Indian J Physiol Pharmacol 2001; 45 (2) : 253-257

REVERSAL OF PACLITAXEL INDUCED NEUTROPENIA BYWITHANIA SOMNIFERA IN MICE

Y. K. GUPTA*, S. S. SHARMA, KAMALA RAI AND C. K. KATIYAR**

*Department of Pharmacology,All India Institute of Medical Sciences,New Delhi - 110 029

and

**Dabur Research Foundation,Shahibabad tu. P.)

(Received on March 1, 2000)

Abstract : The effect of aqueous extract of Withania sominifera (L.Solanaceae) was studied against paclitaxel induced neutropenia in mice.After paclitaxel 1 mg/kg, i.v. administration significant fall in total WBCand absolute neutrophil count was observed on day 3 and day 5. w..Som.inifera (200 mg/kg, p.o.) per se produced significant increase inneutrophil counts. W. somnifera (200 mg/kg, p.o.) when administered for4 days before paclitaxel treatment and continued for 12 days causedsignificant reversal of neutropenia of paclitaxel. The findings of the studysuggest the potential of W. somnifera as an adjuvent during cancerchemotherapy for the prevention of bone marrow depression associatedwith anticancer drugs.

Key words: paclitaxelmice

INTRODUCTION

Paclitaxel is an effective anticanceragent from the bark of Taxus breuifolia Nut(Taxace ae) and is useful in treating severalsolid tumours and soft tissue sarcomas (1).Myelosuppression is a major limiting sideeffect of paclitaxel. It induces troublesomeneutropenia of grade 3 to 4 in the dose rangeof 150-250 mg/m2 in more than 50% of thepatients. To prevent paclitaxel induced

neutropeniaWithania somnifera

myelosuppression the current approach isto use colony stimulating facotors e.g. GM-CSF or G-CSF. However, the high cost isthe major limitation in their use.

W somnifera (Ashwag andha), an Indianherbal plant is being used in manyindigenous preparations as a health tonicand an immunostimulant (2, 3). It has beenreported to have antiinflammatory and anti-arthritic activity (4). It has been shown to

*Corresponding Author

254 Gupta et al

increase the hemoglobin, total red bloodcells and white blood cells in mice withchemically suppressed immunity, andstimulate total WBC in normal Balb/c miceand also reduce leucopenia induced bysublethal dose of gamma radiation (5).Recently, in an experimental study in mice,ashwagandha prevented myelosuppressi oninduced by cyclophosphamide, azathioperineor prednisolone (6, 7, 8). However, effect ofW. somnifera on paclitaxel inducedneutropenia has not been evaluated.Therefore, in the present study, effect ofaqueous extract of W. somnifera wasinvestigated against paclitaxel inducedneutropenia in mice.

METHODS

The study was conducted on healthy(with no apparent infection) male Swissalbino mice (23-25 g). They were housedunder standard laboratory conditions foracclimatization before the experiment andfed standard laboratory dry pallet diet(Golden Feed Company, Delhi) and water adlibitum.

Selection of animals: Total WBC countand differential leukocyte count (DLC) ofmice were done on 3 consecutive days andonly those animals were included in thestudy which showed consistent Total WBCand DLC. The mice which showed anysign of infection were excluded from thestudy.

Drugs and preparation of the solutions:Paclitaxel (courtesy Dabur ResearchFoundation, India) and Recombinant HumanGranulocyte Monocyte Colony Stimulating

Indian J Physiol Pharmacol 2001; 45(2)

Factor (rHuGM-CSF) (Sandoz PharmaLimited, Switzerland) were dilutedwith sterile saline and administeredintravenously and subcutaneouslyrespectively in a volume of 0.1 ml. W.somnifera aqueous extract (courtesy DaburResearch Foundation, India) was suspendedin 1% gum acacia and administered orallyby intragastric tube in a volume of 0.5 ml.All the drug solutions were freshly preparedbefore administration.

Mice were divided randomly intodifferent treatment groups. Each group had10-12 mice each .. Blood (0.2 ml) wascollected by retro-orbital venous sinuspuncture with a capillary tube formeasurement of total WBC and absoluteneutrophil counts.

Effect of paclitaxel on total WBC andabsolute neutrophil counts: A single doseof paclitaxel (1 mg/kg) was administeredintravenously. Total WBC and absoluteneutrophil counts were measured priorto paclitaxel treatment and on day 0, 3, 5,8, 12 and 18 after paclitaxel administration.

