Acta Tropica 69 (1998) 255–260

Causal prophylactic activity of antihistaminicagents against Plasmodium yoelii nigeriensis

infection in Swiss mice

Naresh Singh, S.K. Puri *

Di6ision of Microbiology, Central Drug Research Institute, Lucknow 226001, India

Received 12 September 1997; accepted 24 November 1997

Abstract

The causal prophylactic activity of five tricyclic anti-histaminic agents (histamine H1-recep-tor antagonists) cyproheptadine, ketotifen, loratadine, azatadine and terfenadine was evalu-ated in an experimental murine malaria model. Sporozoite induced infections withPlasmodium yoelii nigeriensis (N-67), a strain innately resistant to choloroquine, wereemployed for the efficacy test and pyrimethamine and primaquine were used as standardreference drugs. Treatment with cyproheptadine or ketotifen at 5 mg/kg and terfenadine at50 mg/kg, orally for 3 days (−1, 0, +1) completely prevented the establishment of patentinfection in mice inoculated with 1×105 sporozoites on day 0. Partial activity was recordedwith lower doses of the above agents as well as with azatadine and loratadine at 10 mg/kgas indicated by marginal delay in the development of patent infection after sporozoitechallenge. None of these agents showed blood schizontocidal activity at doses found effectivein the causal prophylactic test, though initial supression of parasitaemia was observed withcyproheptadine and ketotifen at higher doses. This study is the first report on efficacy ofantihistaminic agents for growth inhibition of pre-erythrocytic stages of any malaria parasite.© 1998 Elsevier Science B.V. All rights reserved.

Keywords: Cyproheptadine; Ketotifen; Terfenadine; Plasmodium yoelii nigeriensis (N-67);Causal prophylaxis

* Corresponding author. Fax: +91 0522 223405; e-mail: root@cscdri.ren.nic.in

0001-706X/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved.

PII S0001-706X(97)00138-1

N. Singh, S.K. Puri / Acta Tropica 69 (1998) 255–260256

1. Introduction

The continued spread of drug resistant Plasmodium falciparum malaria and theappearance of chloroquine resistance in P. 6i6ax malaria underlines the need tosearch novel antimalarial agents for treatment or prophylaxis against malaria. Withan estimated 300–500 million clinical cases suffering from malaria annually and anadditional tens of millions of people from non-malarious zones travelling to theendemic countries, chemoprophylactic intervention of parasite development bykilling the pre-erythrocytic stages appears to be the most feasible as well asefficacious medical approach to check the malaria menace. The declining efficacy ofhitherto used prophylactic agents like chloroquine and other blood schizontocides,has extremely limited the choice of prophylactic drugs for clinical application.Primaquine, a drug of choice for radical cure was demonstrated to possess truecausal prophylactic activity against P. falciparum in limited clinical studies (Powelland Brewer, 1967) but the inherent toxicity limited its wider application and fewnew leads have emerged for potential development as prophylactic agents. In asearch for new causal prophylactic agents, a few tricyclic compounds were evalu-ated, which are in clinical use as antihistaminic drugs (H1-receptor antagonists), fortheir protective efficacy against sporozoite induced P. yoelii nigeriensis (N-67)infection, a murine malaria parasite innately resistant to chloroquine. The antihis-taminic drugs selected for the study were cyproheptadine, ketotifen, azatadine,loratadine and terfenadine. Cyproheptadine and ketotifen have been documented topossess low level of blood schizontocidal activity against P. falciparum in vitro(Zhou et al., 1988), P. berghei in mice (Peters et al., 1990) and P. cynomolgi in themonkey (Huang et al., 1987). In addition, cyproheptadine, ketotifen and azatadinehave been shown to cause a marked reversal of chloroquine resistance against P.falciparum in vitro and P. yoelii in mice (Peters et al., 1990). The causal prophylac-tic activity of these agents in a rodent malaria model is documented in this report.

