AD _ _ _ J1EMOTHERAPY OF RODENT _.44LAR IA EVALUAT 0N/0F4PRUG ACT ION 4A INST NORMAL AND RESISTANT STRAINS, INCLUDING EXO-ERYTHROCYTIC STAGES FINAL TECHNICAL REPT ~ ~ * ~110 ~JX~~~cEPETERS MD, DSc D1 Dec76 Supported by US ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND _____________ has1Q DC. 20314 Contract No DAMD] 7-76-CG-9416 g ~~~DepartmentofPrstlg Liverpool School of Tropical Medicine Pembroke Place, Liverpool L3 5QA, UK 0) LLU Approved for public release; distribution unlimited C-, C=C7
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AD _ _ _
J1EMOTHERAPY OF RODENT _.44LAR IA
EVALUAT 0N/0F4PRUG ACT ION 4A INST NORMAL ANDRESISTANT STRAINS, INCLUDING EXO-ERYTHROCYTIC STAGES
FINAL TECHNICAL REPT ~ ~ *
~110
~JX~~~cEPETERS MD, DSc
D1 Dec76
Supported by
US ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND
_____________ has1Q DC. 20314
Contract No DAMD] 7-76-CG-9416
g ~~~DepartmentofPrstlgLiverpool School of Tropical Medicine
Pembroke Place,Liverpool L3 5QA, UK
0)
LLU Approved for public release; distribution unlimited
C-,
C=C7
_____ ____ _____ ____ _____F__I__M_
S1ECU PITY CLASSIFICATION OF THIS PAGE (W~hen Date Entered) 7REPOT DCUMNTATON AGEREAD INSTRUCTIONS,
REPOR DOCMENTTIONPAGEBEFOR~E COMPLETING FORINIREPORT NUMBER 2.GOVT ACCESSION No. 3. RECIPIENT'S CATALOG NUMBER
4. TITLE (and Subtitle) 5. TYPE OF REPORT &PERIOD COVEREDChemotherapy of Rodent Malaria Evaluation of Drug Final Technical Report4Action against Normal and Resistant Strains,______________Including Exo-erythrocytic Stages 6. PERFORMING ORG. REPORT NUMBER
7. AUTHOR(a) S. CONTRACT ORGRANT NUMBER()
Wallace Peters, M.D., D.Sc. DAMD 17-76-G-9416
_ _ _ _ _ _ _ _ _ _ _fV't
9. PERFORMING ORGANIZATION NAME ANO ADDRESS /10. PROGRAM ELEMENT, PROJECT, TASK
Department of Parasitology ARE AOKUINMBRLiverpool School of Tropical Medicine 36275 9A82.0 66Pembroke Place, Liverpool L3 5QA, UKA675A2.0.3
1I. CONTROLLING OFFICE NAME AND ADDRESS Q2. REPORT DATE
US Army Medical Research and Development CommandDembr17Washington, D. C. 20314 13. NUMBER OF PAGES
14. MONITORING AGLNCY NAME & ADDRESS(If dllforvit froum ControllinA Office) IS. SECURITY CLASS. (of this rept)
Unc lass if ied
15a. DECLASSI F1CATION/ DOWNGRADINGSCHEDULE
16. DISTRIBUTION STATEMENT (of this Report)
Approved for public release; distribution unlimited
17. DISTRIBUTION STATEMENT (of the abstract entered in Stock 30, It different from Report)
Is. SUPPLEMENTARY NOTESt
19. KEY WORDS (Continue on fevers, side it necessary and Identify by block number)
(U) MALARIA (U) BLOOD SCHIZONTOCIDAL ACT? VI TY(U) 8-AMINOQUINOLINES (U) TISSUE SCHIZONTOCIDAL ACTI VITY(U) MEFLOQUI NE (U) DRUG RESISTANCEI(U) CHLOROQUINE
20. iXDsrRAcT (Contrate a reverse atide ft nwce~saty and Identify by block number)
Data are provided on the blood schizontocidal action of 8 WRAIR compounds and the tiss~.ueschizontocidal activity of a total of 1 10 WRA IR 8-aminoquinolines plus 29 other compounds.Emphasis is laid on the comparison of a new series of 5-substituted 8-aminoquinolines andcomparison has been made between data obtained in our rodent models and reports on workIin simian models from other investigators. A good parallel was found with tissue sChizonto-cidalI activity.The administration of mixtures of mefloquine with pyrimethamine, sulphaphenazole or
DD 6AN 1473 EDITION OF I NOV 65 IS OBSOLETE
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SECURITY CLASSIFICATION OF THIS PAGEnon Daita Entered)
I 4primaquiiie has been shown to enhance the development of resistance by P. bergheito the individual components.
