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

Am. J. Trop. Med. Hyg., 58(5), 1998, pp. 625–629Copyright ᭧ 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, 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 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 ofchloroquine plus promethazine (1 ϫ 10Ϫ6 M) reversed chloroquine resistance in standard P. falciparum clones andpatient parasite isolates from Nigeria. The combination reduced the 50% inhibitory concentrations (IC s) for chlo- roquine against resistant parasites by 32–92%. Coadministration of promethazine with chloroquine also demonstrateda dose-dependent effect in Aotus monkeys infected with chloroquine-resistant P. falciparum. Monkeys were given achloroquine 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 lowestpromethazine 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 63days 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 ofclinically achievable doses of promethazine on parasites susceptibilities in vitro. Plasma samples taken at hourlyintervals from patients given a single oral dose of 25 mg of promethazine decreased the IC 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 optimizethe dose regimen in patients for antimalarial efficacy. Historic use of this drug combination for treatment or preventionof chloroquine-associated pruritus or as an antiemetic suggest that the combination is safe and effective when usedat standard dosages. The results from this study demonstrate that promethazine is a potent modulator of chloroquineresistance. 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- Parasites and drug susceptibility testing. Ten isolates of
notype of drug-resistant Plasmodium falciparum.1 In the past P. falciparum obtained from patients in Nigeria were tested decade, numerous compounds have been shown to reverse in vitro for susceptibilities to chloroquine, desethylchloro- resistance in vitro to chloroquine in parasite isolates from quine, quinine, mefloquine, and halofantrine alone and in various geographic areas;2–9 some of these compounds also combination with promethazine or verapamil. The chloro- reverse chloroquine resistance in animal models.3, 10–12 Al- quine-susceptible west African clone D6 and the multidrug- though limited clinical studies have failed to demonstrate a resistant Indochina clone W215 were used as reference par- reversal of chloroquine resistance in human infections,7, 13 asites. Parasites obtained from patients at the University Col- potential clinical application of the phenomenon remains lege Hospital in Ibadan, Nigeria were adapted to continuous valid and provides a potentially innovative strategy to treat culture using standard techniques at the Walter Reed Army Institute of Research where these tests were conducted.15 The Promethazine is an antihistamine that acts by competing parasites were cultured in human erythrocytes (type Aϩ, with histamine for H-1 receptor sites on effector cells. This 6.0% hematocrit) in vitro in RPMI 1640 culture medium H-1 antagonist is also used as adjunct therapy in the treat- supplemented with 10% human plasma.16, 17 Each culture was ment of malaria in English-speaking west African countries.
maintained in 50-ml sealed culture flasks (Corning Glass The drug is given as an antiemetic with chloroquine to pre- Works, Corning, NY) at 37ЊC in an atmosphere of 3–5% O , 2.5–4.0% CO , and 90% N (premixed bottled gas; Potomac vent or alleviate chloroquine-associated pruritus.14 Common- Airgas, Hyattsville, MD). A modification of the semiauto- ly, a dose of 5.0–10 mg is given simultaneously, or just prior mated microdilution technique in which the hematocrit was to administration of chloroquine in children with falciparum 1.5% and the initial parasitemia was 0.5–0.8% was used to malaria in Nigeria; in adults, daily doses of 25 mg are well test the parasites’ susceptibilities to drugs. Suspensions of tolerated. In this report, the in vitro and in vivo effects of parasites, drug(s), and 3H-hypoxanthine were incubated in promethazine on chloroquine-resistant P. falciparum were microtitration plates for 42–46 hr at 37ЊC as described pre- evaluated. The data from both in vitro and in vivo experi- viously.4, 5, 18 Inhibition of 3H-hypoxanthine incorporation by ments demonstrate that promethazine is a potent modulator 50% (IC ) was determined using a nonlinear regression of chloroquine resistance in falciparum malaria.
analysis of the concentration-response curve.