2. Materials and methods

2.1. Parasite

The gametocyte producing strain of P. yoelii nigeriensis (N-67) maintained in thelaboratory bred Swiss mice by syringe passage of infected blood or by cyclictransmission through Anopheles stephens i mosquitoes has been used for the study.

2.2. Host

Random bred Swiss mice (2092 g) of either sex were procured from theInstitute’s animal facility for experimental studies. The animals were maintained ona commercial pellet diet and water ad libitum under standard housing conditions.

N. Singh, S.K. Puri / Acta Tropica 69 (1998) 255–260 257

2.3. Animal inoculation

Sporozoites of P. yoelii nigeriensis (N-67) were harvested from infected Anophelesstephens i mosquitoes fed on gametocyte carrying golden hamster and maintainedfor 10–12 days in insectorium under controlled conditions of temperature (2792°C) and relative humidity (70–80%) as detailed elswhere (Singh et al., 1995). Atthe time of inoculation of sporozoites, aseptically separated mosquito thoraces werelightly homogenized in a glass grinder and diluted with chilled normal salinecontaining 25% serum. Each mouse was inoculated intravenously in the tail veinwith approximately 1×105 sporozoites suspended in 0.20 ml volume on day 0(Singh et al., 1995).

2.4. Causal prophylactic acti6ity

For the causal prophylactic activity, treatment with test compounds was adminis-tered orally to groups of mice at various dose levels for 3 consecutive days (−1, 0,+1). Primaquine and pyrimethamine have been used as the reference drugs.Giemsa stained blood smears from experimental animals were prepared from day 3onwards up to day 28 to record the occurrence of patency. Mice which remainednegative till day 28 were recorded as cured.

2.5. Blood schizontocidal acti6ity

The blood schizontocidal profile of the test agents was evaluated after a 4 doseadministration (day 0–3) to mice inoculated with 1×106 parasitized erythrocyteson day 0.

2.6. Drugs

Samples of cyproheptadine (Alvaetra Lab., India), ketotifen (Sun Pharma, India),azatadine (Fulford, India), loratadine (Cadila Health Care, India) and terfenadine(Cipla, India) were obtained commercially. The test agents were dissolved in waterand administered orally in 1-ml volumes. Pure samples of primaquine andpyrimethamine were obtained from WRAIR, Washington DC.

3. Results and discussion

The data on the causal prophylactic tests with five antihistaminic agents and thereference drugs are summarized in Table 1. The results show that cyproheptadine 5mg/kg, ketotifen 5 mg/kg and terfenadine 50 mg/kg showed 100% prophylacticactivity as none of the mice under these regimens developed patent infection.Efficacy tests with lower doses showed that 2 out of 10 mice treated withcyproheptadine at 2.5 mg/kg were cured and the remaining mice became patentbetween day 5–7. Ketotifen exhibited 50% protection at 2.5 mg/kg and patency in

N. Singh, S.K. Puri / Acta Tropica 69 (1998) 255–260258

another 5 mice was delayed to between 7 and 10 days. The pre-patent period wasalso extended in mice treated with a 1.25 mg/kg dose of the above two agents asalso in groups treated with terfenadine at 25 and 12.5 mg/kg dose levels. Azatadineand loratadine were not curative up to 50 mg/kg dose tested, however, thepre-patent period was extended over control values. Mice treated with Primaquineat 32 mg/kg and pyrimethamine at 0.1 mg/kg were cured while all the vehiclecontrol mice became patent between day 3–5.

