The Role of the Parasite1 Uptake Mechanisms and Metabolic Interference in Parasites As Related to Chemotherapy1' THOMAS C

PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY

The Control of Parasites: The Role of the Parasite1 Uptake Mechanisms and Metabolic Interference in Parasites as Related to Chemotherapy1' THOMAS C. CHENG Institute for Pathobiology, Center for Health Sciences, Lehigh University, Bethlehem, Pennsylvania 18015

ABSTRACT: By employing examples selected from among intra- and intercellular protozoan and helminth parasites, the idea that the specific uptake mechanisms of these dependent organisms may select for or against the delivery of drugs is being advanced. Furthermore it is being pointed out that parasiticidal properties of certain drugs may be based on their ability to alter the uptake mechanisms of parasites. Although not generally considered to be effectual by the pharmaceutical industry, the idea of searching for specialized metabolic pathways unique to parasites and inhibiting these to their detriment is being reemphasized to be a more scientifically challenging approach to the development of new drugs. Also, the idea of taking advantage of quantitative differences favoring disrup- tion of biochemical and physiological processes in the parasite should be considered in drug development.

In the control of parasites by employing drug. In view of this, a brief review of the drugs, the selective physiologic vulnerability uptake mechanisms of selected parasites is and chemical receptibility of the parasites play presented. important roles. In other words, ideally, the drug should readily enter and adversely affect Uptake Mechanisms the parasite and at the same time not be dele- INTRACELLULAR PROTOZOANS. The uptake terious to the host. In this contribution I have mechanisms by intracellular protozoan para- attempted to focus in on some of the proper- sites are intimately associated with how they ties of selected parasites which conceivably acquire nutrients. Among members of the could serve to regulate or control their re- classes Telosporea, Piroplasmea, and Zoo- ceptivity to drugs. mastigophorea that are intracellular parasites, Ecologically speaking the host's internal en- there is a cytostome through which molecules vironment is the endoparasite's habitat. Con- from their habitat can be ingested; however, sequently, in order for a drug to enter the among intracellular members of the classes parasite, it must be taken up from its habitat. Microsporea and Haplosporea, such an aperture In the case of most protozoans and astomate is absent and hence other uptake mechanisms helminths, this means molecular uptake must be operative. through the body surface. On the other hand, The cytosomes of telosporeans and piro- in the case of such helminths as the digenetic plasmeans are essentially circular indentations trematodes and nematodes, the uptake of drugs in the pellicle (Fig. 1). When observed en theoretically could occur either as the result face, each cytostome appears as two electron- of ingestion, passage through the body surface, dense concentric rings (Fig. 2), and when or both. Thus the uptake mechanism employed viewed in cross-section, the lining of each by a specific parasite most probably influences cytostome has been interpreted to consist of or governs its selective receptivity to a specific two electron-dense membranes (Fig. 3). The inner of these is continuous with the parasite's surfacial unit membrane while the outer one is attached to the inner one. The diameter of the cytostome varies not only among members of different genera but also among different species of the same genera (Aikawa, 1971).

Figure 1. Electron micrograph of intracellular sporozoite of Eimeria ninakohlyahimoyae showing cytostome (Ct). (After Kelley and Hammond, 1972). Figure 2. Electron micrograph of en face view of cytostome of trophozoite of Plasmodium cathermcrium (arrow) showing electron-dense concentric rings surrounding aperture. Negative staining technique. (Alter Aikawa, 1967). Figure 3. Electron micrograph of portion of uninucleate trophozoite of Plasmodium cathemerium in- gesting host cell cytoplasm through cytostome (Ct). (After Aikawa et al., 1966).

Nevertheless, the basic architecture is similar Aikawa et al. (1966) and Sterling and Aikawa among telosporeans and piroplasmeans. (1973) have found that the ingested material According to Aikawa et al. (1966), Ham- is taken into the parasitic cell by small vacuoles mond et al. (1967), and Sampson and Ham- pinched off from the cytostomal cavity, and mond (1971), the cytostome only becomes these are subsequently incorporated into a functional after these parasites become estab- digestive vacuole where digestion of the in- lished in their intracellular positions. Further- gested material occurs (Fig, 4). more, there appear to be differences as to Another variation is that found in Leuco- how cytostomes function. For example, Aikawa cytozoon simondi. Sterling and Aikawa (1973) et al. (1967) have reported that in Plasmodium have found that the cytostome in this haemo- elongatum most of the ingested material is sporidian appears to be a transient structure. taken into the parasitic cell by the cytostomal Specifically, they have reported that the cyto- cavity, after which the cavity is pinched off stomal rings are only temporary in this para- from the surrounding host cytoplasm by closure site of birds, being only visible during the of the cytostomal orifice, followed by the for- earliest stages of cytostomal ingestion, and mation of a membrane over the orifice. Sub- disappearing as ingestion advances. Further- sequent to these events, the food vacuole, i.e., more, the digestion of the ingested material the original cytostomal cavity, migrates to and occurs within the cytostomal cavity rather than is eventually incorporated within a digestive in a separate digestive vacuole. vacuole where complete digestion of the in- It is noted that a cytostome does not occur gested material occurs. On the other hand, in in oocysts of any of the members of the class several other species of avian haemosporiclians, Telosporea that have been studied. These

