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A cycloguanil-resistant strain of Plasmodium gallinaceum was produced relatively rapidly by passage through chicks treated with low but effective doses of the drug, the dose being increased as resistance developed.The strain was cross-resistant to proguanil but not to pyrimethamine or chloroquine.A strain highly resistant to proguanil was resistant to cycloguanil but only slightly resistant to pyrimethamine.A strain highly resistant to pyrimethamine was resistant to proguanil and cycloguanil.Passage for 20 months through birds treated with doses of cycloguanil which suppressed infection for relatively long periods failed to change the sensitivity of the strain to this drug or to proguanil. Although the relatively large dose did not eradicate the infection in any of the birds, subinoculations demonstrated that parasites were absent from the blood for a period in some of the birds, though infections finally developed.I am indebted to Parke Davis and Company for the supply of cycloguanil, to Imperial Chemical Industries Ltd. for the proguanil hydrochloride and chloroquine phosphate and to Burroughs Wellcome and Company for the pyrimethamine base.

Cycloguanil pamoate repository was given intragluteally to 50 patients with dermal and mucocutaneous infections of Leishmania braziliensis . In 41 patients there was clinical and parasitological cure with one injection of the drug; 3 others were cured by 2 injections. The cure rate was therefore 88% with cycloguanil pamoate alone. 5 patients received antimony treatment after failing to respond to cycloguanil pamoate alone. One patient with mucocutaneous manifestations continued to be positive for L-D bodies throughout the study. Systemic and other reactions to the drug were minimal and did not preclude its use.

Summary and Conclusions The repository antimalarial cycloguanil pamoate, known as CI-501 (Camolar®) was administered to 173 subjects living on a malarious island near Rabaul, New Guinea. It was given as a deep intragluteal injection at a dosage of 9.3 to 11.0 mg per kg body weight to children and at a total dose of 350 mg to subjects weighing over 32 kg-Concurrently, a single oral dose of a 4-aminoquinoline (amodiaquine) was given on the basis of 600 mg base for adults and proportionately less for children. The drug was well accepted by the community and there were no observed systemic side-effects. Many subjects showed some local injection site reactions but these were mostly mild in nature and subsided quickly. Asexual forms of both Plasmodium falciparum and P. vivax disappeared within 72 hours of drug administration. High attendance was maintained at the monthly follow-up blood surveys. Patent infections of P. vivax first reappeared on day 90, but P. falciparum did not reappear until 150 days after treatment. The entomological observations, as well as the blood surveys conducted on the untreated observation group, showed that transmission of both species occurred throughout the study period. The results from this area indicate that the combined administration of cycloguanil pamoate and a 4-aminoquinoline provided better protection against P. falciparum than against P. vivax. The duration of protection afforded by CI-501 against P. falciparum makes it conceivable that this species could be eliminated from certain isolated communities provided all the inhabitants receive the drug.

The rocker dilution technique was adapted for in vitro testing of antimalarial drugs on five strains of Plasmodium falciparum. Seven antimalarial drugs, chloroquine (concentrations: 0.0075, 0.015, 0.03, 0.06, 0.12, 0.3, 0.6, 1.2 µg/ml culture), amodiaquine (0.12 and 0.3 µg/ml), pyrimethamine (3.6 and 5.5 µg/ml), quinine (2.5 and 5.0 µg/ml), cycloguanil (0.144 µg/ml), trimethoprim (50.0 µg/ml) and dapsone (2.5 µg/ml) were tested. The various strains of P. falciparum were classified as susceptible or resistant to a drug depending upon the concentration of agent required to: a) inhibit maturation of rings to trophozoites (i.e., prevent cytoplasmic growth); b) block formation of schizonts and segmenters (i.e., prevent nuclear multiplication); or c) produce aberrant distribution of chromatin within the parasite. A strain of P. falciparum obtained from Arnold and Martin is resistant to pyrimethamine and quinine, but susceptible to chloroquine. The Malayan-Camp. strain is susceptible to chloroquine. The Vietnam-Oak Knoll strain is resistant to chloroquine. Two strains (the Uganda-Palo Alto and the Vietnam-Monterey) were studied in greater detail. Of the five strains studied, the Uganda-Palo Alto strain proved to be the most susceptible and the Vietnam-Monterey strain the most resistant to chloroquine. Both of these strains are susceptible to amodiaquine, cycloguanil and dapsone. The Uganda-Palo Alto strain is resistant to trimethoprim and pyrimethamine, while the Vietnam-Monterey strain is susceptible to these two drugs.

(1970). The antimalarial activity of some quinolone esters. Annals of Tropical Medicine & Parasitology: Vol. 64, No. 2, pp. 209-222.

