Iron Chelation Therapy for Malaria

Pharmacol. Ther. Vol. 81, No. 1, pp. 53–75, 1999 Copyright © 1998 Elsevier Science Inc.

ISSN 0163-7258/99 $–see front matter PII S0163-7258(98)00037-0

Associate Editor: P. Rosenthal

Iron Chelation Therapy for Malaria: A Review George F. Mabeza,* Mark Loyevsky,† Victor R. Gordeuk†,‡ and Günter Weiss§ *DEPARTMENT OF MEDICINE, UNIVERSITY OF ZIMBABWE MEDICAL SCHOOL, P.O. BOX A178, AVONDALE, HARARE, ZIMBABWE †DEPARTMENT OF MEDICINE, THE GEORGE WASHINGTON UNIVERSITY MEDICAL CENTER, SUITE 428, 2150 PENNSYLVANIA AVENUE NW, WASHINGTON, DC 20037, USA §DEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY OF INNSBRUCK, INNSBRUCK, AUSTRIA

ABSTRACT. Malaria is one of the major global health problems, and an urgent need for the development of new antimalarial agents faces the scientific community. A considerable number of iron(III) chelators, designed for purposes other than treating malaria, have antimalarial activity in vitro, apparently through the mechanism of withholding iron from vital metabolic pathways of the intra-erythrocytic parasite. Certain iron(II) chelators also have antimalarial activity, but the mechanism of action appears to be the formation of toxic complexes with iron rather than the withholding of iron. Several of the iron(III)-chelating compounds also have antimalarial activity in animal models of plasmodial infection. Iron chelation therapy with desferrioxamine, the only compound of this nature that is widely available for use in humans, has clinical activity in both uncomplicated and severe malaria in humans. pharmacol. ther. 81(1):53–75, 1999. © 1998 Elsevier Science Inc. KEY WORDS. Malaria, iron chelators, cytokines, nitric oxide. CONTENTS 1. INTRODUCTION . . . . . . . . . . . . . . 1.1. THE PROBLEM OF MALARIA . . . . . 1.2. IRON WITHHOLDING AS AN ANTIMALARIAL STRATEGY . . . . . 2. IRON METABOLISM OF PLASMODIUM FALCIPARUM . . . . . . . . . . . . . . . 2.1. LIFE CYCLE OF THE MALARIA PARASITE . . . . . . . . . . . . . . 2.2. ACQUISITION OF IRON BY THE ERYTHROCYTIC PARASITE . . . . . 2.2.1. PLASMA TRANSFERRIN . . . 2.2.2. ERYTHROCYTE FERRITIN . . 2.2.3. LABILE INTRAERYTHROCYTIC IRON . . . . 2.2.4. HOST HEMOGLOBIN . . . . . 2.3. METABOLIC PROCESSES THAT ARE DEPENDENT ON IRON . . . . . . . . 3. ANTIMALARIAL ACTIVITY OF IRON CHELATORS IN VITRO . . . . . . . . . . 3.1. TWO MAJOR MECHANISMS OF ACTION . . . . . . . . . . . . . . 3.1.1. WITHHOLDING IRON FROM

3.2.

3.3.

. 54 . 54 . 54 . 54 . 54 . 55 . 55 . 55 . 55 . 56 . 56 . 56 . 56

PLASMODIAL METABOLIC PATHWAYS . . . . . . . . . . . 3.1.2. FORMING TOXIC COMPLEXES WITH IRON . . . . . . . . . . . POTENTIAL MAJOR EFFECTS OF IRON WITHHOLDING . . . . . . . . . . . . 3.2.1. RIBONUCLEOTIDE REDUCTASE . . . . . . . . . . 3.2.2. d-AMINOLEVULINATE SYNTHASE . . . . . . . . . . . PHYSICAL PROPERTIES THAT AFFECT THE ANTIMALARIAL ACTIVITY OF IRON CHELATORS . . . . . . . . . . . 3.3.1. HYDROPHILIC/HYDROPHOBIC BALANCE . . . . . . . . . . . .

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3.3.2. AFFINITY FOR IRON . . . . . . 3.3.3. SELECTIVITY FOR IRON VERSUS OTHER CATIONS . . . 3.3.4. SELECTIVITY FOR IRON(III) VERSUS IRON(II) . . . . . . . . 3.3.5. NUMBER OF COORDINATION SITES . . . . . . . . . . . . . . 3.4. INFORMATION ON SPECIFIC ANTIMALARIAL IRON CHELATORS . . 3.4.1. DESFERRIOXAMINE . . . . . . . 3.4.2. N-TERMINAL DERIVATIVES OF DESFERRIOXAMINE . . . . . 3.4.3. REVERSED SIDEROPHORES . . . 3.4.4. HYDROXYPYRIDINONES . . . . 3.4.5. ACYLHYDRAZONES . . . . . . . 3.4.6. AMINOTHIOLS . . . . . . . . . 3.4.7. IRON CHELATOR COMBINATIONS . . . . . . . . 4. ANTIMALARIAL ACTIVITY OF IRON CHELATORS IN LABORATORY ANIMALS . . . . . . . . . . . . . . . . . . 4.1. DESFERRIOXAMINE . . . . . . . . . . 4.2. DEFERIPRONE . . . . . . . . . . . . . 4.3. REVERSED SIDEROPHORES . . . . . . . 4.4. COMBINATION OF DESFERRIOXAMINE

56 58

5.

58 58 58 58 6.

‡Corresponding

author.

AND SALICYLALDEHYDE ISONICOTINOYL HYDRAZONE . . . . . IRON CHELATION THERAPY FOR HUMAN MALARIA . . . . . . . . . . . . . . . . . . 5.1. IRON CHELATORS IN ADULTS WITH ASYMPTOMATIC PLASMODIUM FALCIPARUM INFECTION . . . . . . . 5.1.1. DESFERRIOXAMINE . . . . . . 5.1.2. DEFERIPRONE . . . . . . . . . 5.2. DESFERRIOXAMINE IN SYMPTOMATIC, UNCOMPLICATED FALCIPARUM AND VIVAX MALARIA . . . . . . . . . . . . IRON CHELATORS AND IMMUNITY IN THE SETTING OF MALARIA . . . . . . . . . . . 6.1. IRON AND IMMUNE FUNCTION . . . .

59 59 59 59 59 59 60 60 60 61 61 61 62 62 62 62 62 62 63 63 63 63 63 63

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G. F. Mabeza et al. 6.2. NITRIC OXIDE AND IRON IN MALARIA. . . . . . . . . . . . . . . . 6.2.1. BENEFICIAL EFFECTS OF NITRIC OXIDE IN MALARIA . . 6.2.2. DETRIMENTAL EFFECTS OF NITRIC OXIDE IN MALARIA . . 6.3. HELPER T-CELL TYPE 1 AND HELPER T-CELL TYPE 2 CYTOKINES IN MALARIA. . . . . . . . . . . . . . . . 7. IRON CHELATORS AND PROTECTION FROM PEROXIDANT TISSUE DAMAGE IN THE SETTING OF MALARIA . . . . . . . . . 7.1. FREE RADICAL-MEDIATED TISSUE DAMAGE IN MALARIA . . . . . . . . .

7.2. EFFECT OF DESFERRIOXAMINE ON 64 65 65 66 67

RECOVERY FROM COMA IN CHILDREN WITH CEREBRAL MALARIA . . . . . . . . . . . . . 7.3. RELATIONSHIP OF OUTCOME IN CEREBRAL MALARIA TO TRANSFERRIN SATURATION . . 7.4. EFFECT OF DESFERRIOXAMINE ON MORTALITY IN CEREBRAL MALARIA . . . . . . . . . . . . . 8. CONCLUSIONS AND DIRECTIONS FOR THE FUTURE . . . . . . . . . . . . . REFERENCES . . . . . . . . . . . . . . .

. . . 67 . . . 68 . . . 68 . . . 69 . . . 69

67

ABBREVIATIONS. BAT, ethane-1,2-bis(N-1-amino-3-ethylbutyl-3-thiol); cGMP, cyclic GMP; CSF, cerebrospinal fluid; DFO, desferrioxamine B (desferal); HNFBH, 2-hydroxy-1-naphthylaldehyde m-fluorobenzoyl hydrazone; IFN, interferon; IL, interleukin; IRE, iron-responsive element; IRP, iron regulatory protein; LPS, lipopolysaccharide; NO, nitric oxide; NOS, nitric oxide synthase; SIH, salicylaldehyde isonicotinoyl hydrazone; STNFrec75, soluble TNF receptor Type II; TAT, N9,N9,N9-tris(2-methyl-2-mercaptopropyl)1,4,7-triazacyclononane; Th-1, helper T-cell Type 1; Th-2, helper T-cell Type 2; TNF, tumor necrosis factor; UTR, untranslated region.

1. INTRODUCTION 1.1. The Problem of Malaria In the 5th century BC, Hippocrates, in his “Airs, Waters and Places,” described a disease of intermittent fevers associated with enlargement of the spleen mainly affecting people who lived near marshes, a description of what, in retrospect, was probably malaria (Bruce-Chwatt, 1988). At the present time, malaria is one of the most geographically widespread and devastating infections in humans. The disease potentially affects about 40% of the world’s population, or more than 2 billion people in some 100 countries (Gilles, 1991). Of the four protozoan species that regularly infect humans, Plasmodium falciparum is responsible for the most severe clinical consequences, including coma, profound anemia, renal failure, and death. It is estimated that falciparum malaria causes 2 million deaths per year (World Health Organization, 1995). Earlier in the century, the widespread application of effective insecticides and antimalarial drugs led to a decline in the incidence of malaria, and some countries were rendered malaria free. Over the past two decades, global resistance to both insecticides and antimalarials has emerged, the incidence of malaria has increased, and the disease has become more widespread (Clyde, 1987). Although early tests of malaria vaccines in human volunteers may have some promise (Patarroyo et al., 1988; Stoute et al., 1997), clinically applicable vaccines will not be available for a number of years (Tanner et al., 1995), and their importance in controlling malaria is uncertain. In this setting, antimalarial chemotherapy remains the principal means available for reducing the morbidity and mortality of malaria, and the task of developing new antimalarial drugs with new mechanisms of action is important (Anonymous, 1984). 1.2. Iron Withholding as an Antimalarial Strategy The first evidence that the withholding of iron from vital metabolic pathways of the parasite is a potential antimalar-

ial chemotherapeutic strategy was provided by Dr. Simeon Pollack and colleagues 16 years ago. In their examination of the growth of P. falciparum in cultured erythrocytes in the presence of the iron-chelating agent desferrioxamine (desferrioxamine B, deferoxamine, desferal, DFO), these investigators demonstrated that iron is an essential nutrient for the asexual erythrocytic phase of the parasite, that the withholding of iron inhibits parasite growth and replication, and that iron chelation exerts its maximal effect at the stage of the late trophozoite (Raventos-Suarez et al., 1982). The study also raised numerous questions that have been addressed with varying degrees of success over the past decade. How does the parasite acquire iron? What is the exact role of iron in the metabolism of the erythrocytic malaria parasite? What metabolic processes of the parasite does the withholding of iron disrupt? Does the withholding of iron have clinically important effectiveness in the treatment of human malaria? In this article, we review the information that has emerged to the present time in response to these questions. 2. IRON METABOLISM OF PLASMODIUM FALCIPARUM 2.1. Life Cycle of the Malaria Parasite The pathogenesis of falciparum malaria is related to the capability of the parasite in the red cell to reproduce at an extremely rapid rate. The erythrocytic phase of the parasite is preceded by sexual reproduction in the intestine of the Anopheles mosquito, asexual replication in the mosquito with storage of sporozoites in the salivary gland, and asexual proliferation in the hepatocyte of the human host, which is infected as the result of the bite of a parasitized mosquito. The asexual, erythrocytic stage of the parasite’s life cycle, which follows the hepatic phase, is responsible for the clinical manifestations, morbidity, and mortality of the disease. In just 48 hr, a single P. falciparum merozoite enters the red cell, matures into a trophozoite, undergoes DNA replica-