Suishave continued on the modesofatoofmflqie horun ndunn.Mefloqu',ne has been shown to have relaively litl meffeoqne uptake ofadequoine.TL synergistic action of chloroquine and erythromycin against chioroquine-resistantparasites has benfurther investigated. The mechanism of this synergism is still obscure.Other work has involved the study of electron transport and, cathepsins of rodent malariaparasi tes.
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CHEMOTHERAPY OF RODENT MALARIA
EVALUATION OF DRUG ACTION AGAINST NORMAL AND
RES I STANT STRAINS, INCLUDING EXO-ERYTHROCYTIC STAGES
FINAL TECHNICAL REPORT
by
WALLACE PETERS, MD, DSc
December 1976
Supported by
US ARMY MEDICAL RESEARCH AND DEVELOPMENT COMMAND
Washington, DC. 20314
Contract No. DAMD17-76-G-9416
X; Department of ParasitologyLiverpool School of Tropical Medicine
Pembroke Place,Liverpool L3 5QA, UK
Approved for public release; distribution unlimited
TABLE OF CONTENTSPage
1. INTRODUCTION I
2. CYCLICAL PASSAGE OF RODENT MALARIA STRAINS I
3. PRODUCTION OF DRUG-RESISTENT LINES I
3.1 NS type lines of P. berghei I3.2 Polytherapy in the prevention of drug-resistance 2
4. CHEMOTHERAPY STUDIES 3
4.1 Blood schizontocides 3
4.1.1 New compounds 34.1.2 Drug combinations 3
4.2 Causal prophylaxis - the value of the rodent screen 3
4.3 Sustained release of drugs 44.4 Mode of drug action 5
4.4.1 Chloroquine and mefloquine 54.4.2 Chloroquine and erythromycin 54.4.3 Pyrimethamine 6
5. PHYSIOLOGY AND BIOCHEMISTRY 6
5.1 Electron transport of intra-erythrocytic P. berghei 65.2 Cathepsins of parasitized erythrocytes 65.3 The effects of PABA on sporogonic development in P. berghei 7
6. CONCLUSIONS AND RECOMMENDATIONS 7
7. LITERATURE CITED 8
8. PAPERS PUBLISHED 9
9. APPENDICES 12
1 I. INTRODUCTION
In this Report we re -w work carried out between the termination of ourlast contract on 31 October 1975 through 31 December, 1976. Details of thiswork have been provided in Quarterly Reports Numbers 1 through, 3.
The main emphasis of this year's studies has been the evaluation in depthof the causal prophylactic potential of new WR compounds and a comparison ofthe data obtained in our rodent malaria screen with that of Dr. Leon Schmidt'sr hesus-P. cynomolgi system1 .
Another major item of our programme has been the follow-up of studiesto evaluate the effect of polytherapy in 'educing the rate at whicl'P. berghei
develops resistance to mefloquine. Details ofthis study are now ready forpublication and' summaries are included in the-following pages.
Further studies-have been made on the effect of several compounds againstthe Sprogonic stages of rodent malaria, andon the fundamental biochemical!processesof the blood stages, especially glycolysis.
2. CYCLICAL PASSAGE OF RODENT MALARIA STRAINS
We have continued to rely on the .yoelii nigerieisis-Anophelei stephensicombination for the supply of infected mosquitoes for chemotherapy investgations.
A temporary falloff in oocyst development was finally traced down to fluctuationsin the temperature of the insectary due to a defective thermostat. Replacement ofthis has brought our cyclical transmission back to normal levels.
3. PRODUCTI ON OF DRUG-RESISTANT LI NES
3.1 NSo-type lines of P. berghei
After several years of" research and soul-seeking we have been ableto reach the conclusion that several strains of P. berghei from highland areas ofthe Katanga region of Zaire are in fact a mixture of two species. The importanceof this will be appreciated when it is pointed out that one of the mixture,P. berghei sensu stricto, is chloroquine sensitive, and the other which we call"P. yoelii ssp. " is innately chloroquine-resistant. These conclusions mustinfluence our interpretation of all work carried out up to now on isolates ofrodent malaria from this part of Africa. The following is the surnmary of a papernow submitted for publication on this topic2 .
'Under chloroquine selection pressure a number of chloroquine-resistant linesof rodent malaria have been selected from "strains" of "P. berghei" originatingin the Katanga highlands. They are called the "NS lines". No resistant parasitewas obtained from a clone under drug pressure, nor from two other highland P. bergheiand a strain of P. v. vinckei.