This report pertains to the pharmacology of a new antimalarial compound identified as WR 99,662 (2-[3-(dimethylamino) propylamino]-4-(trichloromethyl)-6-(α,α,α-trichloro- m -tolyl)-2-triazine). Its toxicity was compared with that of cycloguanil in three animal species: mouse, rat, and dog. The differences between the two triazines are as follows: (a) The lethal doses for cycloguanil are smaller than those for WR 99,662: in mice the LD50 is 54 mg/kg for cycloguanil and 1350 mg/kg for WR 99,662; in dogs the LD100 is 24.3 ± 2.7 mg/kg for cycloguanil and 159 ± 39 for WR 99,663. (b). The chronic effects of cycloguanil in rats include reduction of the reticulocyte count, while WR 99,662 has no such hematologic effect. (c) Cycloguanil increases cardiac output in dogs, but WR 99,662 does not produce this effect. (d) Sublethal doses of cycloguanil in anesthetized dogs increase pulmonary resitance, whereas sublethal doses of WR 99,662 stimulate respiration and reduce aortic blood pressure and cardiac output. (e) In rats the mortality from cycloguanil can be reduced by concurrent injection of leucovorin. The mortality from WR 99,662 could not be reduced by leucovorin indicating that deaths from WR 99,662 cannot be due to antifolic acid property. However, the possibility that antimalarial activity and other pharmacodynamic effects of WR 99,662 are exerted by inhibiting folic acid has not been ruled out.

Summary A repository preparation of the dihydrotriazine metabolite of chlorguanide, known as CI-501 (Camolar®), given as a single intramuscular injection at a dose of 5 mg (base) per kg body weight, was tested for its antimalarial activity against Plasmodium falciparum in 14 prisoner volunteers. The medicated volunteers were exposed to infection with a Southern Rhodesian strain of P. falciparum, either by the intravenous inoculation of parasitized homologous blood and/or by the bites of heavily infected Anopheles freeborni mosquitoes. The number of challenges (i.e., exposures to infection) ranged from 1 to 6. The intervals of time between exposures were variable. Twelve volunteers served as controls to prove the infectivity of the challenges. Whereas CI-501 did not protect against infection with this strain when the challenge was by trophozoites (parasitized blood), it did protect through a minimum of 383 days against infection when challenges were by the bites of heavily infected mosquitoes. One volunteer, after his sixth exposure at 446 days after medication, developed patent parasitemia on day 458. These results indicate that CI-501 has the capacity to exert long-term protection against falciparum malaria, as it was previously demonstrated to have for vivax malaria, provided the parasites are sensitive to chlorguanide. Moreover, in the case of falciparum malaria, which is nonrelapsing, complete eradication could be effected by single injections of this preparation provided certain qualifications were fulfilled.

Summary The repository preparation of the dihydrotriazine metabolite of chlorguanide, known as CI-501 (cycloguanil-pamoate, Camolar®) was studied for its urinary excretion by man in 20 inmate volunteers and its antimalarial activity in a total of 36 volunteers. The excretion studies revealed that the rate of excretion of the drug was related directly to particle size. The drug in the form of smaller crystals was excreted at a faster rate than when it was in the form of larger crystals. At 32 to 36 weeks, approximately 90% of the former but only 65% of the latter had been excreted. Our results indicate that as long as drug is present, inferred by its excretion in the urine of medicated volunteers, protection against patent malaria infection obtains. Barring local reaction at the site of the injection which results in increased absorption and therefore a faster excretion of the drug, the average minimum duration of protection of a single 350 mg intramuscular injection against vivax or falciparum malaria was found to be approximately 32 to 36 weeks or about eight to nine months.

More than 600 million people are still exposed to malaria, and eradication is still far away. Protection for some of those at risk—only a fraction of those exposed can therefore be obtained only by chemoprophylaxis. Schizontocide drugs are in 2 categories:— 1. 1. Those acting rapidly, to which resistance appears only slowly—quinine, mepacrine, the 4-aminoquinolines (the best). Dosages of the 4-aminoquinolines used by French workers are higher than those used by Anglo-Saxons—for instance, for prophylaxis 100 mg. daily, and for cure 2100 mg. during 5 days. Resistance (of type R 1) in P. falciparum is not world-wide, and has not been reported in Africa and Madagascar. 2. 2. Those acting slowly, to which resistance appears quickly—the anti-folinic group (pyrimethamine, proguanil, cycloguanil) and the anti-folic group (sulphonamides and sulphones). Partial cross resistance between these groups has been found, but not between categories 1 and 2. Various combinations of these drugs have been used—4-aminoquinolines + 8-aminoquinolines; chloroquine + pyrimethamine; chloroquine + primaquine + DDS; antifolic + antifolinic drugs. Individual chemoprophylaxis is not difficult, in that there are no strains of malaria parasites resistant to all antimalarials. Chemoprophylaxis of indigenous populations, more or less immune, is more complex. P. falciparum is directly responsible for the death of about 5% of children in hyperendemic areas, and 15% in holoendemic areas; chemoprophylaxis is therefore in the sphere of maternity and child health. In the absence of resistance, chloroquine and amodiaquine are the drugs of choice; the object is to prevent death without interfering with the acquisition of immunity, and this is quite possible. If chloroquine resistance exists, various combinations of other drugs are called for.

