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Enhanced efficacy of chloroquine-chlorpheniramine combination in acute uncomplicated falciparum malaria in children
TRANSACTIONS OFTHEROYALSOCIETY
OFTROPICALMEDICINEAND
Enhanced efficacy of chloroquine-chlorpheniramine uncomplicated falciparum malaria in children A. Sowunmil, A. M. J. Oduola’, salako2 ‘Department of Pharmacology versity
of Ibadan,
Ibadan,
0. A. T. Ogundahunsi’,
Nigeria;‘Nigerian
63
HYGIENE(1997)91,63-67
combination
C. 0. Falade’,
in acute
G. 0. Gbotosho’
and
and Therapeutics, Postgraduate Institute for Medical Research and Training, Institute for Medical Research, Edmund Crescent,Yaba, Lagos, Nigeria
L. A. Uni-
Abstract Chlorpheniramine, a histamine HI receptor antagonist, reverses chloroquine resistance in Plasmodium falciaarum in vitro. However. the clinical sianificance of this remains unclear. We have evaluated the efficacv of chloroquine and a chloroquine-chlorbheniramine combination in 112 consecutive children with acute symptomatic uncomplicated falciparum malaria. There was no significant difference in the parasite and fever clearance times in the 2 treatment groups. However, the proportion of patients in whom parasitaemia increased 24 h after commencement of treatment was significantly higher in the chloroquine group than in the chloroquine-chlorpheniramine group (285% vs. 8.3%,x2=6.61, PcO.01). There was also a higher proportion of children with RI1 and RI11 responses to treatment in the chloroquine than in the chloroquine-chlorpheniramine group but the difference was not statistically significant. The cure rate on day 14 was higher in the chloroquine-chlorpheniramine group than in the chloroquine group. Chloroquine and its combination with chlorpheniramine were well tolerated, the only prominent adverse effect being pruritus, with equal incidence in both groups. Chlorpheniramine reversed chloroquine resistance in vitro in a similar manner to verapamil in isolates of I? falciparum obtained from the patients. Failure of a response in vivo to chloroquine correlated with resistance in vitro in patients treated with this drug. In contrast, all but one patient with isolates which were chloroquine resistant in vitro were successfully treated with chloroquine-chlorpheniramine combination. These data suggest the enhanced efficacy of chloroquine-chlorpheniramine combination in treating acute uncomplicated l? falciparum infection in children from an endemic area of Nigeria. Keywords: malaria, Plasmodiumfulciparum,
chloroquine, chlorpheniramine,
Introduction The increasing spread of Plasmodium falciparum resistant to chloroquine necessitates judicious and efficient utilization of the few available antimalarial drugs. Some studies have been focused on development of combination therapy based on various phenomena, including the reversal of resistance by non-antimalarial drugs (MARTIN et al., 1987). Several non-antimalarial compounds of diverse classes have been shown to reverse resistance when combined with chloroquine in vitro or in animal models (BITONTI et al., 1988; PETERS et al., 1989;K~t~ etal., 1993; COUTAUX etal., 1994; RASOANAIVO et al., 1994). To date, none of the combinations has been used successfully in the treatment of chloroquine resistant malaria (BASCO & LE BRAS, 1991; WARSAME et al., 1992). Chlorpheniramine, a histamine H, receptor antagonist, has been shown to reverse chloroquine resistance in vitro in isolates of l? falciparum from south-west Nigeria and other African countries (OMITOWOJ~ et al., 1992; BASCO & LE BRAS, 1994). The drug is commonly prescribed with chloroquine to alleviate chloroquineinduced pruritus in children and adults in Nigeria and the combination is commercially available under several brand names for the treatment of malaria in Nigeria. Although the combination is not designated for drug resistant malaria, its use has become common practice among patients in Nigeria. Chloroquine resistance in viva is present in more than 15% of malaria infections among-children in south-west Nigeria (SOWUNMI et al., 1990). Currentlv, the incidence is closer to 50% (A. Sowunmi, L. A. Salako & A. M. J. Oduola, unpublished observations). In this report, the efficacy of chloroquine alone and chloroquine in combination with chlorpheniramine for the treatment of uncomplicated falciparum malaria in south-west Nigeria is described. The susceptibility in vitro and patterns of parasite clearance in patients treated with chloroquine or a combination of chloroquine and chlorpheniramine are also described. Address for correspondence: Professor A. M. J. Oduola, Postgraduate Institute for Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria.
children, Nigeria
Methods Studies
in vivo
One hundred and twenty-five consecutive children aged 6 months to 12 years presenting with acute symptomatic uncomplicated P. falciparum malaria were studied between February and July 1995. A patient was enrolled into the study if the following criteria were met: history of fever in the 24-48 h preceding presentation or pyrexia at presentation, pure I? falciparum parasitaemia with >500 asexual forms per uL of blood, no history of antimalarial drug administration in the 2 weeks preceding presentation, negative urine test for 4-aminoquinolines (Dill-Glazko) and sulphonamides (lignin), absence of other concomitant illness, and consent of parent or guardian. Children with sickle cell anaemia and those with severe illness requiring parenteral antimalarial drug were excluded from the study. A patient was withdrawn from the study if the drug was vomited, other concomitant illness developed during the followup period, or it was the desire of the parent or guardian to withdraw the patient from the study. The study protocol was reviewed and approved by the joint University of Ibadan/University College Hospital ethical committee. A careful history was taken, followed by thorough physical examination, of each patient before enrolment. Thereafter, each patient was weighed, axillary temperature taken, and thick and thin blood films prepared from a finger-prick and stained with Giemsa’s stain. The physical and other findings were recorded on a specially designed form. The children were randomly allocated to 2 treatment groups in a double-‘blind’, placebo-controlled study. One group received chloroquine base (25 mg/kg body weight) over 3 d (10 mg/kg on days 0 and 1, and 5 mg/ kg on day 2) and placebo every 8 h for 7 d (days O-6). The other group received chloroquine base (25 mg/kg body weight) ovef~3--dand chlorpheniramine 4 mg at presentation followed by 2 mg every 8 h for 7 d (days O-6) if the child was aged 6 months to 5 years, or 8 mg at presentation followed by 4 mg every 8 h for 7 d if the child was over 5 years of age. This dosing regimen was modified from a previous study of the pharmacokinetics
A. SOWiLTNMI
of chlorpheniramine in children (SIMON et al., 1982). All the drugs were administered orally, Chloroquine doses and the first daily doses of placebo or chlorpheniramine were adminstered by a physician and each child was observed for at least 3 h after drug administration in order to ensure that the drug was not vomited. If the drug was vomited within 3 h, the patient was excluded from the study. Subsequent doses, i.e., the doses of placebo or chlorpheniramine every 8 h, were not administered by the physician but by the parent or guardian. Each parent or guardian gave an account of drug administration (at home) each time the child was seen for follow-up. Clinical observations were recorded daily for 8 d (days O-7) and on day 14. Thick and thin blood films for malaria parasite counts were prepared at presentation and follow-up visits. At each visit, the children (and their parents or guardians) were questioned and the children were examined for the presence of adverse drug reactions. Additional management of some children included the administration of an antipyretic, for example paracetamol 10 mg/kg orally every 6-8 h for 24-36 h, and fanning and tepid sponging when necessary. Giemsa-stained blood films were examined under an oil immersion objective of a light microscope at x1000 magnification. Parasitaemia in thick films was estimated by counting parasites relative to