2-"
The isoenzyme typing of these parasites indicates that the. drug-resistantNS parasites are aIiied to P. oeli rather than P. berghei, but that they can bedistinguished from all but the subspecies-P. y. nigeriensis, sharing wiffih-thisGPI"2, 6PGD 4, LDHI 1-and GDH 2. Theresistant organism is therefore labelled
P. yoelii ssp.
The buoyant density of all, P._berghei and P. yoelii subspecies examined isidentical (1 .683 g/ml). DNA-DNA hybridisation studies have shown thatP. yoeliissp. differs significanfly not only from P.- berghei, but also from P-Y. yoelii andP. y. nigeriensis in terms of the base sequence homologies of these parasites.
Cross-immunity experiments indicate that R. .yoelii ssp. differs not onlyfrom P..berghei, but also from P.y.. nigeriensis in the absence of reciprocal
cross-protection, although P. berghei itself (as well as the mixtures containingP.. yoelii ssp.).provide a broad and relatively non-specific protection againstthe other parasites.
Evidence is presented to refute the suggestion that the "NS lines".havebecome accidentally mixed with-P. berghei in the laboratoy. On the contrary,the evidence indicates that the NS lines are not P. y. nigeriensis but a. newsubspecies in the P. yoelii complex. How such mixtures of P. berghei and P. yoeliissp. have survived through many years and countless syringe passages in the laboratoryis unknown, but this is not an isolated instance in -the.history of the rodentmalarias.
The existence of P. yoelii ssp. may account for a number of previously unaccountableobservations in the laboratory suchas some of the apparent "variability" of P. berghei"strains" under-a variety of experimental conditions.
The importance of these findings is discussed in relation to the zoogeographyof the rodent malarias.'
3.2 Polytherapy in the prevention of drug-resistance
We have already reported the relative ease-with which resistancecan be developed to mefloquine, and preliminary data on our attempts to decreasethe rate at which this resistance develops through the use-of drug mixtures. Using
a relapse technique resistance of P. berghei N strain-to mefloquine can be developedslowly (Fig. 1), but the progress is more rapid in the chloroquine-resistant NS lines.Mefloquine resistance in all these lines is very unstable in the absence of drugselection pressure (Fig. 2).
Resistance develops also when the N strain is submitted to slowly increasingmefloquine dosage in consecutive passages (Fig. 3), the resulting parasites having asimilar morphology at light microscope level to chloroquine-resistant P. bergheiRC line parasites. Like the latter they occupy polychromatophilic red blod cellsalmost exclusively.
-qI
2.
-3 -When the parasites are exposed tomixturesot mefloquine with pyrimethamine,
sulphaphenazole or primaquine, resistance to each componentof the-mixtures developsmore slowly than to the individual components.used alone. t. -
It is strongly recommended that mefloquine should only be deployed forthe prevention of treatment of malaria in man caused by chIoroquine-resistantP. falciparum. -For large-scale use meflgquine should not be employed until asecond antimalarialhas been identified that will minimise the risk-ofparasitesbecoming resistant to this potentia[ly valuable new compound.
3A full report on this work' asbeen submitted for publication
4. CHEMOTHERAPY STUDJES
4.1 Blood schizontocides
4.1 .1, New-compounds
The evaluation of new-compounds for blood schizontocidalactivity has been restricted to some 8 compounds on which data wereprovided inour 3rd-Quarterly Report. Three compounds showed good activity against.thedrug-sensitive N strain of P. berghei, namely WR 219,930i 194,965, and225,449, the-last two being-somewhat more effective po than sc. No.studies weremade in. drug-resistant lines with these compounds.
4.1.2 Drug combinations
The main purpose of our drugcombination work this year wasto determine whether those combinations with mefloquine that we have shown toreduce the rate of resistance development possessed additive or-even potentiatingproperties. The details were given in our 3rd Quarterly Report and are to bepublished4 .
The data summarised in Fig. 7 indicate that there is certainly no potentiationbetween mefloquine and primaquine, or mefloquine and sulphaphenazole. There isa slight indication of potentiation between mefloquine and pyrimetharnine butpossibly only of the order that could be anticipated if the two compounds influenceeach other's pharmacokinetics in the host.
4 2 Causal prophylaxis- the value of the rodent.screen
The greater part of our work has been devoted to examining thecausal prophylactic potential of new WR compounds in our rodent malaria model.We have examined altogether some 110 WRAIR 8-aminoquinolines plus 5 from othersources, 6 naphthyridines and 18 miscellaneous compounds that are covered in thepesent report. Other miscellaneous compounds have been dealt with in previousQuarterly and Final Reports over the years.