Iron Chelation Therapy for Malaria

tion, and divides into 8–32 daughter cells. The red cell then ruptures and releases these merozoites for almost immediate infection of other red cells (Garnham, 1988). The trace element iron is essential for this explosive proliferation of P. falciparum. 2.2. Acquisition of Iron by the Erythrocytic Parasite The intra-erythrocytic parasite lies within a parasitophorous vacuole, the membrane of which is partially derived from the red blood cell membrane (Aikawa, 1988a). The merozoite enters the erythrocyte by attaching to sialic acid residues on the red cell surface (Hudson-Taylor et al., 1995), followed by invagination of the erythrocyte membrane (Aikawa, 1988a). Within the red blood cell, the parasite first appears as a ring form and then matures into a trophozoite. The trophozoite obtains nutrients by ingesting host cell cytoplasm, including hemoglobin, by means of a cytostome (Aikawa, 1988a). The parasite also enhances the permeability of the host red cell membrane to a number of metabolites (Cabantchik, 1990) and possibly may take up molecules from the outer medium directly through a parasitophorous duct (Pouvelle et al., 1991; Loyevsky et al., 1993). Once taken into the parasite, host cytoplasm is transported in vesicles to the central food vacuole. This acidic organelle is the site of primary hemoglobin proteolysis. A cysteine proteinase, falcipain, appears to be involved in the dissociation of the hemoglobin tetramer and the release of heme from globin (Gamboa de Dominguez and Rosenthal, 1996). Two aspartic proteases, plasmepsins I and II (Kolakovich et al., 1997; Francis et al., 1997), cleave hemoglobin into small peptides. The additional cleavage of these oligopeptides (an average of 8.4 amino acids) to individual amino acids apparently requires the activity of an aminopeptidase and, most likely, occurs after their translocation to the parasite cytoplasm (Kolakovich et al., 1997). The heme liberated by the proteolysis of hemoglobin is polymerized in the food vacuole to form inert hemozoin (Slater and Cerami, 1992). How the intra-erythrocytic phase parasite acquires iron for its metabolism has not been determined yet, and several possible sources have been postulated, including plasma transferrin-bound iron, iron derived from red blood cell host ferritin, a labile intra-erythrocytic iron pool, and iron derived from the catabolism of host hemoglobin in the food vacuole of the parasite. 2.2.1. Plasma transferrin. Some early clinical studies suggested a protective effect of iron deficiency against human malaria (Murray et al., 1978; Oppenheimer et al., 1986b) and an enhanced susceptibility to malaria infection with increased nutritional iron (Murray et al., 1975; Oppenheimer et al., 1986a). These studies seemed to be consistent with the possibility that plasma transferrin may be a source of iron for the intra-erythrocytic parasite. More recently, a study of school children in Papua New Guinea did not find any evidence that human iron status affects malarial infection (Harvey et al., 1989). Moreover, experimentally induced iron deficiency and iron overload do not af-

55

fect parasitemia with P. berghei in rats (Hershko and Peto, 1988). Similarly, despite earlier reports that in contrast to normal mature erythrocytes, mature parasitized red blood cells express transferrin receptors (Rodriguez and Jungery, 1986; Haldar et al., 1986), further evidence indicates that transferrin receptors do not exist on parasitized erythrocytes (Pollack and Schnelle, 1988; Sanchez-Lopez and Haldar, 1992). The possibility that nonspecifically bound transferrin is taken up from plasma into parasitized erythrocytes has been proposed (Pollack and Flemming, 1984; Pollack and Schnelle, 1988; van Zyl et al., 1993), but the bulk of the evidence indicates that transferrin iron is not taken up by parasitized red cells (Peto and Thompson, 1986; Hershko and Peto, 1988). 2.2.2. Erythrocyte ferritin. Although the mature erythrocyte cannot synthesize ferritin, it does contain residual ferritin that was produced during the earlier erythroblast phase (Cazzola et al., 1983). This residual ferritin, if fully saturated with iron, may account for about 4.8 mM iron (Gabay and Ginsburg, 1993). The acquisition of iron by the parasite within the parasitophorous vacuole from ferritin in the cytoplasm of the erythrocyte is a theoretical, but uninvestigated, possibility. The fact that iron deficiency is associated with low red blood cell ferritin concentrations (Cazzola et al., 1983) without inhibition of intra-erythrocytic parasite growth (Hershko and Peto, 1988) might be regarded as evidence against erythrocyte ferritin as the source of iron for the plasmodium. Nevertheless, red cells from iron-deficient subjects do contain detectable amounts of ferritin (Cazzola et al., 1983), and the possibility remains that the parasite takes up iron from ferritin across the parasitophorous vacuole membrane and parasite plasma membrane, or through the process of cytostomal ingestion and transport to the food vacuole. 2.2.3. Labile intra-erythrocytic iron. With evidence against plasma transferrin as the source of iron for the intra-erythrocytic trophozoite, it was proposed that the parasite uses a labile pool of iron in the cytoplasm of the erythrocyte for its metabolism (Hershko and Peto, 1988). In support of this hypothesis, gel filtration and ultrafiltration studies on hemolysates of rat red cells parasitized with P. berghei revealed a labile pool of iron that is chelatable by preincubation of the intact cells with DFO (Hershko and Peto, 1988). Furthermore, it was observed that the property of being lipophilic, or ability to cross cellular membranes, correlates with the effectiveness of iron chelators to inhibit parasitemia with P. falciparum (Yinnon et al., 1989; Cabantchik et al., 1996). On the other hand, two studies have shown that when iron-chelating agents are introduced into the cytoplasm of erythrocytes, but not into the parasite compartment within the parasitophorous vacuole, no plasmodial growth inhibition occurs (Scott et al., 1990; Loyevsky et al., 1993). It is possible that both host labile iron and another source of iron, such as host hemoglobin iron, are used by the parasite and that the abrogation of only one source will not prevent parasite growth.

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G. F. Mabeza et al.

2.2.4. Host hemoglobin. The intra-erythrocytic parasite derives a major portion of amino acids necessary for protein synthesis from the catabolism of host hemoglobin (as described in Section 2.2). The heme released during this process contains a substantial amount of iron, which, if liberated from heme, might be available for the parasite’s metabolic needs (Hershko and Peto, 1988; Gabay and Ginsburg, 1993). Host hemoglobin does seem to be a likely source of iron for the parasite (Gabay et al., 1994), although a heme oxygenase activity has not been identified in the majority of Plasmodium species, with the exception of the murine P. berghei (Srivastava and Pandey, 1995). Free heme within the food vacuole is polymerized into the stable and inert pigment hemozoin (Slater and Cerami, 1992). Furthermore, malaria parasites synthesize their own heme (Surolia and Padmanaban, 1992; Wilson et al., 1996; Bonday et al., 1997), suggesting that host heme would not be necessary for their metabolism. Although firm evidence that the parasite utilizes iron derived from host heme is lacking at present, the possibility that a small amount of heme in the food vacuole is degraded in a controlled manner to release iron for the metabolic processes of the parasite represents one of the plausible sources of iron for the intra-erythrocytic parasite. 2.3. Metabolic Processes that are Dependent on Iron Many metabolic processes of the erythrocytic malaria parasite are dependent on iron, and a partial listing of such processes are presented in Table 1. From the information provided in Table 1, it can be inferred that the withholding of iron from the parasite by iron chelators conceivably could disrupt the metabolism of the parasite by preventing DNA synthesis, interfering with carbohydrate metabolism, disrupting proteolysis of host hemoglobin, and inhibiting de novo synthesis of heme, normal mitochondrial function, and electron transport. 3. ANTIMALARIAL ACTIVITY OF IRON CHELATORS IN VITRO 3.1. Two Major Mechanisms of Action Several classes of iron-chelating compounds have been shown to suppress the growth of P. falciparum in erythro-

cytes in vitro, as shown in Table 2. A number of these compounds are naturally occurring siderophores, molecules produced by microorganisms to acquire iron from the environment. Numerous studies have shown that the degree of antimalarial activity of iron chelators correlates with the degree of lipophilicity, or the ability to cross cell membranes, of the compound (Yinnon et al., 1989; Hershko et al., 1991; Shanzer et al., 1991; Cabantchik et al., 1996). The antimalarial iron chelators can be placed into two major categories, depending on the predominant mechanism of inhibition of parasite growth. 3.1.1. Withholding iron from plasmodial metabolic pathways. The mechanism of antimalarial action of one group of iron chelators appears to be the sequestration of iron necessary for plasmodial replication rather than a direct toxic effect on the parasite or the withholding of other essential trace metals. The possibility that iron withholding serves as the antimalarial mechanism for this group of compounds is supported by experiments showing that the inhibitory effect on plasmodial growth is completely negated when equimolar concentrations of iron are added to the incubation mixtures to saturate the chelators. This effect has been documented for DFO (Raventos-Suarez et al., 1982; Heppner et al., 1988; Whitehead and Peto, 1990; Loyevsky et al., 1993), methyl-anthranilic DFO (Loyevsky et al., 1993), desferrithiocin, desferricrocin (Fritsch et al., 1987), a-ketohydroxypyridones (Heppner et al., 1988), pyridoxal isonicotinoyl hydrazone (Clarke and Eaton, 1990), salicylaldehyde isonicotinoyl hydrazone (SIH) (Tsafack et al., 1996), daphnetin (Yang et al., 1992), and two aminothiol compounds recently reported to have antimalarial activity (Loyevsky et al., 1997). In addition to their ability to bind iron, many iron chelators also bind copper and calcium, which are indispensable for parasite metabolism (Scheibel and Rodriguez, 1989; Gabay and Ginsburg, 1993). 3.1.2. Forming toxic complexes with iron. Compounds of the second group, which includes aromatic metal chelators such as 8-hydroxyquinoline, seem to have an antiparasitic effect other than the withholding of iron. In the case of 8-hydroxyquinoline, it appears that a complex with iron is formed extracellularly, which subsequently enters the para-

TABLE 1. Iron-Dependent Pathways of the Erythrocytic Trophozoite Metabolic pathway DNA synthesis Pyrimidine synthesis Glycolysis Pentose phosphate shunt CO2 fixation Proteolysis of hemoglobin Heme synthesis Mitochondrial electron transport

Enzyme Ribonucleotide reductase Dihydroorotate dehydrogenase Glycolytic enzymes Pentose phosphate shunt enzymes Phosphoenol pyruvate carboxykinase Proteolytic enzymes d-Aminolevulinate synthase Cytochrome oxidase

