International Journal of Pharmacy and Pharmaceutical Sciences

ISSN- 0975-1491 Vol 3, Issue 2, 2011

ResearchArticle

CANLEVAMISOLEALONEMAINTAINTHEIMMUNITY?
DIVYENSHAHa*,VAISHALILONDHEa,R.MAZUMDERb,RIMAPARIKHc
SchoolofPharmacy&TechnologyManagementStudies,SVKMsNMIMS,Mumbai,India,bDepartmentofPharmaceuticalTechnology, NoidaInstituteofEngineeringandTechnology,Noida,India,cSchoolofPharmacy&TechnologyManagementStudies,SVKMsNMIMS, Shirpurcampus,Maharashtra,IndiaEmail:divyenshah@gmail.com
a

Received:26Dec2010,RevisedandAccepted:29Jan2011 ABSTRACT Theobjectiveofthepresentstudyistochecktheabilityoflevamisoletomaintaintheimmunitywhengivenaspretreatmentforchronicuse.Mainly medicinalplantsareusedtomaintainimmunity,butduetolackofscientificevidencetheyarenotgivenasmaintherapy.Theanimalsweregiven pretreatment of levamisole for 15, 30 and 45 days before being immunosuppressed by cyclophosphamide and evaluated for heamagglutination titre, delayed type hyper sensitivity reaction, cyclophosphamide reaction and neutrophil adhesion test. It was found that at least 30 days pretreatmentcanpreventimmunosuppression. Keywords: Sheep red blood cells (SRBC), Heamagglutination titre (HA titre), Delayed hypersensitivity sensitivity reaction (DTH reaction), Cyclophosphamideinducedmyelosuppression,neutrophiladhesiontest INTRODUCTION Lack of immunity is the main cause of many diseases like cancer, AIDS,autoimmunedisorderandmanyinfections.Lymphnodeplays an important role in maintenance of immunity.1There are many researches going on development of vaccines to prevent immunosuppression for particular diseases, but they are specific. Newer vaccines include highly purified subunit antigens that are weaklyimmunogenic.Vaccineformulationsoftenrequireadjuvants for increased immunological efficiency and better vaccination schedules.2Ithasbeenobservedthatmanyplantsareusedfornon specific activation of immune system. A number of plants used in traditionalmedicinesforrejuvenationtherapyandchronicailments havebeenshowntostimulateimmuneresponsesandseveralactive substances have been isolated. Plants like ashwagangha, shankpushpi, amala, indian ginseng, etc. are proved to be immunostimulantinimmunosuppressedcondition.3Butithasbeen observed that the active content in plants vary from place to place and seasonal to seasonal with a huge controversy of heavy metal contents. Even the immunostimulant effect can be due to some synergism. Theuseofmedicinal plantsinmodern medicinesuffers fromthefactthatthoughhundredsofplantsareusedintheworldto prevent or to cure diseases, scientific evidence in terms of modern medicineislackinginmostcases.Howevertoday itisnecessaryto providescientificproofaswhethertojustifythe use of plant or its active principles.4Therefore there is a strong need of synthesized drug, which can have a constant known therapeutic effect. Levamisoleisoneoftheidealcandidatesforthispurposebecauseof itsimmunomodulatoryeffect. Levamisole is mainly used as anthelmintic agent in veterinary purpose.5But in some countries its use is limited to immunomodulatory agent in humans in some cancers.6It is having immunostimulating effect in immunosuppressed condition.7It is usedasastandarddruginimmunosuppressedcondition.Butithas been found that it is also having useful effect in autoimmune diseaseslikenephroticsyndromeandrheumatoidarthritis.Ithelps tomakesteroidfreeperiodforupto6monthsto1yearinnephrotic syndrome.8,9 The objective of the present research work is to check effect of chronic pretreatment with levamisole for a particular period on prevention of immunosuppression, when given immunosuppression. MATERIALSANDMETHODS Animals: Unused Male Wistar rats (150200 g) were purchased from Hafkine institute, Mumbai, India. The rats were housed in polypropylene cages under standard conditions of temperature, humidity and light at animal house of School of Pharmacy & TechnologyManagement,Mumbai.Acclimationperiodof7dayswas given prior to experimentation. Water and feed were given ad libitum. The animal experiments were approved by institutional animalethicscommittee(IAEC)ofSchoolofPharmacy&Technology Management, SVKMs NMIMS University, Mumbai in accordance Committee for the purpose of control and supervision on experimentsonanimals(CPCSEA). Chemicals: Levamisole hydrochloride was gift sample from Ashish Life Science (Mumbai, India). Endoxan (Baxter, USA) a marketed formulation of cyclophosphamide was purchased. The other materials were of pharmaceutical and analytical grade and were procuredfromS.D.FineChemLtd(Mumbai,India). Antigen: Fresh blood was collected in Alsvers solution in 1/1 proportion from local slaughter house where sheep are sacrificed. Sheepredbloodcells(SRBCs)wereseparatedbycentrifugationand were washed three times in normal saline and adjusted to a concentrationof1109cells/mlforimmunizationandchallenge. Experimentaldesign:Theanimalsweredividedintofivegroups. Controlgroup 15dayspretreatment 30dayspretreatment 45dayspretreatment Negative control group (For cyclophosphamide induced myelosuppressiononly)