leucocytes; either 500 asexual forms of I? falciparum or the number of such parasites corresponding to 1000 leucocytes were counted, whichever occurred first. From this figure, the parasite density was calculated using the patient’s known white cell count determined on admission. The parasite and fever clearance times and cure rates were defined as described previously (SOWUNMI et al., 1990). The cure rate was defined as the proportion of patients who remained free of parasitaemia at the fourteenth day of follow-up. Treatment was considered a failure if parasitaemia on day 3 was greater than 25% of the day 0 value, if parasitaemia did not clear by day 7, or if parasitaemia cleared before day 7 but reappeared before dav 14 WHO. 1994). All treatment failures were decoded and were either re-treated with the original drugs or with intramuscular artemether or intramuscular sulfadoxineipyrimethamine, depending on the clinical condition of the child. Studies in vitro Venous blood (3 mL) was obtained from all children at presentation and divided into 2 aliquots. One aliquot was used to evaluate parasite sensitivity to chloroquine alone and chloroquine in combination with verapamil or chlorpheniramine, using a modification of the World Health Organization (WHO) schizont inhibition assay (RECKMAN et al., 1978). The modification involved addition of verapamil or chlorpheniramine (1 .O xl Om6M) as resistance reversing agents in wells containing serial dilutions of chloroquine as described previously (OMITOWOJU et al., 1992; ODUOLA et al., 1993). Briefly, three-fold dilutions of chloroquine (0.66-500 ng/ mL) were prepared in duplicate columns of a 96 wells microtitre plate using an automated liquid handling system. The first row of the plate (row A) contained the highest concentration of chloroquine while the last row (row H) contained no drug and served as the control. Five identical test plates were prepared from this plate bv transferring 25 uL of the contents of each well to corresponding wells in each test plate. Twenty-five FL of buffered RPM1 1640 medium were added to the first 2 columns of the testglate. An identical volume (25 FL) containing 1.0x 1O- M chlorpheniramine or verapamil was added to successive duplicate columns of the test plate. Two hundred ltL of a suspension of patient’s blood sample (1: 10 in buffered RPM1 1640) were added to the contents of each well of the plate and incubated at 37°C for 24 h. Thick films were then prepared from the contents of the control well to monitor devel-
ETAL.
opment of the ring forms to schizonts. The contents of each well on plates on which schizonts had developed in the control well were spread on microscope slides, air dried, stained with Giemsa’s stain and schizonts were counted using standard WHO techniaues tRIECKJK4N et al., 1978). -Isolates were considered;0 be resistant if schizogony had occurred in wells containing 33.048 ngi mL or more of chloroquine (WHO standard) or if the minimum inhibitory concentration (MIC) was reduced when chloroquine was combined with verapamil. Data and statistical analysis Data not conforming to normal distribution, for example parasite counts, were log-transformed. Percentage reduction of parasitaemia at day 1 was calculated from the log-transformed data. Data analysis was done using Student’s t test, the x2 test with Yates’s correction, or the Mann-Whitney U test. Values are given as meansfstandard deviations. Values of P less than 0.05 were taken as significant. Attempts were made to correlate sensitivity in vivo and in vitro and to determine the relationship between sensitivity in vivo and reversal in vitro. Results Tests in vivo Sixty-four children received chloroquine while 61 were treated with chloroquine-chlorpheniramine. All patients who completed a minimum follow-up period of 4 d (days 0, 1, 2 and 3) were evaluated. A total of 112 children met the criteria and their data were evaluated; 59 received chloroquine and 53 were treated with chloroquine-chlorpheniramine. Forty-nine and 48 of the children treated with chloroquine and chloroquine-chlorpheniramine, respectively, completed at least 8 d of follow-up. The clinical and laboratory characteristics at enrolment of the children who completed a minimum followup period of 8 d are shown in Table 1. Their characterTable 1. Enrohnent clinical and laboratory data of children with acute uncomplicated falciparum malaria treated with chloroquine or a combination of chloroquine-chlorpheniramine Chloroquinea 49 No. 5e2.9 Age (years) Weight (kg) 17.4k6.4 Duration of fever (d) l-7 Temperature (“C) 384& 1.3 Parasitaemia (per l.tL) 25978 Geometric mean 504-458900 Range Haematocrit (%) Mean 31.2f6.0 Range 18-40 No. ~30% 16b Heart rate (per min) Mean 137f30 Range 84-200 Enlarged organ(s) Splenomegaly only Hepatomegaly only z Hepatosplenomegaly 15 aMearkSD %l=30. Cn=34.
except
where
Chloroquinechlorpheniraminea 48 6.9k3.2 20.3f8.1 l-7 38.4+ 1.1 34182 512-608400 31.9k4.8 19-40 8c 143f25 88-190 9 19
stated
istics at enrolment were similar except for anaemia. There was a significantly higher proportion of children with anaemia (haematocrit ~30%) among those treated
CHLOROQUINE-CHLORPHENIRAMINE
IN ACUTE
65
MALARIA
Table 2. Therapeutic responses of patients with acute uncomplicated falciparum malaria treated with chloroquine or a combination of chloroquine-chlorpheniramine Chloroquine No. 49 Fever clearance time (d) Mean 2.2+ 1.9 l-6 Range Parasite clearance time (d) 4.4*0.95a Mean Range l-6 No. with increased parasitaemia on day 1 13 Response (no. of patients) Cured 37 1
Chloroquinechlorpheniramine 48 1.6fl.l l-6 4.5+ 1.1 l-6 4 41 2
9 2
:,
75.5
85.4
21
RI11 Cure rate at day 14 (%)
sponses in the 2 groups was not statistically significant (22.4% vs. 10.4%. r2=2.63. -0.1). Re-treatment with chloroquine was &ried out in 2 patients with RI1 responses to initial treatment with chloroquine, and these failed to clear during a 14 d period of follow-up (RI1 response). One patient with an RI11 response among those treated with chloroquine became comatose, developing cerebral malaria 1.5 d after chloroquine treatment. Parasitaemia in this patient increased from 74 965 asexual forms per ltL of blood at day 0 to 202 125 l.tL at 1.5 d. The patient was successfully treated with intramuscular artemether. Prompt resolution of coma occurred 12 h after treatment and was followed by clearance of parasitaemia and fever by day 2 of artemether therapy. The other patient with an RI11 response (whose parasitaemia was 15 267 asexual forms/p1 on day 0 and 54 375&L blood on day 4) was treated with intramuscular sulfadoxine-pyrimethamine. Fever in this patient cleared within 3 d but parasitaemia failed to clear by day 7 (RI1 response to sulfadoxine-pyrimethamine). The patient subsequently responded to intramuscular artemether, given on day 14, with parasitaemia clearing within 2 d of treatment. Re-treatment with chloroquine-chlorpheniramine was carried out on day 14 in one patient with an RI1 response to chloroquine. Parasitaemia cleared in this patient by day 5, with no recurrence by day 7. The cure rate among all the children was higher in patients treated with chloroquine-chlorpheniramine than in those treated with chloroquine alone, but the difference was not statistically significant ~*=1.57,130~05). Seventeen children (11 treated with chloroquine and 6 treated with chloroquine-chlorpheniramine) did not complete 8 d of follow-up. Parasitological response in these children was evaluated up to days 3-5. Parasitaemia did not clear by day 3 in 82% (9111) of children in this group treated with chloroquine. Similarly, parasitaemia in 83% (516) of children treated with chloroquine-chlorpheniramine in the group did not clear by day 3. The difference in the 2 proportions was not statistically significant (DO.05). Seventeen children (11 treated with chloroquine and 6 treated with chloroquine-chlorpheniramine) did not complete 8 d of follow-up. Parasitological response in these children was evaluated up to days 3-5. Parasitaemia did not clear by day 3 in 82% (9111) of children in the group treated with chloroquine. Similarly, parasitaemia in 83% (516) of children treated with chloroquine-chlorpheniramine in the group did not clear by
%=38. bl=43.
