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Resistance is readily produced in rodent malaria using the single-dose, '2%-relapse technique' (2%RT) against the individual compounds chlorproguanil (CPG), chlorcycloguanil (CCG), cycloguanil, dapsone (DDS) and artesunate (ASN). Using the '4-day test', a low level of synergism or a simple additional action between CPG and DDS was observed with multiple dosing of these two compounds in a combination. Resistance to a 1 : 3 combination of CPG-DDS was selected in each of three parasite lines: Plasmodium berghei NK65, P. yoelii ssp. NS and P. chabaudi AS. Of these lines, P. chabaudi AS was found to be the most sensitive to the 1 : 3 combination in the 2%RT (and was also previously found to be the most sensitive when the compounds were used individually). Plasmodium chabaudi AS was also the line found most sensitive to a 7 : 21 : 300 combination of CPG-DDS-ASN (CDA). In mice infected with P. chabaudi AS, compared with the use of the individual components, the CPG-DDS combination only a gave a modest level of protection (as indicated by the increase in the time required to select resistance in the 2%RT) but the triple CDA combination was totally effective over the duration of the experiment. New pharmacokinetic data to be reported elsewhere indicate, however, that the antimalarial action of CPG in mice is exerted by a mechanism that is not associated with the drug's conversion to the antifolate triazine, CCG. The question thus arises as to how, in the present model, the protective action of CDA was effected. The present results nevertheless reinforce the hypothesis that a CDA combination, appropriately proportioned for human use, should be of practical value, in protecting the individual components, when used for the treatment of multidrug-resistant P. falciparum, and possibly other Plasmodium species, in endemic areas. Clinical trials, both with a CPG-DDS combination (Lapdap) and CDA, are currently under way in tropical Africa. Further studies are now required to determine whether DDS, CPG or an as-yet unidentified metabolite of CPG interact with ASN, and whether a simple double combination of ASN with one or other of these would be as protective, against the selection of resistance, as CDA.

In recent years infection with chloroquine-resistant Plasmodium falciparum has been combatted with two long-acting antimalarials, pyrimethamine and sulfadoxine, in the combination known as Fansidar that exerts a strong, synergistic action on the asexual stages of the parasite. This second-line regimen, however, is failing increasingly because of the selection of resistant clones in endemic areas, and effective, safe, alternative drugs or drug combinations that are also affordable are urgently needed. Antimalarial drugs with shorter half-lives than those of pyrimethamine or sulfadoxine are likely to be slower to select resistant parasites. In the experiments reported here, the baseline in-vivo responses of rodent malarial parasites to chlorproguanil and proguanil and their active metabolites, chlorcycloguanil and cycloguanil, as well as those to dapsone and artesunate, were explored. In general, the most sensitive parasite to all of these compounds was P. chabaudi. When the drugs were used, individually, to select resistance via the '2%-relapse technique', relatively stable resistance to each was obtained in P. chabaudi as well as in P. berghei and P. yoelii ssp. NS, the last of these being also highly resistant to chloroquine. Of most concern was the rapidity and high level of resistance developed by P. chabaudi to artesunate. The experiments also validated the use of chlorcycloguanil or cycloguanil as surrogates for chlorproguanil or proguanil. Further studies to investigate the possible value of administering chlorproguanil-dapsone, with or without artesunate, are under way and will be reported separately.

The influence of combinations containing the blood schizontocides chloroquine (CQ) or mefloquine (MEF), together with the 8-aminoquinolines (8AQ) primaquine (PQ) or the new, long-acting compound, tafenoquine (TAF), on the rate of selection of resistance to the individual compounds was examined using the asexual, intra-erythrocytic stages in rodent malaria models. The two main procedures used were a 'serial technique' (ST) and the '2%- relapse technique' (2%RT). The ST provided evidence for the contention that a combination with PQ slowed the selection of resistance to CQ or MEF; it has been shown previously that synergism exists between CQ and either PQ or TAF in rodent malaria. Data obtained with the 2%RT, and three parasite lines derived from Plasmodium berghei N (the 238B line), P. chabaudi ASS (the 238C line) or P. yoelii ssp. NS (the 238Y line), indicated that resistance to TAF used alone is acquired rapidly under drug pressure and that this resistance is stable when selection pressure is removed. In the 2%RT, resistance to CQ developed when another line of P. chabaudi (AS15) was exposed to that compound alone, although more slowly than the development of resistance to TAF in the 238C line. However, treatment of a TC line of P. chabaudi, developed in a 2%RT using a combination of CQ with TAF, led to little resistance to either compound. A totally unforeseen phenomenon was the appearance of a high level of resistance to CQ in the 238C line of P. chabaudi that had been exposed only to TAF; this was not observed with the 238B or 238Y lines. Attention has been refocused recently on the use of 8AQ for prophylaxis in man. It remains to be determined if resistance in the asexual intra-erythrocytic forms is carried over to the other stages of the malarial life-cycle, especially the hepatic, pre-erythrocytic schizonts. The implications of the present results for the possible clinical deployment of 8AQ in the future are discussed. It is concluded that, whereas use of an 8AQ alone carries a high risk of selecting resistance, combinations containing 8AQ may have a place in the protection of blood schizontocides that are to be deployed in endemic areas. Furthermore, the inherent gametocytocidal action of the 8AQ should promote the reduction of transmission.