Sulfalene has a marked inhibitory action against a drug-sensitive strain of P. berghei in mice. The ED90 ranges between 0.25 and 1.0 mg/kg (given intraperitoneally daily for 4 days). The action is potentiated when the sulphonamide is given in a 1:1 mixture with pyrimethamine. This mixture shows a high level of potentiation against strains resistant to chloroquine, primaquine and sulphaphenazole, as well as against strains resistant to cycloguanil or pyrimethamine. These observations provide an experimental rationale for the application of such combinations in the treatment of individuals infected with multiple-resistant strains of P. falciparum. Other factors must be taken into consideration where large-scale administration is concerned.

It has been shown that quinine, chloroquine, and mepacrine, which have a suppressive effect on the schizogonous or vertebrate phase of Plasmodium vivax infections have no discernible effect on the sporogonous phase of the same parasite in anopheline mosquitoes. Similarly, drugs with no significant antimalarial activity are also wihout effect on the sporogonous cycle. On the other hand, proguanil and cycloguanil, and pyrimethamine, which have a prophylactic effect on the schizogonous cycle of P. vivax have a comparable prophylactic, or oocysticidal effect on the sporogonous cycle, in that these drugs interrupt the normal development of the oocysts and prevent the formation of sporozoites. Compounds which have a curative effect on the vertebrate phase of this infection, namely, primaquine administered in combination with either quinine, chloroquine, or mepacrine, have a toxic, or sporocidal effect on the sporozoites of the sporogonous cycle of P. vivax , eventually producing destruction of these forms and sterilizing the mosquito of its infection. On the basis of these evidences of drug activity on the sporogonous cycle, it has been postulated that, insofar as parasite reaction to drugs is concerned, specific developmental stages of the sporogonous cycle of the malarial parasite have their physiological counterparts in specific developmental stages of the schizogonous cycle of the same parasite. Thus, the oocysts resemble the primary exoerythrocytic stages, while the sporozoites resemble the secondary tissue phases of the parasite. It is evident, therefore, that the sporogonous cycle provides an efficient and reliable system, pharmacologically related to the schizogonous cycle of the same parasite, for evaluating the prophylactic and curative capabilities of antimalarial compounds.

Cycloguanil pamoate has been used in 71 cases of cutaneous leishmaniasis, for the first time, in Iraq and the Middle East. It was given as deep intramuscular injections in the gluteal muscles according to the described dosage schedule.

SOME REGRESSION of early lesions of dermal leishmaniasis has been observed by one of the authors (A.P.C.) in malaria patients treated orally with chloroguanide hydrochloride. The extent of improvement in the leishmanial lesions was not great, however, possibly because of the short duration of chloroguanide hydrochloride administration. The antimalarial activity of chloroguanide hydrochloride in man is due mainly to a dihydrotriazine metabolite of the drug. 1-5 Cycloguanil pamoate (CI-501; Camolar) 6 is a poorly water-soluble salt of this dihydrotriazine. An intramuscular dose of cycloguanil pamoate acts as a repository antimalarial drug in animals 6,7 and man. 8-11 Therefore, it seemed worthwhile to evaluate the efficacy of cycloguanil pamoate in patients with dermal leishmaniasis, caused by Leishmania braziliensis . This preliminary report gives the results obtained with cycloguanil pamoate in 30 such Costa Rican patients. Materials and Methods The patients who received treatment ranged in age from 7 months to 60 years

1. 1. In hospital trials with Gambian children suffering from acute attacks of falciparum malaria the dihydrotriazine derivative and folic acid antagonist BRL 50216 proved effective in eradicating asexual parasitaemias within 2 or 3 days in doses equivalent to 3 mg./kg. daily X 3; 2 daily doses and single doses were unsatisfactory. No toxic effects were encountered. 2. 2. Given in combination with sulphadimethoxine or sulphafurazole potentiation was such that 3 daily doses of BRL 50216 0·3 mg./kg. were equally effective. 3. 3. P. malariae trophozoites proved sensitive to BRL 50216 alone and in combination with sulphadimethoxine but gametocytes persisted for a week or more. 4. 4. Any sporontocidal effect of BRL 50216 alone was not seen but in combination with sulphonamide, sporogony appeared to be interrupted at the early stage of oocyst development. 5. 5. It is concluded that BRL 50216 is an effective antimalarial but offers no advantages over other folic acid antagonists such as pyrimethamine or proguanil. Rapid excretion excludes single dose therapy.

