625 Am. J. Trop. Med. Hyg., 58(5), 1998, pp. 625–629 Copyright  1998 by The American Society of Tropical Medicine and Hygiene IN VITRO AND IN VIVO REVERSAL OF CHLOROQUINE RESISTANCE IN PLASMODIUM FALCIPARUM WITH PROMETHAZINE A. M. J. ODUOLA, A. SOWUNMI, W. K. MILHOUS, T. G. BREWER, D. E. KYLE, L. GERENA, R. N. ROSSAN, L. A. SALAKO, AND B. G. SCHUSTER Department of Pharmacology and Therapeutics, Postgraduate Institute for Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria; Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, District of Columbia; Gorgas Memorial Laboratory, Panama City, Panama Abstract. 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  10 -6 M) reversed chloroquine resistance in standard P. falciparum clones and patient parasite isolates from Nigeria. The combination reduced the 50% inhibitory concentrations (IC 50 s) for chlo- roquine 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 chlo- roquine 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/l 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 IC 50 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 suscep- tibility, 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 com- bination for treatment of uncomplicated chloroquine-resistant malaria. The reversal of chloroquine resistance by compounds with little intrinsic antimalarial activity is a well established phe- notype of drug-resistant Plasmodium falciparum. 1 In the past decade, numerous compounds have been shown to reverse resistance in vitro to chloroquine in parasite isolates from various geographic areas; 2–9 some of these compounds also reverse chloroquine resistance in animal models. 3, 10–12 Al- though limited clinical studies have failed to demonstrate a reversal of chloroquine resistance in human infections, 7, 13 potential clinical application of the phenomenon remains valid and provides a potentially innovative strategy to treat chloroquine-resistant malaria. Promethazine is an antihistamine that acts by competing with histamine for H-1 receptor sites on effector cells. This H-1 antagonist is also used as adjunct therapy in the treat- ment of malaria in English-speaking west African countries. The drug is given as an antiemetic with chloroquine to pre- vent or alleviate chloroquine-associated pruritus. 14 Common- ly, a dose of 5.0–10 mg is given simultaneously, or just prior to administration of chloroquine in children with falciparum malaria in Nigeria; in adults, daily doses of 25 mg are well tolerated. In this report, the in vitro and in vivo effects of promethazine on chloroquine-resistant P. falciparum were evaluated. The data from both in vitro and in vivo experi- ments demonstrate that promethazine is a potent modulator of chloroquine resistance in falciparum malaria. MATERIALS AND METHODS Parasites and drug susceptibility testing. Ten isolates of P. falciparum obtained from patients in Nigeria were tested in vitro for susceptibilities to chloroquine, desethylchloro- quine, quinine, mefloquine, and halofantrine alone and in combination with promethazine or verapamil. The chloro- quine-susceptible west African clone D6 and the multidrug- resistant Indochina clone W2 15 were used as reference par- asites. Parasites obtained from patients at the University Col- lege Hospital in Ibadan, Nigeria were adapted to continuous culture using standard techniques at the Walter Reed Army Institute of Research where these tests were conducted. 15 The parasites were cultured in human erythrocytes (type A + , 6.0% hematocrit) in vitro in RPMI 1640 culture medium supplemented with 10% human plasma. 16, 17 Each culture was maintained in 50-ml sealed culture flasks (Corning Glass Works, Corning, NY) at 37°C in an atmosphere of 3–5% O 2 , 2.5–4.0% CO 2 , and 90% N 2 (premixed bottled gas; Potomac Airgas, Hyattsville, MD). A modification of the semiauto- mated microdilution technique in which the hematocrit was 1.5% and the initial parasitemia was 0.5–0.8% was used to test the parasites’ susceptibilities to drugs. Suspensions of parasites, drug(s), and 3 H-hypoxanthine were incubated in microtitration plates for 42–46 hr at 37°C as described pre- viously. 4, 5, 18 Inhibition of 3 H-hypoxanthine incorporation by 50% (IC 50 ) was determined using a nonlinear regression analysis of the concentration-response curve.