with chloroquine than among those treated with chloroquine-chlorpheniramine (53.3% vs. 24.0%, x*=4.97, BO.02). The therapeutic responses of infections to treatment among the children are summarized in Table 2; 24.8% and 20.5% of baseline parasitaemia (parasite density on admission) was cleared by day 1 in children treated with chloroquine and chloroquine-chlorpheniramine, respectively. There was no significant difference in parasite clearance by day 1 using the 2 treatment regimens (Mann-Whitney test, 2~0.53, eO.59). Thirteen (26.5%) and 4 (8.3%) of the patients treated with chloroquine and chloroquine-chlorpheniramine, respectively, had increased parasitaemia on day 1 of treatment; this difference was statistically significant (x*=6.61, PcO.01). Parasites did not clear from the blood of 11 patients treated with chloroquine (9 RI1 responses and 2 RIII) or from the blood of 5 patients treated with chloroquine-chlorpheniramine (all RII). The difference in this proportion of RI1 and RI11 re-
Table 3. Sensitivity data in vitro and treatment outcome in patikents treated with chloroquine or a combination of chloroquine-chlorpheniramine
with
In viva
NCP NCP NCP NCP NCP NCI? NCP NCP NCP NCP NCP NW NCP NCP NCP NCP NCP
Treatmenta
18 24 6 50 17 44 41 45 30 34 51 32 21
1 33 38 52
2;
CQ E$
CQ EaQ CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP
Outcome
CQ
Successful Successful Successful Failed Failed Failed Failed Failed Successful Successful Successful Successful Successful Successful Failed Successful Successful
18.5 18.5
aCQ=chloroquine, CP=chlorpheniramine,VER=verapamil. bMIC=minimum inhibitory concentration. CSensitive status in vitro based on the reversal phenomenon
uncomplicated
In MIC
Patient no.
acute
55.6 55.6 6.2 18.5 55.6 55.6 18.5 18.5 18.5 18.5 18.5 55.6 55.6 166.7 500.0
using
verapamil.
(ng/mL)a>b CQ-CP 6.2 18.5 166.7 55.6 2.1 18.5 6.2 18.5 6.2 18.5 55.6 55.6 6.2 18.5 55.6
-
18.5
malaria
vitro
CQ-VER 18.5 18.5 55.6 55.6 2.1 6.2 6.2 18.5 18.5 18.5 18.5 18.5 6.2 6.2 18.5 2.1 500.0
Response to CQ Sensitive Sensitive Sensitive Sensitive Resistant Resistant Resistant Resistant Sensitive Sensitive Sensitive Sensitive Resistant Resistant Resistant Resistant Sensitive
A. SOWUNMI
66
day 3. The difference in the 2 proportions was not statistically significant (Z+O.OS). Side effects were mild in most cases and did not necessitate interruption of treatment in either group. Vomiting occurred between 6 and 24 h in 6 patients, 3 each in the chloroquine and chloroquine-chlorpheniramine groups. Drowsiness occurred in 24 of 47 patients (51%) after the first dose of chlorpheniramine. Pruritus was the most commonly observed adverse effect, occurring in 10.4% (5) and 10.6% (5) of patients in the chloroquine and chloroquine-chlorpheniramine groups, respectively. The itching was severe in 1 patient treated with chloroquine and in 2 patients receiving chloroquine-chlorpheniramine. Pruritus usually subsided within 48-72 h of its onset. Haematological profiles were similar and normal during the course of follow-up in both groups. in vitro Twenty-one isolates obtained from patients were successfully evaluated for susceptibility in vitro. Treatment outcome could not be determined in 4 of the patients from whom the isolates were obtained because they did not satisfy the criteria for evaluation. The sensitivity data in vitro and details of treatment outcome for 17 patients treated with the combination of chloroquine and chlorpheniramine (9) or chloroquine (8) are presented in Table 3. The MICs of chloroquine with parasites isolated from the patients ranged from 6.2 ng/mL to 500 ng/mL. The MICs were reduced by 66.1% to 98.7% when chloroquine was combined with verapamil for 8 of the 17 isolates, which were therefore considered to be resistant to chloroquine. Combination of chloroquine with verapamil did not change the MIC of chloroquine for the remaining 9 isolates (Table 3). When chloroquine was combined with chlorpheniramine, the MIC of chloroquine was reduced by 66.1% to 96.3% for 8 of the 16 isolates successfully evaluated. Combination of chlorpheniramine with chloroquine increased the MIC of chloroquine for 3 isolates, but did not change it for 5 isolates (Table 3). Four of the 8 isolates which were chloroquine resistant in vitro were obtained from patients treated with chloroquine-chlorpheniramine. Parasitaemia in 3 of these patients was-cleared following treatment with the combination. The third uatient failed treatment with the combination, despite an initial reduction in parasitaemia. The remaining 4 isolates resistant in vitro were obtained from patients treated with chloroquine alone. Infections in these 4 patients also failed to respond to chloroquine therapy. Treatment failure in the chloroquine group was associated with parasites which were shown to be chloroquine resistant in vitro by the reversal phenomenon. Treatment with chloroquine or chloroquine-chlorpheniramine was successful in 8 patients from whom isolates which were chloroquine sensitive in vitro had been obtained. Tests
Discussion
This study showed that the efficacy of a combination of chloroquine and chlornheniramine was better than chloroqume alone for the treatment of acute I? j&ipar-urn malaria infections. Eighty-five percent of the children treated with the combination, comnared to 75% of those treated with chloroquine alone, were cured in an area that is witnessing a gradual increase in resistance to chloroquine. Treatment failure among the children treated with chloroquine was associated with parasites resistant to chloroquine in vitro. In contrast, some isolates which demonstrated chloroquine resistance in vitro were obtained from children successfully treated with the combination of chloroquine and chlorpheniramine. Chloroquine-resistant parasites accounted for 15% of all infections in Ibadan in 1989 (SOWUNMI et al., 1990), and this figure has risen to nearly 50% recently (FALADE
ETAL.