In order to rule out the residual effect of these drugs against emerging exo-ery-throcytic merozoites and intra-erythrocytic stages, groups of mice were adminis-tered identical regimens of cyproheptadine (5 mg/kg), ketotifen (5 mg/kg) andterfenadine (50 mg/kg) for 3 consecutive days and animals were challenged with

Table 1Causal prophylactic activity of antihistaminic agents against sporozoite induced infection of Plasmod-ium yoelii nigeriensis (N-67) in Swiss mice

No. of miceDrug/dose (mg/kg) Response to treatment

% ProtectionDay of patency

Mean9S.E.Range

Ketotifen01.25 10 6–11 8.2090.51

502.5 10 7–10 8.6090.545.0 −10 − 100

Cyproheptadine01.25 10 5–6 5.5090.16

202.5 10 5–7 6.1390.21100−−5.0 10

Azatadine7–11 010 9.0090.3710.0

10 10.0090.32 050.0 8–11

Loratadine10 010.0 8.0090.456–10

050.0 10 7–11 9.2090.37

Terfenadine06.1090.225–712.5 10

10 7.0090.40 025.0 6–1010 −50.0 − 100

Primaquine10 − 10032.0 −

Pyrimethamine10 −0.1 100−

3–510 3.8090.19Control 0

−, Mice remained negative during the entire observation period.

N. Singh, S.K. Puri / Acta Tropica 69 (1998) 255–260 259

Table 2Response of cyproheptadine, ketotifen and terfenadine against blood induced Plasmodium yoeliinigeriensis (N-67) infection in Swiss mice

No. of miceDrug/dose Mean % parasitaemia (9S.E.) on day No. cured/treated(mg/kg)

7 104

Ketotifen0/1217.5091.635 9.1791.1912 2.6790.45

6.6791.0510 12 0.6790.19 1.3390.19 0/12Cyproheptadine

32.7892.26 0/125 12.5091.0512 3.0890.272.5890.30 21.4292.4410 12 0/12Nil

0/123.7590.5920 Nil12 Nil40 12 Toxic

Terfenadine61.6793.60 0/1250 31.4094.3812 9.6791.17

0/12Control 12 10.7590.79 33.2992.96

1×105 blood stage parasites, 24 h after the last dose to coincide with the time ofmaturation and rupture of intrahepatic schizonts. No significant difference in thecourse of parasitaemia in drug treated and control animals showed that antihis-taminic agents did not exert residual effect. In addition, the blood schizontocidalaction of ketotifen, cyproheptadine and terfenadine was also evaluated after astandard four-dose regimen and results are summarized in Table 2. Mice treatedwith terfenadine at 50 mg/kg dose developed parasitaemia similar to the untreatedcontrol while ketotifen and cyproheptadine also did not show any activity at dosesfound effective in causal prophylactic test. Tests at higher doses with ketotifen andcyproheptadine produced dose–dependent supression of parasitaemia as comparedto the control, though none of the mice were cured.

The low level of inherent blood schizontocidal activity of antihistaminic agents aswell as their chloroquine resistance modulation capacity has been documentedearlier (Peters et al., 1990). This report adds to their hitherto known antimalarialspectrum. However, the mode of action for any of these activities is entirelyunknown. The study also adds tricyclic antihistaminic H1-receptor antagonists tothe limited chemical classes known to exhibit causal prophylactic activity againstrodent malaria (Davidson et al., 1981). This new lead is intended to boost theefforts to find new prophylactic agents for prevention against drug resistant strainsand also to stimulate SAR studies to delineate antihistaminic and antimalarialactivities for developing a safer agent for mass chemoprophylaxis.

N. Singh, S.K. Puri / Acta Tropica 69 (1998) 255–260260

Acknowledgements

The authors are grateful to Dr C.M. Gupta, Director and Dr B.S. Srivastava,Head of the Division of Microbiology, Central Drug, Research Institute, Lucknow,India for constant encouragement and support. The authors are also thankful toMr G.P. Dixit, Mr K.K. Singh and Mr Sant Ram for technical assistance.Financial assistance in the form of fellowship under CDRI-WRAIR CollaborativeProject DAMD 17-93-J-3019 to one of the authors (NS) is acknowledged. This iscommunication no. 5710 from CDRI.

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