,4*.: Figure 4. Ingestion and digestion of host cytoplasm by Plasmodium elongatum. (a) Food vacuole (phagosome) (arrow) migrating to digestive vacuole (Dv). (b) Several food vacuoles (arrows) within a digestive vacuole (Dv). (After Aikawa et al., 1967). oocysts are surrounded by a thick electron- this can occur or not and what the facilitating dense tunic, from the inner surface of which mechanism(s) is are other aspects of cell biology are pinched off small spherical bodies of me- that parasitologists should be concerned with dium electron density (Fig. 5). Terzakis et al. (Fig. 6). For example, it is of interest to spec- (1967) and Vanderberg et al. (1967) have ulate whether macromolecules could also be suggested that these bodies may supply nutri- taken into host cells by phagocytosis if these ents to the oocyst. cells are leucocytes. For example, the host cells Measurements of the cytostomal aperture of of Leucocytozoon simondi are known to be various species of intracellular protozoans that duck leucocytes, which are capable to phago- possess cytostomes in published electron micro- cytosis. graphs by various authors (Aikawa, 1967; It is noted that although the uptake of Aikawa et al., 1967; Kelley and Hammond, molecules via a cytostome has been reported 1972; Sterling and Aikawa, 1973; Aikawa to occur in the exoerythrocytic stages of and Sterling, 1974) have revealed that the Haemoproteus metchnikovi by Sterling and diameters range between 0.05 and 0.25 /xm. DeGiusti (1972) and those of Plasmodium These dimensions are sufficiently large to per- gallinaceum by Aikawa et al. (1968), it re- mit the uptake of molecules of high molecular mains to be ascertained whether this mech- weights, at least as high as 7 X 104 or more. anism occurs in the exoerythrocytic stages of This figure is based on the molecular weight other species of haemosporidians. According of mammalian hemoglobin, which is known to to Hepler et al. (1966), simple diffusion is be taken in by the cytostomes of haemosporid- responsible for the entry of certain molecules ians. Consequently the intake of most drugs into the exoerythrocytic stages of Plasmodium is possible through the cytostomal mechanism. fallax. This mechanism undoubtedly is the one It must be noted, however, that since the cyto- that occurs among the microsporeans and stome is primarily an organelle of intracellular haplosporeans, which are without a surfacial parasitic protozoa; specifically, members of the aperture. Furthermore, simple diffusion may Telcosporea, Piroplasmea, and Zoomastigo- and probably does complement other special- phorea, molecules that are taken in by this ized uptake mechanisms in those species with mechanism by necessity must pass through the a cytostome. It remains unknown, however, surface membrane of the enveloping host cell whether simple diffusion is a useful mechanism before they can reach the parasite. Whether for the uptake of drugs, since the relatively

cytostome pmocytosis

phagocytosis

Figure 6. Diagrammatic drawing illustrating known mechanisms by which intracellular protozoans can take in molecules and the lack of information on how such molecules, including drugs, enter the host cell.

cell surfaces. Scalzi and Bahr (1968) and Cox and Vickerman (1966) have demonstrated the occurrence of small pinocytotic vesicles along the surfaces of the erythrocytic merozoites of Plasmodium chabaudi and P. vinckei, although these malarial parasites also utilize cytostomes for uptake. Also, Stehbens (1966) and Snigirevskaya and Cheissin (1969) have demonstrated that Lankesterella and Eimeria apparently ingest material in the surrounding Figure 5. Electron micrograph of oocyst of parasitophorous vacuole by pinocytosis, respec- Plasmodium berghei showing capsular material tively. It is noted that Hammond et al. (1967) being pinched off from the internal surface (ar- have also reported the occurrence of pinocy- rows). (After Aikawa and Sterling, 1974). totic vesicles along the lining of small V-shaped invaginations in the pellicle of Eimeria auburn- high molecular weights as well as other physio- ensis macrogametocytes. Of course, pinocytosis chemical features, such as surface charges, of could serve as a portal mechanism for the in- these compounds may prevent their passage take of drugs. But, again, since these parasites through membranes by diffusion. are intracellular, the drug must be able to pass While on the topic of surface permeability, through the delimiting surface of the host cell. it is of interest to note that the cidal mechanism One could speculate whether compounds which of certain drugs is known to involve interfer- would inhibit or retard development of cyto- ence with carbohydrate absorption by host stomes and pinocytotic vesicles would be dele- cells or by the parasite surfaces. For example, terious to intracellular parasites and could be Cenedella and Jarrell (1970) have reported considered in chemotherapy. However, before such compounds can be synthesized, we need that 4,4/-diaminodiphenylsulfone is cidal to Plasmodium berghei as a result of disarrange- to know a great deal more about the cell bi- ment of the glucose transport mechanism in the ology of these organelles. membrane of the host erythrocyte. An example EXTRACELLULAR PROTOZOANS. Prior to the of the alternative mechanism has been contrib- discovery by Steinert and Novikoff (1960) uted by Ghosh and Chatterjee (1961) who that there is a "cytostome" in cultured amasti- have reported that the antibiotic nystatin gotes of Tnjpanosoma mega, it was generally possibly interferes with the absorption of essen- thought that this and related extracellular tial exogenous substrates by Leishmania. hemoflagellates took in exogenous materials by Another method by which unicellular para- diffusion through their pellicles. It is now sites may take in molecules of relatively high known, that in addition to absorption, cyto- molecular weight is by pinocytosis along their stomes play a role in uptake. However, the

