Susceptibility of Panamanian Aotus lemurinus lemurinus to sporozoite-induced Plasmodium falciparum (Santa Lucia) infection.

Aotus monkeys are good models for erythrocyte-induced Plasmodium falciparum and P. vivax infections and have been extensively used in malarial drug and vaccine development. Recently, it has been shown that certain species of Aotus can be infected with sporozoites, and that the degree of susceptibility varies among species. We demonstrate here that Panamanian Aotus lemurinus lemurinus are susceptible to a sporozoite-induced infection, opening the possibility that this species of Aotus could be used as models for testing the efficacy of pre-erythrocytic P. falciparum vaccines and drug candidates directed at the pre-erythrocytic stages of P. falciparum and P. vivax malaria. In this species, we compared sporozoite infection rates. Two of four animals splenectomized prior to infection with sporozoites developed patent parasitemias. Seven of eight animals splenectomized either 7 or 35 days after infection became parasitemic. Additionally, we used a P. falciparum-specific polymerase chain reaction (PCR) method to detect the early appearance of parasitized erythrocytes in the blood prior to detection by conventional microscopy, and found that the parasitemia was detected first in five animals by the PCR method, first in three animals by blood film, with one parasitemia detected simultaneously. We also demonstrated the feasibility of infecting monkeys located in Panama with sporozoites isolated at an insectary in Atlanta, thus documenting the feasibility of similar studies where the insectary and monkey colony are not in the same location. A subsequent attempt to infect these monkeys using sporozoites was not successful, suggesting that this model of human malaria is not yet ready for routine use in vaccine or drug efficacy screening. This model merits further study because of the importance of testing pre-erythrocytic P. falciparum malaria vaccines and drugs in animals.

In vitro and in vivo reversal of chloroquine resistance in Plasmodium falciparum with promethazine.

The effect of combining promethazine with chloroquine was examined against Plasmodium falciparum in vitro in the Aotus-P. falciparum model and in bioassays from volunteers given promethazine. The combination of chloroquine plus promethazine (1 x 10(-6) M) reversed chloroquine resistance in standard P. falciparum clones and patient parasite isolates from Nigeria. The combination reduced the 50% inhibitory concentrations (IC50s) for chloroquine against resistant parasites by 32-92%. Coadministration of promethazine with chloroquine also demonstrated a dose-dependent effect in Aotus monkeys infected with chloroquine-resistant P. falciparum. Monkeys were given a chloroquine dose (20 mg/kg of body weight for seven days), which normally has no effect on parasitemia, plus 10, 20, 40, or 80 mg of promethazine/kg of body weight. In one monkey, parasitemia was suppressed at the lowest promethazine dose, but re-treatment with 20 mg/kg resulted in clearance of parasitemia. Initial treatment with chloroquine and 20 or 40 mg/kg of promethazine cleared parasitemia in some animals followed by recrudescence. Re-treatment at higher doses cured one monkey and resulted in initial clearance and delayed recrudescence 28 or 63 days after treatment in two monkeys. Recrudescent parasitemia in the two monkeys was low (10 parasites/microl of blood) and subsequently cleared without re-treatment. An in vitro bioassay model was developed to examine the effects of clinically achievable doses of promethazine on parasites susceptibilities in vitro. Plasma samples taken at hourly intervals from patients given a single oral dose of 25 mg of promethazine decreased the IC50 values for chloroquine by 20-58% with the most significant reductions occurring in plasma obtained from volunteers 3-4 hr after ingestion. Plasma obtained from two volunteers 6 hr after ingestion of the drug demonstrated no effect on chloroquine susceptibility, suggesting that study of the pharmacokinetic disposition and potential interaction is warranted to optimize the dose regimen in patients for antimalarial efficacy. Historic use of this drug combination for treatment or prevention of chloroquine-associated pruritus or as an antiemetic suggest that the combination is safe and effective when used at standard dosages. The results from this study demonstrate that promethazine is a potent modulator of chloroquine resistance. Clinical evaluation of therapeutic regimens is required to validate clinical efficacy of this promising combination for treatment of uncomplicated chloroquine-resistant malaria.

WR 238605, chloroquine, and their combinations as blood schizonticides against a chloroquine-resistant strain of Plasmodium vivax in Aotus monkeys.

