Craig J. Canfield

Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.

A rapid, semiautomated microdilution method was developed for measuring the activity of potential antimalarial drugs against cultured intraerythrocytic asexual forms of the human malaria parasite Plasmodium falciparum. Microtitration plates were used to prepare serial dilutions of the compounds to be tested. Parasites, obtained from continuous stock cultures, were subcultured in these plates for 42 h. Inhibition of uptake of a radiolabeled nucleic acid precursor by the parasites served as the indicator of antimalarial activity. Results of repeated measurements of activity with chloroquine, quinine, and the investigational new drug mefloquine demonstrated that the method is sensitive and precise. Several additional antimalarial drugs and compounds of interest were tested in vitro, and the results were consistent with available in vivo data. The use of P. falciparum isolates with known susceptibility to antimalarial drugs also permitted evaluation of the cross-resistance potential of each compound tested. The applications and expectations of this new test system within a drug development program are discussed.

Kinetics of a new antimalarial, mefloquine.

The kinetics of mefloquine hydrochloride were studied in 20 healthy adult male subjects after single oral doses of 250, 500, 1,000, and 1,500 mg. Whole blood concentrations of the drug were measured periodically for 83 days after drug administration. The resulting concentration/time data were analyzed to determine the rates of absorption and elimination of the drug. There was considerable variation in all of the estimated kinetic parameters, but differences did not appear to be related to the administered dose. Absorption was slow after administration of a tablet of the drug (mean ka, 4.37/day). When the drug was given in an aqueous suspension it was more rapidly and more completely absorbed, as indicated by a mean ka of 15.34/day and an area under the blood concentration/time curve 35% greater than with the same dose in tablet form. The volume of distribution was quite large (mean value for V/f, 13.30 1/kg). The therapeutic efficacy and prolonged duration of prophylactic effect of a single dose of mefloquine was consistent with the finding of a mean whole blood half‐life (t½) of 13.89 days. There was considerable variation in the t½ within each dose group, with a range of 6.48 to 22.65 days. Because of the wide individual variation in the kinetics of mefloquine, it is anticipated that occasional treatment failures may occur when the drug is used for single‐dose therapy on a large scale.

Atovaquone-Proguanil Compared with Chloroquine and Chloroquine-Sulfadoxine-Pyrimethamine for Treatment of Acute Plasmodium falciparum Malaria in the Philippines

This randomized, open-label clinical trial compared a fixed-dose combination of atovaquone and proguanil (n=55) with chloroquine (n=23) or a combination of chloroquine, sulfadoxine, and pyrimethamine (n=32) for treatment of acute falciparum malaria in the Philippines. Patients were hospitalized for 28 days to ensure medication compliance and prevent reinfection. Atovaquone-proguanil produced a significantly higher cure rate (100%) compared with that for chloroquine (30.4%; P<.0001) or chloroquine-sulfadoxine-pyrimethamine (87.5%; P<.05). Treatments did not differ significantly with respect to parasite clearance time (mean: 46.7 h for atovaquone-proguanil, 60.0 h for chloroquine, and 42.8 h for chloroquine-sulfadoxine-pyrimethamine) or fever clearance time (mean, 38.8, 46.8, and 34.5 h, respectively). Adverse events were typical of malaria symptoms; the most frequently reported events were vomiting (18% for atovaquone-proguanil, 17% for chloroquine, and 9% for chloroquine-sulfadoxine-pyrimethamine), abdominal pain (15%, 17%, and 3%, respectively), anorexia (11%, 13%, and 0%, respectively), and headache (6%, 17%, and 3%, respectively). Atovaquone-proguanil was well tolerated and more effective than chloroquine or chloroquine-sulfadoxine-pyrimethamine for treatment of multidrug-resistant falciparum malaria in the Philippines.