Figure 7. Electron micrograph showing cytostome (Ct) of Trypanosoma cruzi (= Schizotrypanum ciuzi) extending deep into the protozoan's cytoplasm. (After Aikawa and Sterling, 1974). Figure 8. Electron micrograph of a portion of the surface of Trypanosoma brucei showing electron- dense noncellular material (arrow) overlaying surface membrane. X 48,000. C, collecting membrane system; M, mitochondrion; N, nucleus; T, collecting tubule. (After Langreth and Balber, 1975). Figure 9. Electron micrograph of a portion of the surface of Leishmania hoogstraali promastigote showing trilaminate cell membrane (in) and subpellicular microtubules (s). The arrows indicate where the microtubules are connected to the endoplasmic reticulum. X 351,000 (After Lewis, 1975). so-called cytostomes of trypanosomes are struc- take. Although no evidence is available at this turally different from those of intracellular time relative to drugs, it would appear that protozoans. Specifically, each cytostome con- this is a possible vehicle for the introduction sists of a cylinder of fibrils that are continuous of such compounds into these extracellular with the pellicular fibrillar system. This in- protozoan parasites. vagination invades deeply into the cytoplasm, As stated, in addition to employing cyto- and the cell membrane delimiting the opening stomes and associated pinocytotic vesicles for to the exterior is depressed, forming a conical the uptake of molecules, absorption through pit (Fig. 7). Most of the length of the tubular the pellicular surface also occurs. Conse- cytostome is filled with cytoplasm. Further- quently, some consideration is being given to more, minute pinocytotic vesicles, each measur- the fine structure of the pellicle of flagellates. ing about 0.2 /xm in diameter, and delimited by In the bloodstream form of trypanosomes a unit membrane, commonly occur along its and most other parasitic flagellates, there is length. Steinert and Novikoff (1960) and sub- a coat of electron-dense noncellular material sequent investigators have demonstrated that overlaying the surface unit membrane (Vicker- ferritin incorporated into the medium will enter man and Luckins, 1969, and others) (Fig. 8). the tubular cytostome and eventually become This layer is not as substantial in the intra- concentrated in these pinocytotic vesicles, thus cellular flagellates, such as the promastigotes indicating that they serve as organelles for up- of Leishmania (Lewis, 1975). In either case,

this surface coat is chemically a polysaccharide plasmic layer known as the tegument, rather considered by Luft (1971) to be a constituent than a noncellular cuticle. Information pertain- of the surface coats of nearly all animal cells. ing to the fine structure of the tegument has The cell membrane is typically trilaminate, and been reviewed by Lee (1966, 1972) and lying immediately beneath it is an array of Cheng (1973), among others. In brief, the longitudinally arranged subpellicular micro- general functional architecture is as follows. tubules (Fig. 9). The exact organization of The digenean tegument is comprised of two these microtubules has been described in Try- zones. The outer zone, which is separated panosoma brucei and T. gambiense by Fuge from the environment by a unit membrane, (1968), and the pattern appears to hold true consists of cytoplasmic syncytium (Fig. .1.0). in other species. There are cross connections Embedded in this layer are mitochondria, between parallel microtubules, and in most endoplasmic reticulum, various types of vacu- species there are "lollipop-like" structures ar- oles, and, in some instances, glycogen granules ranged along the length of the microtubules. and other types of inclusions. According to Brooker (1971), who studied The outer surface is usually thrown into Crithidia fasciculata, these structures are as- folds to form microvilli. These undulations not sociated with the uptake of macromolecules only serve to increase the absorptive surface such as proteins. but pinocytotic vesicles are also formed in As far as I have been able to determine, the crypts between adjacent microvillae which although no mechanistic studies have yet been presumably serve for the intake of large mole- carried out on how drugs enter through the cules and particulate materials. Also embedded pellicle of protozoans, such compounds obvi- in the outer zone of some species are tegumen- ously do, since the story of drug-resistant forms tary spines. These are overlayed with the sur- of extracellular protozoans is well documented. facial plasma membrane and undoubtedly This, of course, would necessitate molecular serve as ancillary holdfast mechanisms in situ. contact between the drug and the organism's The outer syncytial zone is connected by DNA to result in mutations. Furthermore, as cytoplasmic bridges to nucleated cells, known Rohatgi and Krawiec (1973) and Kay et al. as cytons, embedded deeper in the paren- (1974) have demonstrated, such antimicrobial chyma. The cytons, collectively designated as compounds as chloramphenicol and ethidium the inner tegumentary zone, include vacuoles, bromide will enter the pellicle and the mito- endoplasmic reticulum, mitochondria, Golgi chonclrial membranes of such protozoans as bodies, glycogen deposits, and various types of Tetrahymena pyriformis to effect morpho- vesicles in addition to the nucleus (Fig. 10). metric changes. Thus, there is little doubt that In the region between the outer and inner the fine structure of protozoan pellicles is tegumentary zones are found several other physiologically associated with the permeation types of tissues. Specifically, lying immediately of molecules, and conceivably could serve as a mediad to the outer tegumentary zone, and mechanism which could influence entry of separated from it by a unit membrane, is a drugs. thin basal lamina. Beneath this are found a DIGENEA. The mechanisms utilized by hel- series of circular muscles and mediad to these minths, including digenetic trematodes, for the are the fasicles of longitudinal muscles (Fig. uptake of materials from their environments 10). have been investigated extensively at the fine Investigations into the chemical composition structural and physiological levels. This, as of the adult cligenean tegument have revealed is the case with protozoans, has come about the presence of glycogen, nonglycogenic poly- primarily because of interest in how these saccharides, lipids, acid mucopolysaccharides, parasites obtain their nutrients. Nevertheless, and mucoproteins (Pantelouris, 1964; Ohman, the discovered uptake mechanisms are equally 1965; and others). The occurrence of acid applicable to other categories of molecules, in- mucopolysaccharides is of particular signifi- cluding drugs. cance since these molecules are known to be It is now "ancient history" that the surfaces capable of inhibiting various digestive enzymes of digeneans are covered with a syncytial cyto- and their presence and possible secretion onto