A field trial of the suppression of malaria for a year by 3 injections at 4-monthly intervals of the repository antimalarial agent Dapolar (a 1·1 combination of cycloguanil pamoate and 4,4′-sulfonylbisacetanilide) was carried out amongst the inhabitants of an African village (pop. 693) in the Gambia. Subjects aged 6 months or older were randomly assigned to 2 treatment groups, 100 to receive injections of Dapolar alone and 100 to receive single, therapeutic doses of amodiaquine in addition at the time of Dapolar injections. Dosage was as follows: Dapolar 6 mo.–4 yrs. 88 mg. of each component drug (1·25 ml.) 5–10 yrs. 150 mg. of each component drug (2·0 ml.) 11 yrs. and older 225 mg. of each component drug (3·0 ml.) Amodiaquine Under 50 kg. (body weight) 10 mg. per kg. Over 50 kg. (body weight) 600 mg. per kg. 56% of the subjects in the 2 treatment groups were positive for malaria parasites, mostly P. falciparum, just prior to the 1st round of treatment. Blood surveys made 1 week and monthly thereafter for 4 months after each injection showed that malaria suppression was achieved in almost 90% of subjects after the 1st and 2nd injections and in 100% after the 3rd injection. The 1st injection was carried out in May towards the end of the long dry season when malaria transmission was at a low ebb, the 2nd, in September when malaria was hyperendemic and the 3rd in January when malaria transmission was again reduced. Concomitant amodiaquine therapy made no difference to the efficacy of the repository injections. Surveys amongst groups of control subjects chosen from the rest of the village population indicated a continuing high level of malaria infection with crude infection rate of over 60% in children under 11 years of age. Breakthrough parasitaemias (mstly at the 3rd or 4th month after the 1st and 2nd injections) showed no evidence of resistance to the 3rd injection of Dapolar which in every case cleared these parasitaemias. 186 subjects submitted to the 1st injection, 153 to the 2nd and 106 to the 3rd; 100 subjects had all 3 injections of which 53 had concomitant amodiaquine. No systemic reactions occurred but local tissue reactions consisting of tender swelling around the injection site, in 4 cases with suppuration, were present in 10 and 14% of subjects having the 1st, and 2nd injections and in 7·5% of those having the 3rd injection. Reduction in the number of local reactions to the 3rd injection was achieved by reducing the bore of needle from 18 to 20 gauge. However, the number of these local reactions occurring was sufficient to cause many subjects to refuse the 3rd injection.

Summary CI-564, a 1:1 mixture of cycloguanil pamoate (CI-501) and 4,4′-diacetylaminodiphenyl sulfone (CI-556), was evaluated as a therapeutic agent against blood-induced infections of the Chesson strain of Plasmodium vivax. With a dose of 5 mg per kg of body weight given intramuscularly, cure was effected in each of two volunteers infected with the chlorguanide-sensitive strain. Clearance of patent parasitemia required 3 to 4 days. In two other volunteers infected with the chlorguanide-resistant strain, the clinical and parasitologic courses were modified, but parasitemia was not cleared after 14 and 28 days.

CI-564 (Dapolar®), a mixture of cycloguanil pamoate and diacetyldiamino-diphenylsulfone (DADDS), was used as a repository antimalarial in a test of its suppressive and prophylactic effectiveness. Children of 12 years of age or less, in an area of endemic malaria in the Amazon valley in Brazil, were treated; CI-564 was given to 300 children and CI-564 plus amodiaquin to 300 children; 300 other children to whom no suppressive medication was given served as a control. Initial CI-564 injections were repeated twice at 4-month intervals, and children who had also received amodiaquin were given single doses of this drug with the second and third injections. In the group that received CI-564, ring forms of Plasmodium falciparum disappeared from the peripheral blood within 3 days, and all circulating forms of Plasmodium vivax within 7 days. Throughout the trial there was no significant difference between the treated groups in the prevalence of parasitemia, while there was significantly more parasitemia in the control group. CI-564 alone or with amodiaquin exerted a significant prophylactic effect against both P. falciparum and P. vivax infections. During the 4-month follow-up periods between injections, there were 22 instances of breakthrough by P. falciparum, of which 18 (81%) occurred 90 to 120 days after the previous injection. No breakthrough by P. vivax occurred before Day 90, while there were 39 failures thereafter. Local reactions to CI-564 injections were moderate and transitory, induration and tenderness being most common. There were no allergic reactions after the second and third injections. We conclude that CI-564 can be a practical prophylactic against malaria in suitable situations.

Nineteen Zambian patients with the tropical splenomegaly syndrome and sinusoidal lymphocytosis on liver biopsy were studied. The association of macrobulinaemia with the tropical splenomegaly syndrome has again been confirmed. Sixteen patients were treated with antimalarials—12 with cycloguanil pamoate alone, 3 with cycloguanil and proguanil, and 1 with proguanil alone. Twelve patients were observed for periods of sufficient length for the drug effect to be assessed, and in 11 there was a good response in terms of decrease in spleen size. Cycloguanil pamoate may be of value both for prophylaxis and treatment in areas where tropical splenomegaly syndrome is endemic.