Population pharmacokinetics of atovaquone in patients with acute malaria caused by Plasmodium falciparum

The population pharmacokinetics of atovaquone were examined in 458 black, Oriental, and Malay patients with acute Plasmodium falciparum malaria receiving atovaquone alone or concomitantly with other drugs. Oral clearance (CLF) showed a 0.674 power relationship with weight and is similar in Oriental and Malay subjects but 58.5% lower in black subjects. On the basis of mean body weight, the population estimate of CLF is 3.28, 8.49, and 9.13 L/hr in black, Oriental, and Malay subjects, respectively. The relationship between apparent volume of distribution (VareaF) and weight was linear and similar in all three races at 7.98 L/kg. The population estimate of VareaF is 345, 383, and 428 L in black, Oriental, and Malay subjects, respectively. The bioavailability of the high and low doses of atovaquone was similar. Neither CLF nor VareaF were significantly affected by age, gender, and thecoadministration with chloroguanide (proguanil), pyrimethamine, and tetracycline. Half‐life (t12) showed a 0.326 power relationship with weight; thus, the population estimate of t12 in black, Oriental, and Malay subjects is 72.9, 31.3, and 32.5 hours, respectively. The final magnitudes of interpatient variability in CLF and VareaF were 68% and 49%, respectively.

Failure of Doxycycline as a Causal Prophylactic Agent against Plasmodium falciparum Malaria in Healthy Nonimmune Volunteers