nner zone of tegument

m cv Figure 10. Diagrammatic drawing showing tine structure of the tegument ot a digenetic trematode. bl, basal lamina; cb, cytoplasmic bridge; cm, circular muscle; cv, cytoplasmic vacuole; er, endoplasmic reticulum; g, Golgi apparatus; 1m, longitudinal muscle; m, mitochondrion; mv, microvillus; p, paren- chyma; pv, pinocytotic vesicle; sp, tegumental spine. the body surface may account for why the in- in an essentially liquid environment. It is testinal trematodes are not digested by their noted that information pertaining to the fine hosts' enzymes. structure of the tegument of Schistosoma, both In addition to the chemical entities listed, larval and adult, has been reviewed compre- several enzymes have been detected in the hensively by Hockley (1973). trematode tegument. Both acid and alkaline Scanning electron microscopy has revealed phosphatases have been reported (Yamao, that the surfaces of adult schistosomes are 1954; Lewert and Dusanic, 1961), and ester- different between species as well as between ases have also been detected (Nimmo-Smith sexes. However, all have a basic spongy ap- and Standen, 1963). In certain specialized pearance, i.e., there are ridges and pits (Fig. cases, other hydrolytic enzymes are associated 11). Furthermore, spines are interspersed with certain regions of the tegument. For over the surfaces. When this topography is example, Erasmus and Ohman (1963) have analyzed by transmission electron microscopy, reported the occurrence of aminopeptidase as- the pits have been determined to be tortuous sociated with the body surface in the adhesive channels, which may be branched and inter- organ region of Cyathocotyle bushiensis, a connected (Fig. 12). Hockley (1973), as the caecal parasite of ducks. In this case the result of incubating S. mansoni in colloidal iron enzyme is secreted by underlying cells and is or thorium, has demonstrated that these chan- active in extracorporeal digestion, i.e., the cells nels are open to the exterior, thus providing of the host's caecum are partially predigested a large surface area. It is noted that the outside of the parasite's body prior to inges- schistosome tegument is thinner than that of tion. most other digenetic trematodes, being approx- A few additional words should be said about imately 4 //.m thick in S. mansoni and S. japoni- the tegument of adult schistosomes, which live cum (Morris and Threadgold, 1968; Smith et

to be said about the architecture of the membrane overlying the outer tegumental layer of schistosomes. Smith et al. (1969) and Hockley and Mc- Laren (1973) are responsible for the elucida- tion of the structure of the covering plas- malemma of schistosomes. Specifically, although the outer membrane, which extends over the entire pitted tegumental surface, has been described as being trilaminate and approximately 10 nm thick, Smith et al. (1969) have found that this membrane in the area of the gynocophoric groove is pentalaminate. This observation has been extended by Hockley and McLaren (1973) who, as a result of fixing specimens of S. mansoni with uranyl acetate, discovered that the surface membrane is essentially heptalaminate throughout, and is approximately 17 nm thick. Moreover, the heptalaminate nature of this membrane has its origin in the schistosomule and persists in the adult. According to Hockley's (1973) reason- ing, the more complex nature of the surfacial membrane suggests that it is less efficient as an uptake surface. The surface membrane of the caeca syncytium is typically trilaminate. It is noted that although it would appear from electron microscopical studies that the caecal cells are more efficient from the stand- Figure 11. Scanning electron micrograph of point of uptake, this hypothesis may apply portion of the tegument of adult male Schistosoma only to macromolecules. Molecules of rela- mansoni showing pits, ridges, and spines. (After tively low molecular weights, including the Hockley, 1973). amino acids glycine, proline, methionine, argi- Figure 12. Transmission electron micrograph of nine, cysteine, glutamate, and tryptophan are body surface of adult male Schistosoma mansoni showing profiles of interconnected tortuous chan- taken into S. mansoni adults primarily through nels. (After Hockley, 1973). the tegument (Asch and Read, 1975b). Furthermore, kinetic studies by Asch and Read have revealed that these amino acids are al., 1969; Silk et al, 1969; Hockley, 1970; taken in by different mechanisms. For ex- Inatomi et al., 1970). For comparison, it is ample, cysteine is taken up solely by diffusion, noted that the tegument of Fasciofa hepatica proline is taken up only by active transport, measures 15-20 /tin thick (Threadgold, 1963). while the other amino acids studied are taken Based on these somewhat specialized fea- up through a combination of diffusion and tures of the schistosome tegument, it was gen- active transport. There is a highly specific erally assumed that, as is the case with other transport locus for proline, and one for acidic species of digeneans, the tegument functions amino acids, and there are probably at least as an absorptive surface. However, Hockley (1973), as the result of comparing the struc- two transport systems for most of the neutral ture of the schistosome tegument with that of amino acids. It is noted that earlier, Asch and its digestive epithelium, i.e., the syncytium Read (1975a) had reported that 80-100% of lining the intestinal caeca, has concluded that glycine and proline are taken up through the the caecal wall is probably more efficient as tegument, and Isseroff et al. (1972) have uptake surfaces. However, a few words need demonstrated that monosaccharides are also