The activities of artemisinin (QHS) and a number of its semi-synthetic analogues, as well as Fenozan B07 (B07), a synthetic 1,2,4-trioxane, and arteflene (ATF), a synthetic surrogate of yingzhaosu, were compared in mice infected with drug-sensitive Plasmodium berghei or chloroquine-resistant P. yoelii ssp. NS. The studies were stimulated by the observation that B07, in certain aqueous preparations, appears to be equipotent by the subcutaneous (sc) or oral (po) routes in the rodent model but not in a simian model. In the rodent model, B07 was found to undergo rapid alteration (with a half-life of <24h) in an aqueous stock solution prepared using dimethyl sulphoxide (DMSO) to pre-dissolve the drug. Therefore, for all later experiments with aqueous preparations, the test material was newly formulated each day. In a carboxymethylcellulose formulation used as a 'standard suspending vehicle' (SSV), B07 and dihydroartemisinin (DIHYD) were found to be, respectively, one sixth and one 10th as active po as when the drugs were pre-dissolved in DMSO and then diluted with water. ATF in DMSO given po was less than one 20th as active as when used sc in the rodent model, and this drug in SSV was almost inactive po. The relatively low oral activity of these three compounds (especially DIHYD and ATF) may be attributable to extensive first-pass metabolism in the mouse. Oral beta-artemether (AM) and beta-arteether (AE) were highly active when used in SSV. ATF has been found to have low activity in simian models and clinical trials because of its poor absolute bio-availability. In in-vivo studies of the blood schizontocidal action of anti-malarials, in rodent malaria models, the data collected on the structure-activity relationships (SAR) of the drugs must be viewed critically when selecting specific compounds from a chemical series for further development. A study of the influence of drug formulation on the activity of other, novel antimalarials is crucial to the evaluation of the drugs, and merits high priority.

Several compounds in current clinical use as antihistaminic agents, among them cyproheptadine (CYP), have been shown, in experimental models, to reverse resistance of the asexual, intra-erythrocytic stages of rodent or human malarial parasites to chloroquine (CQ). Although preliminary clinical trials with CYP failed to confirm such activity in subjects with naturally acquired infection with Plasmodium falciparum, Nigerian investigators have reported that another antihistaminic, chlorpheniramine, significantly restores the blood schizontocidal action of CQ in semi-immune patients with CQ-resistant P. falciparum, when the two compounds are administered together. The rates at which resistance to CYP can be produced, in mice infected either with CQ-resistant P. yoelii ssp. NS or CQ-resistant P. yoelii nigeriensis, when drug-selection pressure is exerted with this compound alone have now been compared with the rate and extent to which resistance develops in infected animals that are treated with various combinations of CYP and CQ. The data indicate that, in both parasites, stable resistance develops slowly to CYP alone and that exposure to a combination of CYP plus CQ significantly impedes the selection of resistance to CYP. Although the antimalarial action of CYP is reported to extend to the pre-erythrocytic hepatic stages, there was no evidence of gametocytocidal activity in the present study. The future implications of these observations are discussed in relation to the clinical potential of CYP + CQ and similar combinations and possible future research.