With the growth of international travel, increasing numbers of nonimmune travelers may be at risk for exposure to Plasmodium falciparum malaria [1-3]. Since the emergence and spread of multidrug-resistant P. falciparum parasites [4, 5], the choice of drug to prevent malarial infections has become problematic [6, 7]. In most areas endemic for malaria, chloroquine is no longer the drug of choice. Chemoprophylaxis with Fansidar (Roche Laboratories; Nutley, New Jersey) has been discouraged in recent years after reports of severe life-threatening side effects [8] and the emergence of parasite resistance to this combination drug [9, 10]. The Centers for Disease Control and Prevention (CDC) list of alternative preventive agents for travelers to areas with chloroquine-resistant P. falciparum malaria includes mefloquine, doxycycline, or the combination of proguanil plus chloroquine [11]. Mefloquine is currently the drug of choice. However, reports of increasing parasite resistance to this drug [12-14] and possible mefloquine-associated neuropsychiatric side effects [15-17] may limit its usefulness as a prophylactic agent. Proguanil, although ineffective alone, is widely used overseas in combination with chloroquine. It is not, however, commercially available in the United States. Doxycycline is recommended for prevention of malaria in persons traveling for short periods to areas with chloroquine-resistant P. falciparum malaria who cannot tolerate mefloquine or for whom the drug is contra-indicated [11]. In addition, travelers to areas where mefloquine resistance has been identified, such as the Thai-Burmese border area, may be particularly good candidates for doxycycline prophylactic therapy. Current recommendations advise starting the drug 1 to 2 days before travel and taking it daily throughout the period of potential exposure to infected mosquitoes and for 4 weeks after returning from an endemic area. The terminal use of doxycycline for 4 weeks after possible exposure is intended to eliminate any erythrocytic parasites. This use is based on doxycyclines presumed activity as a suppressive prophylactic agent, that is, an agent effective against blood stages of the parasite. Continued daily drug ingestion for 4 weeks in the absence of symptoms, however, requires well-disciplined compliance and is expensive. It is important, therefore, to ensure that the use of doxycycline for 4 weeks after potential exposure has ceased is necessary. A drug that is active against the pre-erythrocytic liver stage, thereby preventing erythrocytic malaria, is called a causal prophylactic drug. If it is effective against P. falciparum parasites, such a drug prevents symptomatic malaria and needs to be taken only during the hepatic phase of infection, normally about 1 week from the time of mosquito bite. Studies in the early 1970s [18, 19] suggested that tetracycline compounds were effective as causal prophylactic agents. Minocycline, a semi-synthetic, long-acting tetracycline (T1/2, 18 h 4 h) (mean SD), when administered as 100 mg daily for 7 days, starting 1 day before exposure to P. falciparum parasites, prevented parasitemia in four of four persons [18]. Two other persons receiving 100 mg on the day of mosquito challenge and on day 3 after challenge were also protected. Doxycycline, another long-acting tetracycline analog (T1/2, 16 h 6 h) [20], has been shown in field trials to have prophylactic activity [21, 22]. Whether its mechanism of action was causal or suppressive, however, could not be determined. We designed our study to determine whether oral doxycycline, 100 mg administered daily, is effective as a causal prophylactic agent against P. falciparum malaria. If so, it would justify shortening the duration of terminal prophylactic therapy from 4 weeks to 1 week. Methods Volunteers were recruited under a protocol approved by the Human Use Review Committee, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, and the Surgeon Generals Human Subjects Research Review Board of the Department of the Army. Eighteen healthy men (age, 21.7 2.9 [SD] years) were selected after giving written informed consent. The screening evaluation included a baseline medical history, physical examination, electrocardiogram, and chest roentgenogram. Exclusion criteria included a history of malaria; splenectomy; allergy to doxycycline, tetracycline, or chloroquine; abnormal results of a complete blood count or tests for aspartate aminotransferase, alanine aminotransferase, bilirubin, lactic dehydrogenase, alkaline phosphatase, blood urea nitrogen, or creatinine; hematuria or proteinuria; glucose-6-phosphate-dehydrogenase deficiency; antibody to hepatitis B surface antigen; or antibody to human immunodeficiency virus. Informed Consent Prospective volunteers received a briefing and a written information sheet describing the procedures and potential risks associated with the study. Those willing to participate signed a consent form. None of the volunteers received compensation. They were assured of their right to withdraw from the study at any time without penalties. The study was designed to minimize the number of volunteers that might develop malaria. The number enrolled was based on the presumption that doxycycline protection rate would exceed 90% when administered as a causal prophylactic agent. Such a level of efficacy would have justified, in our opinion, changing the current CDC recommendation for terminal prophylactic therapy. The number of volunteers assigned to receive placebo was the minimum needed to ensure infectivity of the mosquitoes. To minimize morbidity, all participants were closely monitored throughout the study. Monitoring was intensified during periods of greatest likelihood of emergence of parasitemia by hospitalizing the participants on the medical ward. Given the close monitoring and the early treatment when parasitemia was low, the risk to the participants was felt to be minimal. Study Design The study was a randomized, double-blind, placebo-controlled trial. Doxycycline, 100 mg daily, was administered orally, starting 3 days before and ending 6 days after exposure to P. falciparum-infected mosquitoes. The study was implemented in two phases, 2 months apart. In phase 1, eight persons (group 1) were randomly assigned to receive doxycycline (six persons) or placebo (two persons). In phase 2, ten persons (group 2) were randomly assigned to receive either doxycycline (seven persons) or placebo (three persons). The drug used was doxycycline hyclate (Vibramycin, Pfizer Inc.; New York, New York), which was given in 100-mg capsules; identical placebo capsules were prepared by the University of Iowa Pharmaceutical Services Division. After an overnight fast the participants were hospitalized on the medical ward for 24 hours. Within 30 minutes after breakfast, either a doxycycline or a placebo capsule was administered to each participant. Nine samples for plasma doxycycline level measurements were then collected from each person during the first 24 hours, with eight additional samples collected during the following 12 days. On the afternoon of day 4 of drug administration, each participant was exposed to Anopheles stephensi mosquitoes infected with chloroquine-sensitive P. falciparum malaria. Giemsa-stained malaria smears were prepared daily from day 5 to day 15 after exposure and at least once weekly for 2 months. Thick and thin malaria smears were examined by two of the investigators. When present, parasitemia was quantified on a thick smear by the method of Earle and Perez [23]. All volunteers were hospitalized during week 2 after exposure, the time of highest risk for developing malaria. Those who remained well were discharged on day 15 and followed as outpatients. Persons who became febrile during the outpatient period were rehospitalized and evaluated for malaria with twice-daily blood smears. If parasites were found, standard chloroquine therapy, 1500-mg base for 48 hours, was administered. The participants were discharged after their blood films were clear of parasites and their symptoms resolved for 3 days. Induction of Malaria Infection Anopheles stephensi mosquitoes were used to transmit NF54 strain P. falciparum parasites. Female mosquitoes, 4 to 7 days after emergence, were membrane-fed on a mixture of cultured gametocytes [24], defibrinated blood, and human serum negative for hepatitis B surface antigen and human immunodeficiency virus antibody. Thirty-five days and 21 days after membrane feeding, the mosquitoes inoculated the first and second group of participants, respectively. To increase the survival rate of the mosquitoes in the first colony to 35 days, the environmental temperature was decreased from room temperature to 20 C in the last 2 weeks before human exposure. Mosquitoes used to induce infection in the second group were given a second blood meal when they were found to have low oocyst counts on day 6 after membrane feeding. On day 4 of drug administration, 6 to 8 hours after the morning medication, a cage containing five A. stephensi mosquitoes was placed on the forearm of each volunteer for 5 minutes. Those mosquitoes ingesting a blood meal were dissected and the sporozoite density of the paired salivary glands was quantified on a log-based categorical scale of 0 to 4. Each volunteer was then exposed to additional mosquitoes, as necessary, until five mosquitoes with sporozoite densities of 2 or greater had successfully taken a blood meal. Previous experience in our laboratory using these procedures resulted in an infection rate of 100% [25-27]. Doxycycline Plasma Concentration Seventeen plasma samples were collected for pharmacokinetic analysis: a sample before dosing and at 1, 2.5, 3, 4, 6, 9, 12, 24, 72, 75, 168, 215, 218, 240, 264, and 288 hours after the first dose. Three of the samples were collected at times corresponding to steady-state trough levels. The last three sam