Reference Wrigglesworth and Baum, 1980; Raventos-Suarez et al., 1982 Bezkorovainy, 1980; Scheibel and Sherman, 1980 Scheibel et al., 1979; Scheibel and Adler, 1980 Bailey-Wood et al., 1975 Wrigglesworth and Baum, 1980 Cook et al., 1961 Bonday et al., 1997 Scheibel and Sherman, 1988

Iron Chelation Therapy for Malaria

sitized red cell to produce a rapidly lethal free radical-mediated intracellular reaction (Scheibel and Adler, 1980; Scheibel and Stanton, 1986). The alkylthiocarbamates (Scheibel and Adler, 1980), 29,29-bipyridyl, and certain aminophenols are other examples of iron-chelation compounds whose inhibitory antimalarial effect cannot be abrogated by precomplexation with iron before addition to malaria cultures (M. Loyevsky, unpublished data). For both of these groups of iron-chelating agents, regardless of whether the effect is iron deprivation or toxicity via generation of free radicals by the iron-chelator complex, an

57

interaction with iron is the focus of the antimalarial activity. Of note, a number of other antimalarial compounds generally are not recognized as iron chelators, but nevertheless have the ability to bind iron (M. Loyevsky, unpublished observations). These agents include anion-transport inhibitors (derivatives of stilbenic acid) and bioflavonoid glycosides (Cabantchik et al., 1983; Silfen et al., 1988). The studies summarized in Table 2 demonstrating a suppressive effect on the growth of P. falciparum by iron chelators were performed on the erythrocytic phase of the parasite’s life cycle. It was also reported that DFO and desferrithiocin

TABLE 2. Iron-Chelating Compounds that Inhibit Growth of Plasmodium falciparum in Cultured Erythrocytes Class of compound and specific agents

ID501

Agents that inhibit parasite growth by withholding iron Hydroxamate siderophores and derivatives DFO

Methyl-anthranilic DFO Circular DFO Nitrilo-DFO Desferrithiocin Desferricrocin Reversed siderophores Rhodotorulic acid Mycobactin Catecholamide and catecholate siderophores Vibrobactin Parabactin g-Amino butyric acid N4-Nonyl-N1,N8-bis(2,3-dihydroxybenzoyl)spermidine hydrobromide (compound 7) a-Ketohydroxypyridones Deferiprone CP96 Dihydroxycoumarins Daphnetin (ash tree bark extract) Polyanionic amines N9,N9-Bis(2-hydroxybenzyl)-ethylenediamine-N9,N9-diacetic acid Acylhydrazones Pyridoxal isonicotinoyl hydrazone SIH HNFBH Aminothiols BAT TAT Agents that inhibit parasite growth by forming toxic complexes with iron 2,29-Bipyridyl

4–35 mM

3–5 mM 5–9 mM 14–20 mM 25 mM 30–40 mM 0.3–70 mM

2–5 mM 2–3 mM 4–5 mM 0.17–1.0 mM 15–45 mM 5–45 mM 25–40 mM 5 mM 30 mM 18–30 mM 0.17–0.26 mM 6–9 mM 3–4 mM 12–14 mM

8-Hydroxquinoline 1Concentrations

of iron chelator that produce 50% growth inhibition after 48–72 hr of culture.

8.3 nM

Reference

Raventos-Suarez et al., 1982; Peto and Thompson, 1986; Heppner et al., 1988; Yinnon et al., 1989; Whitehead and Peto, 1990; Scott et al., 1990; Hershko et al., 1991; van Zyl et al., 1992; Jambou et al., 1992; Loyevsky et al., 1993 Loyevsky et al., 1993 Glickstein et al., 1996 Glickstein et al., 1996 Fritsch et al., 1987 Fritsch et al., 1987 Shanzer et al., 1991; Lytton et al., 1991, 1993b Scheibel and Stanton, 1986 Scheibel and Stanton, 1986 Scheibel and Rodriguez, 1989 Scheibel and Rodriguez, 1989 Scheibel and Rodriguez, 1989 Pradines et al., 1996 Heppner et al., 1988; Hershko et al., 1991 Hershko et al., 1991 Yang et al., 1992 Yinnon et al., 1989 Clarke and Eaton, 1990 Tsafack et al., 1996 Tsafack et al., 1996 Loyevsky et al., 1997 Loyevsky et al., 1997 Jairam et al., 1991; van Zyl et al., 1992 Scheibel and Adler, 1980; Scheibel and Stanton, 1986

58

are able to inhibit the growth of the exo-erythrocytic hepatic phase of P. falciparum in a culture system employing human hepatocytes (Stahel et al., 1988). This finding indicates that iron chelation represents a potential antimalarial strategy, with effectiveness against both the erythrocytic and hepatic phases of the parasite. 3.2. Potential Major Effects of Iron Withholding 3.2.1. Ribonucleotide reductase. As mentioned in Section 2.1, the pathogenesis of malaria is a function, in part, of the ability of the intra-erythrocytic parasite to reproduce at an extremely rapid rate, requiring a high rate of trophozoite DNA synthesis. The morphology and stage specificity of the antiplasmodial action of DFO suggest that the chelator may interfere with DNA synthesis (Raventos-Suarez et al., 1982; Atkinson et al., 1991). One of the trophozoite enzymes essential for DNA synthesis is ribonucleotide reductase, an iron-containing enzyme that catalyzes the reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, the precursors of DNA (Reichard and Ehrenburg, 1983). In fact, the function of ribonucleotide reductase is rate limiting for DNA synthesis (Cavanaugh et al., 1985; Nyholm et al., 1993; Rubin et al., 1993). Eukaryotic ribonucleotide reductase, including the P. falciparum enzyme, contains two a- and two b-subunits (Rubin et al., 1993). The two dimers, a2 and b2, are known as protein B1 (or R1) and protein B2 (R2), respectively. The large a-subunits contain the substrate-binding sites, but both B1 and B2 contribute to the active site of the enzyme (Nordlund et al., 1990). B2 contains an essential tyrosyl radical that is stabilized by an adjacent dinuclear iron center and is thought to initiate the radical-based reaction of the conversion of ribose into deoxyribose. The iron in this dinuclear iron center is in equilibrium with intracellular iron in the labile iron pool (Nyholm et al., 1993). DFO inhibits ribonucleotide reduction and cell growth not by directly attacking the iron-radical center of the B2 protein, but instead by chelating the intracellular iron pool. This prevents the regeneration of the iron-radical center both in newly synthesized apo-B2 protein and in apo-B2 protein continuously formed from active B2 protein by the loss of iron (Nyholm et al., 1993). In vitro, iron chelation reversibly inhibits ribonucleotide reductase (Reichard and Ehrenburg, 1983; Cavanaugh et al., 1985; Nyholm et al., 1993) and produces a potent inhibition of DNA synthesis in various cellular systems (Cavanaugh et al., 1985). Thus, iron chelators such as DFO may exert their antiplasmodial action through the inhibition of parasite ribonucleotide reductase activity (Hoffbrand et al., 1976; Raventos-Suarez et al., 1982). 3.2.2. d-Aminolevulinate synthase. P. falciparum and other plasmodial species synthesize heme de novo, despite the fact that the parasite is located within the red blood cell, a virtual “red sea” of hemoglobin. The de novo synthesis of heme might represent a novel target for antimalarial therapy (Surolia and Padmanaban, 1992; Bonday et al., 1997). To synthesize heme, it appears that the parasite synthesizes the

G. F. Mabeza et al.

first enzyme in the pathway, d-aminolevulinate synthase. It also appears that other enzymes in the heme synthetic pathway, such as d-aminolevulinate dehydrase, coproporphyrinogen oxidase, and ferrochelatase, are of host origin and are transported into the parasite from the host red blood cell compartment (Bonday et al., 1997). In human erythroid cells, iron chelators cause a down-regulation in d-aminolevulinate synthase synthesis (Fuchs and Ponka, 1996), resulting in an inability to synthesize heme. If iron chelators exert a similar effect in the erythrocytic trophozoite, this would prevent cytochrome synthesis. The cytochromes in the electron-transport chain of almost all species contain heme as a prosthetic group. Inhibition of plasmodial heme synthesis by iron chelators could possibly disrupt electron transport, arrest mitochondrial respiration and accumulation of ATP, and eventually result in the death of the parasite. 3.3. Physical Properties that Affect the Antimalarial Activity of Iron Chelators The iron withheld by iron chelators in the process of inhibiting the growth of intra-erythrocytic malaria parasites most likely resides within the parasitic compartment of the infected red blood cell in the ferric [iron(III)] form (Hershko and Peto, 1988; Scott et al., 1990; Loyevsky et al., 1993). One would thus predict that an effective antimalarial iron chelator would have the ability to cross lipid membranes well, would have a high affinity for iron, would selectively bind iron as compared with other trace metals, and would selectively bind iron(III) rather than iron(II) (Scheibel and Rodriguez, 1989; Hershko et al., 1991). 3.3.1. Hydrophilic/hydrophobic balance. The hydrophilic/ hydrophobic balance of a compound is related to the partition coefficient of the drug in a system of n-octanol and water, and is an important factor in the movement of a compound across a lipid-containing membrane to enter a cell (Scheibel and Rodriguez, 1989; Cabantchik et al., 1996). The hydrophobic/hydrophilic balance, or relative lipophilicity of the iron chelator, would be expected to be an important factor in determining the usefulness of a potential iron chelator as an antimalarial agent (Scheibel and Rodriguez, 1989). The direct positive correlation between the degree of lipophilicity of a compound and its inhibitory action against malaria has been demonstrated experimentally for the reversed siderophores (Shanzer et al., 1991), the N-alkyl derivatives of 3-hydroxypyridine-4-one (Hershko et al., 1991), and several aminothiol compounds (Loyevsky et al., 1997). For more details, see Section 3.4. Another example of this property is the fluorescent compound calcein (an analogue of EDTA), which binds iron(III) with an affinity constant of 1024, but is impermeable to cell membranes and only marginally inhibits parasite growth in culture (IC50 1 mM). The hydrophobic and permeant derivative of calcein, acetomethoxyl-calcein, has an IC50 of 5 mM, or 200 times