The compound WR 238605 is a primaquine analog being developed by the U.S. Army as an antimalarial drug. Currently, there is no established treatment for Plasmodium vivax parasitemias that are not cured by chloroquine. This study tested WR 238605, chloroquine, and their combinations against a chloroquine-resistant strain of P. vivax (AMRU 1) in Aotus monkeys. A total dose of 3 mg/kg of WR 238605 given at a dosage of 1 mg/kg/day for three days cleared patent parasites in all eight monkeys but recrudescence of parasitemia occurred 15-25 days after initiation of treatment. A total dose of 9 mg/kg of WR 238605 over a three-day period cured all three monkeys of their infections. A total dose of 30 mg/kg of chloroquine did not clear patent infections in three monkeys, whereas a total dose of 60 mg/kg generally (two of three) cleared patent parasitemia but did not cure. Whereas total doses of 30 mg/kg of chloroquine or 3 mg/kg of WR 238605 given alone failed to cure, both drugs given in combination at these dosages cured two of three infections. These results indicate that WR 238605 may be an alternative treatment for chloroquine-resistant vivax malaria.

New, antimalarial, tricyclic 1,2,4-trioxanes: evaluations in mice and monkeys.

We have concluded initial preclinical studies with synthetic trioxanes numbered 3-9 and have compared them with artemisinin (numbered 1) using CD-1 mice infected with Plasmodium berghei. Based on their antimalarial effectiveness in mice, two of these synthetic trioxanes were selected for evaluation in Aotus monkeys infected with multidrug-resistant (MDR) P. falciparum. Trioxane numbered 8 (12 and 48 mg/kg), trioxane numbered 9 (12 and 48 mg/kg) and arteether (numbered 2, 48 mg/kg) were administered intramuscularly in three 12-hr doses to A. lemurinus lemurinus (Panamanian owl monkeys) infected with the Vietnam Smith/RE strain of P. falciparum and monitored for parasitemia. Trioxane numbered 8 at 12 mg/kg cleared parasitemia in two monkeys, but recrudescence occurred in one animal. Treatment of the recrudescent infection with 48 mg/kg was curative. Infections in two monkeys treated initially with 48 mg/kg were cured (six-month follow-up). Trioxane numbered 9 produced a similar outcome: 12 mg/kg suppressed parasitemia in two monkeys but was not curative; however, 48 mg/kg cured infections in all four monkeys treated. These preliminary observations show synthetic trioxanes numbered 8 and 9 to be as effective as arteether (numbered 2) against MDR in P. falciparum in the Aotus monkey.

PS-15: a potent, orally active antimalarial from a new class of folic acid antagonists.

A new, orally-active inhibitor of dihydrofolic acid reductase (DHFR), PS-15 (N-(3-(2,4,5-trichlorophenoxy)propyloxy)-N'-(1-methylethyl)- imidocarbonimidic diamide 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.

Protective immunity induced in Aotus monkeys by a recombinant SERA protein of Plasmodium falciparum: adjuvant effects on induction of protective immunity.

We report the results of vaccination trial 2 of Panamanian Aotus monkeys with a recombinant blood-stage antigen, SERA 1, of the malaria parasite Plasmodium falciparum. Monkeys were immunized with SERA 1, a 262-amino-acid fragment (amino acids 24 to 285) of the 989-amino-acid SERA protein produced by the Honduras 1 strain of the parasite. Immunization mixtures contained 100 micrograms of recombinant SERA 1 protein per dose mixed with one of five different adjuvants. The protein mixed with either Freund's adjuvant or MF75.2 adjuvant stimulated protective immunity. When other P. falciparum antigens were included in the SERA 1-Freund's adjuvant mixture, no protective immunity was observed, although high anti-SERA 1 antibody titers were produced. Three other adjuvants mixed with SERA 1 failed to induce a protective immune response. These results, their relationship to those reported previously in the first vaccination trial (trial 1), and their relationships to the quantitative measurement of anti-SERA 1 antibodies in enzyme-linked immunosorbent assays provided insights into the induction of a protective immune response in vaccinated monkeys.

Comparison of beta-artemether and beta-arteether against malaria parasites in vitro and in vivo.