Evaluation of in vitro enhancing activities of prometh-
azine. Quantitative analysis of the increased activity of the
quinoline-containing antimalarial drug when combined with
promethazine was done by comparing concentration-re-
sponse curves for chloroquine alone and in the presence of
several fixed, subinhibitory concentrations of promethazine.
Effects of each fixed concentration of promethazine on the
response of the parasites (IC ) to the antimalarial drugs were
expressed as the response modification index (RMI).5 TheRMI was calculated by the following formula: RMI ϭIC , where drug A is a quinoline-containing an- timalarial and B is promethazine. An RMI of 1.0 representsno change in the IC combined with promethazine. The RMI values Ͻ 1.0 rep-resent the degree of potentiation or synergism.
Determination of biologic activity. A bioassay was used
at the University of Ibadan to measure the enhancing effectof promethazine in plasma from volunteers that had receivedpromethazine. Three volunteers 19–25 years old were given25 mg of promethazine each as a single oral dose (equal toa total daily dose for an adult). Similar blood samples wereobtained from two additional volunteers who did not takeany drug and used as controls. Blood samples (5 ml) were Effects of serial concentrations of promethazine on obtained from each volunteer prior to ingestion of the drug the susceptibility of chloroquine-sensitive (clone D6 -Ⅵ-), resistant- (if any) and every hour for 6 hr after ingestion of the drug.
(clone W2 -ⅷ-), and Nigerian isolates (NIG-82 -᭡-) and (NIG-60 The volunteers were observed for tolerance and any reaction -ࡗ-) of Plasmodium falciparum to chloroquine in vitro. The re-sponse modification index (RMI) is the ratio of the 50% inhibitory to the drug. Plasma from each volunteer was collected and concentrations (IC s) for chloroquine plus the concentration of pro- used in the bioassay to determine the ability of promethazine methazine, and chloroquine. An RMI of 1.0 represents no change in in the volunteer plasma to reverse chloroquine resistance in for chloroquine when combined with promethazine. The clones of P. falciparum in vitro. The effect of promethazine RMI values Ͻ 1.0 represent the degree of potentiation or synergism.
in plasma from volunteers was evaluated by using a modi-fication of the semiautomated microdilution technique.18 TheIC for chloroquine versus chloroquine-resistant clone W2 asite inoculation in each monkey when parasitemia ranged was measured in the presence of plasma routinely used for from 0.1 to 5.0ϫ 103 parasites/ml. Additional treatment was continuous culture of P. falciparum in the laboratory. Sim- initiated if there was recrudescence of infection during the ilar data were obtained for plasma obtained from volunteers 100 days of follow-up. Each treatment consisted of daily oral before taking promethazine. Reduction of the IC administration of a combination of chloroquine and pro- used as an index of promethazine concentration in the plas- methazine. Doses of promethazine (10 mg/kg, 20 mg/kg, 40 ma and its biological activity for reversing resistance to chlo- mg/kg, or 80 mg/kg) were combined with the standard dose roquine in vitro. The protocol for the studies in the volun- of chloroquine (20 mg/kg) daily for seven days. Two of the teers and patients was reviewed and approved by the Joint monkeys treated with chloroquine (20 mg/kg) alone were University College Hospital/University of Ibadan Ethical Committee. Potential volunteers were given both oral andwritten explanations of the study and their informed consent was obtained prior to enrollment in the study.