Iron Chelation Therapy for Malaria

greater than the impermeant calcein (M. Loyevsky, unpublished observations). 3.3.2. Affinity for iron. A high affinity for iron is an important prerequisite of antimalarial activity of an ironchelating drug (Scheibel and Rodriguez, 1989; Hershko et al., 1991; Cabantchik et al., 1996). This activity is realized through chemical groups that coordinate with iron, for example the hydroxamate groups of the ferrichromes, the catecholate groups of enterobactin (Neilands, 1995), and the synthetic hydroxypyridinones (Hershko et al., 1991; Singh and Hider, 1994). The affinity constants of antimalarial iron chelators for iron(III) range from 1024 for acetomethoxyl-calcein to 1028 for the acylhydrazones (Ponka et al., 1994), 1031 for DFO (Goodwin and Whitten, 1965), 1036 for hydroxypyridine-4-ones (Hershko et al., 1991), and 1038 for the 8-hydrohyquinolines (Albert, 1981). 3.3.3. Selectivity for iron versus other cations. The selectivity of a chelator for iron versus other cations has importance for two reasons. First, malaria parasites have a limited capability to recover after iron deprivation compared with mammalian cells (Cabantchik et al., 1996), making it reasonable to target iron as compared with other essential metals for which the ability to recover from deprivation has not been studied. Second, removal by iron chelators of the other biometals, such as zinc, calcium, and magnesium, may be detrimental for the mammalian host as well. In this regard, the hydroxamate siderophores have a favorable profile, for their affinity for iron is at least one order of magnitude greater than their affinity for zinc, and several orders of magnitude higher than for calcium (Lytton et al., 1993b). 3.3.4. Selectivity for iron(III) versus iron(II). Ligands that bind iron(III) may have more potential as iron chelators than those that bind iron(II). In experiments with aminoterminal derivatives of DFO, certain hydrophobic derivatives such as methylanthranilic-DFO had preservation of the ability to bind iron(III), but substantially reduced ability to bind iron(II) in comparison with the parental unmodified DFO (Glickstein et al., 1996). These compounds retained high activity against P. falciparum in culture while producing virtually no inhibition of growth of mammalian cells. This result is consistent with the possibility that the iron in the parasite’s labile pool is predominantly in the iron(III) (ferric) form. It is worth noting that iron(II) chelators such as 29,29-bipyridyl may exert their antiparasitic effects through the formation of chelator-iron complexes that generate free radicals rather than through the withholding of iron. 3.3.5. Number of coordination sites. The iron atom has six coordination sites, and hexadentate chelators would be expected to form the most stable complexes with the metal. Pentadentate and quadridentate chelators would leave one or two coordination sites of the iron atom unbound and potentially available to participate in toxic reactions that could damage host tissues. Tridentate and bidentate chela-

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tors could fully occupy the coordination sites of iron by forming 2:1 or 3:1 complexes with the metal, but, especially at low chelator concentrations, partial dissociation from iron might easily occur and expose coordination sites to participate in toxic reactions. 3.4. Information on Specific Antimalarial Iron Chelators 3.4.1. Desferrioxamine. DFO is the only agent now available for clinical use as an iron chelator in most countries. As can be seen from Table 2, more work has been done to examine the antimalarial effect of this compound than of any other chelator. DFO is a naturally occurring trihydroxamic acid derived from cultures of Streptomyces pilosus that has been used extensively for over 25 years for iron chelation therapy in patients with iron-loading anemias; it is remarkably safe and nontoxic. DFO (molecular weight in the mesylate form 5 657) is an hexadentate chelator that complexes with iron in a 1:1 molar ratio to form ferrioxamine with a molecular weight of 713. To be effective, the drug must be given by continuous parenteral infusion. Administered in this way, daily doses of up to 150 mg/kg are tolerated (Modell and Berdoukas, 1984; Brittenham, 1988). When DFO was given by continuous parenteral infusion to 6 non-iron-loaded adults at 100 mg/kg/day, the mean plasma concentration achieved was 20.0 6 2.4 mM (Summers et al., 1979). As shown in Table 2, when given as a single agent, DFO suppresses the growth of P. falciparum in parasitized erythrocytes in vitro in concentrations achieved and tolerated in the blood of patients. DFO has an additive inhibitory effect on the in vitro growth of P. falciparum when it is combined with classical antimalarials (van Zyl et al., 1992), although in a single report, it failed to enhance the activity of chloroquine (Basco and Le Bras, 1993). Certain reports have shed light on the site, stage specificity, and lethality of DFO on the erythrocytic P. falciparum parasite. Studies with a high molecular weight dextran derivative of DFO indicated that iron chelation external to the parasitized red cell has no antiplasmodial effect and that iron chelation within the red cell, but external to the parasite, has no activity (Scott et al., 1990). Rather, the antimalarial activity of DFO appears to be related to its ability to enter the erythrocytic trophozoite and to chelate a pool of parasite-associated iron It has been suggested that DFO may enter the parasite directly through a parasitophorous duct that invaginates from the red cell membrane and communicates with the parasitophorous vacuole (Pouvelle et al., 1991), thus by-passing the host red cell cytoplasm (Loyevsky et al., 1993). In confirmation of the early impression that DFO damages the late trophozoite and interferes with schizogony (Raventos-Suarez et al., 1982), experiments with synchronized in vitro cultures of P. falciparum showed that DFO has a cytocidal effect on late trophozoites and early schizonts, and that the critical duration of exposure may be as short as 6 hr at this stage of parasite development (Whitehead and Peto, 1990). Ultrastructural studies have produced corroborating findings (Atkinson et al., 1991). When DFO was

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added to synchronized cultures containing early rings, parasites developed normally until the late trophozoite stage, when all growth ceased. Ultrastructural lesions included the breakdown of the nuclear envelope into small membranous fragments and progressive vacuolization of the nucleoplasm. Other organelles, including food vacuoles and mitochondria, were not affected. The addition of DFO to synchronized cultures of schizonts had similar effects on the nuclei of early schizonts, but little or no effect on mature schizonts and segmenters. Erythrocyte invasion by merozoites proceeded in the presence of the chelator. These findings provide evidence that DFO acts specifically during the late trophozoite and early schizont stages of parasite maturation, and suggest that the chelator may prevent nuclear division. The zinc-DFO complex may enter into parasitized erythrocytes better than the free chelator, and, compared with free DFO, DFO-zinc complexes were shown to have greater antiparasitic activity in in vitro cultures of P. falciparum (Chevion et al., 1995). Zinc-DFO complexes may penetrate the cell and exchange bound zinc for ferric ions because the affinity of DFO for iron is greater than its affinity for zinc. This process might then render the iron unavailable for vital parasite functions (Chevion et al., 1995). In summary, the available studies indicate that the action of DFO on the intra-erythrocytic parasite is both stagespecific and cytocidal. In contrast, in mammalian cells, this compound displays only a cytostatic inhibitory effect, which is reversed upon the removal of the drug from the suspension (Lytton et al., 1994; Glickstein et al., 1996). The differential effect of DFO on malaria-infected erythrocytes and mammalian cells provides the basis for the selective action of DFO as an antimalarial. 3.4.2. N-Terminal derivatives of desferrioxamine. One of the major disadvantages of DFO as an antimalarial agent is its poor permeability into parasitized red cells. To render hyroxamate-based drugs such as DFO more permeant to infected cells and thereby improve their antimalarial activity, Shanzer and Cabantchik modified hydroxamate-based chelators to produce two classes of compounds with improved permeability properties (Shanzer et al., 1991; Cabantchik, 1995; Loyevsky et al., 1993; Glickstein et al., 1996). The first group of chelators consists of N-terminal derivatives of DFO, whose improved membrane permeability was attained by coupling hydrophobic moieties to the parent core of DFO with no diminution in the iron(III) binding capacity (Glickstein et al., 1996). These N-terminal derivatives of DFO differentially affect the growth and replication of intra-erythrocytic parasites. Methylanthranilic-DFO, the least hydrophilic (and most membrane-permeant) member of this group of compounds, reduces parasite proliferation with an IC50 of 4 6 1 mM. The parental DFO, the most hydrophilic of these compounds, displays the greatest IC50 (21 6 7 mM). Cyclic-DFO and nitrilo-DFO, N-terminal derivatives with intermediate hydrophilicity, have intermediate IC50s of 7 6 2 mM and 17 6 3 mM, respectively.

G. F. Mabeza et al.

Methylanthranilic-DFO has strikingly selective activity against malaria parasites as compared with mammalian cells. This agent inhibits the proliferation of mammalian cells (human K562 erythroleukemia cells and human HEPG2 hepatocarcinoma cells) with an IC50 of .100 mM, but as noted above, methylanthranilic-DFO inhibits the growth of malaria parasites with an IC50 of only 4 6 1 mM. 3.4.3. Reversed siderophores. The second group of hydroxamate-based chelators that was developed by Shanzer and Cabantchik consists of synthetic hydrophobic ferrichrome siderophores, termed “reversed siderophores” (Shanzer et al., 1991; Lytton et al., 1993b). Siderophores are microbial ferric ion binders of a hydrophilic character that take up iron in the medium and transport it into the cell via receptor-mediated mechanisms (Neilands, 1995). The “reversed siderophores” were produced by modifying ferrichrome molecules in a way that preserved their ironbinding properties, but replaced their hydrophilic envelopes with hydrophobic ones to facilitate penetration into infected erythrocytes. Because the function of the modified compounds was iron withholding from cells versus the original function of iron delivery, they were named reversed siderophores (Shanzer et al., 1991). The hydrophobic/hydrophilic balance of reversed siderophores was controlled by the lipophilic character of the amino acid chains emerging from the basic poly-hydroxamate structure, while the molecular dimensions were modulated by changing the size of the connecting arms (Shanzer et al., 1991; Lytton et al., 1993b). The design of reversed siderophores as antimalarial agents relied on hydroxamate groups as iron(III)-binding sites because hydroxamate-based chelators poorly scavenge iron(III) from transferrin (Stahel et al., 1988). While fully retaining iron(III) binding capacity, the permeation properties of these compounds across biological membranes were increased (Cabantchik et al., 1996). The antimalarial activity of these modified ferrichromes correlates with their lipophilicity, and the antimalarial activity is abolished when these chelators are applied to cultures of Plasmodia as iron(III) complexes. The reversed siderophores are effective against all stages of parasite growth and against a variety of multidrug-resistant strains of P. falciparum. The most potent agent of this synthetic ferrichrome series, SF-ileu, is not toxic to mammalian cells in culture and has 15-fold more antimalarial potency and 20fold faster antimalarial action than DFO (Shanzer et al., 1991). In vitro, reversed siderophores have a cytotoxic effect on rings and cytostatic effects on trophozoites and schizonts, in contrast to DFO, which has major cytotoxic effects only on trophozoites and early schizonts (Lytton et al., 1994; Loyevsky et al., 1993). These interesting observations provided the basis for studying combinations of iron chelators as antimalarial regimens (see Section 3.4.7). 3.4.4. Hydroxypyridinones. Hydroxypyridin-4-ones are neutral bidentate ligands with a high specificity for ferric iron. The stability constant for the iron complex (log Ka 5

Iron Chelation Therapy for Malaria

37) is 6 orders of magnitude higher than that of DFO. Unlike DFO, hydroxypyridinones are effective in the treatment of iron overload when administered orally (Porter et al., 1986; Kontoghiorghes and Hoffbrand, 1986). In vitro, they exhibit a dose-related suppression of P. falciparum growth (Heppner et al., 1988; Pattanapanyasat et al., 1997). The dimethyl compound of this group, 1,2-dimethyl-3-hydroxypyridin-4-one, also known as deferiprone (L1 or CP20), inhibits the growth of P. falciparum by more than 50% at concentrations ranging from 5 to 100 mM when exposure to the chelator is continuous (Heppner et al., 1988; Hershko et al., 1991, 1992). 3.4.5. Acylhydrazones. Two members of the acylhydrazone family (Ponka et al., 1994), SIH and 2-hydroxy-1naphthylaldehyde m-fluorobenzoyl hydrazone (HNFBH), were tested on malaria cultures in vitro, either as single drugs or in combination with DFO (Tsafack et al., 1996). The effects of the chelators were assessed on synchronized parasite cultures, either in the presence of the chelators or after their removal from the cultures. SIH and HNFBH were effective in suppressing parasite growth at all developmental stages, with mean (6 SD) IC50s of 24 6 6 mM and 0.21 6 0.04 mM, respectively. SIH and HNFBH produced a dose-dependent inhibition of growth when continuously exposed to cells. 3.4.6. Aminothiols. Two compounds from a family of multidentate aminothiol chelators, ethane-1,2-bis(N-1amino-3-ethylbutyl-3-thiol) (BAT) and N9,N9,N9-tris(2methyl-2-mercaptopropyl)1,4,7-triazacyclononane (TAT), inhibit the growth of P. falciparum cultured in erythrocytes, and they outperform the inhibitory action of DFO more than 5-fold (Loyevsky et al., 1997). The IC50s are 7.6 6 1.2 mM for BAT and 3.3 6 0.3 mM for TAT. Both agents appear to affect the trophozoite and schizont stages of parasite development, and they display selective cytotoxicity to malaria parasites versus mammalian cells. The inhibitory effects of these aminothiols seem to be related mainly to their iron-withholding action, because pre-complexation with iron fully reverses the antiparasitic effect (Loyevsky et al., 1997). The greater antiparasitic properties of TAT compared with BAT correlate with its higher degree of lipophilicity and greater number of coordination sites for iron (6 coordination sites in the TAT molecule versus 4 coordination sites in BAT). Also, the inhibitory effect of TAT seems to be more persistent than the effects of BAT or DFO, and TAT displays earlier onset of parasite growth inhibition (Loyevsky et al., 1997). In addition to their ironbinding properties, the aminothiols are capable of preventing free-radical formation (M. Loyevsky, unpublished observations). This feature most likely is due to the thiol groups present in the molecules, and suggests that these compounds may have promise in blocking the oxidative damage to tissues that occurs in patients with severe malaria.