The antimalarial activity of beta-artemether and beta-arteether was compared in three test systems: in vitro against chloroquine-resistant and chloroquine-sensitive Plasmodium falciparum parasites, in mice infected with P. berghei, and in Aotus monkeys infected with chloroquine-resistant P. falciparum. In vitro, the mean 50% inhibitory concentration (IC50) for beta-artemether was 1.74 nM (range 1.34-1.81 nM), and this value for beta-arteether was 1.61 nM (range 1.57-1.92 nM). They were approximately 2.5-fold more potent than artemisinin, which had a mean IC50 of 4.11 nM (range 3.36-4.60 nM). In the mouse model, the 50% curative doses (CD50) of beta-artemether and beta-arteether had a mean value of 55 mg/kg (32-78 mg/kg). The 50% effective curative doses (ED50) in the Aotus monkey were 7.1 mg/kg (95% confidence interval [CI] = 3.7-13.5) for beta-artemether and 11.8 mg/kg (95% CI = 6.5-21.3) for beta-arteether. Overall, the activities of the two drugs were comparable.

Reversal of Plasmodium falciparum resistance to chloroquine in Panamanian Aotus monkeys.

An Aotus-Plasmodium falciparum model was used to determine if chloroquine resistance could be reversed in vivo. The putative resistance modulators tested all reverse chloroquine resistance in vitro and included verapamil, chlorpromazine, prochlorperazine, cyproheptadine, ketotifen, a tiapamil analog (Ro 11-2933), and a chlorpromazine analog (SKF 2133-A). Combinations of chloroquine plus chlorpromazine or prochlorperazine confirmed reversal of chloroquine resistance as exhibited by cures obtained in six Aotus monkeys infected with chloroquine-resistant P. falciparum (Vietnam Smith/RE strain) and rapid clearance of parasitemia, followed by recrudescence in six additional monkeys. The results indicate the following order of in vivo efficacy for reversing chloroquine resistance in Aotus: chlorpromazine > prochlorperazine >> desipramine >> Ro 11-2933 (tiapamil analog) > ketotifen. Cyproheptadine and verapamil were not effective in reversing chloroquine resistance and probable drug toxicity was observed with these drugs in combination with chloroquine.

Protective immunity induced in Aotus monkeys by recombinant SERA proteins of Plasmodium falciparum.

We describe the vaccination of Panamanian monkeys (Aotus sp.) with two recombinant blood stage antigens that each contain a portion of the N-terminal region of the SERA (serine repeat antigen) protein of the malaria parasite Plasmodium falciparum. We immunized with either a 262-amino-acid SERA fragment (SERA I) that contains amino acids 24 to 285 of the 989-amino-acid protein or a 483-amino-acid SERA fragment (SERA N) that contains amino acids 24 to 506 as part of a fusion protein with human gamma interferon. The recombinant proteins were shown to stimulate protective immunity when administered with complete and incomplete Freund adjuvant. Four of six immunized monkeys challenged by intravenous inoculation with blood stage P. falciparum developed parasitemias that were reduced by at least 1,000-fold. Two of six immunized monkeys developed parasitemias which were comparable to the lowest parasitemia in one of four controls and were 50- to 1,000-fold lower than in the other three controls.

Anatomical distribution of developing trophozoites and schizonts of Plasmodium vivax in Aotus lemurinus lemurinus and Saimiri sciureus.

Organ distribution of developing trophozoite- and schizont-infected erythrocytes of Plasmodium vivax and night monkeys, Aotus lemurinus lemurinus, and squirrel monkeys, Saimiri sciureus, was determined. The primary site for the infection in both species was the splenic vasculature. Secondary organ involvement differed between hosts although some overlapping did occur.

Growth and immunity conferred by a Plasmodium falciparum temperature sensitive mutant in Panamanian owl monkeys.

We have compared the growth of the wild type Plasmodium falciparum strain Honduras 1 and a previously isolated temperature sensitive mutant of it, AP1-16, in Panamanian owl monkeys. We examined serially infected splenectomized and normal animals that were initially infected with cultured parasites that had been grown in a mixture of owl monkey and human erythrocytes. Initial infections in splenectomized monkeys were marked by multiple recrudescences. The mutant grew less well than the wild type in the splenectomized monkeys, as determined by lower peak and total parasitemias. In the splenectomized monkeys tested by rechallenge with the wild type parasite, the mutant stimulated a comparable degree of protection. That protection was manifested in 2 ways. There was a marked reduction in the level of the primary parasitemia in the rechallenged monkeys and an absence of recrudescent parasitemias after the primary parasitemia. The potential value of generating and studying temperature sensitive P. falciparum strains that show attenuated growth is considered.

Reversal of chloroquine resistance in malaria parasite Plasmodium falciparum by desipramine.