It has been shown that in common with other malarial species the drug response in the sporogonous cycle of Plasmodium cynomolgi parallels in a special way the drug response in the schizogonous cycle. Thus, prophylactic drugs produce deteriorating changes in the oocysts while curative drugs have a destructive effect on the sporozoites. A comparison of the effects of prophylactic drugs on the sporogonous and schizogonous cycles of the four malarial species studied thus far suggests the presence of more than one pre-erythrocytic developmental stage in the schizogonous cycles of P. vivax and P. cynomolgi following the initial invasion by the sporozoite.

Series of strains of Streptococcus faecium ATCC 8043, Lactobacillus casei ATCC 7469, and Pediococcus cerevisiae ATCC 8081 with increasing resistance to the active antifolate antimalarial drugs chlorguanide triazine (CGT), pyrimethamine (PM), and trimethoprim (TMP) were isolated. These mutant strains, stable for at least 3 to 5 years, were examined for cross-resistance and collateral susceptibility to the above compounds and to methotrexate (MTX). Generally, they exhibited cross-resistance to all four compounds, but resistance of a strain to one compound did not predict accurately its resistance to another drug. Unexpectedly, L. casei resistant to CGT exhibited collateral susceptibility to MTX, TMP, and PM varying from 5- to 20-fold. P. cerevisiae developed resistance to CGT readily but maintained its susceptibility to PM and TMP after prolonged exposure to these compounds. Resistance to these antimalarial antifolates was accompanied by only low-grade cross-resistance to MTX, a representative antileukemic antifolate agent.

Summary This paper has dealth with evaluation of an antimalarial agent, CI-564, a 1:1 mixture of cycloguanil pamoate (CI-501) and 4,4′-diacetyl-aminodiphenylsulfone (CI-556) against three different multiresistant strains of falciparum malaria, Malayan III, Malayan IV, and Thai II. The CI-501 component, given intramuscularly, at a dosage of 5 mg per kg of body weight, had no protective effect against sporozoite-induced infections. CI-564, also given intramuscularly, at a dosage of 5 to 7.5 mg per kg of body weight, protected eight of nine volunteers exposed to bites of mosquitoes infected with one of the three multiresistant strains, but protection was lost in less than 70 days. When used as a therapeutic agent, radical cures were realized in only 10 of 15 infections. As a consequence of the above, we believe that CI-564 has limited possibilities as an antimalarial agent.

Summary The effectiveness of cycloguanil pamoate in the treatment of a series of 26 cases of cutaneous leishmaniasis from Panama was reported. Complete healing was accomplished in 19 cases; seven cases failed to respond. The time required for healing ranged from 38 to 138 days. More than half of the cases required more than 90 days.

Summary Maturation of trophozoites of Plasmodium falciparum into schizonts was observed during studies in which defibrinated parasitized blood was placed in vials containing glucose and incubated without agitation for 24 hours. Chloroquine or quinine added to this system in vitro inhibited maturation at successively earlier stages of development as parasites were exposed to increasing amounts of these two drugs. In contrast, the most striking morphologic effect detected after the addition of the dihydrotriazine metabolite of chlorguanide (DHT) reflected formation of abnormal-appearing schizonts, with uninhibited maturation of parasites to the preschizont stage. Studies of the effects of chloroquine, of quinine, and of DHT upon maturation of asexual parasites in vitro were performed with two strains of P. falciparum that had been studied in vivo: a chloroquine-sensitive strain from Uganda and a chloroquine-resistant strain from Malaya. Chloroquine, quinine, and DHT inhibited the maturation of trophozoites of the Ugandan strain to a greater extent than the maturation of trophozoites of the Malayan strain. The system employed to assess the effects of drugs in vitro may prove to be of practical value in a number of ways, including the differentiation of drug-sensitive from drug-resistant strains of P. falciparum and the preliminary appraisal of blood schizonticidal effects of prospective antimalarial agents.

To assess their potential value as antimalarial drugs, we studied CI-679 (base) and CI-679 acetate extensively in oral form and parenteral repository form in mice infected with Plasmodium berghei and in rhesus monkeys infected with Plasmodium cynomolgi. All infections were induced with blood forms of the parasites. Suppressive activity was assayed against four drug-resistant lines of P. berghei. The two drug forms had sufficiently similar effects to permit their being summarized together. The drug was highly effective in mice and monkeys when given either orally or parenterally in repository form. Orally, one large dose or several doses rapidly suppressed the parasitemia and either terminated the infections or suppressed growth of the parasites for many days. The drug caused both nuclear and cytoplasmic damage in asexual forms of P. berghei and P. cynomolgi. A repository dose was active for several weeks in mice and for several months in monkeys. Given orally against P. berghei, the drug 1) was several hundredfold more active than quinine, 2) was effective against a group of lines that collectively are resistant to all suppressive drugs in use and thus appeared to represent a new mode of action, 3) acted synergistically with 4,4′-diaminodiphenylsulfone without apparent commensurate increase in toxicity for mice, and 4) was partially antagonized by para-aminobenzoic acid but not by folinic acid or sodium folate.