-4-
We have presented these data in tabular-form (Tables 1 through 14) andin these we include our own interpretation of Minimum Fully Curative Doses (MCFD)presented in Schmidt's Final Report 6f1976.; The figures we give-for the MCFDvalues take into account dose levels at which cure was obtained only in someanimals but not in all treated-at any particular level, and the values are convertedto uM basekg. In our -odeht model animals were routinely treated on a singleocccsion, by the sc route and'it is only recently that we have started' to use alsothe oral route. For this reason there are still many blanks in Tables 1 through 14that we will be filling in on the basis of ongoing studies. This makes a directcomparison of the 145 compounds that both we and Schmidt have examined difficultat the present' time. As we have pointed out-orevidusly it is, in a way, surprisingthat there should be any correlation between our data. Schmidt uses a differentparasite, different host, different route of administration and different dosage schedulefrom ourselves. Nevertheless on the basis of the primaquine indices as we havecalculated them so far we find a remarkably good' corelation between our data in themajority of the 8-aminoquinolines series.
There is however one important area in which we differ considerably andthat is the area represented by poorly-soluble and' (probably) poorly orally absorbablecompounds such as menoctone (WR 49,808) and-WR 226,626. This type of compoundwe believe to be extremely interesting since those members that we have examinedhave proved to possess not only tissue schizontocidal properties but also good activityagainst both drug-sensitive and drug;-resistant blood stages of P. berghei. In thissense we-believe that the rodent malaria screen offers advantages over the rhesus-P. cynomolgi' screen. While appreciating the great value of the simian modelwe do feel that many important leads may'be missed if total reliance is placed onthis and that the rodent screen has a most vaiuable contribution to make as wehave indicated elsewhere5 .
Data on compounds still receiving preliminary screening are presented inTable 15 through 38, while completed sheets not yet forwarded to WRAIR formTables 39 through 53.
4.3 Sustained release of drugs
The work on the sustained release of antimalarials from poly-dimethylsiloxane capsules reported in the 3rd Quarterly Report 1976 (2.4) hasbeen continued and expanded. To date pyrimethamine filled capsules having a A
wall thickness of 0.63 mm and an internal surface area of 105 sq. mm. haveafforded complete protection to mice against P. berghei (N strain) challenge fora period of 102 days. Capsules with an internal surface area of approximately25 sq. mm. gave mice a survival time of between 55 and 65 days though all micehad patent parasitaemias from approximately day+38 onwards. Very similar results Awere obtained with capsules having an internal surface area of approximately50 sq. mm.
No antimalarial effect was observed with encapsulated cycloguanil,WR 99209 and WR 99210. Promising preliminary results have been obtained withanother cycloguanil analogue and menoctone and these compounds are keing
studied in greater detail.
Current experiments are designed to ufilise drug capsules prepar:d bymixing drug and pre-vulcanised silastic as described by Fu et al. 6 and drugincorporated1intb biodegradab le polymers.
4.4 Mode of drug action
4.4.1 ChlOroquine and mefloquine
The method by which chloroquine kills the malaria parasiteis still not known. Its short-term effect in causing the clumping of haerozoinhas been investigated in considerable depth, but haemozoin clumping itself doesnot kill the parasite. We are therefore investigating the time at which the parasitedies after treatment with chloroquine and the way in which the-action. of-mefloquihediffers from that of chloroquine.
The clumpng-of malaria pigment by chl6ro uine (10- 6M)-is complete within-80 minutes but there is no effect within this time-on growth of" the parasite, as,measured by the incorporation of radio-active adenosine. Six hours after treatmentof P. bergheizinfected mice with chloroquine (60 mg/kg) incorporation of adenosine-was-reducedby about 25%, but after 12 houi the-synthesis of nucleic acids hadfallen' sharply. These results suggest that P. berghei dies orly after about 12 hoursexposure to chloroquine and closely parallel those obtainedby -Davies and Howells(unpublished) in experiments on the viability of the parasite after varying times ofexposure in vivo to chloroquine. Radioactive chloroquine taken up by P. bergheiparasitized cells maintains its maximum intracellular concentration for at leastthree hours in vitro. It seems that-the clumping of I.:- ozoin is unrelated to thedeath-of the parasite.
The effect of mefloquine on the erythrocytic stages of P. brghei has beenreexamined at the light and electron microscope levels. The most obvious effectsof the compound were observed in the haemozoin vesicles of the parasite, with theultrastructural changes being broadly similar to those described in-P. bergheitreated with WR 122455 and quinine7 . Within 3 hours of exposure to a singlesubcutaneous dose of 60 mgm/kg primary pigment clumps are formed, but notautophagosornes. With longer periods of exposure to the compound pigment grains-becam6 increasingly finer and electron translucent, with only poorly definedhaemozoin grains being found in trophozoites 24 hours after treatment. Theseobservations amplify the very slow plasmodicidal action of this compound and !' ggestthat the drug is-not solubilizing haemozoin, as was suggested for WR 122455 andquinine by Davies et al.7 but interferes with the catabolism of haemogl6bin and/orthe formation of haemozoin. Mefloquine (10- 5 M) only slightly reduced theincorporation of adenosine by parasitized cells in one hour. A detailed study isproceeding.
