Search

A total of 248 Plasmodium falciparum isolates were sampled in travelers with malaria who came to Marseille, France from Comoros to investigate in vitro activities of antimalarial drugs and molecular markers of drug resistance. Of the 248 isolates, 126 were maintained in culture. Of these, 53% were resistant to chloroquine, and 3% had reduced susceptibility to quinine, mefloquine, and atovaquone; 1% had reduced susceptibility to halofantrine and dihydroartemisinin; 7% had reduced susceptibility to monodesethylamodiaquine; 37% had reduced susceptibility to cycloguanil; and none had reduced susceptibility to lumefantrine. Resistance-associated point mutations were screened in 207 isolates. No mutations in the cytochrome b gene were found. Of the 207 isolates, 119 (58%) had a mutation in the P. falciparum dihydrofolate reductase (Pfdhfr) gene at codon 108, 6 (5%) had mutations in both Pfdhfr codon 108 and the P. falciparum dihydropteroate synthase codon 437, and 115 (56%) had the chloroquine resistance–associated K76T mutation in the P. falciparum chloroquine resistance transporter gene. This study represents a unique opportunity to improve surveillance of P. falciparum drug resistance in Comoros with consequences for treatment and chemoprophylaxis guidelines.

The chemosusceptibility and genetic polymorphism of Plasmodium falciparum populations from 48 patients hospitalized for malaria at the Hospital Principal in Dakar, Senegal were investigated during the 2002 malaria transmission season. Sixty-two percent of the isolates collected were from patients with severe malaria and 38% were from patients with mild malaria. In vitro activities of chloroquine, quinine, cycloguanil, atovaquone, mefloquine, halofantrine, and artesunate were evaluated. The prevalence of mutations in the Plasmodium falciparum dihydrofolate reductase (dhfr) and dihyropteroate synthetase (dhps) genes and the P. falciparum chloroquine resistance transporter (Pfcrt) gene associated with cycloguanil, pyrimethamine, sulfadoxine, and chloroquine resistance were estimated. The genetic polymorphism of the parasite populations was evaluated by analysis of the highly polymorphic regions of merozoite surface protein 1 (msp1) block 2 and msp2. Seventy percent of the isolates were assessed by an in vitro assay. Fifty-two percent of the isolates were chloroquine resistant, 45% were cycloguanil resistant, and 24% were atovaquone resistant. Four percent had low susceptibility to quinine. The Pfcrt and dhfr mutations were associated with in vitro chloroquine- and antimetabolic drug-resistant isolates, respectively. Approximately 70% of the isolates contained two or more clones. Genetic diversity of P. falciparum was high. The prevalence of allelic family K1 of msp1 was 68%. Isolates of P. falciparum were highly resistant to chloroquine, cycloguanil and atovaquone. The transmission rate of malaria in Dakar is low but a high degree of genetic polymorphism can increase severe malaria, as shown by persons coming to Dakar from areas highly endemic for malaria. Areas with urban malaria should use vector control measures and efficient chemoprophylaxis for non-immune populations.

Chloroquine-proguanil combination is one of the options for chemoprophylaxis. The rapid evolution of drug resistance status requires a constant upgrade of epidemiologic data. Due to various difficulties in conducting prospective clinical studies on the prophylactic efficacy of the drug combination, especially in highly chloroquine-resistant zones, in vitro drug sensitivity assays and specific molecular markers for chloroquine (Plasmodium falciparum chloroquine-resistance transporter, pfcrt) and cycloguanil (a biologically active metabolite of proguanil; dihydrofolate reductase, dhfr) resistance were evaluated as an alternative approach in this study. Of 116 isolates, 62 (53.4%) were doubly resistant in vitro to chloroquine (IC50 > or = 100 nM) and cycloguanil (IC50 > or = 15 nM). Likewise, 62 of 118 isolates (52.5%) carried both the mutant Thr-76 pfcrt allele and at least one dhfr mutant allele (1 with a single Asn-108 allele, 8 with double Arg-59 and Asn-108 mutations, and 53 with triple Ile-51, Arg-59, and Asn-108 mutations). The in vitro drug response corresponded with the presence or absence of key mutation(s) in the pfcrt and dhfr genes. These results suggest the high proportion of P. falciparum isolates in southern Cameroon that may not respond to chloroquine-proguanil combination.