al., 1997); it was 25% in the present study. The data obtained in vitro suggest that use of the combination of chloroquine with chlorpheniramine is beneficial in the treatment of malaria. Treatment outcome and the susceptibility in vitro of parasites isolated from the patients highlighted the enhanced efficacy of chloroquine when combined with chlorpheniramine. The children were successfully cured of infections with the combination despite resistance in vitro of isolates from some of the children and persistence of parasites on day 5 in one patient. Patterns of parasite clearance in the patients are also of interest with regard to the beneficial effects of combining chloroquine with chlorpheniramine. Parasitaemia in 2 patients was significantly higher than 25% of the pre-treatment count on day 3, indicating resistance based on the clinical standard for determining sensitivity in vivo. However, one of these patients was cured with the combination while the other, who received chloroquine alone, was not. Parasitaemia in the cured patient cleared on day 7. Although the role of immunity in potentiating the treatment cannot be discounted, the absence of a similar outcome in patients treated with chloroquine alone supports the enhanced efficacy of the combination. Absence of RI11 resistance. and the lower number of patients showing RI1 resistance, among those treated with the combination may also be an indication of its superior efficacy. The rationale for the better efficacy of this combination of chloroquine and chlorpheniramine is not readily apparent. However, the reduced capacity of chlorpheniramine to bind plasma proteins (SIMON et al., 1982) may have been an advantage compared with previous unsuccessful combinations (BASCO & LE BRAS, 1991; WARSAME et al., 1992). Failure of the previous trials was attributed to pharmacokinetic factors including high protein binding (BOULTER et al., 1993). A better understanding of the potential role of protein binding in the pharmacokinetic disposition of the 2 drugs in a combination and in treatment outcome may be useful in further efforts to apply the phenomenon clinically. The initial description (MARTIN et al., 1987) and subsequent confirmation (PETERS et al., 1989; O~WTOWOTU et al.. 1992; COUTAUX et al., 1994; RASOANAIVO et al., 1994) of the reversal phenomenon in vitro with I? falciparum provided new prospects of combination therapy against malaria. Although the phenomenon has been confirmed in animal models (BITONTI et al., 1988; KYLE et al., 1993), studies on its application in human infections have not realized the promise of the experiments in vivo (BASCO & LE BRAS. 1991: WARSAME et al., 1992). However, the results of the present study are encouraging and atErm the need to support continued work on the clinical application of the phenomenon. Although the value of the combination against drug-resistant malaria was not assessed in this study, the advantages seem obvious. Additional studies on dosage requirements will be necessary to establish the therapeutic efficacy of this combination of chloroquine and chlorpheniramine in the management of drug resistant malaria. et
Acknowledgements
These studies received financial support from the UNDPi World Bar&WHO Special Programme for Research and Training in Tropical Diseases. A. S. was in receipt of a WHO/ TDR Career Development Grant Award and A. M. J. 0. acknowledges a Rockefeller Biotechnology Career fellowship. We thank Mrs Peggy Bell and Mrs Olatoye for assistance in preparation of the paper. References
Basco, L. K. & Le Bras, J. (199 1). Reversal of chloroquine resistance with cyproheptadine in ‘wild’ strains of ZYasmodium falciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85,204-205. Basco, L. K. & Le Bras, J. (1994).
In virro reversal of chloro-
CHLOROQUINE-CHLORPHENIRAMINE
IN ACUTE
67
MALARIA
auine resistance with chlornheniramine against African isolates of Plasmodium falcipaFum. Japanese journal of Medical Science and Biology, 41,59-63. Bitonti, A. J., Sjoendoma, A., McCann, P. l?, Kyle, D. E., Oduola. A. M. T., Rossan, R. N., Milhous, W. K. & Davidson, D. E.,ir (1988). Reversal of chloroquinein the malaria parasite Plasmodium falciparum by desipramine. Science, 242, 1303-1305. Boulter, M. K., Bray, l? G., Howells, R. E. & Ward, S. E. (1993).The potential of desipramine to reverse chloroquine resistance of Plasmodium falcioarum is reduced bv its bindina to plasma protein. fiam&tions of the Royal Socieiy of Tropica Medicine and Hygiene, 87,303. Coutaux, A. F., Mooney, J. J. & Wirth, D. F. (1994). Neuronal monoamine reuptakeinhibitors enhance in vitro susceptibility to chloroquine in resistant PZasmodium falciparum. Antimicrobial Agents and Chemotherapy, 38, 1419-1421. Falade, C. O., Salako, L. A., Sowunmi, A., Oduola, A. M. J. & Larcier, P. (1997). Comparative efficacy of halofantrine, chloroquine and sulfadoxine-pyrimethamine in treatment of acute uncomplicated falciparum malaria in Nigerian children. Transactions of the Royal Society of Tropical Medicine and Hygiene, 91, 58-62. Kvle, D. E., Milhous, W. K. & Rossan, R. N. (1993). Reversal sof~Plasm~dium falcibarum resistance-to chloroauine in Panamanian Aorta mon-keys. American Journal of Tropical Medicine and Hygiene, 48, 126-133. Martin, S: K., Oduola, A. M. J. & Milhous, W. K. (1987). Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science, 235, 899-901. Oduola, A. M. J., Omitowoju, G. O., Gerena, L., Kyle, D. E., Milhous, W. K., Sowunmi, A. & Salako, L. A. (1993). Reversal of mefloquine resistance with penfluridol in isolates of Plasmodium falciparum from south-west Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 81, 81-83. Omitowoju, G. O., Ogundahunsi, 0. A. T., Milhous, W. K., Gerena, L., Sownumi, A., Schuster, B. G. & Oduola, A. M.
J. (1992). Chlorpheniramine: a resistance reversing agent with ootential clinical aunlication. American Society- of- Tram.. cal Medicine and Hygiene, Annual Meeting, Atlanta, Georgt’a, November l-4,1992, abstract no. 379. Peters, W., Ekong, R., Robinson, B. L. & Warhurst, D. C. (1989). Antihistaminic drugs that reverse chloroquine resistance in Plasmodium falciparum. Lancet, ii, 334-335. Rasoanaivo, P., Ratsimamangra-Urverg, S., Milijaona, R., Rafatro, H., Rakoto-Ratsimamanga, A., Galeffi, C. & Nicoletti, M. (1994). In vitro and in viva chloroquine-potentiating action of Strychnos myrtoides alkaloids against chloroquine-resistant strains of Plasmodium malaria. Planta Medica, 60, 13-16. Rieckmann, K. H., Campbell, G. H., Sax, L. J. & Mrema, J. (1978). Drug sensitivity of Plasmodium falciparum: an in vitro microtechnique. Lancet, i, 22-23. Simon, R. E. R., Luciuk, G. H. & Simons, K. J. (1982). Pharmacokinetics and efficacy of chlorpheniramine in children. Journal of Allergy and Clinical Immunology, 69,376-38 1. Sowunmi, A., Salako, L. A., Walker, 0. & Ogundahunsi, 0. A. T. (1990). Clinical efficacy of mefloquine in children suffering from chloroquine-resistant I’lasmodium falciparum malaria in Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 84, 761-764. Warsame, M., Wernsdorfer, W. H. & Bjorkman, A. (1992). Lack of effect of desipramine on the response to chloroquine of patients with chloroquine-resistant falciparum malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86, 235-236. WHO (1994). Antimalarial drug policies: data requirement and uncomplicated malaria and management of malaria in pregnancy. Geneva: World Health Organization, mimeographed documentWHO/MAU94.1070.