A4.4.2 Chloroquine and erythromycin
The synergistic action of erythromycin and chloroquine onchloroquine-resistant parasites does not appear to be due to an increased uptakeof chloroquine in the presence of the antibiotic. In vitro, slightly more chloroquinewas taken up by RC strain P. berghei in the presence of erythromycin but treatmentof infected mice with erythromycin before measurement of chloroquine uptake in vitro,dramatically reduced the uptake of chloroquine.
No obvious effects on the ultrastructure ofP.: berghei (N strain) wereobserved following treatment with erythromycin. Studies are in progress on theeffects of dhloroquine and erythromycii, alone and- in combination, on theRC strain P. berghei.
4.4.3 Pyrimethamine
The effects of-pyrimethamine on the erythroc tic stages ofP. berghei have been examined at the electron microscop level. These effectswere described in the 3rd Quarterly Report oF 1976.
5. PHYSIOLOGY AND BIOCHEMISTRY
5.1 Electron transport of intra-erythrocytic P. berghei
P. berghei appears to depend for energy production-on a Form ofelectron transport which differs from that of the host8 . It therefore provides apotential target-for chemotherapeutic attack. Conventional inhibitors of electrontransport and uncouplers not only inhibit chloroquine-induced pigment clumping8
but also reduce the incorporation of denosine into the parasites' nucleic acids(data given in the 3rd Quarterly Report 1976). The donor of electrons to thechain is not known.
Preliminary results have shown that treatment of the parasitized erythrocyteswith menadione (10*4 M) or n-heptyl-4-hydroxyquinoline-N-oxide (10- 5 M) reducedonly slightly the utilization of glucose and the production of lactate by parasitizedcells in vitro. This suggests that glucose metabolism may not be tightly linked toelectron transport.
5.2 Cathepsins of parasitized erythrocytes
Our failure to isolate from P. bergbei-parasitized cells acathepsin D which was indisputably of parasite origin, and the presence ofparasitized cells of a cathepsin D indistinguishable from that of mouse reticulocytes,
prompted us to investigate the relationship between the- number of parasites andthe cathepsin activity of parasitized mouse red cells. Although results weresomewhat variable (Table 54), there was little indication of an increase incatheptic activity as the parasitaemia rose. Instead, the apparent activity perparasitized cell fell, although the total activity for all cells remained approximatelyconstant. This suggests that in the conditions used, littie of the measured cathepticactivity was due to the parasites. It is therefore probable that the parasites do notcontain cathepsin D, and that other cathepsins must be present.
l I
-7-
5.3 The effects of PABA on sporogonic developmenf, in P. berghei
As outlined in -ihe 3rd Quarterly Report of 1976 (2.3.4) thesestudies Were initiated to attempt to obtain results more statistically significantthanithose obtdined by Ramkaran 9 . Initiblly, difficulties were encountered in themosquito colony during our attempts to repeat this work. Abnormally high mortalityrates and low infection rates in the mosquitoes were caused by excessive fluctuationsof temperature and humidity in the insectaries. Examination of samples of themosquito populations at the electron microscope level has not revealed the-presenceof concomitant, viral or microbial oinfections which might contribute to the vagariesin the malarial infections.
In recent experiments acceptable infection rates have been obtained-in the:mosquitoes but variations in the oocyst numbers within experimental batches are Jso' great as to preclude the obtentior of statistically significant results.
6. -CONCLUSIONS AND RECOMMENDATIONS
The additional information gathered from our rodent causal prophylaxisstudies has confirmed our beliefl that this is a valid trel for tissue schizontocidalaction. Taking intoaccount the differences betw~erour technique and that ofDr. L. H. Schmidt, thereisa 'remarkably goOd parallel in our joint findings.The use of parenteral-route in our rodent model will, webelieve, permit us todetect activity in certain chemical groups (e.g. menoctone) the activity of whichwould be missed in the simian model.
During the coming year we will extend these observations and consolidateour data using both oral and parenteral routes of drug administration.
Extension of our long-term studies on drug combinations has provided.useful. leads for the possible protection of such, promsing new compounds asmefloquine. This work will be continued.
The mode of action of the antimalarials is still being investigated andfundamental gaps in our knowledge of parasite biocheistry are being exposedby our exploration of the drugs. Further work on these matters with specialreference to mefloquine will be carried out during-the coming year.
Studies will also be extended on the development of slow-releasepreparations of selected drugs.
7. LITERATURE CITED-
1 Schmidt, L. H. (1976). The use of Actus.trivirgatus and Macaca mulattaas tools for studies on prevention and therapy of infections withPlasmodium falciparum and Plasmodiumvivax.Final Progress Report to US Army Medical Research and DevelopmentCommand.