The search for combinations of antimalarial drugs that will impede the selection of drug resistance, especially in Plasmodium falciparum, is currently focused on the use of a member of the artemisinin family, with a short half-life, in association with a relatively long-acting blood schizontocide. Experiments with such 'third-generation' combinations, in mice infected either with chloroquine-sensitive P. berghei or P. chabaudi, or chloroquine-resistant P. yoelii ssp. NS, have produced interesting results. The data collected, using the '2% relapse technique' (2%RT), indicate that a combination of artemisinin with mefloquine can impede to a significant degree, although by no means completely, the selection of resistance to both compounds in P. berghei and in P. yoelii ssp. NS. Similarly, a combination of artesunate with pyronaridine impedes the selection of resistance to these compounds in P. berghei. Parallels are drawn between observations with such combinations in man and in the rodent models which, it is argued, once again demonstrate their value in predicting the protective value of using different types of antimalarials together. Evidence is presented that resistance to single compounds may emerge more rapidly when a high dose is employed in the 2%RT than a lower dose. It is noted also that the rate at which resistance to pyronaridine is selected by a given dose varies with the species of rodent Plasmodium, and the relevance of this to the malarial parasites of human is discussed.

Chloroquine-sensitive Plasmodium berghei N and chloroquine-resistant P. yoelii ssp. NS were exposed to selection pressure, in the '2% relapse technique', from artemisinin, artesunate, a bicyclic, synthetic endoperoxide Ro 41-3823 (an analogue of arteflene) or Fenozan B07, a synthetic 1,2,4-trioxane endoperoxide. Whereas resistance against artemisinin did develop to a moderate level in both parasites, only a low level of resistance or none developed to the other compounds, and resistant parasites readily lost resistance once drug-selection pressure was withdrawn. The relevance of these observations and the experience of other investigators are discussed in relation to the possible risk that resistance may be developed in nature once endoperoxides are deployed widely against multidrug-resistant P. falciparum.

Two interactions of two potent blood schizontocides, pyronaridine and artemisinin, were assessed in mice infected with chloroquine-resistant Plasmodium yoelii ssp. NS or one of two lines derived from it, namely ART, which is resistant to artemisinin and SPN, which is resistant to pyronaridine. While the drug combination proved to be only additive in its action against P. yoelii ssp. NS, a marked potentiation between the two compounds was observed against the ART and SPN lines. The implications of the findings in terms of the impeding of drug resistance when these compounds are deployed for the treatment of multi-drug-resistant P. falciparum are discussed.

Fenozan B07, a 1,2,4-trioxane endoperoxide with potent blood schizontocidal activity against drug-sensitive and drug-resistant rodent malaria parasites, exerted a modest potentiating action when administered with chloroquine (CQ) to mice infected with parasites of the CQ-resistant P. yoelii ssp. NS, but not when given to mice infected with the CQ-sensitive P. berghei N strain. The reason why this potentiation may be of particular value in the treatment of severe falciparum malaria is discussed. Mefloquine and halofantrine displayed a similar level of potentiation with Fenozan B07 against the CQ-resistant parasites. However, antagonism was shown by combinations of Fenozan B07 with pyronaridine or the 8-aminoquinoline WR 238 605 when used against CQ-resistant parasites. Mefloquine with Fenozan B07 is also antagonistic against a highly mefloquine-resistant line of P. yoelii ssp. NS. The reasons behind such antagonism are not known. The importance is stressed of using carefully selected drug combinations of novel antimalarials, rather than single drugs, in order to impede the selection of drug-resistant parasites, but only after adequate, preclinical, toxicity testing.

Fenozan B07, a difluorinated 3,3'-spirocyclopentane, 1,2,4-trioxane, is a novel, second-generation antimalarial endoperoxide which is a potent blood schizontocide against strains of rodent malaria that are highly resistant to a wide spectrum of classical antimalarials. Like compounds of the artemisinin series, its action is limited to the intra-erythrocytic stages, both asexual and sexual, and it is devoid of causal prophylactic activity. Both Fenozan B07 and the artemisinins are potent gametocytocides. In contrast to arteether, in a model using synchronous infection with Plasmodium vinckei petteri, Fenozan B07 inhibits the development of all asexual stages except preschizonts, as well as gametocytes. The activity of the artemisinin series in rodent-malaria models is limited to the rings and young trophozoites. The combined effect of Fenozan B07 with artesunate against P. v. petteri was only additive. A slight degree of potentiation was found in mice infected with asynchronous, drug-sensitive P. berghei but the combination was only additive against CQ-resistant P. yoelli ssp. NS. On the other hand, a significant degree of synergism was observed when mice infected with the artemisinin-resistant ART line of P. yoelii ssp. NS received combinations of Fenozan B07 with artemisinin. The conclusion is drawn from these and other data that there are significant differences between the blood schizontocidal actions of Fenozan B07 and the artemisinins. The basis of these differences remains to be determined.