Received publication
18 April 1996; revised 6 August 1996
5 August
1996;
for
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OFTROPICALMEDICINEAND
Enhanced efficacy of chloroquine-chlorpheniramine uncomplicated falciparum malaria in children A. Sowunmil, A. M. J. Oduola’, salako2 ‘Department of Pharmacology versity
of Ibadan,
Ibadan,
0. A. T. Ogundahunsi’,
Nigeria;‘Nigerian
63
HYGIENE(1997)91,63-67
combination
C. 0. Falade’,
in acute
G. 0. Gbotosho’
and
and Therapeutics, Postgraduate Institute for Medical Research and Training, Institute for Medical Research, Edmund Crescent,Yaba, Lagos, Nigeria
L. A. Uni-
Abstract Chlorpheniramine, a histamine HI receptor antagonist, reverses chloroquine resistance in Plasmodium falciaarum in vitro. However. the clinical sianificance of this remains unclear. We have evaluated the efficacv of chloroquine and a chloroquine-chlorbheniramine combination in 112 consecutive children with acute symptomatic uncomplicated falciparum malaria. There was no significant difference in the parasite and fever clearance times in the 2 treatment groups. However, the proportion of patients in whom parasitaemia increased 24 h after commencement of treatment was significantly higher in the chloroquine group than in the chloroquine-chlorpheniramine group (285% vs. 8.3%,x2=6.61, PcO.01). There was also a higher proportion of children with RI1 and RI11 responses to treatment in the chloroquine than in the chloroquine-chlorpheniramine group but the difference was not statistically significant. The cure rate on day 14 was higher in the chloroquine-chlorpheniramine group than in the chloroquine group. Chloroquine and its combination with chlorpheniramine were well tolerated, the only prominent adverse effect being pruritus, with equal incidence in both groups. Chlorpheniramine reversed chloroquine resistance in vitro in a similar manner to verapamil in isolates of I? falciparum obtained from the patients. Failure of a response in vivo to chloroquine correlated with resistance in vitro in patients treated with this drug. In contrast, all but one patient with isolates which were chloroquine resistant in vitro were successfully treated with chloroquine-chlorpheniramine combination. These data suggest the enhanced efficacy of chloroquine-chlorpheniramine combination in treating acute uncomplicated l? falciparum infection in children from an endemic area of Nigeria. Keywords: malaria, Plasmodiumfulciparum,
chloroquine, chlorpheniramine,
Introduction The increasing spread of Plasmodium falciparum resistant to chloroquine necessitates judicious and efficient utilization of the few available antimalarial drugs. Some studies have been focused on development of combination therapy based on various phenomena, including the reversal of resistance by non-antimalarial drugs (MARTIN et al., 1987). Several non-antimalarial compounds of diverse classes have been shown to reverse resistance when combined with chloroquine in vitro or in animal models (BITONTI et al., 1988; PETERS et al., 1989;K~t~ etal., 1993; COUTAUX etal., 1994; RASOANAIVO et al., 1994). To date, none of the combinations has been used successfully in the treatment of chloroquine resistant malaria (BASCO & LE BRAS, 1991; WARSAME et al., 1992). Chlorpheniramine, a histamine H, receptor antagonist, has been shown to reverse chloroquine resistance in vitro in isolates of l? falciparum from south-west Nigeria and other African countries (OMITOWOJ~ et al., 1992; BASCO & LE BRAS, 1994). The drug is commonly prescribed with chloroquine to alleviate chloroquineinduced pruritus in children and adults in Nigeria and the combination is commercially available under several brand names for the treatment of malaria in Nigeria. Although the combination is not designated for drug resistant malaria, its use has become common practice among patients in Nigeria. Chloroquine resistance in viva is present in more than 15% of malaria infections among-children in south-west Nigeria (SOWUNMI et al., 1990). Currentlv, the incidence is closer to 50% (A. Sowunmi, L. A. Salako & A. M. J. Oduola, unpublished observations). In this report, the efficacy of chloroquine alone and chloroquine in combination with chlorpheniramine for the treatment of uncomplicated falciparum malaria in south-west Nigeria is described. The susceptibility in vitro and patterns of parasite clearance in patients treated with chloroquine or a combination of chloroquine and chlorpheniramine are also described. Address for correspondence: Professor A. M. J. Oduola, Postgraduate Institute for Medical Research and Training, College of Medicine, University of Ibadan, Ibadan, Nigeria.
children, Nigeria
Methods Studies
in vivo
One hundred and twenty-five consecutive children aged 6 months to 12 years presenting with acute symptomatic uncomplicated P. falciparum malaria were studied between February and July 1995. A patient was enrolled into the study if the following criteria were met: history of fever in the 24-48 h preceding presentation or pyrexia at presentation, pure I? falciparum parasitaemia with >500 asexual forms per uL of blood, no history of antimalarial drug administration in the 2 weeks preceding presentation, negative urine test for 4-aminoquinolines (Dill-Glazko) and sulphonamides (lignin), absence of other concomitant illness, and consent of parent or guardian. Children with sickle cell anaemia and those with severe illness requiring parenteral antimalarial drug were excluded from the study. A patient was withdrawn from the study if the drug was vomited, other concomitant illness developed during the followup period, or it was the desire of the parent or guardian to withdraw the patient from the study. The study protocol was reviewed and approved by the joint University of Ibadan/University College Hospital ethical committee. A careful history was taken, followed by thorough physical examination, of each patient before enrolment. Thereafter, each patient was weighed, axillary temperature taken, and thick and thin blood films prepared from a finger-prick and stained with Giemsa’s stain. The physical and other findings were recorded on a specially designed form. The children were randomly allocated to 2 treatment groups in a double-‘blind’, placebo-controlled study. One group received chloroquine base (25 mg/kg body weight) over 3 d (10 mg/kg on days 0 and 1, and 5 mg/ kg on day 2) and placebo every 8 h for 7 d (days O-6). The other group received chloroquine base (25 mg/kg body weight) ovef~3--dand chlorpheniramine 4 mg at presentation followed by 2 mg every 8 h for 7 d (days O-6) if the child was aged 6 months to 5 years, or 8 mg at presentation followed by 4 mg every 8 h for 7 d if the child was over 5 years of age. This dosing regimen was modified from a previous study of the pharmacokinetics
A. SOWiLTNMI
of chlorpheniramine in children (SIMON et al., 1982). All the drugs were administered orally, Chloroquine doses and the first daily doses of placebo or chlorpheniramine were adminstered by a physician and each child was observed for at least 3 h after drug administration in order to ensure that the drug was not vomited. If the drug was vomited within 3 h, the patient was excluded from the study. Subsequent doses, i.e., the doses of placebo or chlorpheniramine every 8 h, were not administered by the physician but by the parent or guardian. Each parent or guardian gave an account of drug administration (at home) each time the child was seen for follow-up. Clinical observations were recorded daily for 8 d (days O-7) and on day 14. Thick and thin blood films for malaria parasite counts were prepared at presentation and follow-up visits. At each visit, the children (and their parents or guardians) were questioned and the children were examined for the presence of adverse drug reactions. Additional management of some children included the administration of an antipyretic, for example paracetamol 10 mg/kg orally every 6-8 h for 24-36 h, and fanning and tepid sponging when necessary. Giemsa-stained blood films were examined under an oil immersion objective of a light microscope at x1000 magnification. Parasitaemia in thick films was estimated by counting parasites relative to leucocytes; either 