Yoelii Ssp Research Articles

The chemotherapy of rodent malaria. LXIII. Drug combinations to impede the selection of drug resistance, part 6: the potential value of chlorproguanil and dapsone in combination, and with the addition of artesunate

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.

The chemotherapy of rodent malaria. LXII. Drug combinations to impede the selection of drug resistance, part 5: rates of development of resistance to some inhibitors of folate metabolism and to artesunate

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 chemotherapy of rodent malaria. LXII. Drug combinations to impede the selection of drug resistance, part 5: rates of development of resistance to some inhibitors of folate metabolism and to artesunate

The enzymatic profiles of 22 Jordanian Leishmania isolates obtained from humans, Psammomys obesus and Phlebotomus papatasi were determined using starch-gel electrophoresis and a 15-enzyme system. Thirteen of the isolates were typed as L. major and the other nine, all from Mediterranean or sub-Mediterranean regions, as L. tropica. The two zymodemes of L. major encountered, MON-26 and MON-103, differed in terms of purine nucleoside phosphorylase 2. The MON-26 isolates came from the Jordanian plateau whereas those of MON-103 were only collected from the Jordan valley. The four zymodemes of L. tropica observed (MON-7, MON-137, MON-200 and MON-265) were identical for only two of the 15 enzymes studied (i.e. isocitrate dehydrogenase and glucose phosphate isomerase), confirming the high level of enzymatic polymorphism of L. tropica. So far, MON-200 and MON-265 have only been found in Jordan.

A short synthesis and biological evaluation of potent and nontoxic antimalarial bridged bicyclic beta-sulfonyl-endoperoxides.

The syntheses and in vitro antimalarial screening of 50 bridged, bicyclic endoperoxides of types 9-13 are reported. In contrast to antimalarial trioxanes of the artemisinin family, but like yingzhaosu A and arteflene, the peroxide function of compounds 9-13 is contained in a 2,3-dioxabicyclo[3.3.1]nonane system 6. Peroxides 9 and 10 (R(1) = OH) are readily available through a multicomponent, sequential, free-radical reaction involving thiol-monoterpenes co-oxygenation (a TOCO reaction). beta-Sulfenyl peroxides 9 and 10 (R(1) = OH) are converted into beta-sulfinyl and beta-sulfonyl peroxides of types 11-13 by controlled S-oxidation and manipulation of the tert-hydroxyl group through acylation, alkylation, or dehydration followed by selective hydrogenation. Ten enantiopure beta-sulfonyl peroxides of types 12 and 13 exhibit in vitro antimalarial activity comparable to that of artemisinin (IC(50) = 6-24 nM against Plasmodium falciparum NF54). In vivo testing of a few selected peroxides against Plasmodium berghei N indicates that the antimalarial efficacies of beta-sulfonyl peroxides 39a, 46a, 46b, and 50a are comparable to those of some of the best antimalarial drugs and are higher than artemisinin against chloroquine-resistant Plasmodium yoelii ssp. NS. In view of the nontoxicity of beta-sulfonyl peroxides 39a, 46a, and 46b in mice, at high dosing, these compounds are regarded as promising antimalarial drug candidates.

The chemotherapy of rodent malaria. LXI. Drug combinations to impede the selection of drug resistance, part 4: the potential role of 8-aminoquinolines.

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 chemotherapy of rodent malaria. LX. The importance of formulation in evaluating the blood schizontocidal activity of some endoperoxide antimalarials.

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.

The chemotherapy of rodent malaria. LIX. Drug combinations to impede the selection of drug resistance, Part 3: Observations on cyproheptadine, an antihistaminic agent, with chloroquine.

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 chemotherapy of rodent malaria. LVIII. Drug combinations to impede the selection of drug resistance, Part. 2: The new generation--artemisinin or artesunate with long-acting blood schizontocides.

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.

The chemotherapy of rodent malaria. LVII. Drug combinations to impede the selection of drug resistance, part 1: which model is appropriate?