A comparison was made between the blood schizontocidal action in mice of racemic mefloquine hydrochloride and the free bases of its (+)- and (-)-enantiomers (Ro 13-7224 and Ro 13-7225) against chloroquine-resistant Plasmodium yoelii ssp. NS. The racemic hydrochloride was two to three times as active against this parasite in mice as either of the enantiomer free bases, which were of similar activity to each other. Under drug selection pressure, the parasites acquired resistance in approximately the same time for each of the three compounds.

WR 238,605, a novel 3-phenoxy-substituted 8-aminoquinoline, possesses causal prophylactic, blood schizontocidal and gametocytocidal activity against rodent malaria parasites. Against the asexual, intra-erythrocytic stages of drug-sensitive Plasmodium berghei N strain, it is about nine times as active as primaquine (PQ). It is from four to 100 times as active as PQ against lines of P. berghei or P. yoelii that are resistant to currently used antimalarials. WR 238,605 is three times as active as PQ against the pre-erythrocytic stages of P. y. nigeriensis but it has very poor gametocytocidal action and no sporontocidal activity against this parasite. In combination with chloroquine (CQ), WR 238,605 and PQ display a synergistic or 'resistance-reversing' action against CQ-resistant P. yoelii NS parasites. No such effects are seen when WR 238,605 is deployed with mefloquine against a mefloquine-resistant line or with artemisinin against an artemisinin-resistant line and it appears to be antagonistic to halofantrine against a halofantrine-resistant parasite. It is suggested that WR 238,605 is a good candidate compound for clinical trials against polyresistant P. falciparum, possibly in combination with CQ.

A novel difluorinated 3,3'-spirocyclopentane 1,2,4-trioxane ('Fenozan-50F') is a potent blood schizontocide against drug-sensitive and drug-resistant rodent malaria parasites. It also exerts some action against pre-erythrocytic schizogony, is a potent gametocytocide, and exerts a direct sporontocidal effect in infected mosquitoes. In the '4-day test' the ED90s are 6.8 and 6.0 mg/kg/day for four consecutive days by the subcutaneous and oral routes respectively against drug-sensitive Plasmodium berghei N, and 6.3 and 25 mg/kg against chloroquine-resistant P. yoelii NS in vivo. By the oral route against P. berghei N infection in mice, Fenozan-50F is about half as active as arteether but nearly three times as active as sodium artesunate. The activity of Fenozan-50F is retained against a wide spectrum of drug-resistant parasite lines, although those highly resistant to quinine or to artemisinin are less responsive at the ED90 level. At the ultrastructural level the compound, when administered to infected mice, causes marked changes in the membranes and ribosomes of trophozoites and young schizonts and of immature gametocytes, although few changes are apparent in mature gametocytes. Its toxicity appears to be very low when it is administered to mice by either the oral or subcutaneous route. Fenozan-50F is considered to be a good candidate for eventual use as a therapeutic agent for infection with polyresistant malaria in man.

The new Chinese antimalarial blood schizontocide, artemisinin, derived from the plant Artemisia annua, displays a high level of activity against polyresistant Plasmodium falciparum. Several synthetic 1,2,4-trioxanes were examined in a search for compounds that exhibit a similar type of action against drug-resistant parasites. This paper, the first of a series, describes the examination of these trioxanes against drug-sensitive and drug-resistant malaria parasites in a rodent model, using artemisinin and arteether as comparison standards. Cis-fused cyclohexeno-1,2,4-trioxanes (10-17) substituted with various side-chains revealed for the most part variable but weak antimalarial activity. On the other hand, cis-fused cyclopenteno-1,2,4-trioxanes (18-19) showed greater activity, 19 showing about 1/30th of the activity of arteether against drug-sensitive Plasmodium berghei in vivo, thereby providing a clue to the structure-activity relationship.

The activity of 51 synthetic cis-fused cyclopenteno-1,2,4-trioxanes has been examined against drug-sensitive and chloroquine-resistant malaria parasites in vivo. Some of them display high levels of blood schizontocidal activity when administered orally or subcutaneously. They retain their activity against lines of parasites that are resistant to widely differing antimalarials such as 4-aminoquinolines, aminoalcohols, dihydrofolate reductase inhibitors and artemisinin. The most potent compound of the present series is cis-(+/-)-4a,7a-dihydro-6,7a-di(p-fluorophenyl)spiro [cyclopentane-3,3'-7H-cyclopenta-1,2,4-trioxin], otherwise known as Fenozan-50F.