Atovaquone and proguanil versus pyrimethamine/sulfadoxine for the treatment of acute falciparum malaria in zambia

Atovaquone and proguanil hydrochloride are blood schizonticides that demonstrate in vitro synergy against drug-resistant strains of Plasmodium falciparum. When coadministered, they may therefore be effective for the treatment of malaria in regions where there is known or suspected drug resistance. In an open-label, randomized, parallel-group, clinical trial conducted in Zambia, 163 patients (age range, 14 to 54 years) with acute P falciparum malaria were randomly assigned to receive treatment with atovaquone and proguanil hydrochloride (1000 and 400 mg, respectively, administered orally at 24-hour intervals for 3 doses; n = 82) or pyrimethamine/sulfadoxine (75/1500 mg administered orally as a single dose; n = 81). Efficacy was assessed by cure rate (the percentage of patients in whom parasitemia was eliminated and did not recur during 28 days of follow-up), parasite clearance time (PCT), and fever clearance time (FCT). Safety was determined by sequential clinical and laboratory assessments over 28 days. Cure rates did not differ significantly between patients treated with atovaquone and proguanil (100%) and those treated with pyrimethamine/sulfadoxine (98.8%). Patients in the atovaquone and proguanil group had a significantly shorter FCT than patients in the pyrimethamine/sulfadoxine group (mean, 30.4 vs 44.9 hours; P < 0.05) but a longer PCT (mean, 64.0 vs 51.4 hours; P < 0.05). Both treatments were well tolerated; adverse events and laboratory abnormalities were typical of those normally observed in patients with malaria. In this study, the combination of atovaquone and proguanil was equally effective and as well tolerated as pyrimethamine/sulfadoxine for the treatment of acute, uncomplicated, drug-resistant falciparum malaria in Zambia.

Combination atovaquone and proguanil hydrochloride vs. halofantrine for treatment of acute Plasmodium falciparum malaria in children.