Summary Twenty cases of American Cutaneous leishmaniasis were treated with intramuscular injection of 2.5 ml cycloguanil pamoate (Camolar®). Improvement was observed in all cases, and complete clinical and parasitologic cure was obtained in 16 (80%), although the healing response was often delayed, and viable parasites were demonstrated up to 15 weeks after injection of the drug. Second injections were necessary in nine patients, and one received a third injection. In three of the four failures of treatment there had been prior unsuccessful treatment with other drugs. No adverse reactions to the drug were encountered, and cycloguanil pamoate effected dramatic reduction in lost-duty time and hospital costs compared with patients treated with antimonials.

A SUSTAINED effort to develop antimalarial substances with prolonged action was initiated in these laboratories more than seven years ago. Recently, we reported the synthesis of cycloguanil pamoate (‘CamolarR’, CI-501) (refs. 1 and 2), a pamoic acid salt of 4,6-diamino-1-(p-chlorophenyl)-1,2-dihydro-2,2-dimethyl-s-triazine (DHT), and various other dihydrotriazine1,3 and pyrimethamine3,4 salts that exhibit remarkable repository antimalarial properties2,4,5. A single intramuscular dose of cycloguanil pamoate has an unusual capacity to protect man for many months against challenges with susceptible strains of Plasmodium vivax and P. falciparum6. Cycloguanil pamoate has so far not shown a liability to induce rapid resistance during numerous observations on P. cynomolgi in monkeys2,5, although parasites known to be resistant to proguanil, DHT, or pyrimethamine are less susceptible to it7,8. A systematic search has been continued for a well-tolerated repository preparation that would destroy both normal and DHT-resistant parasites and simultaneously block the emergence of resistance denovo. Efforts were directed toward the synthesis of long-acting antimalarials possessing a different mode of action from DHT or pyrimethamine, both of which apparently inhibit the conversion of folic acid to folinic acid9.

The review of antimalarial pharmacology and the examination of results in recent studies of malaria in Vietnam revealed that (1) quinoline derivatives have multiple actions which include interference with nucleic acid replication and glucose metabolism; (2) sulfonamides and sulfones competitively antagonize aminobenzoic acid (PABA); and (3) folic acid reductase inhibitors interfere with the conversion of folic acid to folinic acid. No single agent produced satisfactory cure rates in recent trials with malaria in Vietnam. A combination of quinine and pyrimethamine appeared to give additive results. A combination of pyrimethamine and a sulfonamide gave superior results consistent with their proved synergism. A combination therapy of a long-acting sulfonamide with pyrimethamine, possibly with the addition of quinine, is recommended as the most rational therapy at this time.

A parent line of Plasmodium berghei and three drug-resistant derivatives inadvertently became mixed with Eperythrozoon coccoides. Oxophenarsine hydrochloride (Mapharsen?) was used to eradicate E. coccoides. The parent line was cleared by repeated passage through mice maintained on 0.05% or 0.1% drug diets or given 10 mg/kg doses of the drug daily by parenteral administration. Examinations several months after 0.05% drug-diet treatment indicated that the plasmodia were not resistant to oxophenarsine. The 0.1% drug-diet treatment also was used in clearing a line highly resistant to cycloguanil hydrochloride. P. berghei lines that were highly resistant to 4,4'-diaminodiphenylsulfone or to chloroquine were cleared by the repeated parenteral administration of 5 mg/kg doses daily. The maintenance of disease agents in mice by serially transferring blood or tissues provides repeated opportunities for the agents to be mixed with such natural organisms of mice as Eperythrozoon coccoides. The type of disease then induced may differ markedly from that produced by the experimental agent alone. Stansly (1965) has reviewed instances of synergism with the agents of mouse hepatitis, lymphocytic choriomeningitis, and lactic dehydrogenase. He also reviewed the independent induction of splenomegaly by E. coccoides, which has greatly complicated studies of neoplastic diseases in mice. A competitive action by E. coccoides has been described with Plasmodium berghei by Kretschmar (1963) and Peters (1965) and with Babesia rodhaini by Peters (1965). E. coccoides was detected recently in several of our P. berghei lines. Both theoretical considerations and practical difficulties emphasized the importance of freeing these parasites of the contaminant. Neoarsphenamine has been used to cure mice of E. coccoides (Bruynoghe and Vassiliadis, 1929; Thurston, 1953). Kretschmar (1963) has described the elimination of E. coccoides from a line of P. berghei in mice by sequential treatment with neoarsphenamine and oxytetracycline. Inasmuch as neoarsphenamine is not readily available in this country, we elected to try the related trivalent arsenical oxophenarsine hydrochloride. This report deReceived for publication 14 March 1966. 674 scribes the successful use of this drug with a parent line and three drug-resistant lines of P. berghei. MATERIALS AND METHODS The 12A strain of Plasmodium berghei and three drug-resistant derivatives used in this work were maintained in female CF-1 mice. All mice were sprayed with a disinfectant (1:300 Kreso Dip?) upon arrival and no lice were ever seen on any of the animals. Infections were passed weekly by inoculating parasitized blood intraperitoneally. In some instances a known inoculum (15 X 106 parasitized cells) was injected but usually from 0.1 to 0.3 ml of whole blood was used, roughly in accordance with the parasite density. The drug-resistant parasites included lines that had been induced, by long periods of partially effective treatment, to grow in mice given large daily doses of the drugs subcutaneously (Thompson et al., 1965a, b). The drugs and maintenance doses were as follows: 4,6diamino1( p-chlorophenyl ) -1,2-dihydro-2,2-dimethyl-s-triazine hydrochloride (cycloguanil HC1), 25 mg/kg; 4,4'-diaminodiphenylsulfone (DDS), 25 mg/kg; and chloroquine diphosphate, 30 mg/kg. Each line was more than 30-fold more resistant to the homologous drug than the parent line. The resistant lines were maintained by serial passage through treated mice (usually five to ten), on a schedule designed to provide daily exposure of the parasites to the drugs. Each passage also included the infection of a side group of five to ten untreated mice. Oxophenarsine hydrochloride (3-amino-4-hydroxyphenyl arsineoxide hydrochloride, Mapharsen?) was given either in a powdered diet ad lib. or parenterally. The first parenteral dose was given subcutaneously, to avoid local effects on the parasites injected during the same day into the periThis content downloaded from 157.55.39.220 on Fri, 02 Sep 2016 05:18:44 UTC All use subject to http://about.jstor.org/terms THOMPSON AND BAYLES-ERADICATION OF EPERYTHROZOON FROM P. BERGHEI MICE 675 toneal cavity; all subsequent doses were given intraperitoneally, to avoid skin lesions at the injection site by high doses. All drug dosages and diet concentrations were expressed in terms of the free base. Parenteral doses were given as aqueous preparations in volumes of 5 to 10 ml/kg of body weight. The effects of treatment were assessed by examining Giemsa-stained blood films. Smears made on the 5th day were used mostly in examining the E. coccoides, and 7th-day smears were routinely used for P. berghei studies.