The activities of Mannich base antimalarials, including pyronaridine, have been explored against drug-sensitive (Plasmodium berghei N) and chloroquine-resistant (Plasmodium yoelii NS) rodent malaria parasites in vivo. Lines of these parasites have been developed with resistance to pyronaridine, amodiaquine, or WR 228,258. The responses and patterns of cross-resistance of these lines to Mannich bases and other blood schizontocides are inconsistent. It is concluded that some Mannich bases may prove still to be of value inthe treatment of chloroquine-resistant Plasmodium falciparum infection.

Fresh Giardia lamblia trophozoites were isolated by in vitro excystation of purified cysts from the faeces of Saudi patients. The method consisted of mixing one part of the cyst suspension with nine parts of 1 N hydrochloric acid, at pH 2, then culturing the excysted trophozoites in modified TYI-S-33 medium in sealed glass vials. During the excystation process most cysts developed a space between the trophozoite and the cyst wall, and after several steps of enlargement the trophozoite emerged at the posterior end of the cyst. Keeping the cysts at 4 degrees C for more than 10 days did not affect the excystation process. Trophozoites obtained by this method can be cryopreserved and recultured without significant loss of activity. This is a simple and rapid method of obtaining G. lamblia trophozoites from cysts in stools or from other sources.

Multiple drug resistance in Plasmodium falciparum is already showing evidence of extending to mefloquine, which at present is one of the few alternative antimalarials for the prevention or treatment of infection with such parasites. Neither verapamil nor cyproheptadine, which reverse chloroquine (CQ) resistance in P. falciparum and in rodent malaria parasites, reverse resistance to mefloquine (MEF) in the MEF-resistant NS/1100 line of P. yoelii ssp. NS. On the other hand, such resistance is clearly reversed when mefloquine is administered to infected mice together with penfluridol.

The inherent blood schizontocidal activities of five antihistaminic compounds, cyproheptadine hydrochloride (CYP), ketotifen hydrogen fumarate (KET), pizotyline hydrogen maleate (PIZ), azatadine maleate (AZAT) and loratadine (LOR) were examined against the following organisms: chloroquine-sensitive (CS) Plasmodium berghei and chloroquine-resistant (CR) P. yoelii ssp. NS in mice; and CS Tak 9 clone 96 and CR K1 strain of P. falciparum in vitro. Chloroquine, verapamil and desipramine were used as comparison standards. CYP, KET, PIZ were active against the CS strain in vivo with ED90 levels between 20 and 30 mg kg-1 (given sc daily for four days). They were slightly more active against the CR strain. AZA was active, but much less so than the other compounds. LOR, verapamil and desipramine were inactive in vivo at the doses tested. Against CS P. falciparum in vitro, all five antihistaminics and desipramine were active at EC50 concentrations ranging from about 50-80 mumol l-1, while verapamil was only active at 175 mumol l-1. Against the CR strain of this parasite, CYP, PIZ and LOR were slightly more active than against the CS strain, but KET, AZAT, desipramine and verapamil were significantly less active. The action of all these compounds in combination with chloroquine was then examined both in vivo and in vitro. The ability of verapamil and desipramine to reverse chloroquine resistance in vitro was confirmed, but only a low level of reversal was seen with these compounds in vivo. However, CYP, KET, PIZ and AZAT produced a marked reversal of chloroquine resistance both in vivo and in vitro. The implications of these observations in relation to further laboratory and clinical research are discussed.

The electrophoretic variation of the enzymes MDH, GPI, G6PGH, 6PGDH were determined for 68 strains of Leishmania isolated in the Aethiopian zoogeographical region. Other characters determined were the DNA buoyant density of nuclear and kinetoplast DNA and the excreted factor serotype. The strains could be readily identified on the basis of these characters thus assisting in the elucidation of epidemiological problems. On the basis of these characters the strains could be divided into six groups. Three of the groups corresponded to L. donovani s.l., L. major and L. aethiopica. The three other groups identified at present have no specific name associated with them.

The relationships between rodent malarias were examined by means of DNA buoyant density determinations and DNA--DNA hybridization data. Current views of the existence in this group of four species: Plasmodium berghei, P. yoelli, P. vinckei and P. chabaudi were confirmed. The identity of two chloroquine-resistant lines derived from P. berghei N strain was established. One of these, the NS line, was found to be a subspecies of P. yoelii; the implications of this finding are discussed.