BACKGROUND Malaria is a major cause of pediatric mortality in sub-Saharan Africa. Worldwide estimates of mortality among children with Plasmodium falciparum malaria range from 1 to 2 million deaths per year. Management of malaria is increasingly difficult because of the global spread of drug-resistant strains of P. falciparum. There is an urgent need for safe and effective new therapies to treat multidrug-resistant malaria. METHODS This open label, randomized trial compared atovaquone and proguanil hydrochloride with halofantrine for treatment of acute, uncomplicated P. falciparum malaria in children age 3 to 12 years (84 patients per group). Study drug dosages were adjusted by weight (approximately 20 and 8 mg/kg daily for three doses for atovaquone and proguanil hydrochloride and 8 mg/kg every 6 h for three doses for halofantrine). Patients were monitored by serial clinical and laboratory assessments for 28 days after starting treatment. RESULTS Both regimens were effective (cure rate, 93.8% for atovaquone and proguanil hydrochloride and 90.4% for halofantrine) and produced prompt defervescence. Mean parasite clearance times were 50.2 h for halofantrine and 64.9 h for atovaquone and proguanil hydrochloride. More adverse experiences were reported in children treated with halofantrine (119) than with atovaquone and proguanil hydrochloride (73). CONCLUSIONS In Kenyan children the combination of atovaquone and proguanil hydrochloride has efficacy comparable with that of halofantrine for treatment of acute uncomplicated multidrug-resistant falciparum malaria and is associated with a lower rate of adverse events.

New Experimental Antimalarial Drugs

Publisher Summary Malaria remains a serious worldwide health problem, and chemotherapy is one of the few effective measures for control of the disease. Quinine, a natural product obtained from the cinchona bark, was the first chemotherapeutic agent proven effective against malaria, and it retains an important role in the clinical management of this parasite infection. However, the periodic scarcities of quinine, especially during wartime, coupled with the emergence of plasmodia1 strains that are relatively resistant to this drug have stimulated the search for effective substitutes—especially among the synthetic chemicals. Although, all three major synthetic drug development programs have been highly successful, several factors have complicated the search for newer and more effective antimalarials: (1) The malaria parasite has shown a remarkable ability to develop resistance to antimalarial drugs, and many new drugs show cross-resistance to those previously developed; (2) novel leads are increasingly difficult to uncover, and efforts to do so often result in more complex organic molecules, with attendant synthetic problems; and (3) the development of new drugs must be accompanied by more sophisticated animal toxicity testing prior to clinical trials to minimize potential toxicity to humans. To meet these problems, continual work is required at various levels to develop new synthetic drugs and to develop new techniques for treating the disease.

Treatment of Falciparum Malaria from Vietnam with a Phenanthrene Methanol (WR 33063) and a Quinoline Methanol (WR 30090)

Two new investigational antimalarial drugs developed by the U.S. Army Malaria Research Program were tested in patients with multi-drug-resistant falciparum malaria from Vietnam. WR 33063, a phenanthrene methanol, cured 13 patients treated in the United States. All of these patients had suffered multiple recrudescences after treatment with standard antimalarial drugs. In addition, 23 of 25 patients with acute attacks of falciparum malaria treated in Vietnam were cured. The rate of clinical response was prompt. WR 30090, a quinoline methanol, similarly cured eight patients with multiple recrudescences in the United States and 23 of 26 patients in Vietnam. Adverse effects associated with the drugs were not seen. These drugs signify a major advance in the chemotherapy of drug-resistant falciparum malaria.

Trypanosoma rhodesiense: Semiautomated microtesting for quantitation of antitrypanosomal activity in vitro

Abstract A rapid, semiautomated microdilution method was developed to measure the antitrypanosomal activity of large numbers of compounds against blood form Trypanosoma rhodesiense at 37 C in vitro . Parasites harvested from infected rats were incubated for 3 hr in microtitration plates with serial dilutions of test compounds. Inhibition of uptake of radiolabeled thymidine and l -leucine by the parasites served as indicators of antitrypanosomal activity. Repeated measurements of the activity of ethidium bromide demonstrated the sensitivity and precision of the method. Several additional antitrypanosomal drugs were tested in vitro and all except tryparsamide (a pentavalent arsenical) and suramin (a complex polyanion) were consistent with in vivo activity. Antimicrobial agents which are not effective antitrypanosomal drugs failed to show activity in vitro . Other compounds active in vitro included allopurinol and a number of aromatic diamidines, carbamodithioic acid derivatives, and 2-acetylpyridine thiosemicarbazones. This in vitro microtest system was developed as a primary screen for the selection of active compounds in a new Antitrypanosomal Drug Development Program.