Copyright © 2011, The Helminthological Society of Washington 10 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY primarily taken up through the tegument of formation of small surface outgrowths from S. mansoni. the tegument, followed by increasing vacuola- What has been said about schistosomes tion of the tegument, and the development of relative to uptake probably also holds true larger, balloonlike surface exudates. Eventu- for Fasciola hepatica and other digenetic trem- ally, the host's phagocytic cells become as- atodes since Mansour (1959) has shown that sociated with these exudates, and as de- ligation of the mount of F. hepatica does not generation progresses, the phagocytes infiltrate effect the rate of absorption of glucose, and through the parasite's degenerate tegument, Isseroff and Read (1969) have reported that and invade all of the tissues of the schisto- amino acids are taken up by this parasite some. Standen has expressed the opinion that through the tegument. More recently, Hanna the drug exerts a deleterious effect on the (1976) has reported that the monosaccharid.es permeability and architecture of the parasite's galactose and glucose are also taken up pri- tegument, and as a consequence, the uptake marily through the tegument of F. hepatica. of nutrients through the tegument is partially The point being made is that there is ample or totally inhibited and this leads to the rapid evidence that the teguments of S. mansoni and senescence of the worm. F. hepatica are actively involved in the uptake Bueding et al. (1967) have reported that a of molecules of low molecular weights; how- subcurative dose of tris(p-aminophenyl) car- ever, larger molecules are most probably taken bonium chloride administered to mice infected up through the caecal epithelium. Conse- with S. mansoni results in a reduction in the quently, in delivering drugs, which are com- amount of glycogen in the dorsal tubercles of monly compounds of relative high molecular male worms, and this results in the flattening weights, to schistosomes and liver flukes, con- and eventual disappearance of the tubercles, sideration must be given to the fact that its but there is no direct effect on the tegument. effective uptake can only occur through the Finally, Hockley (1973) has reported that digestive epithelium after ingestion. preliminary electron microscopical studies on It is noted that certain drugs are known to S. mansoni recovered from a monkey 1 hour effect the target helminth parasites by inter- after treatment with the antimonial compound fering with their surfacial uptake mechanisms. Triostam (sodium antimonygluconate) has re- For example, Bueding (1959, 1962) has dem- vealed balloonlike swellings on the outer sur- onstrated that the chemotherapeutic activity face of the tegument. These swellings are 3-4 of alkyldiphenylamines can be explained by /Am in diameter and each is jointed to the their ability to inhibit glucose absorption by normal tegument by an isthmus. The outer Schistosoma mansoni. Similarly, Bueding et al. membrane of the tegument is continuous over (1961) have reported that the therapeutic the swellings, but the inclusions in the tegu- activity of a cyanine dye, dithiazanine, is the mental syncytium do not extend into them. result of its ability to inhibit uptake of glucose A few studies are also available on the ef- by Trichuris vulpis, and Strufe and Gonnert fects of drugs on the tegument of Fasciola (1967) are of the opinion that the cestodecidal hepatica. Dawes (1966a, b, 1967) has re- mechanism of such drugs as dichlorophen and ported that there is vacuolation of the tegument Yomesan most probably is also based on such of this parasite after the rat host had been a phenomenon. treated with Bithinol, and Thorsell and Bjork- At this point it would appear of interest to man (1966) have reported similar vacuolation point out that there is some information avail- of the tegument of this fluke after in vitro able on the effect of drugs on the tegument of treatment with hexachlorophene and its di- schistosomes. Gonnert (1955) has reported methylether. that Miracil D causes the vacuolation of S. The question that must be raised is whether mansoni tegument, Standen (1962) has re- these pathological alterations of helminth tegu- ported that l:7-bis(p-aminophenoxy) heptane, ments after drug treatment represent the direct when injected into S. mansoni-intected mice, effect of the drugs or reflect the moribund causes the parasite's tegument to disintegrate. condition of the worms. That the latter is the Specifically, he found that initially there is the answer appears to be supported by Dawes's