500 asexual forms of I? falciparum or the number of such parasites corresponding to 1000 leucocytes were counted, whichever occurred first. From this figure, the parasite density was calculated using the patient’s known white cell count determined on admission. The parasite and fever clearance times and cure rates were defined as described previously (SOWUNMI et al., 1990). The cure rate was defined as the proportion of patients who remained free of parasitaemia at the fourteenth day of follow-up. Treatment was considered a failure if parasitaemia on day 3 was greater than 25% of the day 0 value, if parasitaemia did not clear by day 7, or if parasitaemia cleared before day 7 but reappeared before dav 14 WHO. 1994). All treatment failures were decoded and were either re-treated with the original drugs or with intramuscular artemether or intramuscular sulfadoxineipyrimethamine, depending on the clinical condition of the child. Studies in vitro Venous blood (3 mL) was obtained from all children at presentation and divided into 2 aliquots. One aliquot was used to evaluate parasite sensitivity to chloroquine alone and chloroquine in combination with verapamil or chlorpheniramine, using a modification of the World Health Organization (WHO) schizont inhibition assay (RECKMAN et al., 1978). The modification involved addition of verapamil or chlorpheniramine (1 .O xl Om6M) as resistance reversing agents in wells containing serial dilutions of chloroquine as described previously (OMITOWOJU et al., 1992; ODUOLA et al., 1993). Briefly, three-fold dilutions of chloroquine (0.66-500 ng/ mL) were prepared in duplicate columns of a 96 wells microtitre plate using an automated liquid handling system. The first row of the plate (row A) contained the highest concentration of chloroquine while the last row (row H) contained no drug and served as the control. Five identical test plates were prepared from this plate bv transferring 25 uL of the contents of each well to corresponding wells in each test plate. Twenty-five FL of buffered RPM1 1640 medium were added to the first 2 columns of the testglate. An identical volume (25 FL) containing 1.0x 1O- M chlorpheniramine or verapamil was added to successive duplicate columns of the test plate. Two hundred ltL of a suspension of patient’s blood sample (1: 10 in buffered RPM1 1640) were added to the contents of each well of the plate and incubated at 37°C for 24 h. Thick films were then prepared from the contents of the control well to monitor devel-
ETAL.
opment of the ring forms to schizonts. The contents of each well on plates on which schizonts had developed in the control well were spread on microscope slides, air dried, stained with Giemsa’s stain and schizonts were counted using standard WHO techniaues tRIECKJK4N et al., 1978). -Isolates were considered;0 be resistant if schizogony had occurred in wells containing 33.048 ngi mL or more of chloroquine (WHO standard) or if the minimum inhibitory concentration (MIC) was reduced when chloroquine was combined with verapamil. Data and statistical analysis Data not conforming to normal distribution, for example parasite counts, were log-transformed. Percentage reduction of parasitaemia at day 1 was calculated from the log-transformed data. Data analysis was done using Student’s t test, the x2 test with Yates’s correction, or the Mann-Whitney U test. Values are given as meansfstandard deviations. Values of P less than 0.05 were taken as significant. Attempts were made to correlate sensitivity in vivo and in vitro and to determine the relationship between sensitivity in vivo and reversal in vitro. Results Tests in vivo Sixty-four children received chloroquine while 61 were treated with chloroquine-chlorpheniramine. All patients who completed a minimum follow-up period of 4 d (days 0, 1, 2 and 3) were evaluated. A total of 112 children met the criteria and their data were evaluated; 59 received chloroquine and 53 were treated with chloroquine-chlorpheniramine. Forty-nine and 48 of the children treated with chloroquine and chloroquine-chlorpheniramine, respectively, completed at least 8 d of follow-up. The clinical and laboratory characteristics at enrolment of the children who completed a minimum followup period of 8 d are shown in Table 1. Their characterTable 1. Enrohnent clinical and laboratory data of children with acute uncomplicated falciparum malaria treated with chloroquine or a combination of chloroquine-chlorpheniramine Chloroquinea 49 No. 5e2.9 Age (years) Weight (kg) 17.4k6.4 Duration of fever (d) l-7 Temperature (“C) 384& 1.3 Parasitaemia (per l.tL) 25978 Geometric mean 504-458900 Range Haematocrit (%) Mean 31.2f6.0 Range 18-40 No. ~30% 16b Heart rate (per min) Mean 137f30 Range 84-200 Enlarged organ(s) Splenomegaly only Hepatomegaly only z Hepatosplenomegaly 15 aMearkSD %l=30. Cn=34.
except
where
Chloroquinechlorpheniraminea 48 6.9k3.2 20.3f8.1 l-7 38.4+ 1.1 34182 512-608400 31.9k4.8 19-40 8c 143f25 88-190 9 19
stated
istics at enrolment were similar except for anaemia. There was a significantly higher proportion of children with anaemia (haematocrit ~30%) among those treated
CHLOROQUINE-CHLORPHENIRAMINE
IN ACUTE
65
MALARIA
Table 2. Therapeutic responses of patients with acute uncomplicated falciparum malaria treated with chloroquine or a combination of chloroquine-chlorpheniramine Chloroquine No. 49 Fever clearance time (d) Mean 2.2+ 1.9 l-6 Range Parasite clearance time (d) 4.4*0.95a Mean Range l-6 No. with increased parasitaemia on day 1 13 Response (no. of patients) Cured 37 1
Chloroquinechlorpheniramine 48 1.6fl.l l-6 4.5+ 1.1 l-6 4 41 2
9 2
:,
75.5
85.4
21
RI11 Cure rate at day 14 (%)
sponses in the 2 groups was not statistically significant (22.4% vs. 10.4%. r2=2.63. -0.1). Re-treatment with chloroquine was &ried out in 2 patients with RI1 responses to initial treatment with chloroquine, and these failed to clear during a 14 d period of follow-up (RI1 response). One patient with an RI11 response among those treated with chloroquine became comatose, developing cerebral malaria 1.5 d after chloroquine treatment. Parasitaemia in this patient increased from 74 965 asexual forms per ltL of blood at day 0 to 202 125 l.tL at 1.5 d. The patient was successfully treated with intramuscular artemether. Prompt resolution of coma occurred 12 h after treatment and was followed by clearance of parasitaemia and fever by day 2 of artemether therapy. The other patient with an RI11 response (whose parasitaemia was 15 267 asexual forms/p1 on day 0 and 54 375&L blood on day 4) was treated with intramuscular sulfadoxine-pyrimethamine. Fever in this patient cleared within 3 d but parasitaemia failed to clear by day 7 (RI1 response to sulfadoxine-pyrimethamine). The patient subsequently responded to intramuscular artemether, given on day 14, with parasitaemia clearing within 2 d of treatment. Re-treatment with chloroquine-chlorpheniramine was carried out on day 14 in one patient with an RI1 response to chloroquine. Parasitaemia cleared in this patient by day 5, with no recurrence by day 7. The cure rate among all the children was higher in patients treated with chloroquine-chlorpheniramine than in those treated with chloroquine alone, but the difference was not statistically significant ~*=1.57,130~05). Seventeen children (11 treated with chloroquine and 6 treated with chloroquine-chlorpheniramine) did not complete 8 d of follow-up. Parasitological response in these children was evaluated up to days 3-5. Parasitaemia did not clear by day 3 in 82% (9111) of children in this group treated with chloroquine. Similarly, parasitaemia in 83% (516) of children treated with chloroquine-chlorpheniramine in the group did not clear by day 3. The difference in the 2 proportions was not statistically significant (DO.05). Seventeen children (11 treated with chloroquine and 6 treated with chloroquine-chlorpheniramine) did not complete 8 d of follow-up. Parasitological response in these children was evaluated up to days 3-5. Parasitaemia did not clear by day 3 in 82% (9111) of children in the group treated with chloroquine. Similarly, parasitaemia in 83% (516) of children treated with chloroquine-chlorpheniramine in the group did not clear by
%=38. bl=43.
