloroquine against resistant strains of P. falciparum in vitro. None of these

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MALARIA: Obstacles and Opportunities compounds have yet been tested in clinical trials. These drugs may act by blocking the active efflux of chloroquine from the parasite, a process that can be measured in vitro (Krogstad et al., 1987). Similar compounds inhibit the efflux of anticancer drugs from resistant tumor cells. A number of compounds with pharmacokinetics similar to that of chloroquine, including verapamil (and its analogs), desipramine, ketotifen, cyproheptadine, nifedipine, diltiazem, and chlorpromazine, have been tested in animals (Bitonti et al., 1988; Peters, 1990b). All but chlorpromazine have been found wanting because of toxicity, an inability to cure infection, or both (Vennerstrom et al., 1991). The combination of chloroquine and chlorpromazine can cure experimental P. falciparum infections in aotus monkeys (Rossan et al., 1990). Like chloroquine, chlorpromazine has a long half-life. A great deal of clinical experience has been obtained with this drug. It has a good safety profile at the doses required to treat malaria and is inexpensive. Other phenothiazines should be evaluated for their ability to reverse chloroquine resistance. If the efficacy of a chloroquine-chlorpromazine combination is supported by field studies, the addition of chlorpromazine or similar drugs to preparations of chloroquine could have tremendous potential for treating falciparum malaria. It should also be determined whether the chloroquine-chlorpromazine combination is useful for chloroquine-resistant P. vivax malaria. Agents in Preclinical Development Three classes of compounds are in various stages of pre-human testing. These include analogs of floxacrine, which has unsuitable toxicity; quinazoline folate antimetabolites; and 1,2,4-trioxane derivatives, which are synthetic analogs of artemisinin. Pharmaceutics and Clinical Pharmacology The discovery and subsequent development of novel antimalarial drugs have little value unless those compounds reach the people who can benefit from their use. The way drugs are manufactured, packaged, stored, distributed, and marketed is crucial to this end. Although these processes are dependent to some degree on the management of malaria control programs and the infrastructure of malarious countries, they are very much part of the drug development process. These issues are too broad to discuss in detail here, but it is essential to recognize that the development of antimalarial drugs is futile unless those drugs reach the populations that require them: the residents of malarious regions. Great advances in the use of an antimalarial drug can be expected once

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MALARIA: Obstacles and Opportunities the compound is made available. Studies in clinical pharmacology often lead to better ways of using drugs, better formulations or delivery systems, the optimization of dosing regimens in various populations, and the recognition and alleviation of side effects. A thorough understanding of clinical pharmacology is not achieved during drug development, but support for such studies, devoted to both established and new drugs, can be expected to improve the quality of care for those who suffer from or are at risk of contracting malaria. RESEARCH AGENDA Artemisinin The need for new classes of antimalarial drugs is urgent because of the spread of resistance to most existing classes of compounds. RESEARCH FOCUS: Acceleration of the clinical development of artemisinin and its derivatives. Drug Mechanisms of Action With the exception of the dihydrofolate reductase inhibitors, the mechanisms of action of antimalarial drugs are poorly understood. Identification of these mechanisms is important because it may reveal new targets for drug discovery and provide specific screens for new compounds that operate by the same or similar mechanisms. In this way, drugs might be developed with an eye toward avoiding parasite resistance while retaining the antiparasite selectivity of the prototype. RESEARCH FOCUS: The basic pharmacological mechanisms of action of all available antimalarial drugs. RESEARCH FOCUS: A mechanism-based screening strategy that identifies compounds of interest by their ability to bind to or otherwise inhibit specific proteins (enzymes or receptors) or processes thought to be critical for parasite viability, development, or reproduction. Quinoline Resistance The emergence of resistance to the quinoline family of schizonticidal antimalarial drugs (chloroquine and its analogs; mefloquine, halofantrine, and quinine) has had a major impact on the prophylaxis and treatment of

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MALARIA: Obstacles and Opportunities falciparum malaria. Although enhanced drug efflux does appear to play a role in this phenomenon, uncertainties remain about the functional mechanism of antimalarial drug resistance. Certain compounds containing protonatable amines reverse resistance both in vitro and in vivo, but there is a lack of conclusive data on how this effect is mediated. RESEARCH FOCUS: The molecular biological basis of parasite resistance to the quinoline antimalarial drugs. New Drug Targets While great benefit can be obtained from further discovery efforts aimed at known parasite targets of drug action, it is equally important to identify new targets. This may require determining the functional role of the parasite mitochondria, the functional physiology and biochemistry of the food vacuole (including proton pumps, digestive enzymes, and the bio-chemistry of pigment formation and iron metabolism), the enzymology of lipid synthesis and membrane formation, the enzymology of glycolysis, and the molecular biology of parasite development. RESEARCH FOCUS: Evaluation of parasite biochemistry and physiology, with the goal of cloning key parasite proteins—such as phosphofructokinase, tubulin, and the vacuolar proton pump—that might be useful drug targets. These proteins can then be compared with their human homologues. Transformation To get the most out of studies of mechanisms of drug action and of parasite drug resistance, a method by which genes can be introduced into P. falciparum is required. The ability to clone the genes responsible for resistance would be greatly enhanced if they in turn could be transferred into (or “transform”) drug-sensitive strains. RESEARCH FOCUS: Develop a transformation system for P. falciparum. Plasmodium vivax Culture System Unlike P. falciparum, P. vivax cannot be grown in the laboratory. This fact makes conducting basic studies on P. vivax biochemistry and pharmacology problematic and hinders other important research activities, such as

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MALARIA: Obstacles and Opportunities analysis of chloroquine uptake and efflux in resistant strains and development of a convenient assay system for screening candidate antimalarial drugs for P. vivax activity. RESEARCH FOCUS: Development of an in vitro culture system for P. vivax. Drug Delivery Some antimalarial medications must be administered by injection, a method that has several drawbacks. For one, the use of contaminated hypodermic needles is a significant risk factor for contracting a number of serious diseases, including AIDS and hepatitis, in many areas of the world. The discomfort and inconvenience of injections also reduces patient compliance with drug regimens. RESEARCH FOCUS: New drug delivery approaches, especially transdermal (skin patch) methods. Botanical Preparations Two of the most important antimalarial drugs, quinine and artemisinin, were derived from plants, and many indigenous populations have developed botanical preparations to treat the symptoms associated with malaria. While not all of these preparations contain medically useful substances, it is not unreasonable to believe that novel compounds with potential antimalarial activity could be found through an organized screening effort. RESEARCH FOCUS: A systematic method for identifying plants of interest, screening them, and characterizing the structures or compounds responsible for their antimalarial activity. Secondary Metabolites The secondary metabolites of bacteria and fungi have many interesting pharmacologic properties. Microbial fermentations, which contain a rich variety of organic molecules, have not yet been exploited for their potential antimalarial properties. While performance of such assays is not difficult and can be contracted out, considerable research on the chemistry of natural products is needed to identify active components. RESEARCH FOCUS: Extension of the technology of de-

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