Evaluation of in vitro activities in a monkey model. The
Potentiation of chloroquine in vitro. Simultaneous ex-
effect of promethazine plus chloroquine on infection with posure of isolates and cloned strains of P. falciparum to the chloroquine-resistant Vietnam Smith/RE strain of P. fal- chloroquine plus subinhibitory concentrations of prometha- ciparum in owl monkeys (Aotus lemurinus lemurinus) was zine increased the intrinsic schizontocidal activities of chlo- evaluated at the Gorgas Memorial Laboratory as described roquine against the resistant parasites. This enhanced effect previously.3, 11 Six monkeys were each inoculated intrave- on chloroquine occurred in the presence of 17.8–284 ng/ml nously with 5.0ϫ 106 trophozoites of the parasite. The mon- of promethazine and increased the intrinsic schizonticidal keys were maintained following procedures and husbandry activity of chloroquine against the resistant isolates by 32– practices outlined in the Guide for the Care and Use of Lab- 92% (Figure 1 and Table 1). Identical concentrations of pro- oratory Animals as described previously.19 Infection in each methazine increased intrinsic activities of desethylchloro- monkey was monitored by microscopic examination of quine and quinine against the same parasites by 49–89%, Giemsa-stained blood smears prepared daily for 15 consec- and 29–79%, respectively. The IC s for desethylchloroquine utive days after inoculation; enumeration of parasitemia was against the isolates ranged from 7.08 ng/ml to 56.89 ng/ml done using the Earle-Perez method.20 Response of infection and were reduced to 6.3–35 ng/ml in the presence of 284 to treatment regimen was followed for 100 days in each monkey. Primary treatment was initiated five days after par- against the parasites ranged from 20.38 ng/ml to 57.73 REVERSAL OF CHLOROQUINE RESISTANCE WITH PROMETHAZINE Reversal of chlorquine resistance with promenthazine or verapamil Susceptibility of chloroquine-resistant reference clone W2 of Plas- in Nigerian isolates and reference cloned strains of Plasmodium modium falciparum to chloroquine in the presence of plasma from human volunteers given 25 mg of promethazine and a controlvolunteer that did not take any drug* ϭ 50% inhibitory concentration. Values in parentheses are the percent reductions † Plasma obtained from volunteer at designated time interval. The plasma was not heat inactivated to maintain drug integrity. Variation in IC is probably due to the lack of heat inactivation and antiparasitic activities in the semi- ‡ NA ϭ not available (sample was contaminated with bacteria).
ϭ 50% inhibitory concentration. Values in parentheses are the percent reductions † 1 ϫ 10Ϫ6 M promethazine.
‡ 1 ϫ 10Ϫ6 M verapamil.
were reduced by 45%, 54.7%, and 58%, respectively (Table3). Combination of plasma samples obtained from the con-trol volunteers at identical times did not reduce the IC ng/ml, and were reduced to 5.9–26.5 ng/ml in the presence ues for chloroquine against the parasites.
of promethazine (284 ng/ml). Similar combinations did not Activities in a monkey model. Response of infection in
have significant effect on susceptibilities of sensitive para- Aotus monkeys treated with chloroquine alone or a combi- sites to chloroquine, desethylchloroquine, or quinine. Incu- nation of chloroquine and promethazine showed that primary bation of parasites with identical concentrations of prometh- treatment with the combination ameliorated infection while azine alone reduced growth of both chloroquine-resistant and chloroquine alone had no effect on parasitemia (Table 4).
-sensitive parasites by 10% or less. Combination of pro- Additional treatment with higher doses of promethazine re- methazine with mefloquine or halofantrine did not have any sulted in cure of two monkeys with an infection that is nor- significant effects on the parasites susceptibilities to the an- mally chloroquine resistant. Parasitemia was cleared in three of the four monkeys treated initially with the standard dose either chloroquine-sensitive or -resistant parasites ranged of chloroquine combined with either 10 mg/kg (one monkey) from 443 ng/ml to 2,500 ng/ml (Table 2), confirming that or 20 mg/kg (two monkeys) of promethazine. Infection in the drug is not a potent antimalarial drug when used alone.