The antifolate proguanil is commonly used in the prophylaxis and treatment of Plasmodium falciparum malaria. A series of point mutations in the dihydrofolate reductase (DHFR) gene has been linked to differential susceptibility of varied P. falciparum clones or isolates to this drug. To survey the efficiency of proguanil prophylaxis in an African endemic region, and to evaluate the level of proguanil resistance in the corresponding parasite population, we performed drug susceptibility assays with P. falciparum isolates from Senegal, Kenya, and Niger. In parallel, we developed a mutation-specific polymerase chain reaction assay that enabled us to characterize mutations in the DHFR gene of the same isolates without in vitro parasite cultivation. We confirm previously available data showing that parasites harboring a point mutation from Ser108 to Asn present a decrease in susceptibility to cycloguanil (the active metabolite of proguanil), and we show that mutations in codons 51 and 59 appear to modulate the level of resistance to cycloguanil. No mutations in codons 16 and 164 were detected in resistant parasites, in contrast with results from some previous studies.

The objective of this study was to examine the disposition of proguanil in malaria-infected Thai patients with acute uncomplicated falciparum malaria. Eleven patients were administered 500 mg of proguanil twice a day for three days (total dose = 3,000 mg). Four patients were tentatively classified as extensive metabolizers (EMs) and seven as poor metabolizers (PMs). The mean plasma clearances of proguanil for EMs and PMs were 1.31 and 1.10 L/hr/kg, respectively. The mean elimination half-life of proguanil was statistically longer in PMs than EMs (19.6 hr versus 16.1 hr; P = 0.01). Plasma clearance and elimination half-life of proguanil in the malaria patients were comparable with those reported in the literature for healthy Thai volunteers. In contrast to other ethnic groups. Thai EM patients had relatively low plasma concentrations of cycloguanil, the active metabolite of proguanil. None of the 11 patients treated with proguanil were cured of malaria and their phenotype status did not affect the treatment outcome. Although high levels of parasite resistance to cycloguanil were probably responsible for the poor response to proguanil treatment, the inability of Thai EM and PM patients to produce cycloguanil may have also contributed to the treatment outcome.

Plasmodium falciparum isolates from 24 Papua New Guinean patients with symptomatic malaria were tested for susceptibility to pyrimethamine and cycloguanil. Thirteen isolates were sensitive to both agents and the remainder exhibited varying degrees of resistance. No isolates were found to be resistant to one agent yet sensitive to the other and a positive correlation suggesting cross-resistance was found. Parasite DNA extracted from the patients' stained blood slides was amplified and sequenced to examine point mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase genes (DHPS) associated with antifolate resistance. All resistant isolates possessed mutations in the DHFR gene at codon 108, the majority changing from Ser to Asn, but one isolate from Ser to Thr, a change not previously reported in field isolates. A second mutation of the DHFR gene at Cys-59 to Arg was present in isolates with higher level resistance, but not exclusively so. Sequencing the DHPS gene, as a predictor of sulfadoxine resistance, revealed only one example that was different from DHPS alleles of sensitive isolates.

A rapid and simple method to detect pyrimethamine susceptibility of Plasmodium falciparum by analyzing DNA from whole blood is presented. Samples from cultured isolates and from patients infected with P. falciparum were spotted onto filter paper disks, dried, and stored for subsequent analyses. After extracting the P. falciparum DNA using Chelex-100 ion-chelating resin (Bio-Rad, Richmond, CA), the polymerase chain reaction (PCR) was used to amplify the dihydrofolate reductase (dhfr) gene. The PCR product of 727 basepairs was digested with the Alu I restriction endonuclease to detect whether the isolates were sensitive or resistant to the antimalarial drugs pyrimethamine and cycloguanil. This reaction endonuclease digests only DNA from pyrimethamine-sensitive parasites because the recognition locus of Alu I is changed by mutations giving rise to pyrimethamine and cycloguanil resistance. This method is simple and sensitive and could therefore bu used to study the epidemiology of pyrimethamine resistant in P. falciparum. The DHFR gene of pyrimethamine-resistance clones from Vietnamese patients showed three amino acid changes that were previously found in pyrimethamine-resistant isolates. Two other clones, T9/94 and T9/96, originally isolated from a single malaria patient from Thailand, had different DHFR gene sequences. The nucleotide sequence of the DHFR gene from T9/96 was identical to the wild-type DHFR sequence, whereas T9/94 possessed amino acid substitutions at positions 16 and 108 that have been described in cycloguanil-resistant parasites.