All the above groups are given cyclophosphamide (30 mg/kg, i.p.) for last 10 days during their pretreatment with levamisole (2.5 mg/kg,p.o.).Theparametersevaluatedafter chronic treatment are asfollow: 1. 2. 3. 4. Heamagglutinationtitre(HAtitre) Delayedhypersensitivityreaction(DTHreaction) Cyclophosphamideinducedmyelosuppression Neutrophiladhesiontest

Thedetailmethodforeachevaluationparameterisgivenbelow: 1. Heamagglutinationtitre(HAtitre)10,11,12,13,15,16,17:

Each group is immunized by i.p. administration of 0.2 ml of 1x109 SRBC/ml suspension after their pretreatment. Blood sample is collectedandserumisseparatedfromthebloodsampleforprimary antibodydeterminationafteroneweekofimmunizationwithSRBC. Aftercollectionofbloodsample,eachgroupischallengedby0.2mlof 1109SRBC/ml.Again,bloodsamplesarecollectedafteraweekand secondaryantibodydeterminationisdone.

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IntJPharmPharmSci,Vol3,Issue2,2011,161164 Neutrophiladhesion%= 100

Antibodylevelsweredeterminedbyheamagglutinationtechnique. Briefly, equal volume of individual serum samples of each group was pooled. Two fold dilutions of pooled serum samples were made in 25 l volumes of normal saline in 96 well plate microtitration plates and to it 25 l of 1% suspension of 110 9 SRBCinsalineisadded.Aftermixing,theplateswereincubatedat 37C for 2 h. and examined for agglutination. The highest no. of dilutionofserumshowingheamagglutinationhasbeenexpressed asHAtitre. 2. Delayedtypehypersensitivity(DTH)response3,14,17 WhereNIuisneutrophilindexofuntreatedbloodsamplesandNI tis theneutrophilindexoftreatedbloodsamples. Statistical analysis: Data were expressed as mean standard deviation(n=6),andstatisticalanalysiswascarriedoutbyoneway ANOVA followed by unpaired students ttest, *p<0.05 was consideredasstatisticallysignificant. RESULTS 1. Heamagglutinationtitre(HAtitre)

In this method, six animals per group are immunized by i.p. administrationof1109 SRBCs/rataftertheirchronicpretreatment and challenged by s.c. administration of 0.25109 SRBCs/rat into righthindfootpad.Thevolumefortheinflammationwasmeasured afterchallengeat24and48hbyPlethysmometer(IITCLifeScience, USA). The differences obtained for pre and postchallenge foot volumes were taken for the measurement of DTH and were expressedin%inflammation. 3. Cyclophosphamideinducedmyelosuppression10,11,13,14

TheHAtitrewasusedtoassesshumoralimmuneresponse.Itwas observedthat as thenumberof thepretreatmentdays increases, the primary and secondary antibody increases in rats. The augmentation of the humoral immune response to SRBC by levamisolesolutionisevidencedbyincreaseintheantibodytitres inthebloodofrat. 2. Delayedtypehypersensitivity(DTH)response

Group I serves as control group receiving saline solution. During pretreatment,allthegroupsreceivecyclophosphamideforlast10 days,whilegroupVisgivencyclophosphamidealoneat30mg/kg i.p. After the pretreatment and 10 days of cyclophosphamide treatment, blood samples were collected from the retroorbital plexus ofindividual animals and analyzed for hematological and serological parameters by automated hematology analyzer (Sysmex,Japan). 4. Neutrophiladhesiontest11,13