(1968) finding that the vacuolation was due to usually means the empirical screening of nu- impending death after drug treatment. This merous compounds. For example, according to interpretation is further supported by Dawes's Standen (1967) approximately 250,000 com- (1963, 1964) reports that invasion of the tegu- pounds have been screened as possible anti- ment by host phagocytes occurs when speci- schistosomal drugs. It is usually only when an mens of F. hepatica are weakened by X-irradia- effective drug is found as the result of mass tion. In other words, drug treatment leads to screening that the underlying cidal mechanism chemical changes on the surfaces of these is investigated. The history of the development worms so that they no longer mimic their hosts of chemotherapeutic agents for vaginal tricho- immunologically (Smithers and Terry, 1967, miasis as presented by Jirovec and Petru 1969; Smithers et al., 1969; Damian, 1962, (1968) and that of the development of chemo- 1964), and consequently are recognized as therapeutic agents against Manson's schisto- nonself and are attacked by phagocytes. In somiasis by Pellegrino and Katz (1968) attest conclusion, it may be said that from the limited to this route in parasiticidal drug research. This data available, drugs known to be cidal to is not to say that empirical screening has not schistosomes and liver flukes apparently do led to the discovery of useful drugs. As an cause cytopathological alterations in the para- example, the development of Miracil D, the sites' teguments but these are secondary mani- first metal-free compound which proved to be festations of the moribund condition of the of therapeutic value in human schistosomiasis parasites rather than the direct effect of the (Kikuth et al., 1946; Kikuth and Gonnert, drugs. Therefore, the route of entry of the 1948) came about by modification of Miracil drug into the parasite has not been revealed A, which, in turn, was selected from the screen- by the studies reviewed above. As stated, from ing of about 4000 substances. The discovery other available information, it is hypothesized of Miracil D has led to the synthesis of Hy- that the drugs, all with relatively high molec- canthone (l-N-/?-diethyleminoethylamino-4-hy- ular weights, are taken in orally. droxymethylthioxanthone) (Rosi et al., 1965), In summary, it would appear that in the which, despite the controversy, is in use in formulation of antiparasitic drugs, consideration schistosomiasis endemic areas. should be given to: (1) whether the molecule In this section is reiterated the concept of will permeate the surfaces of the specific para- metabolic interference in the development of site, and (2) whether it will elicit normal func- drugs for chemotherapy of parasitic diseases. tion by the specialized uptake mechanisms of This, of course, requires understanding the re- of the parasite, e.g., cytostomal uptake, pino- lationship between drugs, hosts, and parasites cytosis, and other similar processes. Alter- at the biochemical level. natively, the drug could be of such a nature CHEMOTHERAPY OF PROTOZOAN DISEASES. It that it would interfere with the uptake of es- is not my intent to present a critical review of sential exogenous substrates by the parasite. either the metabolism of protozoan parasites or the available information on chemotherapy of Metabolic Interference protozoan-caused diseases in this section. The first topic has been reviewed brilliantly by von Ideally, the search for new parasiticidal Brand (1973), and Fletcher and Maegraith drugs should involve the finding of new com- (1972) have reviewed what is known in this pounds \vhich would interrupt some critical area about Plasmodium spp. The second topic biochemical pathway in the parasite and at has been reviewed by Peters (1970) and the same time not effect the host deleteriously. others. Rather, it is my intent to reinforce the In other words, a systematic search for meta- thoughts of many how basic understanding of bolic pathways unique to the target parasite the biochemistry of host and parasite could should be conducted. This, of course, as we lead to a more rational approach to the chemo- all recognize, is a long, tedious, and expensive therapy of diseases caused by protozoa. Un- process which cannot guarantee results. Con- fortunately, the development of new drugs sequently, those in pharmaceutical research based on this assumption has yet to become a know that research on parasiticidal compounds reality. From what is known, the primary rea-

Copyright © 2011, The Helminthological Society of Washington 12 PROCEEDINGS OF THE HELMINTHOLOGICAL SOCIETY son for this appears to be that the parasites zymes involved in the pentose phosphate path- are biochemically "uncooperative," i.e., al- way. One could go one step further and hy- though qualitative and quantitative differences pothesize that if simplified or abbreviated between the metabolism of host and parasite metabolic shunts are to be found in a host- have been recorded, only a few clear-cut in- parasite association, in most, if not all, instances stances are yet known where the parasite in- the modified pathway would occur in the para- cludes a critical metabolic pathway that is site since in their unique type of niche, para- absent from the host. A few selected examples sites are exposed to restricted amounts of of this situation are presented below. exogenous substrates, as are organisms that sur- Considerable interest has been focused on vive under other types of ecological stress, and the pentose phosphate pathway of Plasmodium this type of selective pressure, in my opinion, spp. and their host cells during the past two favors biochemical change from the complex to decades. Theoretically, it would appear that the simple. Consequently as is well docu- the malaria parasite could have an absolute re- mented by available data (von Brand, 1973), quirement for this pathway since this is the there are few clear-cut examples of the occur- principal, if not the only, pathway for the pro- rence of essential metabolic pathways in para- duction of the pentose sugars essential for sites that are absent in the host. nucleic acid synthesis. In view of the fact that Let us look at an example. It is well known degradation of glucose via the pentose phos- that the classical Embden Meyerhoff glycolytic phate pathway leads to the formation of only pathway leading from carbohydrate to lactic 3 moles of ATP per mole of glucose utilized acid is independent of oxygen and therefore indicates that it is less important as an energy- serves as the principal energy-producing path- producing mechanism than for the provision of way under anaerobic conditions. The wide dis- compounds utilized in various syntheses. tribution of many glycolytic enzymes among Studies by Bowman et al. (1961), Fletcher Tnjpanosoma spp. and Entamoeba histolytica and Maegraith (1962), Bryant et al. (1964), (see von Brand, 1973 for review), and the Herman et al. (1966), Theakston and Fletcher demonstration of phosphorylated glycolytic in- (197la, b), and Luzzatto et al. (1969) all have termediates in trypanosomes clearly indicate suggested that the erythrocytic stages of vari- that their glycolytic sequences are in general ous species of Plasmodium are completely typical of the classical ones. However, if there dependent on the enzyme glucose-6-phosphate- is a lack or a very low level of activity of a dehydrogenase and possibly also 6-phospho- specific enzyme, a modification of the classical gluconate-dehydrogenase of the host cells. glycolytic pathway occurs. For example, the These enzymes are essential for the operation low level of enzymatic activity causing reduc- of the pentose phosphate pathway. The point tion of pyruvate to lactate results in the re- I wish to make is that if it was the other way oxidation of NADH formed during triose oxi- around, i.e., the host cells were dependent on dation, and this process prevents the cessation the parasite's enzymes, then an agent which of glycolysis. Now, among monomorphic try- would block the activities of these enzymes panosomes of the brucei group, little or no could theoretically be employed to inhibit the lactic acid is produced, and phosphoglyceralde- pentose phosphate pathway in the parasite hyde (PGAL) acts as the hydrogen acceptor and this presumably would lead to the death for the reoxidation of NADH, a process leading of both the parasite and the infected cell. to the formation of phosphoglycerol. It is The reversed example cited above serves to known that glycerol is one of the main anaero- illustrate what is meant by the biochemical bic end products of these flagellates, and this "uncooperativeness" of parasites. But then, in molecule is the end product of the metabolism view of the nature of parasitism and the evo- of phosphoglycerol mediated by glycerolphos- lutionary events leading to its development phate dehydrogenase and phosphatase, which (Cheng, 1973), it is not surprising that if bio- enzymes have been demonstrated in these hemo- chemical dependency is to be found in a host- flagellates (Harvey, 1949; Grant and Sargent, parasite relationship, it is the parasite that is 1960). In view of this, theoretically, an independent on host erythrocytes for certain en- hibitor of glycerolphosphate dehydrogenase