A simple high-performance liquid chromatographic (HPLC) assay method was developed for the measurement of proguanil (PG) and its major metabolites, cycloguanil (CG) and 4-chlorophenyl-biguanide (CPB), in human plasma and urine. The assay allowed the simultaneous determination of all analytes in 1 ml of plasma or 0.1 ml of urine. The detection limits of PG, CG, and CPB, defined as the signal-to-noise ratio of 3, were 1 and 5 ng/ml for plasma and urine samples, respectively. Recoveries of the analytes and the internal standard (pyrimethamine) were > 62% from plasma and > 77% from urine. Intra-assay and interassay coefficients of variation for all analytes in plasma and urine were < 10% except for the values of CG and CPB, which ranged from 10% to 15% at one or two concentrations among 4-5 concentrations studied. The clinical applicability of the method was assessed by the preliminary pharmacokinetic study of PG, CG, and CPB in six healthy volunteers with the individually known phenotypes (extensive and poor metabolizers) of S-mephenytoin 4'-hydroxylation, suggesting that individuals with a poor metabolizer phenotype of S-mephenytoin have a much lower capacity to bioactivate PG to CG compared with the extensive metabolizers.

The Thai-Cambodian border is a difficult area in which to provide adequate malaria chemoprophylaxis because of multiple drug-resistant Plasmodium falciparum. In 1990–1991, Thai soldiers were randomly selected to receive proguanil (200 mg/day) combined with dapsone (4 mg or 12.5 mg/day) (n = 184) or pyrimethamine/dapsone (12.5 mg and 100 mg/week) (n = 177). Doxycycline (100 mg/day) was given to men with glucose-6-phosphate dehydrogenase deficiency (n = 77). Falciparum malaria attack rates were the same whether proguanil/dapsone (10.3%) or pyrimethamine/dapsone (11.3%) was used. However, proguanil/dapsone was more effective than pyrimethamine/dapsone in preventing vivax malaria (1.6% versus 12.4%). Men receiving doxycycline had falciparum malaria (3.9%) and vivax malaria (1.3%) at low rates. Adjusting the dapsone component from 4 mg to 12.5 mg did not improve the prophylactic effectiveness. Hematologic toxicity was not observed with the proguanil/dapsone combination. We conclude that proguanil/dapsone is not a useful alternative for malaria chemoprophylaxis on the Thai-Cambodian border.

Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatment and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that point mutations at key positions of the dihydrofolate reductase-thymidylate synthase gene are strongly associated with antifolate resistance. However, whether the same or distinct mutations are involved in the development of resistance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase domain of the gene (from 34 of 42 isolates) to analyze the genotype that may distinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrations (IC50s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.60-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid substitution, Asn-108. Only three mutant codons were observed among the antifolate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance between pyrimethamine and cycloguanil that is based on similar amino acid substitutions in dihydrofolate reductase and suggest that two or three mutations, including at least Asn-108, may be necessary for cycloguanil resistance, whereas a single Asn-108 mutation is sufficient for pyrimethamine resistance.

The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area.

Sulfadoxine/pyrimethamine (SP) is considered an alternative treatment for acute uncomplicated malaria caused by Plasmodium falciparum resistant to chloroquine. However, the appearance of resistance to this drug has been reported since its initial use in Colombia. Molecular analysis of the dihydrofolate reductase gene indicates a correlation between in vitro resistance to SP and the Asn-108 point mutation. Little is known about the association of this point mutation and in vivo resistance to SP. We used a mutation-specific polymerase chain reaction strategy to analyze the presence of the Asn-108 point mutation in 48 clinical samples with adequate clinical response (ACR), 2 early treatment failures (ETF), and 1 late treatment failure (LTF). The Asn-108 mutation was detected in 36 of the ACR samples and in all of the ETF and LTF samples. Eleven ACR samples amplified with the wild-type-specific primer and one amplified with the primer for the Thr-108 mutation described for resistance to cycloguanil. These results suggest that the Asn-108 marker may not be useful in predicting SP treatment failure.

Resistance to antifolate antimalarial drugs (cycloguanil, a biologically active metabolite of proguanil, and pyrimethamine) is associated with a Ser- to Asn-108 point mutation in the dihydrofolate reductase-thymidylate synthase gene of Plasmodium falciparum. The frequency of this mutation was studied in 127 clinical isolates obtained in Yaounde, Cameroon using a simple and rapid molecular technique based on the polymerase chain reaction and restriction fragment length polymorphism. Of the 127 isolates, pure wild-type Ser-108 codon, pure mutant-type Asn-108 codon, and mixed codons were observed in 66, 55, and six parasites, respectively. The proportion of antifolate-resistant, pure mutant-type codon, with respect to pure wild-type or mixed alleles, was 43% (55 of 127). The results of the molecular assay were compared with those of semimicro isotopic in vitro assay in 34 isolates. All 17 pure Ser-108 isolates and two isolates with mixed alleles were sensitive to both pyrimethamine (50% inhibitory concentration [IC50] < 100 nM) and cycloguanil (IC50 < 50 nM). Fourteen of 15 isolates with the mutant-type Asn-108 codon were resistant to pyrimethamine and cycloguanil. One isolate with Asn-108 showed a slightly elevated pyrimethamine IC50 (78 nM), which was within the sensitive range. This study provides further evidence that antifolate-resistant P. falciparum isolates are already present in Yaounde, Cameroon.