One line of Plasmodium berghei was made highly resistant to cycloguanil hydrochloride and another to pyrimethamine. The degree of cross resistance of each line was then determined to these two drugs and to four others representing diverse chemical types. This was done by testing each compound at several dose levels for suppressive action in respective groups of 10 mice following infection with the parent and resistant lines. The degree of relative cross resistance varied among the compounds. The composite results indicated that resistance induced by cycloguanil hydrochloride was broader than that induced by pyrimethamine. The data also suggested that the latter two drugs have similar but not identical modes of action. A line of Plasmodium berghei made resistant to cycloguanil hydrochloride* showed high cross resistance to pyrimethamine (Thompson et al., 1967). One might presume therefore that this line could be used in determining whether or not a new substance is likely to be active against parasites that are resistant to pyrimethamine. This would be a safe presumption if cycloguanil and pyrimethamine have identical modes of action or if resistance induced with cycloguanil is not narrower or more specific than that induced with pyrimethamine. This paper deals with these hitherto undetermined points. The approach chosen was determination of the degree of reciprocal cross resistance between these drugs by the use of two resistant lines-one that had been made resistant by exposure to cycloguanil hydrochloride and the other by exposure to pyrimethamine. Four additional compounds also were tested for information on the relative breadth of resistance induced by the two drugs. MATERIALS AND METHODS Carworth (CF-1) female mice of approximately 18 to 22 g weight range were used. The KGB-173 strain of Plasmodium berghei was used as the parent line (P), and two derivatives from it were used as the drug-resistant lines. Infections were induced by the intraperitoneal administration of 15 X 106 parasitized blood cells. Treatment was administered by feeding the drug in the ration (powdered Rockland mouse pellets) for 6 days and was started the day before parasites were injected. The medicated rations were given in food Received for publication 12 January 1968. * 4,6-diamino-1( p-chlorophenyl)-l,2-dihydro-2,2dimethyl-s-triazine hydrochloride. cups designed to minimize spillage and to permit meaningful calculations of drug intakes. The effects of treatment were assessed by comparing the percentage of parasitized cells in Giemsa-stained blood smears, prepared from treated and control mice on the 5th day after parasites had been injected. Estimates of the percentages of parasitized cells were made in the following manner. The number of erythrocytes per field (demarcated by a Howard ocular disc) were first estimated in the portion of the smear selected for study; the erythrocyte estimates in terms of 100 or 200 were made by inspection, based upon much experience in actual counting. When at least 5% of the cells were infected, parasites were counted in a sample of an estimated 200 erythrocytes. When clearly less than 5% of the cells were parasitized but at least two parasites occurred per field, parasites were counted in an estimated 1,000 erythrocytes. In fields containing less than two parasites counting was continued until 10 parasites had been seen or at least 100 fields had been examined; the number of fields examined in such instances ranged from 10 to 100. Groups of 10 mice were infected for each dose level of drug tested and a group of 10 untreated mice were infected as controls for each experiment. The percentage of parasite suppression was calculated for each treated mouse, and from this the mean percentage of suppression and its standard error were computed. These values were plotted against the drug dose (in mg/kg/day) on a logarithmic scale and the doses required for 70% suppression (sD70) were estimated from the graphs. The degree of increase in drug tolerance (resistance or cross resistance) was computed by dividing the SD7o of the resistant lines by the SD7, of the parent line in the same experiment. History of the drug-resistant lines The early history of the cycloguanil-resistant line (T) has been presented previously (Thompson et al., 1967). Briefly, it was derived from the parent KGB-173 strain by 48 weekly passages