2 Peters, W., Chance, M. L., Lissner, R.,. Momen, H. and Warhurst, D. C.(1977). The chemotherapy of rodent malariaj, XXX. The enigmasof the NS lines of R. berghei. Ann. trop. Med. Parasit. (in press).
3 Peters, W., Portus, J. P. and Robinson, B. L. (1977). The chemotherapyof rodent malaria, XXVIII. The developmen" of resistance tomefloquine (WR 142,490). Ann. trop. Med. Parasit.. (in press)
4 Peters, W., Howells, R. E., Portus, J. P., Robinson, B. L., Thomas, S.
and Warhurst, D. C. (1977). The chemotherapy ofi rodent malaria, XXVII.Studies on mefloquine,(WR 142,490). Ann. trop. Med. Parasit.(in press).
5 Peters, W.., Davies, E. E. and Robinson, B. L. (1975). The chemotherapyof rodent malaria, XXIII. Causal prophylaxis, part 11: Practicalexperience with Plasmodium. yoelii nigeriensis in drug screening.Ann. trop. Med., Parasit., 69,. 311-328. .(Cont. :No.. 1325).
6 Fu, J. C., Kale, A. K. and Moyer, D. L. (1973). Drug-incorporatedsilicone discs as sustained release capsules. I. Chloroquinediphosphate. J. Biomed. Mater.-Res., 7,_71-78.
7 Davies, E. E., Warhurst, D. C. and Peters, W. (1975). The chemotherapyof rodent malaria, XXI. Action of quinine and WR 122,455(a 9-phenanthrenemethanol) on the fine structure of Plasmodiumberghei 'in mouse blood. Ann. trop. Med. Parasit., 69, 147-153.(Cont. No. 1286)
8 Homewood, C. A., Warhurst, D. C., Peters, W. and Baggaley, V. C.(1972). Electron transport in intraerythrocytic Plasmodium berghei.Proc. Helminth. Soc., Wash., 39 (Special Issue), 382-386.(Cont. No. 1135).
9 Ramkaran, A. E. (1972). The influence of chemical agents on thetransmission of rodent malarias. Ph.D. thesis: Liverpool.
S. PAPERS PUBLISHED -b
Homewood, C. A., Neame, K. D. and Momen, H. (1975). Permeability oferythirocytes~fromcniiice ,infected With Babesia rodhaini.
Howells, R. E. (1975). Host-parasite relationshtps in ,malaria.
Task Force, WHO, Geneva: November 1975- TDR/CM/WP/75.9
Merkli, B. and* Peters, W. (1976). A comparison of two different -rnethodsfor ,he--selection of primnaquine6 resistance -in P._b. bErgqhei1.Ann. trop. Med. Parasit., 70, 473-474.
Momen, H. (1976). Carbohydrate hietabolism~of some ,apicompk-xan infections-in mouse erythrocytes., Parasitdlogy, 473, (2), xvi.Abstract presented at British Society of Parasitology Meeting, April 1976
Momen, H. (1975). Enzyme variation in rodent Babesia.Trans. R. Soc. trop.,Med. Hyg,., 69, 438.
.Mornen, H. and Chance, M. L. :(1976). DNA buoyant densities-ofrodent piroplasms.Trans. R.<Soc. trap. Med...Hyg.,,70, 13.
Momer,, H., Homewood, C. A. and Neame, K. D. (975). .Effect ofAnthemosoma garnhami infection on the permeability of the mouseerythrocyte. Ann. trop. Med. Parasit., 69, 519-520 .4
Moore, G.,A. and Homnewood, C. A. (1975). -Observations on-malarialand schistosomal pigment. ITrans.,R. Soc.-trop. Med. Hyg., 69, 10.
Neame, K. D.and Homewoodj C. A. (1975). Alterations in the permeabilityof mouse erythrocytes infected with the malaria parasitei1 Plasmodium- berghei-.Int. J. Parasit., 5, 537-540.
Peters, W. (1975). Lacunae in knowledge on the mode of action of antimialaria Is.Paper presented at First Meei:nq of Chemotherapy of Malaria Task Force,WHO, Geneva, November 1975. TDR/CM/WP/75 .1 0
Peters, W. .(1975). Guest editorial on malaria and drug- resistance.J. trop. Med. Hyg., August, 167-170i,
Peters, W. (1976)-. The role of university research departments in the developmentof antiparasitic chemotherapy.Paper presented at 9th InternationalI Congress of Chemotherapy, London 1975.In: "Chemotherapy Progress", Plenum Press: London, Vol. VI, 29-34.
4t,. .. .....~.