one monkey receiving chloroquine plus 10 mg/kg of pro- In vivo/in vitro biologic activities. The effects of plasma
methazine was only suppressed. This monkey was cured fol- obtained from human volunteers who took promethazine on lowing treatment with chloroquine and 20 mg/kg of pro- susceptibilities of the chloroquine-resistant W2 clone in a methazine. Infections in the other three monkeys recru- bioassay for determining reversal of chloroquine resistance desced between seven and 17 days after completing the ini- tial treatment (Table 4). Subsequent treatment in these resistant clone was reduced by 20–58% when the antima- monkeys resulted in cure of the infection in one monkey larial drug was combined with plasma samples obtained at given 20 mg/kg of chloroquine and 80 mg/kg of prometha- specific time intervals after the volunteers ingested prometh- zine. Infection in the other two monkeys was again cleared azine. The most significant reduction in the IC but recrudescence occurred 28 and 63 days after completing curred 3–4 hr after ingestion of the drug. The IC for chloroquine when combined with plasma obtained fromthe three volunteers 3 or 4 hr after ingestion of promethazine Response of chloroquine-resistant infection of the Plasmodium fal- ciparum Smith/RE strain in Aotus monkeys (Aotus lemurinus le- murinus) to treatment with chloroquine and promethazine Comparative susceptibilities of Nigerian isolates and reference cloned strains of Plasmadium falciparum to chloroquine, meflo- * Chloroquine, 20 mg/kg/day for seven days.
† Promethazine (mg/kg) given twice a day for seven days.
‡ Promethazine (mg/kg) given daily for seven days.
¶ CD ϭ animal was cured of drug-resistant infection with the combination.
# CL ( ) ϭ parasitemia was cleared but infection recrudesced in (8) days.
** Parasitemia at recrudescence was low (10 parasites/␮l of blood) and cleared without additional treatment (after 60 and 40 days in #89022 and #86040, respectively).
treatment. Parasitemia in the two monkeys at recrudescence a good candidate for further studies. Unlike cyprohepta- was low, less than 10 parasites/␮l of blood. The recrudescent dine,21 promethazine retained detectable reversing activities infection cleared without re-treatment in the two animals in human plasma after oral administration of drug to human volunteers. In addition, both chloroquine and promethazineare commonly used in combination in anglophone countriesof west Africa (for other reasons) and appear to be safe and well tolerated. Data on pharmacokinetic interactions of the The results of this study demonstrate that promethazine, two drugs are warranted to optimize dosage regimens of pro- an H-1 antagonist, is a potent modulator of chloroquine re- methazine plus chloroquine for a combination therapy in sistance in P. falciparum isolates and clones. Promethazine enhanced the activity of chloroquine against resistant para-sites in vitro and in vivo. Perhaps most importantly, potent Acknowledgments: We thank Dr. James Peggins and Dr. R. Keith chloroquine reversal activity was demonstrated in a bioassay Martin for critical review of the manuscript. We are grateful to Drs.
of plasma from volunteers that had taken promethazine.
O. A. T. Ogundahunsi and G. O. Omitowoju for contributions to this The costs of a complete dosage of mefloquine, halofan- study, and to Margaret Bell for secretarial assistance.
trine, or the derivatives of qinghaosu (artemether and arte- Financial support: This investigation received financial support from sunate) are prohibitively high for most residents of the poor the UNDP/World Bank/WHO Special Program for Research and malaria-endemic countries. This economic reality makes it Training in Tropical Diseases and the Rockefeller Foundation Bio- imperative that cheaper alternatives be available for treat- technology Career Fellowship Program.
ment of malaria. The phenomenon of reversal of chloroquine Authors’ addresses: A. M. J. Oduola, A. Sowunmi, and L. A. Salako, resistance could play an important role in efforts to control Department of Pharmacology and Therapeutics, Postgraduate Insti- drug-resistant malaria at economically feasible costs, es- tute for Medical Research and Training, College of Medicine, Uni-versity of Ibadan, Ilbadan, Nigeria. W. K. Milhous, T. G. Brewer, D.