AMP ATP+P-, carboxylase, the NAD-linked malate dehydro- (PPDK) genase, and malic enzyme could be amoebi- cidal. o ' ^NADPH p. !•> <" CHEMOTHERAPY OF HELMINTHIC DISEASES. H » ppi ~E V "? ^^ Few examples are available of major metabolic E£ V + " (D V NADP differences between helminths and their hosts c°2\6 ^X ^002 (see Bryant, 1970, for review). On the other hand, there appears to be some differences in rwalaretatfi (MDH) >• malatp the sensitivities of certain helminth enzymes NADH NAD and their counterparts in their hosts. This, of Figure 13. Interrelationship between the en/ymc- course, could be capitalized upon in the de- niediated steps involved in glycolysis in Entamoeba velopment of drugs; for example, the inhibi- histolytica. PPDK, phosphoenolpyruvic carboxy- tion of schistosome phosphofructokinase by transphosphorylase; PCTP, phosphopyruvate car- antimonials. Saz and Bueding (1966) have boxylase; MDH, malate dehydrogenase. demonstrated that this enzyme from schistosomes differs from that of mammals in sen- and phosphatase would block the liberation of sitivity to antimonials, with the former being glycerol to the detriment of these parasites. more so. This is most probably the basis for Another example is considered at this point. chemotherapeutic properties of such anti- In most organisms that produce lactic acid, monial compounds as potassium antimony phosphoenolpyruvate is transformed into pryu- tartrate, stibophen, and others. Trivalent anti- vate via a pathway mediated by pyruvate monials are efficient in inhibiting phospho- kinase as follows: fructokinase in these parasites, and this enzyme pyruvate is known to control the rate of glycolysis in kinase schistosomes. Therefore, this is generally c o n - Phosphoenolpyruvate + ADP < sidered to be the cidal mechanism of anti- pyruvate + ATP monials, although these drugs may also affect other mechanisms essential for the survival of This enzyme, however, is lacking in Entamoeba the parasites (Bueding, 1959). histolytica, and the transformation is accom- It is noted that Cu++ is more effective at plished as a result of mediation by pyruvate- inhibiting succinic dehydrogenase activity of phosphate ligase (AMP, phosphoenolpyruvic Ascaris than of the mollusc Biomphalaria carboxytransphorylase), which requires inor- glabrata (Cheng, unpubl.). This fact is being ganic pyrophosphate as the substrate. This is mentioned because it also serves to exemplify illustrated by the following: the concept that enzymes that catalyze the Phosphoenolpyruvate + AMP + PPj *± same process in different species of animals can pyruvate + ATP + P, be different in their sensitivities to inhibitors. The presence of the essential pyrophosphate is It is also of interest to note that Cu++ will retard assured by the occurrence of the enzyme the normal development of larval Schistosoma phosphopyruvate carboxylase, which catalyzes mansoni in. Biomphalaria glabrata when in- the formation of pyrophosphate and oxalacetate fected snails are exposed to 60 ppm of Cu in the form of CuSO4 for 20 hours (Cheng, un- from phosphoenolpyruvate, CO2, and inorganic phosphate. In conjunction with phosphopyru- publ. ) . Although the mechanism for this vate carboxylase, pyruvate and oxalacetate are phenomenon remains uninvestigated, it is, produced from glucose and CO2. The oxal- nevertheless, of interest to note that both anti- acetate subsequently is converted to malate mony and copper, are schistosomidal. Since by a NAD-linked malate dehydrogenase, and the cupric ion is essentially nontoxic to mam- malate is converted to pyruvate by a NADP- mals, except for sheep, at low concentrations, linked malate dehydrogenase known as the i.e., in concentrations of parts per million, malic enzyme. The interplay of the four en- perhaps studies to determine the chemothera- zymes is depicted in Figure 13. Thus, theo- peutic properties of copper compounds should retically, the introduction of inhibitors for be carried out. As far as I have been able to pyruvatephosphate ligase, phosphopyruvate ascertain, such studies have not been done.