Two decades ago 2-hydroxy-1,4-naphthoquinones were reported to have considerable suppressive and prophylactic activity against avian malaria. A few compounds were selected for clinical testing but were ineffective in human malarias presumably because of oxidation in the human body into inactive products. Recently homologs with longer n-alkyl side-chains than those of earlier compounds in the series have been synthesized and tested against rodent malarias. In four of the five series of 2-hydroxy-1,4-naphthoquinones reported in this study, peak activity occurred when the methylene groups adjacent to the alicyclic or terminal methyl group numbered 8 or 9. The higher members of the series have been demonstrated to possess not only schizonticidal activity at a dose level lower than that required for the older hydroxynaphthoquinones but also curative activity at high doses. The discovery of strains of Plasmodium falciparum refractory to either suppressive or therapeutic doses of 4-aminoquinolines (chloroquine, hydroxychloroquine, amodiaquine) and a-aminoacridines (quinacrine, propoquine) made reappraisal of malaria chemotherapy mandatory largely because of the known limitations of alternate drugs (chlorguanide, pyrimethamine, quinine, cycloguanil, primaquine, etc.). In an effort to discover new antimalarial drugs our attention was drawn to a group of 2-hydroxy-l,4-naphthoquinone compounds synthesized by Fieser and his associates at Harvard University's Chemical Laboratory as a part of the antimalarial survey conducted on a national scale during World War II. Two of these naphthoquinones (M-1916 and M-285) were shown to possess suppressive and curative effects against avian plasmodia (P. lophurae and P. gallinaceum), but they did not prove to have significant effects against P. vivax and P. falciparum infections of humans (Fieser et al., 1948a). These and other related naphthoquinone antimalarials received scant attention at that time because of the marked suppressive and therapeutic efficacy of 4aminoquinoline antimalarials at well tolerated dose levels. After the discovery of rodent plasmodial infections (P. berghei, P. vinckei, P. chabaudi), it became possible to screen drugs on a wide scale against mammalian rather than avian plasmodia. Several of the old and a few of the more recently synthesized hydroxynaphthoquinone antimalarials (Fieser et al., 1967a, Received for publication 12 March 1968. b) were screened and evaluated against bloodinduced rodent infections and were found to be effective following oral administration. The discovery by Yoeli and Most in 1960 of means for facilitating the transfer of P. berghei through the bites of infected anophelines under laboratory conditions made it possible for us to test the efficacy of the new hydroxynaphthoquinone derivatives against sporozoiteinduced as well as blood-induced P. berghei infections in mice. This paper describes the antimalarial screening procedures used and presents the activity of several compounds belonging to each of the various series of 3-substituted alkyl-2-hydroxy-1,4-naphthoquinones against blood-induced P. berghei infections in mice. A few selected derivatives from the various series of hydroxynaphthoquinones were administered to mice infected by injection of sporozoites of P. berghei. The details of these tests are the basis for a separate paper. MATERIALS AND METHODS Rodent plasmodia Four strains of Plasmodium berghei (NYU-2, NK-65, KSP-11, and subspecies yoelii), two other rodent plasmodia (P. vinckei and P. chabaudi), and a strain of P. berghei maintained under chloroquine pressure were used in our studies. In this paper the results of screening against two strains of P. berghei (NYU-2 and NK-65) will be presented. The NYU-2 strain was isolated on 6 January 1959 by Dr. Van Braeckel from infected salivary glands of Anopheles dureni caught in the vicinity of Kasapa (near Elizabethville, Congo). Dr. Yoeli of New York University obtained the strain from Professor Jadin of Belgium on 20 February 1959. Although originally a vigorous gametocyte producer, it gradually lost this ability through numer

Chloroquine-resistant infections of malaria are prevalent in many areas of Southeast Asia and South America and their importation into the United States will remain a problem during the foreseeable future. The sensitivity of Plasmodium falciparum to chloroquine and other antimalarial drugs can be determined by a simple in vitro technique. It provides an expedient method for identifying drug-resistant strains and is useful in assessing the blood schizontocidal activity of some antimalarial agents. During studies with the recently solated Vietnam (Marks) strain of P falciparum , parasites showed a marked degree of resistance to chloroquine in vivo and in vitro, but they were significantly more susceptible to amodiaquine. The findings suggest that some P falciparum infections resistant to chloroquine may respond more favorably to treatment with another 4-aminoquinoline.