pecially in Africa. A combination of chloroquine with pro- E. Kyle, L. Gerena, and B. G. Schuster, Division of Experimental methazine, or some other resistance modulator drugs, would Therapeutics, Walter Reed Army Institute of Research, Washington, be substantially cheaper than any of the currently available DC 20307-5100. R. N. Rossan, 9122 W. Viking Road, Las Vegas, antimalarial drugs. Unfortunately, a decade has elapsed since the description of the reversal phenomenon1 without a clearchoice among the potential resistance reversal agents or sig- nificant preclinical studies leading to development of a novelresistance modulator drug. Results of the present studies sug- 1. Martin SK, Oduola AMJ, Milhous WK, 1987. Reversal of chlo- gest that promethazine has potential to fill this need in the roquine resistance in Plasmodium falciparum by verapamil.
Science 235: 899–901.
2. Peters W, Ekong R, Robinson BL, Warhurst DC, Pan X, 1989.
The limited success among compounds tested in a monkey Antihistaminic drugs that reverse chloroquine resistance in model3, 11 and the failure in human volunteers7, 13, 21 has been Plasmodium falciparum. Lancet i: 334–335.
attributed in part to protein binding and pharmacokinetic in- 3. Bitonti AJ, Sjoerdsma A, McCann PP, Kyle DE, Oduola AMJ, teractions of the potential combinations. Both cyprohepta- Rossan RJ, Milhous WK, Davidson DE Jr, 1988. Reversal ofchloroquine resistance in the malaria parasite Plasmodium fal- dine and desipramine reversed resistance in P. falciparum in ciparum by desipramine. Science 243:1301–1306.
vitro and in animal models,2, 3, 7, 11 but failed to enhance an- 4. Oduola AMJ, Moyou-Somo RS, Kyle DE, Martin SK, Gerena timalarial activity of chloroquine in patients infected with P. L, Milhous WK, 1989. Chloroquine-resistant Plasmodium fal- falciparum.7, 13 Evaluation of blood obtained from a volunteer ciparum in indigenous residents of Cameroon. Trans R SocTrop Med Hyg 83: 308–310.
who took cyproheptadine also failed to show reversal of re- 5. Kyle DE, Oduola AMJ, Martin, SK, Milhous WK, 1990. Plas- sistance in vitro.21 The discrepancies between these in vitro modium falciparum: modulation by calcium antagonists of re- and in vivo observations with potential combination drugs sistance to chloroquine, desethylchloroquine, quinine, and are a major concern for a successful clinical application of quinidine in vitro. Trans R Soc Trop Med Hyg 84: 474–478.
the reversal phenomenon. Obviously, the selection of a suc- 6. Basco LK, Le Bras J, 1990. Reversal of chloroquine resistance with desipramine in isolates of Plasmodium falciparum from cessful combination must be based on several factors: these Central and West Africa. Trans R Soc Trop Med Hyg 84: include pharmacokinetics, pharmacodynamics, toxicity, and the ability to reverse chloroquine resistance.
7. Bjorkman A, Wilcox M, Kihamia CM, Mabikwang LF, Philips Binding of drugs to plasma proteins, especially albumin PA, Hakansson A, Warhurst D, 1990. Field study of cypro-heptadine/chloroquine synergism in falciparum malaria. Lan- and acute phase proteins is known to decrease antimalarial drug efficacy22, 23 and has been suggested to account for fail- 8. Sowunmi A, Oduola AM, Ogundahunsi OA, Falade CO, Gbo- ure of desipramine in clinical studies.24 The ability of pro- tosho GO, Salako LA, 1997. Enhanced efficacy of chloro- methazine in volunteers plasma to enhance chloroquine ac- quine-chlorpheniramine combination in acute uncomplicated tivity (after taking drug orally) suggests the H-1 antagonist falciparum malaria in children. Trans R Soc Trop Med Hyg91: 63–67.
might retain its resistance reversal activity in vivo. In addi- 9. Basco LK, LeBras J, 1994. In vitro reversal of chloroquine re- tion, coadministration of promethazine has been shown to sistance with chlorpheniramine against African isolates of Plasmodium falciparum. Jpn J Med Sci Biol 47: 59–63.