The principle has finally been accepted that, whenever possible, antimalarial drugs should be deployed in appropriate combinations in endemic areas, in order to minimize the inevitability that monotherapy will, probably sooner than later, select populations of drug-resistant parasites. Which laboratory models can predict the combinations of old or novel compounds that are likely to be of practical value in minimizing this risk? Very few relevant data on the use of Plasmodium falciparum in vitro have been published. Most research has been carried out with one or other strain of chloroquine-sensitive P. berghei or with chloroquine-resistant P. yoelii ssp. NS in mice. The two most widely used procedures to select for resistance are the ‘serial technique’ (ST), in which drug selection pressure in vivo is gradually increased, and the ‘2% relapse technique’ (2%RT), in which a single, large drug dose is applied at the time of each passage. Both procedures have been used to demonstrate the ability of pairs of drugs (e.g. sulfadoxine with pyrimethamine, cycloguanil with dapsone, pyrimethamine or sulphonamides with chloroquine, mepacrine or mefloquine) or triple combinations (e.g. sulfadoxine-pyrimethamine with chloroquine, mefloquine or pyronaridine) to delay the development of resistance. The relative merits of the ST and 2%RT are discussed and the data obtained by these procedures are compared with the results of deploying drug combinations in man, especially against multidrug-resistant P. falciparum. It is concluded that, even though the rodent malaria models are imperfect, no better alternatives are available at present with which to predict the value of antimalarial combinations for the protection of the individual components. The 2%RT is considered to be the procedure of first choice.

The chemotherapy of rodent malaria. LVII. Drug combinations to impede the selection of drug resistance, Part 1: Which model is appropriate?

The principle has finally been accepted that, whenever possible, antimalarial drugs should be deployed in appropriate combinations in endemic areas, in order to minimize the inevitability that monotherapy will, probably sooner than later, select populations of drug-resistant parasites. Which laboratory models can predict the combinations of old or novel compounds that are likely to be of practical value in minimizing this risk? Very few relevant data on the use of Plasmodium falciparum in vitro have been published. Most research has been carried out with one or other strain of chloroquine-sensitive P. berghei or with chloroquine-resistant P. yoelii ssp. NS in mice. The two most widely used procedures to select for resistance are the 'serial technique' (ST), in which drug selection pressure in vivo is gradually increased, and the '2% relapse technique' (2%RT), in which a single, large drug dose is applied at the time of each passage. Both procedures have been used to demonstrate the ability of pairs of drugs (e.g. sulfadoxine with pyrimethamine, cycloguanil with dapsone, pyrimethamine or sulphonamides with chloroquine, mepacrine or mefloquine) or triple combinations (e.g. sulfadoxine-pyrimethamine with chloroquine, mefloquine or pyronaridine) to delay the development of resistance. The relative merits of the ST and 2%RT are discussed and the data obtained by these procedures are compared with the results of deploying drug combinations in man, especially against multidrug-resistant P. falciparum. It is concluded that, even though the rodent malaria models are imperfect, no better alternatives are available at present with which to predict the value of antimalarial combinations for the protection of the individual components. The 2%RT is considered to be the procedure of first choice.

The chemotherapy of rodent malaria. LVI. Studies on the development of resistance to natural and synthetic endoperoxides.

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.

The chemotherapy of rodent malaria. LV. Interactions between pyronaridine and artemisinin.

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.

The chemotherapy of rodent malaria. LIV. Combinations of ‘Fenozan B07’ (Fenozan-50F), a difluorinated 3,3′-spirocyclopentane 1,2,4-trioxane, with other drugs against drug-sensitive and drug-resistant parasites

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.

The chemotherapy of rodent malaria. XLVI. Reversal of mefloquine resistance in rodent Plasmodium

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 chemotherapy of rodent malaria. XLV. Reversal of chloroquine resistance in rodent and human Plasmodium by antihistaminic agents

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.