Desipramine and several other tricyclic antidepressant drugs reverse chloroquine resistance in Plasmodium falciparum in vitro at concentrations observed in the plasma of human patients treated for depression. Reversal of resistance is associated with increased chloroquine accumulation in the parasite, probably because of inhibition of a putative chloroquine efflux pump. When owl monkeys (Aotus lemurinus lemurinus) infected with chloroquine-resistant Plasmodium falciparum were treated with chloroquine plus desipramine, their parasitemias were rapidly suppressed. Desipramine was found to be one of the most effective compounds yet described for the reversal of chloroquine resistance both in vitro and in vivo.

In vitro and in vivo studies of the effects of halogenated histidine analogs on Plasmodium falciparum.

The effects of four halogenated analogs of histidine on in vitro growth of Plasmodium falciparum malaria parasites were monitored by measurement of the incorporation of 3H-labeled amino acids into parasite proteins and by light and electron microscopy. The uptake of [3H]isoleucine was reduced to 50% of the control value by addition of 70 microM 2-fluoro-L-histidine (2-F-HIS) or 420 microM 2-iodo-L-histidine (2-I-HIS). [3H]histidine uptake into acid-insoluble material was affected equally by these two compounds, 50% inhibition resulting at 200 microM concentration. Morphological analysis of parasite development proved a sensitive assay, since development of mature trophozoites was inhibited 50% by 25 microM 2-F-HIS or 100 2-I-HIS. Electron microscopy studies suggested different mechanisms of action of 2-F-HIS and 2-I-HIS on P. falciparum. 2-F-HIS produced a decrease in knob number at the erythrocyte surface and accumulation of electron-dense material under the parasite membrane. 2-I-HIS had no obvious effect on knobs or electron-dense material but affected parasite morphology. Surprisingly, 2-chloro-L-histidine and 2-bromo-L-histidine did not inhibit P. falciparum in vitro, even though their halogen atom substituents are intermediate in size between F and I atoms. 2-F-HIS and 2-I-HIS were tested in vivo against P. falciparum in owl monkeys (Aotus sp.) but were ineffective at doses that were nontoxic.

Adaptation of the Panama II strain of Plasmodium falciparum to Panamanian owl monkeys.

The Panama II strain of Plasmodium falciparum, acquired at the second passage level in splenectomized Colombian owl monkeys, was adapted to owl monkeys of Panamanian origin. Patent infections were induced in 22 of 27 unaltered and 20 of 21 splenectomized recipients during 19 serial passages. The infections were significantly more virulent in splenectomized than normal Panamanian owl monkeys, however recrudescences in seven normal monkeys achieved peak parasitemias 48 times greater than in the primary attack. These results describe the first reproducible infections of indigenous falciparum malaria in Panamanian owl monkeys.

Desferrioxamine suppresses Plasmodium falciparum in Aotus monkeys.

Clinical observation has suggested that iron deficiency may be protective in malaria, and we have found that desferrioxamine (DF), an iron-specific chelating agent, inhibited Plasmodium falciparum growth in vitro. It was difficult to be confident that DF would be effective in an intact animal, however, because continuous exposure to DF was required in vitro and, in vivo, DF is rapidly excreted. Also, the in vitro effect of DF was overcome by addition of iron to the culture and in vivo there are potentially high local iron concentrations when iron is absorbed from the diet or released from reticuloendothelial cells. We now show that DF given by constant subcutaneous infusion does suppress parasitemia in P. falciparum-infected Aotus monkeys.

Comparison of Plasmodium falciparum infections in Panamanian and Colombian owl monkeys.

Parameters of blood-induced infections of the Vietnam Oak Knoll, Vietnam Smith, and Uganda Palo Alto strains of Plasmodium falciparum studied in 395 Panamanian owl monkeys in this laboratory between 1976-1984 were compared with those reported from another laboratory for 665 Colombian owl monkeys, studied between 1968-1975, and, at the time, designated Aotus trivirgatus griseimembra. The virulence of these strains was less in Panamanian than in Colombian owl monkeys, as indicated by lower mortality rates of the Panamanian monkeys during the first 30 days of patency. Maximum parasitemias of the Vietnam Smith and Uganda Palo Alto strain, in Panamanian owl monkeys dying during the first 15 days of patent infection, were significantly higher than in Colombian owl monkeys. Panamanian owl monkeys that survived the primary attack had significantly higher maximum parasitemias than the surviving Colombian owl monkeys. Peak parasitemias were attained significantly earlier after patency in Panamanian than in Colombian owl monkeys, irrespective of the strain of P. falciparum. More Panamanian than Colombian owl monkeys evidenced self-limited infection after the primary attack of either the Vietnam Smith or Uganda Palo Alto strain. The duration of the primary attacks and recrudescences were significantly shorter in Panamanian than in Colombian owl monkeys. Mean peak parasitemias during recrudescence were usually higher in Panamanian owl monkeys than in Colombian monkeys. Differences of infection parameters were probably attributable, in part, to geographical origin of the two monkey hosts and parasite strains.