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3.4.7. Iron chelator combinations 3.4.7.1. Desferrioxamine and reversed siderophores. The antimalarial action of DFO is limited to mature forms (trophozoites and schizonts), possibly because of the hydrophilic nature of the chelator. To achieve the antiparasitic effect, relatively high doses and prolonged incubation times are required. The disadvantages resulting from these properties are somewhat balanced by the persistent nature of the action of DFO, probably related to retention of the drug within cells once it has gained entrance (Whitehead and Peto, 1990). When DFO is added to malaria parasites cultured in erythrocytes in combination with the more lipophilic and more permeant reversed siderophore RSFilem2, a strong synergistic inhibitory effect is observed (Lytton et al., 1994). This effect may result from the different speeds of permeation of the two chelators through the host and parasite cell membranes. The rapidly permeating lipophilic agent RSFilem2 irreversibly affects ring-stage parasites, whereas the slowly permeating, but persistent, DFO mainly arrests the development of mature parasite stages (Lytton et al., 1994). It, seems therefore, that with the combination of DFO and reversed siderophores, the parasite is vulnerable at all stages of growth and the antimalarial potential of the drugs increases to beyond the theoretical additive effects (Lytton et al., 1994; Golenser et al., 1995; Lytton et al., 1993b). 3.4.7.2. Desferrioxamine and acylhydrazones. Both SIH and HNFBH potentiate the antimalarial effects of DFO in vitro (Tsafack et al., 1996). For the combination of SIH and DFO, the synergistic inhibitory effect may be explained by free shuttling of SIH through the membranes, withholding iron from critical intraparasitic sources, exiting from the cell as an iron-chelator complex, and conveying iron to the slowly permeating DFO, which has a greater affinity constant for iron and, therefore, may serve as an iron sink (Tsafack et al., 1996; Cabantchik et al., 1996). SIH has a dual nature, acting as an iron scavenger from and an iron provider to mammalian cells in culture (Laskey et al., 1986; Ponka et al., 1994). At relatively low concentrations (,20 mM), SIH potentiates the growth of malarial parasites, implying that SIH-extracted iron is recycled and reutilized by the parasite. Prevention of such reutilization by slowly permeating DFO or by impermeant DFO-hydroxyethyl starch, given along with SIH or HNFBH, results in synergistic inhibitory effects on malaria cultures (Tsafack et al., 1996). The nature of the synergistic inhibitory effect for the acylhydrazone and DFO combination is apparently different from the synergistic effect demonstrated for the combination of reversed siderophore and DFO (Lytton et al., 1994), which resides in the ability of each drug to preferentially inhibit the different stages of parasite growth (Cabantchik et al., 1996). 3.4.7.3. Desferrioxamine and deferiprone. When DFO was combined with deferiprone, an additive interaction on plasmodial growth suppression was observed (G. F. Mabeza, unpublished observations), implying that the two iron(III)

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chelators do not antagonize the effects of each other, but rather complement them. 3.4.7.4. Desferrioxamine and 29,29-bipyridyl. As single agents, both DFO and 29,29-bipyridyl independently inhibit the growth of P. falciparum in culture, with IC50s of 5.2 mM and 12.4 mM, respectively (Jairam et al., 1991). The combination of DFO (2 mM) with 29,29-bipyridyl at various concentrations leads to a relative increase in parasitemia compared with 29,29-bipyridyl alone, suggesting that these compounds have antagonistic effects (Jairam et al., 1991). This result might be explained on the basis of the different antimalarial mechanisms of the two agents, with 29,29-bipyridyl apparently acting through binding iron and forming a complex that is toxic to the parasite, while DFO acts through the withholding of iron. DFO may successfully compete with 29,29-bipyridyl for iron binding by virtue of its greater affinity for iron (Ka of 1031 for DFO versus Ka of 1028 for 29,29-bipyridyl), and thereby prevent the formation of toxic 29,29-bipyridyl-iron complexes. 4. ANTIMALARIAL ACTIVITY OF IRON CHELATORS IN LABORATORY ANIMALS 4.1. Desferrioxamine In the only animal study investigating iron chelation therapy to suppress parasitemia with P. falciparum (Pollack et al., 1987), DFO was active against the erythrocytic phase of the parasite in Aotus monkeys. Similar observations were made with P. berghei and P. vinckei petteri infections in rodents (Fritsch et al., 1985; Hershko and Peto, 1988; Yinnon et al., 1989). Iron chelation therapy with DFO, 2,3-dihydroxybenzoic acid, or the phenolic ethylenediamine derivative N9,N9-bis(2-hydroxybenzyl)-ethylenediamine-N9,N9diacetic acid, starting before, on the day of, or 1 day after initiation of the infection, suppressed parasitemia. Animal studies have demonstrated an antimalarial effect of DFO at doses that overlap with acceptable doses in humans (up to 100–150 mg/kg/day). Parasitemia with P. vinckei and P. berghei in rodent models was inhibited by DFO given in parenteral doses of 90—1,000 mg/kg/day (Fritsch et al., 1985; Hershko and Peto, 1988; Yinnon et al., 1989). In these studies, divided doses of DFO, 2–3 times daily, were more effective than single daily doses in reducing parasitemia and mortality in mice, indicating that sustained exposure to the drug is necessary for it to be effective. P. falciparum parasitemia in Aotus monkeys was markedly inhibited with continuous subcutaneous infusions of DFO in doses of 60–120 mg/kg/day beginning 1–2 days after inoculation with parasitized red cells (Pollack et al., 1987). When the same amount of DFO was given by twice daily subcutaneous injections, no suppression of parasitemia occurred, again demonstrating that sustained exposure to the drug is necessary for it to be effective. 4.2. Deferiprone Although in vitro studies suggest that the oral administration of hydroxypyridone (deferiprone) in safe doses might

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result in a clinically detectable antimalarial effect; the single reported animal study of deferiprone proved to be negative. Deferiprone in 3 divided doses of 300 mg/kg/day for 13 days did not suppress P. berghei infection in 6 female Wistar rats (Hershko et al., 1992). The lack of effectiveness of deferiprone in this animal model, as compared with the studies in vitro, was attributed to intermittent attainment of suppressive plasma concentrations with the subcutaneous or oral mode of administration and to the relatively low lipophilicity of deferiprone, which would limit entry into the red cell under these circumstances (Hershko et al., 1992). 4.3. Reversed siderophores To achieve sustained blood levels of the highly lipophilic reversed siderophore RSFileum2, the agent was delivered in fractionated coconut oil (miglyol 840) via subcutaneous injections to mice infected with P. vinckei petteri (Lytton et al., 1993a). Miglyol is an approved cosmetic and pharmaceutical formulation with low viscosity, high thermal stability, and lack of oxidant capacity, especially adaptable for slow release of hydrophobic compounds into the blood. The chelator was administered at a dosage of 370 mg/kg every 8 hr, and no adverse reactions were observed. Repeated injections of the reversed siderophore over a 56-hr period were associated with a significant delay in the increase in parasitemia compared with the controls. Parasitemia relapsed in all mice 24 hr after ceasing the treatment (Lytton et al., 1993a). 4.4. Combination of Desferrioxamine and Salicylaldehyde Isonicotinoyl Hydrazone DFO and SIH were administered as a combination to Swiss mice infected with P. vinckei petteri or P. berghei (Golenser et al., 1997). The drugs were delivered by several routes: single intraperitoneal injection, multiple intraperitoneal injections, or subcutaneous insertion of a drug-containing polymeric device designed for slow, continuous drug release over 7 days. As single agents administered in these ways and in doses of 125–500 mg/kg/day, all 3 agents led to delays and reductions in peak parasitemias and to reduced mortality. In combination with slowly permeating DFO, the inhibitory actions of SIH on ring forms were significantly potentiated in terms of speed of drug action and extent of inhibition. It appears that SIH enters the infected cell, binds iron within the cell, exits the cell as the ironiron chelator complex, and then transfers iron to the extracellular DFO. The antimalarial action of these combinations in vivo was greatest when the drugs were slowly released into the circulation by means of a biodegradable polymer that was implanted subcutaneously. 5. IRON CHELATION THERAPY FOR HUMAN MALARIA The first use of an iron chelator for human malaria can be attributed to the Chinese, who used the bark of ash trees, which are rich in coumarins, as a folk remedy for malaria.

Iron Chelation Therapy for Malaria

One of these coumarins, a dihydroxycoumarin named daphnetin, is an iron chelator with moderate antimalarial activity in vitro (Yang et al., 1992). More recently, Traore et al. (1991) reported the administration of DFO with chloroquine to 6 patients with uncomplicated falciparum malaria, and there was no evidence of toxicity. Larger clinical trials of the use of DFO in adults with uncomplicated malaria have now been conducted in Thailand and Zambia.