As a blood schizontocide, halofantrine is about three times as active against a drug-sensitive line of Plasmodium berghei (N strain) as chloroquine, but it lacks any causal prophylactic effect. This activity is retained against parasites highly resistant to primaquine, cycloguanil, pyrimethamine, sulphaphenazole and menoctone. A marked resistance to halofantrine is shown by parasites resistant to mefloquine, quinine, chloroquine and amodiaquine, although the moderately chloroquine-resistant 'NS line' is sensitive. The artemisinin-resistant line shows a moderately reduced response. A high level of resistance was rapidly developed in the 'NS line' to halofantrine (NS/HAL line) but not in the N strain (N/HAL). The NS/HAL line was cross-resistant to mefloquine, amodiaquine and, to a lesser degree, artemisinin, but remained sensitive to chloroquine and quinine. The implications to a lesser degree, artemisinin, but remained sensitive to chloroquine and quinine. The implications of these observations for the possible future clinical deployment of halofantrine are discussed, and emphasis is laid on the need to find means of impeding the resistance to it of Plasmodium falciparum.

More oocysts of Plasmodium yoelii developed in Anopheles stephensi if the mosquitoes received a supplement of p-aminobenzoic acid (PABA) in their diet prior to their taking an infective blood meal, than in unsupplemented control insects. The optimum concentration was 0.05% PABA in 10% sucrose. This effect was not observed if the blood meal was taken prior to feeding with PABA. Similarly, PABA administered to gametocyte-carrying mice increased the numbers of oocysts developing in mosquitoes fed on them subsequently, the effect also being dose dependent, and not mediated through an increase in gametocyte numbers. Sulphadoxine (which blocks PABA uptake) had the opposite action, reducing the numbers of oocysts either when fed directly to the mosquitoes or to the donor mice. These results are compared with those reported in other host-parasite systems by earlier workers, and certain paradoxical observations are discussed with reference to a possible relationship between sulphonamides and contaminating microorganisms in the vector mosquitoes.

Two new Mannich base antimalarials, WR 194,965 and 228,258 exhibit blood schizontocidal action against Plasmodium berghei in vivo. This action is retained also against the P. berghei NS line which has a low level of resistance to chloroquine. While WR 228,258 is also active against the highly chloroquine-resistant RC line, WR 194,965 is not. Both compounds show slightly reduced activity against the highly mefloquine-resistant N/1100 line. WR 225,448, an 8-aminoquinoline, is as active against blood stages of the NS, RC and N/1100 lines of P. berghei as against the N strain, and more so on a dose/weight basis than the first two compounds. It shows a low level of cross-resistance against the primaquine-resistant P line. WR 225,448 has a clear causal prophylactic effect on the pre-erythrocytic schizonts of P. yoelii nigeriensis. While WR 194,965 and 228,258 also appeared to act on these stages, the effect is most probably due to residual action on emerging erythrocytic merozoites since pre-erythrocytic schizonts in treated animals were morphologically indistinguishable from those of the untreated control.

Light microscope and ultrastructural studies show that WR 225,448, an 8-aminoquinoline related to primaquine, causes substantial damage to developing pre-erythrocytic (EE) schizonts of Plasmodium yoelii yoelii in the liver of young rats. In addition to lesions of the mitochondria and nuclear membranes, secretory granules in the parasite fail to reach the surrounding host cell cytoplasm across the parasite membrane. The effective dose of WR 225,448 is much lower than that of primaquine. However, hepatic damage is also seen in treated animals. This may be the summation of damage produced by the experimental inoculation of a large quantity of infective salivary gland tissue from mosquitoes (which is also seen in untreated animals) plus toxic effects of the drug itself. Particular attention would have to be directed to this aspect in preclinical toxicity testing of the compound.

Artemisinin (Qinghaosu), a poorly soluble sesquiterpene lactone derived from the plant Artemisia annua Linn., and a number of more soluble, semi-synthetic derivatives are rapidly-acting blood schizontocides against Plasmodium berghei and P. yoelii nigeriensis. An oily suspension of artemisinin given s.c. is more effective than aqueous suspensions. The activity is retained against lines resistant to primaquine, cycloguanil, pyrimethamine, sulphonamides, mefloquine and menoctone, but a highly chloroquine-resistant line is much less sensitive. Artemisinin has no causal prophylactic, gametocytocidal or sporontocidal action. Dihydroartemisinin causes the pigment of P. berghei to clump, but in a different fashion from the pigment changes induced by chloroquine or quinine, reflecting a different mode of action of the sesquiterpenes from that of these other antimalarials.