Of course, the inhibition of enzymes in chick-embryo liver as observed electron mi- helminths is not always brought about by croscopically. Am. I. Trop. Med. Hyg. 17: metals. Tetramisole and thiabendazole, both 156-169. nonmetal-containing drugs, interfere directly Asch, H. L., and C. P. Read. 1975a. Trans- with fumarate reductase activity in Ascaris and tegumental absorption of amino acids by male Schistosoma mansoni. J. Parasit. 61: Haemonchus (van den Bossche and Janssen, 378-379. 1967, 1969; Prichard, 1970). , and . 1975b. Membrane trans- Finally, brief consideration is being given to port in Schistosoma mansoni: transport of another mechanism by which certain drugs amino acids by adult males. Exptl. Parasit. cause the death of helminths. It is known that 38: 123-135. succinate production in Ascaris is considerably Bowman, I. B. R., P. T. Grant, W. O. Ker- reduced when treated with piperazine. Bued- mack, and D. Ogston. 1961. Metabolism ing et al. (1959) have shown that this is not of Plasmodium berghei, the malaria parasite due to direct inhibition of one of the metabolic of rodents. II. An effect of mepracrine on the metabolism of glucose by the parasite sepa- steps leading to the production of succinate. rated from its host cell. Biochem. J. 78: Rather, it is due to the fact that piperazine in- 472-478. duces paralysis of the nematodes and this Brooker, B. E. 1971. The fine structure of lowers their energy requirements. Crithidia fasciculata with special reference to In conclusion, my intent has been to point the organelles involved in the ingestion and out that the formulation of effective drugs digestion of protein. Z. Zellforsch. Mikrosk. against parasites could depend on a variety of Anat. 116: 532-563. factors among which (1) entry into the para- Bryant, C. 1970. Electron transport in parasitic site and that portion of the host harboring the helminths and protozoa. Adv. Parasit. 8: 139-172. parasite, (2) alteration of the uptake surface Bryant, C., A. Voller, and M. J. H. Smith. of the parasite, (3) inhibition of some special- 1964. The incorporation of radioactivity ized metabolic pathway unique to the parasite, from glucose-14C into the soluble metabolic and (4) quantitative differences favoring in- intermediates of malaria parasites. Am. J. hibiting of some biochemical or physiological Trop. Med. Hyg. 13: 515-519. process in the parasite are important ones. Bueding, E. 1959. Mechanisms of action of ADDENDUM (added in proof). Since the submission of schistosomicidal agents. J. Pharm. Phanna- this paper, A. B. Clarkson Jr. and F. H. Brohn (1976. col. 11: 385-392. Science, 144:204-206) have published an excellent example of selective destruction of Trypanosoma brucci . 1962. Effects of benzylic diamines on bntcei in the mammalian host by salicyl hydroxamic acid Schistosoma mansoni. Biochem. Phannacol. and glycerol based on differences in host and parasite carbohydrate metabolism. 11: 17-28. Bueding, E., H. J. Saz, and G. W. Farrow. Literature Cited 1959. The effect of piperazine on succinate Aikawa, M. 1967. infrastructure of the pellic- production by Ascaris lumbricoides. Br. J. ular complex of Plasmodium fallax. J. Cell Pharmacol. Chemother. 14: 497-500. Biol. 35: 103-113. Bueding, E., E. Kmetec, C. Swartzwelder, S. . 1971. Plasmodium: The fine structure Abadie, and H. J. Saz. 1961. Biochemical of malarial parasites. Exptl. Parasit. 30: effects of dithiazanine on the canine whip- 284-320. worm, Trichuris vulpis. Biochem. Pharmacol. Aikawa, M., and C. R. Sterling. 1974. Intra- 5: 311-322. cellular Parasitic Protozoa. Academic Press, Bueding, E., E. L. Schiller, and J. G. Bourgeois. New York. 1967. Some physiological, biochemical and Aikawa, M., P. K. Hepler, C. G. Huff, and morphologic effects of tris (p-aminophenyl) H. Sprinz. 1966. The feeding mechanism carbonium salts (TAG) on Schistosoma man- of avian malarial parasites. J. Cell Biol. 28: 355-373. soni. Am. J. Trop. Med. Hyg. 16: 500-515. Aikawa, M., C. G. Huff, and H. Sprinz. 1967. Cenedella, R. J., and J. J. Jarrell. 1970. Sug- Fine structure of the asexual stages of Plas- gested new mechanisms of antimalarial ac- modium elongatum. J. Cell Biol. 34: 229- tion for DDS (4,4'-cliaminodiphenyl sulfone) 249. involving inhibition of glucose utilization by , , and . 1968. Exoerythro- the intraerythrocytic parasite. Am. J. Trop. cytic stages of Plasmodium gallinaceum in Med. Hyg. 19: 592-598.

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The Control of Parasites: The Role of Drugs1

W. C. CAMPBELL Merck Institute for Therapeutic Research, Rahway, New Jersey 07065

". . . the play is the tragedy 'Man', are the prevailing social and political forces in and its hero, the Conqueror Worm" the case of man, and the husbandry practices Edgar Allan Poe in the case of domestic animals. Of the mechanisms invoked intentionally to reduce para- Poe's words were allegorical rather than sitism, biological control and vaccination have parasitological; but parasite control might be played roles that have been overshadowed by regarded, in large measure, as an attempt to the role of chemotherapy. It seems likely that strip the worm of its role as conqueror. antiparasitic drugs will continue to be of im- Undoubtedly the major extrinsic factors in portance for some time. determining the extent and degree of parasitism Antiparasitic drugs actually play many roles —not just because there are many drugs, but because one drug in its time plays many parts. Some are general roles—the parts played by