5.1. Iron Chelators in Adults with Asymptomatic Plasmodium falciparum Infection 5.1.1. Desferrioxamine. To determine if iron chelation therapy has activity against human malaria, DFO (100 mg/ kg/day by continuous 72-hr subcutaneous infusions) was administered to 65 Zambian adult subjects with asymptomatic infection with P. falciparum. Two randomized, doubleblind, placebo-controlled, crossover trials were performed (Gordeuk et al., 1992b, 1993). Compared with placebo, DFO treatment significantly enhanced the rate of parasite clearance. Serum concentrations of DFO 1 ferrioxamine (the iron complex of DFO) were measured in 26 subjects. Mean 6 SEM steady-state concentrations were 6.9 6 0.6 mM at 36 hr and 7.7 6 0.7 mM at 72 hr. These levels are at the lower end of the range of values reported for the ID50 for DFO against P. falciparum, as determined in vitro (Table 2). Parasite concentrations were monitored for up to 7 weeks following therapy in 16 subjects, and the reduction in parasitemia lasted for up to 4 weeks. While results obtained with low levels of parasitemia in partially immune adults cannot necessarily be extrapolated to patients with severe infection, these findings suggested that iron chelation may be a potential chemotherapeutic strategy for human infection with P. falciparum. 5.1.2. Deferiprone. Deferiprone has been used for a number of years in patients with transfusional iron overload, and at doses of 75–100 mg/kg/day, this agent leads to a reduction in iron stores in some patients (Matsui et al., 1991; Olivieri, 1996). When the drug was given orally to ironloaded humans at a dose of 75 mg/kg/day, peak serum concentrations of 94–125 mM were achieved (Matsui et al., 1991). These levels are in the range of the in vitro antimalarial suppressive effects of the agent (Hershko et al., 1992; Heppner et al., 1988). A prospective, double-blind, placebo-controlled crossover trial of deferiprone was conducted in 25 Zambian adults with asymptomatic P. falciparum parasitemia to determine if the oral administration of this iron chelator has a clinically detectable antimalarial effect (Thuma et al., 1998b). Deferiprone was administered daily for 3 or 4 days in divided doses of 75 or 100 mg/kg body weight. No reduction in asexual intra-erythrocytic parasites was observed during or after deferiprone treatment. The mean peak plasma concentration of deferiprone (108.2 6 24.9 mol/L) achieved was within the range demonstrated to inhibit the growth of P. falciparum in vitro. However, the times to reach peak plasma levels and to clear

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the drug from the plasma were short, and plasma levels of deferiprone were only in the range of a modest antimalarial effect for much of the time between the oral doses used in these studies. No evidence of deferiprone-associated hematological toxicity was noted in this short-term study of these subjects, all of whom had clinical evidence of normal body iron stores. Because of the risk of neutropenia and other adverse effects with higher doses or prolonged use of the chelator, additional trials of deferiprone as an antimalarial would not seem to be justified. 5.2. Desferrioxamine in Symptomatic, Uncomplicated Falciparum and Vivax Malaria The use of DFO as a single agent for the therapy of symptomatic, uncomplicated falciparum and vivax malaria has been examined in Thailand (Bunnag et al., 1992). Fourteen adult males with P. falciparum infection and 14 adult males with P. vivax infection were given DFO, 100 mg/kg/day, as a continuous intravenous infusion for 3 consecutive days. All subjects were treated in hospital in Bangkok where malaria transmission does not take place; they were followed on the ward for 3 weeks. The initial geometric mean peripheral blood parasite concentration was 13,540 rings/mL in the subjects with falciparum malaria and 19,392 rings/mL in the subjects with vivax malaria. DFO as a single agent reduced parasitemia to 0 within 57 hr for the falciparum group and 106 hr for the vivax group. DFO, in general, was well-tolerated, but about one-third of the subjects experienced transient visual blurring. Recrudescence was observed in all subjects, occurring on the average 10 days after start of therapy in the falciparum group and 15 days in the vivax group. This study demonstrated that iron chelation with DFO is effective as a single agent in both uncomplicated falciparum and vivax malaria, and that this therapy can clear moderate degrees of parasitemia. It also showed that the dose and duration of iron chelation therapy employed in this study failed to achieve a radical cure (Bunnag et al., 1992).

6. IRON CHELATORS AND IMMUNITY IN THE SETTING OF MALARIA 6.1. Iron and Immune Function The maintenance of cellular iron homeostasis is not only a general requirement for the growth and proliferation of all cells, but it is also of central importance for the regulation of immune function (for reviews, see Means and Krantz, 1992; Brock, 1994; Weiss et al., 1995). Iron deficiency, as well as iron overload, seem to exert subtle effects on the immune system by altering the proliferation of T-cells and B-lymphocytes, although the data available so far are quite controversial (Brock, 1994; Weiss et al., 1995). Furthermore, cellular iron availability may even have a differential influence on the proliferation of helper T-cell Type 1 (Th-1) and Type 2 (Th-2) subsets, thus modulating the activities of these lymphocyte subpopulations and their effector

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mechanisms. In addition, iron plays a critical role in macrophage-mediated cytotoxicity by catalyzing the production of reactive oxygen species (Halliwell et al., 1992; Rosen et al., 1995). As a consequence, imbalances of iron metabolism could substantially alter immune function. Special attention has been paid to the effects exerted by iron on the cytotoxic effector potential of macrophages. Increased intracellular concentrations of non-ferritin bound iron reduce the effect of interferon (IFN)-g on cells of the mononuclear-phagocyte system (Weiss et al., 1992). Compared with exposure of these cells to IFN-g without added iron, exposure to the cytokine in the presence of moderate concentrations of ferric-, ferrous-, or transferrin-bound iron results in up to an 80% reduction of major histocompatibility complex Class II expression. The presence of iron also leads to reduced formation of neopterin, a pyrazino-derivative that is produced in excess by macrophages upon stimulation of GTP-cyclohydrolase I by IFN-g (Huber et al., 1984), and to diminished degradation of tryptophan, a molecule that is cleaved to kynurenine, anthranilic acid, and quinolinic acid by the cytokine-inducible enzyme indoleamine-2,3 dioxygenase (Werner et al., 1989; Weiss et al., 1992). Determination of neopterin and kynurenine is widely used as a measure for monitoring cell mediated (Th-1driven) immune activation in vivo and in vitro (Fuchs et al., 1988). The withdrawal of iron from cells of the mononuclear-phagocyte system by the application of the iron chelator DFO stimulates the metabolic pathways induced by IFN-g. Thus, iron-loaded macrophages seem to have a reduced cytotoxic potential towards invading microorganisms, as demonstrated by impaired host defense against such intracellular pathogens as Listeria monocytogenes, Legionella pneumophila, Ehrlichia chaffeensis, or viruses (Alford et al., 1991; Byrd and Horwitz, 1991; Barnewall and Rikihias, 1994; Karupiah and Harris, 1995). Moreover, iron directly targets the formation of nitric oxide (NO), a central component involved in macrophage-mediated cytotoxicity. 6.2. Nitric Oxide and Iron in Malaria NO is a labile radical that is involved in many biochemical and physiological processes, including vasodilatation, neurotransmission, blood coagulation, and immune defense. This molecule is synthesized upon cleavage of L-arginine to L-citrulline and NO via five consecutive oxidative steps. The reaction is catalyzed by the enzyme NO synthase (NOS), which requires flavin-mononucleotide, flavin-adenine-dinucleotide, heme, and 5,6,7,8-tetrahydrobiopterin as cofactors and oxygen, L-arginine, and reduced nicotinic acid-adenine-dinucleotide-phosphate as co-substrates. Three different isoforms of NOS have been identified: nNOS or NOS I in neuronal cells, inducible NOS or NOS II in macrophages, and eNOS or NOS III in endothelial cells. NOS I and NOS III are expressed constitutively, and their activity can be regulated by the availability of their cofactors or by modulation of intracellular calcium concentration. In contrast, the macrophage type of NOS (NOS II) is transcrip-

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tionally induced and subsequently activated upon stimulation with cytokines such as IFN-g, tumor necrosis factor (TNF)-a, interleukin (IL)-1, and/or lipopolysaccharide (LPS) (for reviews, see Nathan, 1992; Moncada and Higgs, 1993; Bredt and Snyder, 1994). Besides its biochemical and pharmacological role in cardiovascular research, intensive care medicine, and neurology, NO has attracted great interest among immunologists because of its role as a central component in cell-mediated immune effector function. NO is produced and released in excess by activated macrophages, thus leading to growth inhibition and killing of invading microorganisms and tumor cells (Hibbs et al., 1987; Drapier and Hibbs, 1988; Stuehr and Nathan, 1989). Many biological functions of NO can be attributed to its high affinity for iron. Effects of NO in blood vessels or in the brain are transduced by NO-mediated activation of guanylate cyclase. NO targets the catalytic center of this heme enzyme, forming an iron-nitrosyl complex that causes a conformational change of the protein, leading to the formation of cyclic GMP (cGMP). The latter substance then induces further biochemical pathways, such as the phosphorylation of proteins via activation of protein kinases, that lead to the known effects on target cells attributed to NO (Ignarro, 1990; Nathan, 1992; Moncada and Higgs, 1993). In a complementary fashion, NO exerts cytostatic and cytotoxic effects towards invading microorganisms and tumor cells. NO, produced and released in excess by cytokine-activated monocytes/macrophages, targets iron-containing enzymes in tumor cells or microorganisms, thus leading to the formation of iron-nitrosyl complexes (Lancaster and Hibbs, 1990). NO inactivates critical enzymes for mitochondrial respiration, such as NADH:ubiquinone-oxidoreductase and NADH:succinate-oxidoreductase, as well as the Krebs cycle enzyme cis-aconitase, via removal of a labile iron from the central iron-sulfur cluster of these enzymes (Drapier and Hibbs, 1988; Stuehr and Nathan, 1989). This causes an allosteric switch in the protein’s center, leading to impaired enzyme function. Similarly, NO inhibits DNA synthesis in tumor cells by inactivation of the nonheme iron enzyme ribonucleotide reductase (Lepoviere et al., 1990; Kwon et al., 1991). Finally, NO is able to remove nonheme iron from the iron storage protein ferritin (Reif and Simmons, 1990). Most recently, we and others have shown that iron metabolism and NO pathways are functionally connected. NO is directly involved in the orchestration of cellular iron homeostasis. Increased formation of NO in response to IFN-g or LPS treatment of macrophages enhances the binding affinity of iron regulatory proteins (IRP) to iron-responsive elements (IRE) (Weiss et al., 1993; Drapier et al., 1993), which are stem-loop structures present within the 59-untranslated region (UTR) of ferritin and the 39-UTR of transferrin-receptor mRNA (Klausner et al., 1993; Hentze and Kühn, 1996). While NO-induced binding of IRP to IRE within the 59-UTR of ferritin mRNA causes repression of translation of this protein (Weiss et al., 1993), interaction of IRP and IRE within the 39-UTR of transferrin receptor

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mRNA results in mRNA stabilization and increased transferrin receptor mRNA expression (Pantopoulos and Hentze, 1995). The expression of NOS II in macrophages is also regulated by intracellular iron availability (Weiss et al., 1994). Increased concentrations of low molecular weight iron reduce IFN-g/LPS-mediated transcription of NOS II and subsequent formation of NO, while intracellular iron deprivation leads to opposite effects. These results (Weiss et al., 1993, 1994; Drapier et al., 1993) suggest the existence of an autoregulatory loop that links the maintenance of iron homeostasis with the optimal formation of NO for host defense (Weiss et al., 1995). Thus, it appears reasonable that increased intracellular availability of low molecular weight iron reduces the function of activated macrophages, while the withdrawal of iron by iron chelators such as DFO enhances macrophage-mediated cytotoxicity against various pathogens and tumor cells, in part through increased formation of NO. Increased generation of NO by macrophages contributes substantially to host defense in parasitic infections in animals (Liew, 1993; Stenger et al., 1994). Production of NO prevents the development of exo-erythrocytic stages of malaria parasites in the liver, and this effect has been attributed to the production of IFN-g and the involvement of CD81 cells (Mellouk et al., 1994; Seguin et al., 1994). Moreover, resistance of certain mouse strains such as C57BL/6 against infection with plasmodia is pivotally due to IFN-g- and TNF-a-mediated formation of NO (Nüssler et al., 1991; Mellouk et al., 1991; Rockett et al., 1991; Taylor-Robinson et al., 1993; Jacobs et al., 1996). However, in humans, the role of NO in malaria is far from clear. On the one hand, increased production of NO by macrophages or hepatocytes may contribute to a favorable prognosis through enhanced parasite killing (Nüssler et al., 1991; Mellock et al., 1991, 1994; Seguin et al., 1994). On the other hand, it has also been proposed that NO even contributes to the pathology of cerebral malaria via its function as a neurotransmitter and its ability to induce vasodilatation (Clark et al., 1991, 1992). According to the data presented in more detail in Sections 7.2 and 7.4, the treatment of cerebral malaria patients with the iron chelator DFO, in addition to a standard therapeutic regimen including quinine, results in increased parasite clearance and/or a faster recovery from coma when compared with patients receiving no additional iron chelation (Gordeuk et al., 1992b). While this apparently beneficial effect of iron chelation may be partly due to the withholding of the essential growth factor iron from the parasites (Gordeuk et al., 1992a) or to protection of the CNS from iron-mediated peroxidant injury (Gordeuk et al., 1995), it has also been postulated that enhanced antiplasmodial activity of macrophages may also have a role (Weiss et al., 1997). 6.2.1. Beneficial effects of nitric oxide in malaria. An apparently beneficial effect of NO formation in malarial infections has been described by several recent reports in which