Corticosteroids were found to enhance gametocyte production in vitro in two gametocyte-producing chloroquine-resistant strains of Plasmodium falciparum. No significant enhancement was observed in two chloroquine-sensitive strains. Gametocytes were not induced by corticosteroids in a fifth strain which had lost the ability to produce gametocytes.

Infections of Leishmania mexicana in cultured normal mouse peritoneal macrophages show different morphological features depending on whether the parasites invade as promastigote or amastigote forms. Infections derived from promastigote invasion are characterized by parasitophorous vacuoles which develop slowly, and acquire only modest proportions. In contrast, the organisms in amastigote-derived infections lie within parasitophorous vacuoles which develop more rapidly, and attain a much greater size. From observation of promastigotes of different species of Leishmania, it appeared that survival subsequent to endocytosis by normal macrophages depends on the parasites' rapid transformation to the amastigote form. Activation of the macrophage population produced an enhanced parasiticidal effect only against incompletely transformed Leishmania promastigotes. Electron microscope investigations, involving enzyme histochemistry and lysosome labelling techniques, indicate that intracellular Leishmania avoid digestion by interfering with the activity of lysosomal enzymes that are freely delivered to the parasitophorous vacuole. It is proposed that this ability is acquired on transformation to the amastigote, and incidentally induces fluid distension of the parasitophorous vacuole through phenomena recently described by other workers.

Three lines of Leishmania have been grown successfully in tissue culture. L. mexicana mexicana, L.tropica major and L. donovani develop readily as amastigotes in dog sarcoma cells, and in a hamster peritoneal exudate cell line. The procedures used for cultivating both host cells and parasites are described, as are the methods employed for studying the senitivity of the parasites and hosts to drugs. It is concluded that the use of a tissue culture system is a valid way of determining the baseline sensitivity of a Leishmania to chemotherapy and the limitations of the technique are discussed. Illustrative data are presented of the response of two Leishmania species to amphotericin B and to a quinine analogue. L. mexicana mexicana is highly sensitive to amphotericin B and the response is shown to be consistent in duplicate experiments. L. mexicana mexicana and L. donovani are shown to have a different innate sensitivity to the quinine analogue.

Mefloquine (WR 142,490) is a potent blood schizontocide active against drug-sensitive and drug-resistant lines of Plasmodium berghei. The ED50 and ED90 against the P. berghei N strain in albino mice are 1.5 and 3.8 mg/kg respectively. The highly chloroquine-resistant RC line is less sensitive and mefloquine is not fully effective at the maximum tolerated dose in the '4-day test'. Mefloquine has a similar mode of action to quinine both in vitro and (as demonstrated by the morphological changes it induces in P. berghei) in vivo, but is some 100 times more potent. Unlike quinine and WR 122,455 it appears not to interact with DNA. It has no causal prophylactic effect. Mixtures of mefloquine with pyrimethamine, sulphaphenazole or primaquine have an additive effect.

Wistar rats were injected intravenously with sporozoites from Anopheles stephensi infected with Plasmodium yoelii yoelii. The animals were subsequently treated with primaquine at doses of 30, 50 or 100 mg kg-1 at various times. Liver biopsies were made and the exoerythrocytic schizonts examined by light and electron microscopy between 45 and 50 hours. The action of the drug appeared to be principally on the parasite mitochondria, the membranes of which became thickened, then proliferated into multiple layers. Finally the whole organelles swelled up losing all structural organisation. Other parasite membranes were affected similarly and the peripheral enzyme granules disappeared. Following treatment the small, disrupted schizonts were apparently absorbed by their host cells which appeared unaffected by primaquine.

Chloroquine-induced pigment clumping (CIPC) in vitro in Plasmodium knowlesi is inhibited by 7.3 micrograms ml-1 erythromycin. P. knowlesi infections in rhesus monkeys were controlled by doses of erythromycin which gave similar serum levels of the antibiotic. The antimalarial effect was complete but temporary, and recrudescences were noted.