Peters, W. and Porter,,M. (1976). The chemot herapy of rodent mfa laria, XXV I.the potential value of WRA 22,455 (a 9 -phenanthrene-!methanol) againstdrug -resistant. malaria parasites.Ann. 'top. Med. Parasit., 70, 271 -281 . Ctont. No. 1384
Porter,. M. and Peters, W. (1976). The, chemotherapy -of rodent-ma laria,, XXV.Antima laraId activit>'. fVWR 122,455 (a ,9 phenianthrdne-myethanol)in viva and in vitro.Ann. trop.i M'~d. -Parasit. 70, 259-270 Cont. No. 1379.
Warhurst, D. C. and Thomas, S. C. (1975). Pharmacology of the ma lariaparasite. A study of dose-response, relationships in chloroquine-inducedautophagic va cuble, formation in R-lasmhodium 'bergheI.
Warhurst, D. C. and Thomas, S. C. (1975). Localitation of mepacrine inP.. berghei and 'P.-falciparum by- fluarescence, microscopy.Ann. trop. Med.~ P~rasiti, 69, 41 7-420.
Warhurst, ,D. C. and Thomas, S. C. (1975). Pharmacology of Plasmodium berghei.Trans. R. Soc. trop. Ivied. H-yg.',.,69, 428.1
Warhurst, D. C., Robinson, B . L. and Peters, W. (1976). The chemotherapy ofrodent tnalcria,,XXlV. The blood schiziontociddl action of' erythromycinupon P. berghei.-Ann. trap.,Med. Parasit., 70, 263T258, Conit. No.. 1376
Papers in press
Homewood, C. A. (1977). 'Transportf of carbohydrates by, ma lar ia' parasites.Carbohydrate metabolism of malaria parasites.'Papers'prtsented at USAID/WHO Workshop-on the Biology of the MalariaParasite, New York 1977.~
Peters, W. (1977). The role of chemotherapy in the containment of malaria,,with special referenceto drug resistance. Paper presented at FirstNational Congress of Parasitology, Inidia, 1977..
Peters, W. and Howells, R. E. (1977). Drugs, rodents and malaria. .
'in: "Malaria Parasites of Rodents" (Edi. -Killick-Kendrick, R. andPeters, W-.) Academic Press.
3r 1Peters, W., Portus, J1. and Robinson, B . (1977). The chemotherapy of rodentmalaria, XXVIII. The-development of resistance to mefloquine (WR 142,490).Ann. trop. Med. Parasit.
Peters, W.,. Chance, M. L., Lissner, R., Momen, H and Warhurst, D. C.(1977). The chemotherapy of rodent ma laria, XXX. The enigmas4of the NS lines of P-.,-bergheiAnn. trap. Med. Parasit..
-II -
Peters, W., Howells, R. E., Portus, J., Robinson, B. L., Thomas, S. andWarhurst, D. C. (1977). The chemotherapy of rodent malaria, XXVII.
Studies on mefloquine (WR142,490).Ann. trop. Med. Parasit.,
Warhurst, D. C. and Thomas, S. C. (1977). The chemotherapy of rodentmalaria, XXXI. The effect of some nmetabolic inhibitors upon chloroquine-induced pigment clumping (CIPC) in Plasmodium berghei.Ann. trop. Med. Pcrasit.
Papers in preparation
Chance, M. L., Momen, H. and Wa'hurst, D. C. (1977). The chemotherapyof rodent malaria, XXIX. DNA-of rodent malaria parasites.Ann. tr6p. Med. Parasit.
Peters, W. and Ramkaran, A. E. (1977). The chemotherapy of rodent malaria,XXXII. The influence of para-aminobenzoic acid on the transmissionof Plaimodium yoelii andP. berghei by Anopheles stephensi.Ann. trop. Med. Parasit.
aI . ,:
'II
I]'
-12-
9 APPENDICES
Tables 1 through 14 The causal prophylactic potenti,! of newWalter Reed compounds in rodent malaria model.Included are results of 110 Walter Reed 8-amino-
quinolines plus 5 from other sources, 6-naphthyridinesand 18 miscellaneous compounds. These data includean interpretation oi minimum fully curative dosage(MFCD) presented in Schmidt's Final Report 1976.
Tables 15 through 38 The results of preliminary screening of compoundsio causal prophylactic activity in the rodent. screen.
Tables 39 through 53 Summary of data from causal prophylactic test inP. y. nigeriensis
Table 54 The relationship.between parasitaemiaan&catheptic.activity in P. berghei-infected mouse, erythrocytes
Figure 1 A comparison of the rate of acquisition of resistanceto mefloquine by the chloroquine-sensitive-P. bergheiN strain and the NS line which has a low level ofresistance to chloroquine.
Figure 2 The acquisition of resistance to mefloquine byP. berghei NS line passaged tcnder drug pressure(mefloquine 60 mg/kg sc at time of each passage),and its reversion to sensitivity on the release ofdrug selection pressure.
Figure 3 Rate of acquisition by P. berghei N strain of resistanceSr to mefloquine, pyrimethamine and sulphaphenazole
when the drugs are used alone.