New drugs for causal prophylaxis of malaria are needed. A proguanil/sulfamethoxazole combination was investigated using a rhesus monkey model (Macaca mulatta infected with Plasmodium cynomolgi) to determine whether causal prophylaxis could be achieved. When a five-day regimen of proguanil (40 mg/kg/day) combined with sulfamethoxazole (100 mg/kg/day) was used, infection of all animals (6 of 6) was observed, with an extended prepatent period (median 40 days). Two control animals became infected on days 9 and 23 following sporozoite inoculation. Plasma concentrations indicated that proguanil and sulfamethoxazole were adequately absorbed and metabolized to cycloguanil and N-4-acetylsulfamethoxazole, respectively. Analysis of liver biopsy specimens demonstrated that the drugs were present two days following sporozoite inoculation but were not detectable one week later. Proguanil plus sulfamethoxazole does not eliminate exoerythrocytic-stage parasites in the rhesus monkey-P. cynomolgi model.

The in vitro activity of two dihydrofolate reductase (DHFR) inhibitors, pyrimethamine and cycloguanil, was evaluated against African clones and isolates of Plasmodium falciparum using an isotopic, semimicro drug susceptibility test. Three susceptibility levels (susceptible, intermediate, and resistant) were observed in the response of culture-adapted clones and strains to pyrimethamine (50% inhibitory concentration [IC50])100, 100-2,000, and2,000 nM) and cycloguanil (IC5050, 50-500, and500 nM). Based on these susceptibility levels, 73 and 68 of 96 fresh clinical isolates were susceptible to pyrimethamine (mean IC50 15.4 nM) and cycloguanil (mean IC50 11.1 nM), respectively. Thirteen and 18 isolates were resistant to pyrimethamine (mean IC50 9,440 nM) and cycloguanil (mean IC50 2,030 nM), respectively. A highly significant positive correlation was found between pyrimethamine and cycloguanil (r = 0.786), indicating in vitro cross-resistance between these antifolates. The spread of resistance to one of the DHFR inhibitors in Africa may compromise the utility of the other DHFR inhibitor.

A new, orally-active inhibitor of dihydrofolic acid reductase (DHFR), PS-15 (N-(3-(2,4,5-trichlorophenoxy)propyloxy)-N′-(1-methylethyl)-imidocarbonimidicdiamide hydrochloride), has significant activity against drug-resistant Plasmodium falciparum. It is not cross-resistant with other inhibitors of DHFR (e.g., pyrimethamine and cycloguanil). Although it bears similarities to proguanil, PS-15 represents a new antifolate class of drugs that we have named oxyguanils or hydroxylamine-derived biguanides. This compound displays intrinsic antimalarial activity and also is metabolized in vivo to WR99210, an extremely active triazine inhibitor of DHFR. When tested in vitro against drug-resistant clones of P. falciparum, PS-15 was more active than proguanil, and the putative metabolite, WR99210, was more active than the proguanil metabolite cycloguanil. The drug is also more active as well as less toxic than proguanil when administered orally to mice infected with P. berghei. When administered orally to Aotus monkeys infected with multidrug-resistant P. falciparum, PS-15 was more active than either proguanil or WR99210. In 1973, WR99210 underwent clinical trials for safety and tolerance in volunteers. The trials showed gastrointestinal intolerance and limited bioavailability; further development of the drug was abandoned. Because PS-15 has intrinsic antimalarial activity, is not cross-resistant with other DHFR inhibitors, and can be metabolized to WR99210 in vivo, oral administration of this new drug should circumvent the shortcomings and retain the advantages found with both proguanil and WR99210.