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higher NO production, as quantitated by determination of the stable end products of NO, nitrite and nitrate (NO2–/ NO3–), in serum was associated with a more favorable outcome in cerebral malaria patients (Nüssler et al., 1994; Cot et al., 1994; Kremsner et al., 1996). Moreover, in a recent study, our group found a significant increase in serum concentrations of NO2–/NO3– in children with cerebral malaria who received standard quinine-based therapy and additional treatment with DFO, as compared with children treated with quinine alone. Children treated with DFO have been shown to recover faster from coma and to have an increased rate of clearance of plasmodia from the serum when compared with children receiving quinine and placebo (Gordeuk et al., 1992a; see also Sections 7.2 and 7.4). As discussed in Section 6.2, the expression of NOS II in murine macrophages is greatly dependent on intracellular iron availability, and treatment of cells with DFO enhances NO formation (Weiss et al., 1994). If applicable to human macrophages, these results suggest that the increased concentrations of NO2–/NO3– observed in patients during iron chelation therapy may be due to stimulation of cytokine-inducible NO formation in macrophages or hepatocytes by DFO. Increased NO formation might then contribute to enhanced cytotoxic immune effector potential against plasmodia. Although, there is still some uncertainty concerning NO formation by human macrophages, evidence for the existence of the NOS pathway in human macrophages and its importance for antimicrobial effector functions is rapidly accumulating (Nicholson et al., 1996; Sharara et al., 1997; for a review, see Dugas et al., 1995). Therefore, increased formation of NO in the circulation and/or the liver appears to exert protective effects in human malaria. Confirmation for this perspective is provided by a recent report that plasma NO2–/NO3– levels were inversely correlated with disease severity in Tanzanian children with cerebral malaria, with highest levels occurring in subclinical infection and lowest levels in fatal cerebral malaria (Anstey et al., 1996).

6.2.2. Detrimental effects of nitric oxide in malaria. In contrast to the preceding results, evidence exists that in the CNS, increased formation of NO may contribute to the pathogenesis of cerebral malaria. It has been suggested that NO may be involved in the pathology of cerebral malaria, either by induction of vascular relaxation in the brain, thus contributing to increased intracranial pressure, or by direct interference with neuronal function (Clark et al., 1991, 1992). Most of this evidence is based on investigations with various animal models. In a recent study, NO formation was determined in cerebrospinal fluid (CSF) specimens obtained from 130 children with cerebral malaria (Weiss et al., 1998). One hundred and eleven children survived over an observation period of at least 5 weeks, while 19 children died. Concentrations of NO2–/NO3– in CSF were significantly lower in survivors than in children who died. A logistic regression analysis revealed that each 10-point in-

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crease in CSF NO2–/NO3– level was associated with a 2.2-fold increase in the risk of death (95% confidence interval of 1.1–4.3; P 5 0.024), after adjustment for temperature, respiratory rate, and depth of coma at the time of enrollment (Weiss et al., 1998). However, increased NO2–/ NO3– levels in CSF may not have reflected production of NO by cytokine-inducible NOS II because no correlation was found between CSF NO2–/NO3– levels and local CSF formation of immune activation markers, such as neopterin or soluble TNF receptor Type II (sTNFrec75). In accordance with the hypotheses presented by Clark and colleagues (1991), it is more likely that CSF NO2–/NO3– was derived from brain-type NOS (NOS I) or endotheliumtype NOS (NOS III). The question remains why increased concentrations of NO2–/NO3– in CSF are associated with an increased risk of death in children with cerebral malaria. In our opinion there are at least two plausible hypotheses. First, NO, previously identified as endothelium-derived relaxing factor, causes vasodilatation via the formation of cGMP (Ignarro, 1990; Moncada and Higgs, 1993), which may increase cerebral perfusion, but also intracranial pressure (Clark et al., 1991). Observations demonstrating that neuronal death following hypoxia and hypoglycemia is associated with an increase in glutamate and cGMP concentrations would fit with this hypothesis (Garthwaite, 1991). Second, NO may damage neurons directly by altering their cellular energy metabolism and mitochondrial respiration as outlined above (Drapier et al., 1993; Nathan and Xie, 1994; Bolanos et al., 1996). Therefore, as observed in other biological systems (Kröncke et al., 1997), NO appears to exert both beneficial and detrimental effects in human malaria, depending on its localization, concentration, and the surrounding microenvironment. 6.3. Helper T-Cell Type 1 and Helper T-Cell Type 2 Cytokines in Malaria Cytokines originating from helper T-cells, natural killer cells, and macrophages are major players in the body’s response to parasitic infections (Taylor-Robinson et al., 1993; Romagnagi, 1997). Two CD41 helper T-cell subsets exist in humans, each of which produces a typical set of cytokines that regulate different immune effector functions and cross-react with each other (Mosmann and Coffman, 1989; Seder and Paul, 1994; Romagnagi, 1997). Th-1 cells produce IFN-g, IL-2, and TNF-b. These cytokines activate macrophages, thus contributing to the formation of pro-inflammatory cytokines such as TNF-a, IL-1, and IL-6, and the induction of cytotoxic immune effector mechanisms of macrophages. By contrast, Th-2 cells produce IL-4, IL-5, IL-10, and IL-13, which induce a strong antibody response, but also inhibit various macrophage functions (Mosmann and Coffman, 1989; Seder and Paul, 1994; Romagnagi, 1997). The balance between Th-1 and Th-2 cell-mediated immune effector mechanisms is of central importance for the

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host response to parasitic infections (Taylor-Robinson et al., 1993; Romagnagi, 1997). While Th-1-derived cytokines, such as IFN-g and IL-2, are crucial for effective host defense in the acute phase of certain parasitic infections, increased activity of Th-2-derived cytokines, such as IL-4, IL-10, and IL-13, heightens susceptibility to these infections and causes exacerbations. The latter effects may be due to an inhibitory function of IL-4, IL-10, or IL-13 on the production of Th-1 cytokines and on macrophage activation, thus reducing cell-mediated antimicrobial cytotoxicity, e.g., by suppressing the induction of NOS II (Bogdan et al., 1991, 1994; Gazzinelli et al., 1992; Modolell et al., 1995). Evidence for the detrimental role of Th-2-mediated immune function for acute parasitic infections has been shown in several models: (1) IL-4-deficient mice become susceptible to parasitic infections after re-introduction of the IL-4 gene into their genome (Powrie et al., 1993); (2) the inhibitory effect of IL-4 and IL-10 towards cell-mediated immunity is due in part to depression of IFN-g production (Leal et al., 1993); (3) in parasitic infections, Th-1 cells default to the Th-2 pathway in the absence of endogenous IFN-g (Wang et al., 1994); (4) various infections are less severe and have reduced mortality in IL-4-deficient mice (Kanegawa et al., 1993); and (5) plasma levels of IL-10 increase with disease severity in human malaria (Anstey et al., 1996). Based on these and several other studies, Th-1mediated immune effector function appears to be beneficial during early stages of plasmodial infections, while Th-2derived cytokines exert a protective role in chronic infection with plasmodia (Taylor-Robinson et al., 1993; Mellouk et al., 1994; Seguin et al., 1994). Interestingly, iron chelators such as DFO affect not only IFN-g-mediated pathways, such as major histocompatibility complex Class II antigen expression, neopterin production, and NO formation (Weiss et al., 1992, 1994), but also the Th-1/Th-2 balance. Two different studies of Zambian children who were enrolled in placebo-controlled trials of DFO in addition to quinine for cerebral malaria suggested a possible effect of iron chelation on Th-1-mediated immune function. In one study, serum levels of neopterin did not change significantly in children receiving DFO plus quinine, but did decline significantly in children receiving placebo plus quinine (Weiss et al., 1997). In the same study, serum concentrations of NO2– and NO3–, the stable end products of NO metabolism, increased significantly in the children receiving DFO plus quinine, but not in those receiving placebo plus quinine. The IFN-g-inducible compound neopterin is produced by monocytes/macrophages following stimulation with this cytokine. Neopterin, therefore, has turned out to be a valuable parameter for monitoring Th-1 cell-mediated immune function in vitro and in vivo (Huber et al., 1984; Fuchs et al., 1988). Our observation made in patients is compatible with in vitro results demonstrating that DFO enhances neopterin formation by positively modulating IFN-g activity (Weiss et al., 1992). In both studies, serum concentrations of IL-4, a Th-2-related

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cytokine, increased with placebo plus quinine, but not DFO plus quinine (Thuma et al., 1996; Weiss et al., 1997). Taken together, these studies raise the possibility that the beneficial effect of iron chelation therapy in part may result from a strengthening of Th-1 cell-mediated immune function by enhancing IFN-g activity, as reflected by neopterin and NO formation, while reducing the production of Th-2-mediated cytokines such as IL-4. According to the Th-1/Th-2 paradigm, the latter effect could be due to a direct negative regulatory effect of IFN-g, the activity of which is enhanced by the iron chelator, on the production of Th-2 cytokines (Romagnagi, 1997). Therefore, DFO may direct the immune response towards a Th-1 effector mechanism, which should be beneficial in the early phase of parasitic infections (Fig. 1). This notion was supported by a recent study of mice with Candida albicans infection, in which the addition of DFO appeared to promote Th-1 responses and reduce Th-2 effects (Cenci et al., 1997). As with NO, the role of immune function in the CNS in malaria appears to be complex. For example, the development of murine cerebral malaria was associated with increased accumulation of TNF-a and IFN-g mRNA in the brain, while IL-4 mRNA levels were decreased, suggesting that Th-1-mediated immune phenomena may play a role in the pathogenesis of cerebral malaria (Clark et al., 1991; de Kossondo and Grau, 1993). Nevertheless, there is also evidence that just the opposite may be true; namely, that the Th-1 immune response in the CNS may be protective in cerebral malaria (see above). A recent study investigating CSF samples from 130 cerebral malaria children would seem to support this latter notion (Weiss et al., 1998). In this study, no correlation was found between depth of coma or death from cerebral malaria with CSF markers indicating local immune activation in the brain such as neopterin or