Figure 4 Influence of combining mefloquine with pyrimethamineon the rate of acquisition of resistance to each drugby P. berghei in consecutive passages.
Figure 5 Influence of combining mefloquine with sulphaphenazoleon the rate of acquisition of resistance to each drug byP. berghei N strain in consecutive passages.
Figure 6 Influence of combining mefloqjine with primaquineon the rate of acquisition of resistance to each drugby P. berghei in consecutive passages. A
Figure 7 Blood schizontocidal action of drug mixtures againstP. berghei N strain in the "4-Jay test".k -L
I GROUP87-aminoquinolines I Primaquine derivatives (a) variations in 1, 2, 3, 4 positions
S.. po Comments:, 1LIV~~~~~~ N..W No BNN.y as/ MF.E'D. P. I-W. F. E. D. . .. (toxicity, preliminary di
A comparison of the rate of acquisition of resistance to mefloquineby the chioroquine-sensitive P. berghei-N strain and the NS linewhich has a low levelI of resistance to chiloroquine.
0-0, N strain exposed to a single dose of 30 m9Ag mefloquineon the day of passage.
*-o NS line exposed to 60 mg~g mefloquine sc on the day of passage.
The acquisition of resistance to mefloquine by P., berghei NS linepassaged under drug pressure (mefloquine 60 mg/kg sc at time ofeach passage), and ;ts reversion to sensitivity on the release ofdrug selection pressure.
o-o passages under drug pressure.o-0 passages without selection pressure.
~ '-'-'.'~ a ~ A1
... AW.VA4A~a'S"~.~'' 4Se .a ~--~W~tsta~~ ~~ -
100-
E MN LW MEFLOQUINE s.c.
YN __
-J P~-YRIMEHAMINE iLp.I
SN
SULPHAPHENAZOLE s.c.
0.14
F M A M J J A S 0 NTIME SCALE OF PAsSAGES
FIGURE 3.
Rate of acquisition by P. betghei N strain of resistance to mefloquine,pyrirnethamine ad suiphaphenazoleI when the drugs are used alo6ne.Consecutive passages were exposed to increasing drug doses, givendaily for 6 days of each week, the pasaes being made on the 7th day.
-ii
20.IS.
10.
MY
PVyRIMETIHAMI- Lp
0I
0I
o I I I I I | | I I I I I I
0
0
-'
F MY
F M A 11 J J A , 0 N, D J F M A M J
TIME SCALE OF PASSAGES
FIGURE 4
Influence of combining mefloquine with pyrimethamine on therate of acquisition of resistance to each drug byP. berghei inconsecutive passages. Top lines indicate maximum levels ofpyrimethamine and lower lines of mefloquine in the mixture ateach passage (cf Figure 3).
20-
ISm
0
0
Is
ISm
'TIME SCALE OF PASSAGES
a ~ FIGURE 5
Influence of combining mefloquine with-suip haphenazole on the rateof acquisition of resistance to each drug by P. berghei N strain inconsecutive passages. Top lines indicat aiu eeso upahnzland bottom lines of mefloquine in the mixtures at each passage (cf Figure 3).
10 s '[pPPRIMAQUINE
0' J A S 0
0
MP
1.01 MEFLOQUINEI II 0II
D J F M A M JTIME SCALE OF PASSAGES
FIGURE 6
Influence of combining mefloquine with primoquine on the rate ofacquisition of resistance to each drug by P. berghei in consecutivepassages. Top lines indicate maximum level of primaquine in SP lineexposed to primaquine alone. Middle line indicates maximum levels4
of primaquine and bottom of miefloquine in the mixtures at each passage.
1410 16, A B
6 12 0
- 4 0L2 )?
0 0 • • • •
0 -to 0
2 2 4- 0
00o 0-2 0-4 06 0 2 4 6
P (P.METHAMINE ED90 OPWIAQUINEE06
-012 C
0
0
0
01
0 os 1.0 S ;0 2-5 3-0
SLPHAPHENAZOLE ED0
FIGURE 7
Blood schizontocidal action of drug mixtures against.P. berghei N strain
in the "4 day test". The graphs are plotted to show the EDoo-values ofmefloquine when given alone or with different doses of (A) pyrimethamine,(B) primaquine, or (C) sulphaphenazole (o), or of the latter when givenwith different doses of mefloquine (.). (All doses in mg/kg daily x 4).
D)1 TRTBOTION -LIST
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1 copy SuperintendentAcademy ol Health Sciencesi US AffhyATTN: AlUS-COM
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-1 copy DeanSchool of NedicineUnifre Sevce niiiersity of the!!dalth SciencesO-ffice, of the Secretary of Defeiis e6917 Arlingtoi Road.Bethesda,, %,l 20014