The cell mediated immune response was assessed by DTH reaction, i.e. foot pad reaction. The pretreatment showed period dependentincreaseinDTHreactivity.IncreaseinDTHreaction in rat in response to T cell dependent antigen revealed the stimulatoryeffectofpretreatmentoflevamisoleonTcells.Thus, asthepretreatmentincreases,the%edemaisdecreasedinrats. 3. Cyclophosphamideinducedmyelosuppression

Group I was served as control group and received saline solution. Afterthetreatment,bloodsamplesarecollectedbypuncturingretro orbital plexus into vials having EDTA as an anticoagulant and analyzedfortotalleukocytecell(TLC)anddifferentialleukocytecell (DLC) counts using automated hematology analyzer (Sysmex, Japan). After initial counts, blood samples were incubated with 80 mg/ ml of nylon fiber for 15 min. at 37C. The incubated blood samples wereagainanalyzed forTLC andDLC.Theproduct of TLC and % neutrophil gives neutrophil index of blood sample. The percentneutrophiladhesionwascalculatedasshownbelow:

Cyclophosphamideatadoseof30mg/kg,i.p.causedasignificant reduction in the hemoglobin (HGB), RBCs, WBCs and platelets count.Pretreatmentwithlevamisolewasabletorestorethebone marrow activity as compared with cyclophosphamide treatment alone. Though, there was not much immunostimulant action seen with 45 days pretreatment as compared with 15 and 30 days pretreatment. 4. Neutrophiladhesiontest

Pretreatment of drug evoked a significant increase in the in vitro adhesion to nylon fibres, which correlates the increase in % neutrophils. As percentage neutrophil increases, the antigen engulfmentalsoincreases.

Table1:Effectwithpretreatmentoflevamisoleonantibodytitrestoantigenicallychallengedrat Group No.ofthepretreatmentdays Meanheamagglutinationantibody(HA)titre PrimaryHAtitre IControlgroup II III IV 15 30 45 63.464 10.674.62 18.6712.22 26.679.24 SecondaryHAtitre 10.674.62 13.334.62 21.339.23 32.00.00

ValuesareinmeanS.D,n=6.p<0.5whencomparedwithcontrolgroupinallcases(statistics;onewayANOVAfollowedbyunpairedttest) Table2:EffectofpretreatmentoflevamisoleonSRBCinducedDTHinrat Group I II III IV No.ofpretreatment days 15 30 45 Mean%edemaat24 hrs. 51.784.55 37.3214.92 27.916.77 27.977.28 %changeinDTH reactionat24hrs. 27.93 46.10 45.98 Mean%edemaat48 hrs. 48.256.75 35.074.79 23.494.45 23.647.26 %changeinDTH reactionat48hrs. 27.32 51.32 51.00

ValuesareinmeanS.D,n=6.p<0.5whencomparedwithcontrolgroupinallcases(statistics;onewayANOVAfollowedbyunpairedttest) 162

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Table3:Effectofpretreatmentoflevamisoleonbloodcellsofrattreatedwithcyclophosphamidefor10days. Group I II III IV V Hemoglobin concentrationingm. 14.73.66 12.152.62 12.60.565 12.771.27 11.250.07 RBCcountin(%)million (cmm) 8.621.65 7.150.551 7.470.59 7.350.662 6.450.304 WBCcountinthousands (cmm) 11.64.293 3.80.99 6.960.874 8.90.781 0.60.141 Plateletcountsinthousands (cmm) 840.582.73 638.0352.14 761.33179.89 804.66145.22 567.0257/38

GroupIcontrol(withoutanydrugtreatment),GroupII15dayspretreatmentwithlevamisole,GroupIII30dayspretreatmentwithlevamisole, GroupIV45dayspretreatmentwithlevamisole,GroupVCyclophosphamidetreatedgroup. ValuesareinmeanS.D,n=6.p<0.5whencomparedwithcontrolgroupinallcases(statistics;onewayANOVAfollowedbyunpairedttest)

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Table4:Effectofpretreatmentoflevamisoleonneutrophiladhesiontest Group I II III IV TLC(103/ml)[A] UTB FTB 11.42.29 11.072.04 10.858.41 9.96.79 7.940.87 7.931.72 6.850.64 6.10.71 %Neutrophil[B] UTB 21.84.98 38.3520.01 19.677.97 25.96.36 FTB 21.774.77 3820.78 18.477.31 24.654.74 Neutrophilindex[AB] UTB FTB 245.0250.26 238.0449.25 331.9105.58 305.6452.14 152.2747.71 138.4421.75 175.3927.11 148.6911.47 Neutrophil adhesion(%) 2.763.73 10.028.11 11.489.37 14.716.64

