Transdermal administration of primaquine has been found to produce radical cure against sporozoite induced Plasmodium cynomolgi B infection in rhesus monkeys. Formulation containing 7mg/kg primaquine (base) was curative in 12 out of 13 rhesus monkeys as no relapse occurred during the observation period of 100 days

Dutta G.P.

Jain G.K.

Puri S.K.

Sarin J.P.S.

Singh Satyawan

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Trop. Med., 35 (3), 101-104, September, 1993 101Anti-Relapse Efficacy of Transdermal PrimaquineAgainst Plasmodium Cynomolgi B in Rhesus MonkeysS.K. PURI, G.K. JAIN, Satyawan SINGH, J.P.S. SARIN and G.P. DUTTADivision of Microbiology and Division of Pharmaceutics, Central DrugResearch Institute, Lucknow 226 001 INDIAAbstract: Transdermal administration of primaquine has been found to produce radicalcure against sporozoite induced Plasmodium cynomolgi B infection in rhesus monkeys. For-mulation containing 7mg/kg primaquine (base) was curative in 12 out of 13 rhesus monkeysas no relapse occurred during the observation period of 100 days.Key words: Plasmodium cynomolgi, Anti-relapse (antimalarial) efficacy, Primaquine, Transder-mal drug delivery.INTR ODUCTIONPrimaquine is the only 8-aminoquinoline drug in clinical use for treatment of relapses ofPlasmodium vivax infections. The drug is usually administered for 7-14 consecutive days toachieve radical cure and is known to produce adverse side-effect including gastrointestinaldistress, nausea and methaemoglobinemia with cyanosis (Clyde, 1981). Transdermal drugdelivery as an alternative mean of drug administration has gained, in recent years, high pa-tient acceptance in many clinical conditions and the advantages of delivering drugs across theskin for systemic circulation are well-documented (Price et at., 1981; Laufer et al, 1983;Karim, 1985; Shaw, 1983). This mode of drug administration can eliminate some of the side-effects associated with gastrointestinal absorption and also avoid deactivation of the drug byfirst-pass metabolism.Recent studies on the use of transdermal route for drug delivery in malaria are limitedto the preparations with blood schizontocidal agents like sodium artesunate and artemether(Xuan et al., 1988, 1990), and artelinic acid (Klayman et al., 1991). Klayman et at., (1991)recorded per cent curative and prophylactic efficacy with transdermal preparations ofartelinic acid against blood stages of P. berghei in mice. This is the first report on the suc-cessful use of transdermal preparation of tissue schizontocidal drug primaquine to achieveradical cure against sporozoite induced P. cynomolgi infection in rhesus monkeys.Received for Publication September 16, 1993Correspondence: S.K. Puri, Division of Microbiology, Central Drug Research Institute, Lucknow226 001, India.102MATERIALS AND METHODSParasite:Plasmodium cynomolgi bastianelli strain has been used for the present study. Thesporozoite induced infections of P. cynomolgi B in rhesus monkeys are being maintainedroutinely by cyclic transmission through Anopheles stephensi (Puri et at., 1989).Animals:The rhesus monkeys (Macaca mulatto} were obtained from approved contractors andquarantined for one month before experimentation. The monkeys were fed on pellets, bread,seasonal fruits and water was provided ad libitum. During quarantine, monkeys were tuber-culin tested and their blood smears were examined 2-3 times to ensure absence of anynatural blood protozoan infections.Preparation of primaquine transdermal tapesTransdermal tapes were prepared from 1 gm of the matrix filled in molds of 25.6 mmdiameter and 3 mmdepth. The molds were prepared from polypropylene sheets by hot presstechnique using a die of appropriate size. All the molds so prepared had constant surfacearea (5 cm2) available for drug release. The matrix was prepared according to method of Jainet al. (1992) by dissolving 65.5% of polyvinyl pyrolidone and 17% of polyethylene glycol in amixture of 1:1 dichloromethane and ethyl alcohol by aid of slight heat followed by removingthe solvent. To the residue, the required amount of primaquine diphosphate and 0.5% urea in17% propylene glycol were homogenously mixed. Each mold is uniformaly filled with 1 gmof this matrix and kept under vacuumfor 2-3 hrs to remove entrapped air if any. On the sur-face of matrix in each mold, 0.1 gm of azone was uniformaly spread. The matrix was subseq-quently allowed to cure for 24 hrs under vacuumat room temperature.Assay for anti-relapse activity:For efficacy tests, the monkeys were anaesthetized with 5 mg/kg ketaminhydrochloride (i.m.) and their upper arm was carefully shaved to avoid any cuts or abrasionson the skin. The bare skin was cleaned with alcohol before applying the matrix filled moldscontaining 1.75 mg base/kg to 7.00 mg base/kg primaquine. The molds were kept in contactwith monkey's skin by wrapping three-four layers of adhesive tape around the arm. Themolds were applied to the infected monkeys when parasitaemia level of approximately5000/mm3 was attained after sporozoite induced infections. These tapes were kept in placefor 7 days. The anti-relapse activity of primaquine was also established