There is an urgent need to capitalize on the potential che- 10. Peters W, Ekong R, Robinson BL, Warhurst DC, Pan XQ, 1990.
The chemotherapy of rodent malaria. XLV. Reversal of chlo- motherapeutic advantages of the chloroquine resistance re- roquine resistance in rodent and human Plasmodium by an- versal phenomenon for the treatment of drug-resistant ma- tihistaminic agents. Ann Trop Med Parasitol 84: 541–551.
laria. The present observations suggest that promethazine is 11. Kyle DE, Milhous WK, Rossan RN, 1992. Reversal of Plas- REVERSAL OF CHLOROQUINE RESISTANCE WITH PROMETHAZINE modium falciparum resistance to chloroquine in Panamanian semiautomated microdilution technique. Antimicrob Agents Aotus monkeys. Am J Trop Med Hyg 48: 126–133.
12. Tanabe K, Kato M, Izumo A, Hagiwara A, Doi S, 1990. Plas- 19. Rossan RN, Happer III JS, Davidson DE Jr, Escajadillo A, modium chabaudii in vivo effect of Ca2ϩ antagonists on chlo- Christensen HA, 1985. Comparison of Plasmodium falcipa- roquine-resistant and chloroquine-sensitive parasites. Exp rum infections in Panamanian and Columbian owl monkeys.
Am J Trop Med Hyg 34: 1037–1047.
13. Warsame M, Wernsdorfer WH, Bjorkman A, 1993. Lack of ef- 20. Earle WC, Perez M, 1931. Enumeration of parasites in the blood fect of desipramine on the response to chloroquine of patients of malarial patients. J Lab Clin Med 19: 1124–1130.
with chloroquine-resistant falciparum malaria. Trans R Soc 21. Basco LK, Ringwald P, LeBras J, 1991. In vivo-in vitro test for chloroquine potentiation by cyproheptadine against Plasmo-dium falciparum. Trans R Soc Trop Med Hyg 85: 206–207.
14. Sowunmi A, Walker O, Salako LA, 1989. Prutitus and antima- 22. Silamut K, White NJ, Looareesuwan S, Warrell DA, 1985.
larial drugs in Africans. Lancet ii: 213.
Binding of quinine to plasma proteins in falciparum malaria.
15. Oduola AMJ, Weatherly NF, Dowdre JH, Desjardins RE, 1988.
Am J Trop Med Hyg 34: 681–686.
Plasmodium falciparum: cloning by single-erythrocyte micro- 23. Mihaly GW, Ching MS, Kle’u M, Paull J, Smallwood RA, 1987.
manipulation and heterogeneity in vitro. Exp Parasitol 66: Differences in the binding of quinine and quinidine to plasma proteins. Br J Clin Pharmacol 24: 769–774.
16. Trager W, Jensen JB, 1976. Human malaria parasites in contin- 24. Boulter MK, Bray PG, Howells RE, Ward SE, 1993. The po- uous culture. Science 193: 674–675.
tential of desipramine to reverse chloroquine resistance of 17. Haynes JD, Diggs CL, Hines FA, Desjardins RE, 1976. Culture Plasmodium falciparum is reduced by its binding to plasma of human malaria parasites Plasmodium falciparum. Nature protein. Trans R Soc Trop Med Hyg 87: 303.
25. Ehiwmua AO, Komolafe OO, Oyedeji GA, Olamijulo SK, 1988.
18. Desjardins 18. RE, Canfield CJ, Haynes JD, Chulay JD, 1979.
Effects of promethazine on the metabolism of chloroquine.
Quantitative assessment of antimalarial activity in vitro by a Eur J Drug Metab Pharmacokinet 13: 15–17.

Source: http://www.ajtmh.org/cgi/reprint/58/5/625.pdf