with chloroquine than among those treated with chloroquine-chlorpheniramine (53.3% vs. 24.0%, x*=4.97, BO.02). The therapeutic responses of infections to treatment among the children are summarized in Table 2; 24.8% and 20.5% of baseline parasitaemia (parasite density on admission) was cleared by day 1 in children treated with chloroquine and chloroquine-chlorpheniramine, respectively. There was no significant difference in parasite clearance by day 1 using the 2 treatment regimens (Mann-Whitney test, 2~0.53, eO.59). Thirteen (26.5%) and 4 (8.3%) of the patients treated with chloroquine and chloroquine-chlorpheniramine, respectively, had increased parasitaemia on day 1 of treatment; this difference was statistically significant (x*=6.61, PcO.01). Parasites did not clear from the blood of 11 patients treated with chloroquine (9 RI1 responses and 2 RIII) or from the blood of 5 patients treated with chloroquine-chlorpheniramine (all RII). The difference in this proportion of RI1 and RI11 re-
Table 3. Sensitivity data in vitro and treatment outcome in patikents treated with chloroquine or a combination of chloroquine-chlorpheniramine
with
In viva
NCP NCP NCP NCP NCP NCI? NCP NCP NCP NCP NCP NW NCP NCP NCP NCP NCP
Treatmenta
18 24 6 50 17 44 41 45 30 34 51 32 21
1 33 38 52
2;
CQ E$
CQ EaQ CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP CQ+CP
Outcome
CQ
Successful Successful Successful Failed Failed Failed Failed Failed Successful Successful Successful Successful Successful Successful Failed Successful Successful
18.5 18.5
aCQ=chloroquine, CP=chlorpheniramine,VER=verapamil. bMIC=minimum inhibitory concentration. CSensitive status in vitro based on the reversal phenomenon
uncomplicated
In MIC
Patient no.
acute
55.6 55.6 6.2 18.5 55.6 55.6 18.5 18.5 18.5 18.5 18.5 55.6 55.6 166.7 500.0
using
verapamil.
(ng/mL)a>b CQ-CP 6.2 18.5 166.7 55.6 2.1 18.5 6.2 18.5 6.2 18.5 55.6 55.6 6.2 18.5 55.6
-
18.5
malaria
vitro
CQ-VER 18.5 18.5 55.6 55.6 2.1 6.2 6.2 18.5 18.5 18.5 18.5 18.5 6.2 6.2 18.5 2.1 500.0
Response to CQ Sensitive Sensitive Sensitive Sensitive Resistant Resistant Resistant Resistant Sensitive Sensitive Sensitive Sensitive Resistant Resistant Resistant Resistant Sensitive
A. SOWUNMI
66
day 3. The difference in the 2 proportions was not statistically significant (Z+O.OS). Side effects were mild in most cases and did not necessitate interruption of treatment in either group. Vomiting occurred between 6 and 24 h in 6 patients, 3 each in the chloroquine and chloroquine-chlorpheniramine groups. Drowsiness occurred in 24 of 47 patients (51%) after the first dose of chlorpheniramine. Pruritus was the most commonly observed adverse effect, occurring in 10.4% (5) and 10.6% (5) of patients in the chloroquine and chloroquine-chlorpheniramine groups, respectively. The itching was severe in 1 patient treated with chloroquine and in 2 patients receiving chloroquine-chlorpheniramine. Pruritus usually subsided within 48-72 h of its onset. Haematological profiles were similar and normal during the course of follow-up in both groups. in vitro Twenty-one isolates obtained from patients were successfully evaluated for susceptibility in vitro. Treatment outcome could not be determined in 4 of the patients from whom the isolates were obtained because they did not satisfy the criteria for evaluation. The sensitivity data in vitro and details of treatment outcome for 17 patients treated with the combination of chloroquine and chlorpheniramine (9) or chloroquine (8) are presented in Table 3. The MICs of chloroquine with parasites isolated from the patients ranged from 6.2 ng/mL to 500 ng/mL. The MICs were reduced by 66.1% to 98.7% when chloroquine was combined with verapamil for 8 of the 17 isolates, which were therefore considered to be resistant to chloroquine. Combination of chloroquine with verapamil did not change the MIC of chloroquine for the remaining 9 isolates (Table 3). When chloroquine was combined with chlorpheniramine, the MIC of chloroquine was reduced by 66.1% to 96.3% for 8 of the 16 isolates successfully evaluated. Combination of chlorpheniramine with chloroquine increased the MIC of chloroquine for 3 isolates, but did not change it for 5 isolates (Table 3). Four of the 8 isolates which were chloroquine resistant in vitro were obtained from patients treated with chloroquine-chlorpheniramine. Parasitaemia in 3 of these patients was-cleared following treatment with the combination. The third uatient failed treatment with the combination, despite an initial reduction in parasitaemia. The remaining 4 isolates resistant in vitro were obtained from patients treated with chloroquine alone. Infections in these 4 patients also failed to respond to chloroquine therapy. Treatment failure in the chloroquine group was associated with parasites which were shown to be chloroquine resistant in vitro by the reversal phenomenon. Treatment with chloroquine or chloroquine-chlorpheniramine was successful in 8 patients from whom isolates which were chloroquine sensitive in vitro had been obtained. Tests
Discussion
This study showed that the efficacy of a combination of chloroquine and chlornheniramine was better than chloroqume alone for the treatment of acute I? j&ipar-urn malaria infections. Eighty-five percent of the children treated with the combination, comnared to 75% of those treated with chloroquine alone, were cured in an area that is witnessing a gradual increase in resistance to chloroquine. Treatment failure among the children treated with chloroquine was associated with parasites resistant to chloroquine in vitro. In contrast, some isolates which demonstrated chloroquine resistance in vitro were obtained from children successfully treated with the combination of chloroquine and chlorpheniramine. Chloroquine-resistant parasites accounted for 15% of all infections in Ibadan in 1989 (SOWUNMI et al., 1990), and this figure has risen to nearly 50% recently (FALADE
ETAL.