via oral route in 4monkeys which were administered 1 mg (base)/kg drug for 7 days.The test protocol for anti relapse activity established at this institute was followed (Dut-ta and Puri, 1988). Chloroquine at a dose of 5 mg (base)/kg x 7 days was concurrently ad-ministered as the companion blood schizontocide to eliminate blood stage parasites. Giemsastained blood smears from the experimental monkeys were observed to record the occurrenc-ce of relapse. If there was no relapse for 100 days after the last chloroquine dose, themonkeys were taken as cured (Dutta and Puri, 1988).103RESULT AND DISCUSSIONPrimaquine administered orally at 1 mg/kg x 7 days, has been found to produceradical cure against sporozoite induced P. cynomolgi B infection in 4/4 rhesus monkeys. Thisis in agreement with our earlier report (Dutta and Puri, 1988). Transdermal formulation ofprimaquine was evaluated for radical curative activity at 4 dose levels (1.75-7.00 mg base/kg)(Table-1). The tapes containing primaquine diphosphate equivalent to 1.75 mg base/kg werenot curative in any of the four monkeys. The tapes containing drug equivalent to 3.50 mg/kgprotected 1 out of 4 experimental monkeys. The tapes containing 5.25 mg base/kg prima-quine protected 5 out of 7 monkeys (cure rate 71.43%), while the tapes containing 7 mgbase/kg primaquine protected 12 out of 13 monkeys thus giving a cure rate of 92.3%. One ofthe 13 monkeys which was not protected with the highest dose, showed a delayed relapse onday 36. Four monkeys applied with placebo tapes (without primaquine), relapsed betweenday ll-19. The log-dose probit analysis of the present data gave the ED50 and ED95 values of4.38 mg/kg and 6.65 mg/kg respectively.The present study thus establishes that primaquine is active by transdermal route as ananti-relapse drug. Transdermal primaquine formulation is likely to enhance the dose com-pliance at it would eliminate the need of repeated drug intake and avoid gastric irritation.Table 1. Anti-relapse activity of primaquine transdermal tape againstsporozoite induced Plasmodium cynomolgi B infection inrhesus monkeys.No. of Relapse if any No ofPrimaquine* monkeys - monkeys**treated No. of Days after protected (%)mnnlrpw last dosemonkeys of chloroquine0.0 4 4 ll,14,14,19 Nil (0%)1-75 4 4 13,15,17,18 Nil (0%)3-50 4 3 17,17,19 1 (25%)5-25 7 2 18,22 5 (71.43%)7.00 13 1 36 12 (92.3%)Pri maquine1.00mg/kgx 7 4 4 (100%)days (oral)* The tape was applied on monkey's fore arm for 7 days andchloroquine 5 rag base/kgx 7 days was administered orally.** No relapse observed till 100 days after last chloroquine dose.104ACKNOWLEDGEMENTThe authors are grateful to Dr. V.P. Kamboj, Director, for continued encouragementand support. Technical Assistance rendered by Mr. K.K. Singh and Mr. Sant Ram isacknowledged. This is communication number 5060 from Central Drug Research Institute,Lucknow.REFERENCES1 ) Clyde, D.F. (1981): Clinical problems associated with the use of primaquine as a tissue schizontocidaland gametocytocidal drug. Bull. WHO, 59, 463-479.2 ) Dutta, G.P. & Puri, S.K. (1988): Standardization of P. cynomolgi B rhesus monkey model for radicalcurative test and primaquine sensitivity of the relapse stages of the parasite from different cyclicpassages. Ind. J. ParasitoL, 12, 99-107.3 ) Jain, G.K., Singh, S., Puri, S.K., Dutta, G.P. & Sarin, J.P.S. (1992): A process for making an im-proved transdermal device of primaquine diphosphate. Indian Patent Application No. 101/Del./92.4 ) Karim, A. (1983): Transdermal absorption: Unique opportunity for constant delivery ofnitroglycerine. Drug Development & Industrial Pharmacy, 9, 667-689.5 ) Klayman, D.L., Ager, A.L., Fleckenstein, L. & Lin, AJ. (1991): Transdermal artelinic acid: an effec-tive treatment for P. berghei infected mice. Am. J. Trop. Med. Hyg., 45, 602-607.6 ) Laufer, L.R., Faizu, J.L., Lu, J.K.H., Meldrum, D.R., Eggena, P., Sambhi, M.P., Hershman, J.M. &Judd, H. (1983): Estrogen replacement therapy by transdermal estradiol administration. Am. J. Obst.Gynecol., 146, 533-540.7) Price, N.M., Schmitt, L.G., McGuire, J., Shaw, J.E. & Trobough, G. (1981): Transdermalscopolamine in prevention of motion sickness at sea. Clin. Pharmacol. Therap., 29, 414-420.8 ) Puri, S.K., Kamboj, K.K. & Dutta, G.P. (1989): Infectivity studies on A. stephensi using P. cynomolgiB infection in rhesus monkeys. Trop. Med. ParasitoL, 40, 409-411.9 ) Shaw, J. (1983): Development of transdermal therapeutic System. Drug Development & IndustrialPharmacy, 9, 579-603.10) Xuan, W., Zhao, Y., Li, A., Xie, P. & Cai, Y. (1988): Experimental studies on the antimalarial activi-ty estimation by artesunate and artemether preparation per skin absorption. J. Tradit. Chin. Med.,8,282-284.ll) Xuan, W., Zhao, Y., Li, A., Xie, P. & Liu, X. (1990): Effect of artesunate transdermal preparationon P. cynomolgi. Acta Pharm. Sinica, 25, 220-222.

Anti-Relapse Efficacy of Transdermal Primaquine Against Plasmodium Cynomolgi B in Rhesus Monkeys

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Anti-Relapse Efficacy of Transdermal Primaquine Against Plasmodium Cynomolgi B in Rhesus Monkeys

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