WR 122,455, 3,6-bis-(trifluoromethyl)-alpha-(2-piperidinyl)-9-phenanthrenemethanol HCl, suppresses infection with drug-sensitive Plasmodium berghei N strain in mice. It acts rapidly and affects all the stages of the asexual intraerythrocytic parasites, the effective dose levels being about three times those of chloroquine and one-twelfth to one-fifteenth those of quinine. Under the influence of WR 122,455 haemozoin seems to disappear from the affected parasites following an initial coarsening of the fine pigment granules. These changes are similar to those exerted by quinine. Large doses of WR 122,455 have a residual affect due in part, at least, to deposition of insoluble material in the tissues. The drug appears to exert an antagonistic action on chloroquine when both drugs are administered simultaneously. It has no causal prophylactic effect. In vitro WR 122,455 is a competitive antagonist of chloroquine in a similar manner to quinine, and appears to have a dissociation constant (Ki) of 2-26 x 10(-8) M, making it about 18 times as active as quinine. WR 122,455 interacts strongly with calf thymus DNA, but the mechanism of interaction has yet to be defined. Mice tolerate single doses of a saline/Tween 80 suspensions up to about 400 mg/kg but sc administration induces necrotic changes at the injection site. Up to 30 mg/kg daily po for seven consecutive days is well tolerated systemically but local tissue reaction may occur if the drug is given by the sc or ip routes. However, systemically up to 60 mg/kg is tolerated sc or ip. The relation of WR 122,455 to drug resistant malaria will be reported later.

In the search for more effective alternatives to the presently-used antileishmanial drugs, the activity of the major groups of antimycobacterial compounds has been examined, both in vitro and in animal models of infection. In vitro, clofazimine was the most active compound tested, with a mean ED50 of 2.3 mg l-1 against Leishmania mexicana amazonensis, 1.4 mg l-1 against L. donovani and 0.5 mg l-1 against L. major. Other active compounds were the thiosemicarbazone, thiambutosine, and salinazid, a derivative of isoniazid. Isoniazid itself was inactive, and rifampicin only partially active. In vivo, only clofazimine displayed significant activity, and it was most effective against the cutaneous infections. It is concluded that antimycobacterial activity is in general a poor predictor of antileishmanial potency.

In addition to their blood schizontocidal action on Plasmodium berghei in vivo, two Mannich bases WR 194,965 and 228,258 are also active against chloroquine-sensitive and chloroquine-resistant lines of P. falciparum in vitro. The response of the lines to each drug differs but shows no correlation in either case with response to chloroquine. The 8-aminoquinoline WR 225,448 is also active against P. falciparum in vitro but at much higher concentrations than the Mannich bases. Application of the 'chloroquine-induced pigment clumping (CIPC) test' and the study of ultrastructural changes induced in P. berghei in drug-treated mice indicate that WR 194,965 has a mode of action somewhat resembling that of quinine. WR 228,258 in vitro shows a chloroquine-like effect, but not in vivo, suggesting that its mode of action in vivo is different from that of chloroquine. WR 225,448 has no action in the CIPC in vitro and affects primarily mitochondria of the parasites in vivo. It probably acts through a metabolite. Both pre-erythrocytic and erythrocytic stages of rodent malaria parasites are affected by WR 225,448.

A variety of compounds used in the treatment of parasitic or bacterial infections in man, including leishmaniasis itself, were examined for their activity against three lines of Leishmania in tissue culture. The organisms used were L. mexicana mexicana, L. tropical major and L. donovani; they were grown in dog sarcoma and hamster peritoneal exudate cell lines. Leishmanicidal activity was observed in a number of compounds currently in clinical use for the treatment of one or other form of leishmaniasis. Cycloguanil, nifurtimox, amphotericin B and monomycin were effective but pentamidine showed poor activity. In each case marked differences were observed in the level of response in the different parasite lines. Organic antimonials were most active when anmastigotes were exposed to them prior to entry of the parasites into host cells. This suggests that such compounds may exert an effect on amastigotes during their brief extracellular transit from one host cell to another in vivo. A number of antimalarials showed good to moderate leishmanicidal action, particularly against L. mexicana and L.t. major. Several schistosomicidal agents also possessed leishmanicidal properties. The commonly used broad spectrum antibiotics showed little if any activity. In discusssion a comparison is drawn between data published on the action of some of these drugs against L.t. major in mice and our observations with the same strain and L. mexicana in tissue culture. A remarkably good agreement is found for most of the compounds examined. General agreement is also noted between these data and reports of clinical trials although it is not possible to draw too many conclusions because of the failure in most clinical studies to make an accurate identification of the causative Leishmania. It is concluded that, although the tissue culture model is not to be considered as ideal and can probably be improved, data obtained by its use do bear relevance to the action compounds in vivo, and the model may be use in the screening of drugs for leishmanicidal activity.