al., 1997); it was 25% in the present study. The data obtained in vitro suggest that use of the combination of chloroquine with chlorpheniramine is beneficial in the treatment of malaria. Treatment outcome and the susceptibility in vitro of parasites isolated from the patients highlighted the enhanced efficacy of chloroquine when combined with chlorpheniramine. The children were successfully cured of infections with the combination despite resistance in vitro of isolates from some of the children and persistence of parasites on day 5 in one patient. Patterns of parasite clearance in the patients are also of interest with regard to the beneficial effects of combining chloroquine with chlorpheniramine. Parasitaemia in 2 patients was significantly higher than 25% of the pre-treatment count on day 3, indicating resistance based on the clinical standard for determining sensitivity in vivo. However, one of these patients was cured with the combination while the other, who received chloroquine alone, was not. Parasitaemia in the cured patient cleared on day 7. Although the role of immunity in potentiating the treatment cannot be discounted, the absence of a similar outcome in patients treated with chloroquine alone supports the enhanced efficacy of the combination. Absence of RI11 resistance. and the lower number of patients showing RI1 resistance, among those treated with the combination may also be an indication of its superior efficacy. The rationale for the better efficacy of this combination of chloroquine and chlorpheniramine is not readily apparent. However, the reduced capacity of chlorpheniramine to bind plasma proteins (SIMON et al., 1982) may have been an advantage compared with previous unsuccessful combinations (BASCO & LE BRAS, 1991; WARSAME et al., 1992). Failure of the previous trials was attributed to pharmacokinetic factors including high protein binding (BOULTER et al., 1993). A better understanding of the potential role of protein binding in the pharmacokinetic disposition of the 2 drugs in a combination and in treatment outcome may be useful in further efforts to apply the phenomenon clinically. The initial description (MARTIN et al., 1987) and subsequent confirmation (PETERS et al., 1989; O~WTOWOTU et al.. 1992; COUTAUX et al., 1994; RASOANAIVO et al., 1994) of the reversal phenomenon in vitro with I? falciparum provided new prospects of combination therapy against malaria. Although the phenomenon has been confirmed in animal models (BITONTI et al., 1988; KYLE et al., 1993), studies on its application in human infections have not realized the promise of the experiments in vivo (BASCO & LE BRAS. 1991: WARSAME et al., 1992). However, the results of the present study are encouraging and atErm the need to support continued work on the clinical application of the phenomenon. Although the value of the combination against drug-resistant malaria was not assessed in this study, the advantages seem obvious. Additional studies on dosage requirements will be necessary to establish the therapeutic efficacy of this combination of chloroquine and chlorpheniramine in the management of drug resistant malaria. et
Acknowledgements
These studies received financial support from the UNDPi World Bar&WHO Special Programme for Research and Training in Tropical Diseases. A. S. was in receipt of a WHO/ TDR Career Development Grant Award and A. M. J. 0. acknowledges a Rockefeller Biotechnology Career fellowship. We thank Mrs Peggy Bell and Mrs Olatoye for assistance in preparation of the paper. References
Basco, L. K. & Le Bras, J. (199 1). Reversal of chloroquine resistance with cyproheptadine in ‘wild’ strains of ZYasmodium falciparum. Transactions of the Royal Society of Tropical Medicine and Hygiene, 85,204-205. Basco, L. K. & Le Bras, J. (1994).
In virro reversal of chloro-
CHLOROQUINE-CHLORPHENIRAMINE
IN ACUTE
67
MALARIA
auine resistance with chlornheniramine against African isolates of Plasmodium falcipaFum. Japanese journal of Medical Science and Biology, 41,59-63. Bitonti, A. J., Sjoendoma, A., McCann, P. l?, Kyle, D. E., Oduola. A. M. T., Rossan, R. N., Milhous, W. K. & Davidson, D. E.,ir (1988). Reversal of chloroquinein the malaria parasite Plasmodium falciparum by desipramine. Science, 242, 1303-1305. Boulter, M. K., Bray, l? G., Howells, R. E. & Ward, S. E. (1993).The potential of desipramine to reverse chloroquine resistance of Plasmodium falcioarum is reduced bv its bindina to plasma protein. fiam&tions of the Royal Socieiy of Tropica Medicine and Hygiene, 87,303. Coutaux, A. F., Mooney, J. J. & Wirth, D. F. (1994). Neuronal monoamine reuptakeinhibitors enhance in vitro susceptibility to chloroquine in resistant PZasmodium falciparum. Antimicrobial Agents and Chemotherapy, 38, 1419-1421. Falade, C. O., Salako, L. A., Sowunmi, A., Oduola, A. M. J. & Larcier, P. (1997). Comparative efficacy of halofantrine, chloroquine and sulfadoxine-pyrimethamine in treatment of acute uncomplicated falciparum malaria in Nigerian children. Transactions of the Royal Society of Tropical Medicine and Hygiene, 91, 58-62. Kvle, D. E., Milhous, W. K. & Rossan, R. N. (1993). Reversal sof~Plasm~dium falcibarum resistance-to chloroauine in Panamanian Aorta mon-keys. American Journal of Tropical Medicine and Hygiene, 48, 126-133. Martin, S: K., Oduola, A. M. J. & Milhous, W. K. (1987). Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science, 235, 899-901. Oduola, A. M. J., Omitowoju, G. O., Gerena, L., Kyle, D. E., Milhous, W. K., Sowunmi, A. & Salako, L. A. (1993). Reversal of mefloquine resistance with penfluridol in isolates of Plasmodium falciparum from south-west Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 81, 81-83. Omitowoju, G. O., Ogundahunsi, 0. A. T., Milhous, W. K., Gerena, L., Sownumi, A., Schuster, B. G. & Oduola, A. M.
J. (1992). Chlorpheniramine: a resistance reversing agent with ootential clinical aunlication. American Society- of- Tram.. cal Medicine and Hygiene, Annual Meeting, Atlanta, Georgt’a, November l-4,1992, abstract no. 379. Peters, W., Ekong, R., Robinson, B. L. & Warhurst, D. C. (1989). Antihistaminic drugs that reverse chloroquine resistance in Plasmodium falciparum. Lancet, ii, 334-335. Rasoanaivo, P., Ratsimamangra-Urverg, S., Milijaona, R., Rafatro, H., Rakoto-Ratsimamanga, A., Galeffi, C. & Nicoletti, M. (1994). In vitro and in viva chloroquine-potentiating action of Strychnos myrtoides alkaloids against chloroquine-resistant strains of Plasmodium malaria. Planta Medica, 60, 13-16. Rieckmann, K. H., Campbell, G. H., Sax, L. J. & Mrema, J. (1978). Drug sensitivity of Plasmodium falciparum: an in vitro microtechnique. Lancet, i, 22-23. Simon, R. E. R., Luciuk, G. H. & Simons, K. J. (1982). Pharmacokinetics and efficacy of chlorpheniramine in children. Journal of Allergy and Clinical Immunology, 69,376-38 1. Sowunmi, A., Salako, L. A., Walker, 0. & Ogundahunsi, 0. A. T. (1990). Clinical efficacy of mefloquine in children suffering from chloroquine-resistant I’lasmodium falciparum malaria in Nigeria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 84, 761-764. Warsame, M., Wernsdorfer, W. H. & Bjorkman, A. (1992). Lack of effect of desipramine on the response to chloroquine of patients with chloroquine-resistant falciparum malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene, 86, 235-236. WHO (1994). Antimalarial drug policies: data requirement and uncomplicated malaria and management of malaria in pregnancy. Geneva: World Health Organization, mimeographed documentWHO/MAU94.1070.
Received publication
18 April 1996; revised 6 August 1996
5 August
1996;
for
accepted
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