Primary cultures of Macaca mulatta hepatocytes infected with sporozoites of Plasmodium knowlesi, P. cynomolgi (Cambodian strain), and P. cynomolgi bastianellii were exposed in vitro to 7 antimalarial compounds. The number of exoerythrocytic schizonts present after 4-7 days of culture was used to assess the activity. With pyrimethamine, proguanil, cycloguanil, primaquine, and 2 of its analogues (WR242511 and WR238605), marked inhibition of schizont formation could be achieved at concentrations below those causing a cytotoxic effect on the host hepatocytes. Chloroquine had only minimal schizonticidal activity at a concentration that produced severe hepatocyte toxicity. This simian in vitro system provides a reliable model for screening antimalarial compounds and for investigating their effects on the hepatic stage of malaria parasites.

The ease with which Plasmodium falciparum parasites adapt to culture by the method of Trager-Jensen is variable. The strain characteristics of the first three isolates we cultured differed markedly, depending on the ease of adaptation. The West African I, which was cultured readily, proved to be sensitive to all antimalarials assessed by an in-vitro method, caused benign infections in Aotus monkeys, and produced gametocytes both in the cultures and in Aotus monkeys. In contrast, parasites of the Liberian I and Haitian I strains, both of which required an adaptive period of a month or longer proved more tolerant to chloroquine, quinine, and cycloguanil but sensitive to pyrimethamine; commonly produced fatal infections in Aotus monkeys, and did not produce demonstrable gametocytemia. That the differences may be due to the selection of a more virulent and drug tolerant subpopulation is suspected. The implication of these findings, particularly as it may apply to the epidemiology of chloroquine resistant falciparum malaria, is discussed.

A test system that uses infective gametocytes from in vitro cultures was developed for evaluating the sporontocidal activity of antimalarial compounds. In evaluating the system, pyrimethamine and cycloguanil (dihydrofolate reductase inhibitors) and primaquine (8-aminoquinoline) were tested against pyrimethamine-sensitive and pyrimethamine-resistant strains of Plasmodium falciparum. The drugs were administered to Anopheles either in a blood meal containing infective gametocytes or in a noninfective meal 2-4 days later. The mosquitoes were dissected 9-10 days after they received the infective blood meal, and the sporontocidal effect of the drugs was evaluated by the number of oocysts found in the gut. Both cycloguanil and pyrimethamine had marked sporontocidal activity. The susceptibility pattern of the strains to the sporontocidal effect of pyrimethamine and cycloguanil was similar to the susceptibility of their asexual blood stages in vitro to the schizontocidal effect of the compounds. The sporontocidal effect was observed only when the compounds were administered at the same time as the infective blood meal, but not when they were given 2-4 days later. No sporontocidal activity was observed with primaquine. This system permits more reliable quantitative observations than have been possible with previous methods.

Twenty-six Plasmodium falciparum isolates obtained during a prophylaxis study at Jilore primary school, Malindi, Kenya, were adapted to in vitro culture and their susceptibility to 13 antimalarial drugs was tested by a modified radioisotopic method. Pyrimethamine, chloroquine, amodiaquine, cycloguanil, chlorcycloguanil, quinine, quinidine and sulfadoxine, and the experimental compounds MB 35769, mefloquine, WR 184806, parvoquone, and menoctone were used. The isolates could be divided into two groups with significantly different susceptibility to pyrimethamine, shown by a 755-fold difference in the mean ID50 values (2.77 ± 1.98 × 10-10 mol/l and 2.09 ± 1.64 × 10-7 mol/l). The mean susceptibility of the two groups differed 7.7-fold for chlorcycloguanil and 14.6-fold for cycloguanil, but were not significantly different for the other drugs. All isolates were more sensitive to amodiaquine than to chloroquine in vitro. The ratio of the geometric mean ID50 values of chloroquine to amodiaquine was 3.13. The ratio for the chemically related compounds parvoquone to menoctone was 5.63, quinine to quinidine was 5.58, and mefloquine to WR 184806 was 12.16.

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.

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.

Three tetrahydrofolate dehydrogenase (dihydrofolate reductase = EC 1.5.1.3) inhibitors were tested for antimalarial activity against Plasmodium falciparum, using an in vitro radioisotopic technique. Activity of each drug was tested in both normal RPMI medium 1640 and in modified medium (containing no p-aminobenzoic acid and 2.27 × 10-8 M folic acid) after a 24- or 48-hour exposure. Activity was increased 20- to 85-fold using the modified medium and the longer exposure time. Under all conditions, pyrimethamine and cycloguanil were of equal or greater potency than an experimental pyrimethamine analogue, M&B 35769, against pyrimethamine-sensitive strains, but M&B 35769 was more active than either pyrimethamine or cycloguanil against pyrimethamine-resistant strains.

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.

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.