UB:untreatedblood;FTB:fibertreatedblood. ValuesareinmeanS.D,n=6.p<0.5whencomparedwithcontrolgroupinallcases(statistics;onewayANOVAfollowedbyunpairedttest) DISCUSSION Antibodymolecules,aproductofBlymphocytesandplasmacells, are central to humoral immune responses, IgG and IgM are the major immunoglobulin which are involved in the complement activation, opsonization, neutralization of toxins, etc. The augmentation of the humoral immune responses to SRBCs by pretreatment of levamisole, as evidenced by increase in the antibody titre in rats (Table 1) indicated the enhanced responsiveness of T and B lymphocyte subsets, involved in the antibodysynthesis.Thevalueofhaemagglutinatingantibodytitre increase as the no. of pretreatment daysincreases indicates that immunostimulationwasachievedthroughhumoralimmunity. Cellmediated immunity (CMI) involves effector mechanisms carried out by T lymphocytes and their products (lymphokines). CMIresponsesarecriticaltodefenseagainstinfectiousorganisms, infection of foreign grafts, tumor immunity and delayedtype hypersensitivity reactions. Therefore, increase in DTH reactionin rat in response to T cell dependent antigen revealed the stimulatoryeffectbypretreatmentoflevamisoleonTcells(Table 2). Cyclophosphamide at the dose of 30 mg/kg, i.p. caused a significant reduction in the WBCs, RBCs, hemoglobin and platelets count. Combined treatment of cyclophosphamide and levamisole resulted in a restoration of bone marrow activity according to pretreatment as compared with cyclophosphamide treatment alone. Treatment with levamisole indicated that cyclophosphamideinduced immunosuppression was not restored to normal, but the animals treated with levamisole showedmorenormalactivityascomparedtocyclophosphamide treatedgroup.Asshownintable3,thelevamisoletreatedgroup normalizes all the parameters of blood like RBCs, hemoglobin and platelet count level to normal level according to no. of pretreatmentdays. The neutrophil, an end cell unable to divide and with limited capacity for protein synthesis is, nevertheless, capable of a wide range of responses, in particular chemotaxis, phagocytosis, exocytosis and both intracellular and extracellular killing. In the present study, pretreatment with levamisole evoked a significant increase in percent neutrophils. This is indicating that when any immunosuppression is given, the drug will try to prevent this immunosuppression and this may help in increasing immunity of bodyagainstmicrobialinfections. CONCLUSION The present investigation suggests that chronic pretreatment with levamisole may stimulate both cellular and humoral immune responses and thus prevent the immunosuppression in normal rat suggestingitstherapeuticusefulnessindisordersofimmunological origin. Further studies using in vivo and in vitro models of immunomodulation are needed to elucidate the exact immunomaintenance property of levamisoleand its mechanism of action. ACKNOWLEDGEMENT Authors are grateful to the authorities of Ashish Life Science, Mumbai, India for providing free gift sample of Levamisole and to the authorities of Deonar Slaughter House, Mumbai, India for collectionofbloodsampleofsheep. REFERENCES 1. LookM,BandyopadhyayA,BlumJS,FahmyTM.Applicationof nanotechnology for improved response against infectious diseases in the developing world. Advance drug delivery reviews2010;62:378393. GautamM,DiwanayS,GairolaS,ShindeY,PatkiP,Patwardhan B.Immunoadjuvant potential of Asparagus racemosus aqueous extractinexperimentalsystem.JournalofEthnopharmacology 2004;91:251255. Mungantiwar AA, Nair AM, Shinde UA, Dikshit VJ, Saraf MN, ThakurVS,SainisKB.Studiesontheimmunomodulatoryeffects of Boerhaavia diffusa alkaloidal fraction. Journal of Ethnopharmacology1999;65:125131. Singh RP, Padmavathi B, Rao AR. Modulatory influence of Adhatodavasica (Justicaadhatoda)leafextractontheenzyme of xenobiotic metabolism, antioxidant status and lipid peroxidationinrat.MolecularandCellularBiochem2000;213: 99109. Jabbar A, Zafar I, Dominique K, Muhammad G, Khan MN, Afaq M. Anthelmintic resistance: The state of play revisited. Life Sciences2006;79:24132431. Mitchell MS. Immunotherapy as part of combinations for the treatmentofcancer.Internationalimmunopharmacology2003; 3:10511059. Farghali H, Masek K. Immunopharmacologic agents in the amelioration of hepatic injuries. International journal of immunopharmacology1998;20:125139. 163

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