Antimalarial activities and subacute toxicity of RC-12, a 4-amino-substituted pyrocatechol.

RC-12 [1,2-dimethoxy-4-(bis-diethylaminoethyl)-amino-5-bromobenzene] was evaluated for prophylactic, radical curative, and suppressive activities against infections with Plasmodium cynomolgi and subacute toxicity in rhesus monkeys. Applied as a prophylactic agent, RC-12, administered in doses of 6.25 to 25.0 mg/kg daily throughout the incubation period, provided near-complete to complete protection against 10(5) to 10(6) times the minimum infective dose of sporozoites. Applied as a suppressive agent, daily doses of 100.0 mg of RC-12 per kg did not eradicate blood schizonts regularly; hence, the need for concomitant administration of a blood schizonticide, such as chloroquine, in assessments of radical curative activity. In such appraisals, daily doses of 6.25 to 25.0 mg of RC-12 per kg for 14 days, in combination with 2.5 mg of chloroquine per kg daily for 7 days, effected cure of 69 and 93% of established infections, respectively. The curative activity of RC-12 was related to the total dose and could be achieved with a regimen as brief as 4 days. With respect to outward expressions of toxicity, daily doses of 50.0 mg/kg or lower for 15 to 225 days evoked no reactions. Doses of 100.0 or 200.0 mg/kg, scheduled for 15 days, evoked convulsions and depression and were, respectively, lethal to 4 of 17 and 7 of 7 recipients. Doses of 25.0 mg/kg or lower evoked no discrete reactions. Doses of 50.0 mg/kg and higher evoked hepatomegaly, vacuolation of hepatocytes, and elevations of glutamic oxalacetic and glutamic pyruvic transferase activities in serum, reactions related in intensity to dose but not duration of dosage.

Cryopreservation of the blood stages of Plasmodium falciparum and Plasmodium vivax for in vivo studies.

Cryopreservation in Alsevers-glycerol of the blood stages of Plasmodium falciparum (5 strains) and P. vivax (2 strains) indicated that parasite infectivity for Aotus was retained for more than 1,100 days.

Activities of respository preparations of cycloguanil pamoate and 4,4'-diacetyldiaminodiphenylsulfone, alone and in combination, against infections with Plasmodium cynomolgi in rhesus monkeys.

The studies summarized in this report were concerned with the capacities of repository preparations of cycloguanil pamoate (CGT-P) to protect rhesus monkeys against infections with drug-susceptible and pyrimethamine-resistant strains of Plasmodium cynomolgi. Administered intramuscularly as a suspension in an oleaginous vehicle, CGT-P (i) provided long-term protection against single and repetitive challenges of rhesus monkeys with sporozoites of the drug-susceptible B and Ro strains, (ii) effected prompt clearance of parasitemia in established infections, and (iii) delayed relapse. Protection was equated to absence of parasites on thick blood films, negative results when blood was transferred to susceptible recipients, and inability to activate infection by splenectomy. Eventual loss of protection was not related to emergence of parasites resistant to cycloguanil (CGT). Although protection varied from monkey to monkey, its mean duration was related directly to size of CGT-P dose and size of particles in the suspension. Urinary excretion studies indicated that protection persisted as long as the daily output of CGT did not fall below that attained with the parenterally administered hydrochloride salt at a dose equivalent to 0.015 mg of CGT per kg. Studies on infections with the resistant Ro/PM strain showed that the activity of CGT-P was compromised severely by resistance to pyrimethamine. Attempts to minimize this liability by concomitant administration of 4,4'-diacetyldiaminodiphenylsulfone met with limited success. These results suggest that even the best of the repository preparations of CGT-P, with or without 4,4'-diacetyldiaminodiphenylsulfone, would be useful only in areas where Plasmodium falciparum and Plasmodium vivax are fully susceptible to chlorguanide and pyrimethamine.