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TNF receptor (sTNFrec75), thus suggesting that the local Th-1 response is not harmful. It remains to be determined whether the observed beneficial effects of iron chelators in cerebral malaria is due to immune modulation in the CNS, to the chelation of iron and prevention of toxic hydroxyl-radical formation in this compartment, or to a combination of both mechanisms (Halliwell et al., 1992; Gordeuk et al., 1995; Rosen et al., 1995). 7. IRON CHELATORS AND PROTECTION FROM PEROXIDANT TISSUE DAMAGE IN THE SETTING OF MALARIA 7.1. Free Radical-Mediated Tissue Damage in Malaria The obstruction of the cerebral microvasculature by P. falciparum-infected erythrocytes, leading to ischemia and microhemorrhage, may contribute to the development of cerebral malaria (MacPherson et al., 1985; Oo et al., 1987; Aikawa et al., 1990; Berendt et al., 1994). The final common pathway in ischemic and hemorrhagic injury to the brain and other organs is mediated by oxygen-derived free radicals that induce lipid peroxidant damage to cellular and subcellular membranes (McCord, 1985; Henson and Johnson, 1987; Sadrzadeh et al., 1984, 1987). Superoxide and hydrogen peroxide are formed in all aerobic cells, and their concentrations are increased in ischemic and hemorrhagic injury. Starting from these species, iron catalyzes the generation of hydroxyl radicals by means of the Fenton reaction. Hydroxyl radicals are highly reactive and biologically hazardous molecules that cause lipid peroxidation and damage cellular and subcellular membranes (McCord, 1985; Sadrzadeh et al., 1984, 1987). The reperfusion period, when oxygen reenters the ischemic tissue, appears to be an especially vulnerable time for the appearance of free radicals (Ambrosio et al., 1987). Free hemoglobin can serve as a biologic Fenton reagent to provide iron for electron transfer and the generation of the hydroxyl radical (Sadrzadeh et al., 1984). DFO inhibits peroxidant damage to lung tissue in mice (Ward et al., 1983), to the myocardium in rabbits (Ambrosio et al., 1987), and to the CNS in cats (Sadrzadeh et al., 1987). Iron chelation therapy in cerebral malaria, therefore, may be expected to diminish CNS damage by protecting against lipid peroxidation by iron-generated free radicals. 7.2. Effect of Desferrioxamine on Recovery from Coma in Children with Cerebral Malaria

FIGURE 1. Modulation of Th-1/Th-2 balance by the iron chelator DFO. In humans, two types of helper T-cell clones exist. Th-1 cells produce primarily IFN-g and IL-2, thereby activating macrophages and inducing a cytotoxic immune effector mechanism directed against invading pathogens. Th-2 cells produce IL-4, IL-5, IL-6, IL-10, and IL-13, which induce a strong antibody response, but also inhibit macrophage functions. DFO modulates Th-1/Th-2 balance by enhancing IFN-g activity. This enhancement increases the antimicrobial cytotoxicity of macrophages, but also weakens the Th-2 arm of immunity.

Cerebral malaria, one of the most severe complications of infection with P. falciparum, is especially common in young children in Africa. The mortality rate is 15% or more, despite therapy with parenteral antimalarials and attentive management of complications. Advances in treatment are urgently needed, especially in view of the lack of effective vector control and the spread of strains of P. falciparum that are resistant to chloroquine and other antimalarials. Cere-

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bral malaria is diagnosed when asexual forms of P. falciparum are found in the blood of a patient with signs of an acute, diffuse symmetric encephalopathy not attributable to other causes. Pathologically, the condition is characterized by sequestration of parasitized red cells in cerebral venules and capillaries (MacPherson et al., 1985; Aikawa, 1988b). Elevated CSF lactate concentrations indicate that cerebral hypoxia may be present (White et al., 1985), and generalized cerebral edema, ring hemorrhages, and necrosis around cerebral veins are frequent postmortem findings (MacPherson et al., 1985). The pathophysiology of cerebral malaria includes obstruction of the cerebral microvasculature by P. falciparum-infected erythrocytes, local ischemia and microhemorrhage, the release of free hemoglobin and iron, and the generation of oxygen-derived free radicals, which produce lipid peroxidant damage to cellular and subcellular membranes (McCord, 1985; Sadrzadeh et al., 1984). As discussed in Sections 1, 2, 3, and 6, iron is an essential nutrient for the growth of P. falciparum, and the element is a necessary factor in reactions that generate free radicals that mediate ischemic and hemorrhagic injury to the brain (Sadrzadeh et al., 1987). Iron chelation with DFO may protect against damage to the CNS (1) by enhancing parasite clearance through withholding iron from a vital metabolic pathway of the parasite, (2) by inhibiting iron-induced peroxidant damage to cells and subcellular structures of the brain, and (3) by enhancing Th-1 cell-mediated immunity, as discussed in the previous section. A prospective, randomized, double-blind trial of DFO or placebo added to standard quinine therapy was conducted in 83 Zambian children. The goal was to determine if iron chelation speeds recovery of full consciousness in cerebral malaria (Gordeuk et al., 1992a). Entrance criteria included age less than 6 years, P. falciparum parasitemia, normal CSF, and unarousable coma. Each child received quinine, 10 mg/kg, every 8 hr for 5 days and a single dose of sulfadoxine/pyrimethamine (25/1.2 mg/kg). In addition, either DFO (100 mg/kg/day) or placebo was given as a 72-hr intravenous infusion. Time to recovery of full consciousness was examined after stratification according to the depth of coma at Time 0, as suggested by Molyneux and colleagues (1989). In 33 subjects with light coma (scored initially as 3–4), the estimated median time to regain full consciousness among 20 children given DFO was 20.0 hr, as compared with 17.8 hr in 13 who received placebo (P 5 0.997). Among 50 children with deep coma (scored initially as 0–2), the time to regain full consciousness was shorter with DFO therapy (P 5 0.03). Estimated median recovery time was 68.2 hr with placebo (n 5 28) and 24.1 hr with DFO (n 5 22). The estimated relative rate of recovery of full consciousness with DFO was 2.2 times that with placebo (95% confidence interval of 1.1–4.7). Time to parasite clearance could be measured for 69 subjects, and none of 12 baseline variables had a statistically significant effect. The rate of parasite clearance was significantly enhanced (2.0-fold; 95% confidence interval 1.2– 3.6) with the addition of DFO therapy (P 5 0.01). Mortal-

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ity was 16.7% in 42 children receiving DFO versus 22.0% among 41 given placebo (P 5 0.52). These results suggest that iron chelation therapy may hasten clearance of parasitemia and enhance recovery from deep coma in cerebral malaria. The finding that DFO can more than halve the duration of unconsciousness in the most severely affected children with cerebral malaria suggests that iron chelation might have a substantial effect on the underlying pathophysiology of the disorder. This initial study of cerebral malaria examined too few children (n 5 83) to detect an appreciable effect on mortality. 7.3. Relationship of Outcome in Cerebral Malaria to Transferrin Saturation The inflammatory response generated by a severe infection is usually associated with a marked reduction in serum iron concentration, a finding that is mediated by increased release of cytokines such as TNF-a and IL-1 (Dinarello et al., 1986; Alvarez-Hernandez et al., 1989). In severe malaria, several factors, including the presence of hemolysis and dyserythropoiesis, may raise transferrin saturations and potentially lead to the presence of nontransferrin-bound iron and hemoglobin in the plasma of some patients (Pootrakul et al., 1988; Gordeuk et al., 1995). If free-radical generation is important in the pathophysiology of cerebral malaria, more severe or prolonged CNS damage might be expected in patients with elevated transferrin saturations, and iron chelation therapy might be particularly beneficial in this group of patients. An analysis was performed on the children with cerebral malaria described in the previous section to determine if there was any relationship between transferrin saturation on admission and recovery from coma (Gordeuk et al., 1995). Transferrin saturations at presentation were determined retrospectively, and the children were divided into two groups, those with markedly elevated transferrin saturations (over 43%) and those with transferrin saturations less than or equal to 43%. Children with markedly elevated transferrin saturations had a delayed time to recover full consciousness compared with those with transferrin saturations less than or equal to 43%. The addition of the iron chelator DFO seemed to enhance recovery from coma specifically in children with high transferrin saturations, a finding consistent with the hypothesis that iron-generated free radicals may play a role in the pathogenesis of coma in cerebral malaria. 7.4. Effect of Desferrioxamine on Mortality in Cerebral Malaria To examine the effect of iron chelation on mortality in cerebral malaria, 352 children were enrolled into a clinical trial of DFO in addition to standard quinine therapy at two centers in Zambia, one rural and one urban (Thuma et al., 1998a). Entrance criteria included age less than 6 years, P. falciparum parasitemia, normal CSF, and unarousable coma. DFO (100 mg/kg/day infused for a total of 72 hr) or placebo

Iron Chelation Therapy for Malaria

was added to a 7-day regimen of quinine that included a loading dose. Mortality was 18.3% (32/175) in the DFO group and 10.7% (19/177) in the placebo group (adjusted odds ratio, 1.8; 95% confidence interval, 0.9–3.6; P 5 0.074). At the rural study site, mortality was 15.4% (18/ 117) with DFO compared with 12.7% (15/118) with placebo (P 5 0.78, adjusted for covariates). At the urban site, mortality was 24.1% (14/58) with DFO and 6.8% (4/59) with placebo (P 5 0.061, adjusted for covariates). Among survivors, there was a trend to faster recovery from coma in the DFO group (adjusted odds ratio, 1.2; 95% confidence interval, 0.97–1.6; P 5 0.089). This study did not provide evidence for a beneficial effect on mortality in children with cerebral malaria when DFO was added to quinine in a regimen that included a loading dose of quinine. Indeed, at one of two research sites, there was a trend to higher mortality with DFO. It should be noted that there was a trend toward an interaction between experimental treatment and study site (P 5 0.074) in this investigation, and indeed, mortality rates appeared to be substantially different between the two study sites. The results from the urban center are especially enigmatic because the mortality in the placebo group seemed to be substantially lower than mortality rates described in previous studies (Gordeuk et al., 1992b; Molyneux et al., 1989). A careful review of the case records and the study procedures did not reveal any reason for the apparent difference in results between the two study sites except for potential differences with regards to malarial transmission. It is possible that there was an unidentified problem or an error in the conduct of the study at the urban site; it is also possible that DFO may have had a negative effect. The lack of a positive effect of DFO on both parasite clearance and recovery from coma in this study, in contrast to earlier work (Gordeuk et al., 1992a), may be attributable to the impact of a loading dose of quinine used in the present study, but not in the previous one, i.e., a relatively delayed beneficial effect of DFO in cerebral malaria was masked by a substantial beneficial effect of the quinine loading dose. 8. CONCLUSIONS AND DIRECTIONS FOR THE FUTURE Malaria is one of the major global health problems, and an urgent need for the development of new antimalarial agents faces the scientific community. A considerable number of iron(III) chelators, designed for purposes other than treating malaria, have antimalarial activity in vitro, apparently through the mechanism of withholding iron from vital metabolic pathways of the intra-erythrocytic parasite. Several of these agents also have antimalarial activity in animal models of plasmodial infection. Evidence is now available that iron chelation therapy with DFO has clinical activity in both uncomplicated and severe malaria in humans. It is appropriate to further advance our knowledge of the iron metabolism of the malaria parasite and to develop iron chelators specifically designed for the treatment of malaria.

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