Effect of W somnifera on paclitaxelinduced changes in total WBC and absoluteneutrophil counts: In group of animalstreated with W somnifera (200 mg/kg, p.o.for 4 days, total WBC and absoluteneutrophil counts were measured at thebeginning of the treatment. After 4 days ofW somnifera treatment a single dose ofpaclitaxel (1 mg/kg, i.v.) was injected. Theadministration of W somnifera (200 mg/kg)continued until 8 days of post paclitaxelinjection. Total WBC and absoluteneutrophil counts were measured on day 0,

.Indian J Physiol Pharmacol 2001; 45(2)

3, 5, 8, 12 and 18 after paclitaxeladministration.

Effect of GM-CSF on paclitaxel inducedchanges in total WBC and absoluteneutrophil counts: GM-CSF (25 mcg, s.c.)was administered after 5 min. of paclitaxeladministration. It was continued until 8days of post paclitaxel injection. Total WBCand absolute neutrophil counts weremeasured on day 0, 3, 5, 8, 12 and 18 afterpaclitaxel administration.

The total WBC and absolute neutrophilcounts of W somnifera and GM-CSF treatedgroup were compared with paclitaxel controlgroup. Total WBC and absolute neutrophilcounts values were represented inmean ± sem and results were analysed withANOVA and students paired "t" test wasused to calculate statistical significance.

RESULTS

Effect of paclitaxel on total WBC andabsolute neutrophil counts: Afterintravenous injection of a single doseof paclitaxel 1mg/kg a significant fall intotal WBC count was observed on day 3(8875 ± i259) and day 5 (9260 ± 1843) ascompared to counts before paclitaxeladministration (12950 ± 1285) (Fig. 1). It didnot .retu rn to normal even after day 18 ofpaclitaxel injection. Paclitaxel also causedsignificant fall in absolute neutrophilcounts on day 3, 5 and day 8 to 385 ± 79,736 ± 118 and 1052 ± 191 as compared tocounts before paclitaxel administration(2056 ± 103) respectively (Fig. 2). Allthe animals survived till the study period(18 days).

Withania Reversed Paclitaxel Neutropenia 255

8

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~ n a c lit a x e I (1 m g/kg)

--O--W. e o m nifera (200mg/kg)-+ n ac llte x e!--Q--GM-CSF (25mcg/kg)+l'aclitaxel

Fig. 1 :Effect of W. somnifera aqueous extract and GM-CSF on paclitaxel induced changes in total WBCcounts in mice. Each value representsMean ± SEM of total WBC Counts. *P<0.05,**P<O.Ol and ***P<O.OOl Compared topaclitaxel control.

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Fig. 2 :Effect of W. somnifera aqueous extract and GM-CSF on paclitaxel induced neutropenia in mice.Each value represents Mean ± SEM of absoluteneutrophil Counts. *P<0.05, **P<O.Ol and***P<O.OOl Compared to paclitaxel control.

Effect of w. somnifera on paclitaxelinduced neutropenia: Mice that received Wsomnifera (200mg/kg, p.o.) for 4 days beforeand 8 days after paclitaxel administrationshowed significant increase in absoluteneutrophil counts on day 3 (4226 ± 121), day5 (6845 ± 188) and day 8 (1657 ± 147) ascompared to paclitaxel treated group(385 ± 79, 736 ± 118 and 1052 ± 191)respectively (Fig. 2). W somnifera (200 mg/kg, p.o.) per se produced significantincreases in absolute neutrophil counts.Maximum increase was observed after 8

256 Gupta et al

days of W. somnifera treatment. The countswere increased to 8450 ± 235 as comparedto day 0 counts (2845 ± 195).

Comparative effect of W. somnifera withGM-CSF on paclitaxel induced neutropenia:Mice received GM-CSF (25 mcg/kg, s.c.) for8 days after paclitaxel administrationcompletely prevented neutropenia ofpaclitaxel. Reversal of paclitaxel inducedneutropenia by W. somnifera (200 mg/kg,p.o.) was similar to GM-CSF (25 mcg/kg, s.c.)(Fig. 2). Similar reversal was observed onpaclitaxel induced changes in total WBCcounts. GM-CSF per se also producedsignificant increase in neutrophil counts aswith W. somriifera.

DISCUSSION

In the present study, anticancer drug-paclitaxel (1 mg/kg, i.v.) significantlydecreased total WBC count and absoluteneutrophil counts. Aqueous extract of W.somnifera per se produced significantincrease in neutrophils counts. Others havealso shown that W. so m n.ifer a inducesneutrophilia (5). Four days pretreatmentfollowed by 8 days posttreatment with W.somnifera significantly reversed paclitaxelinduced neutropenia and changes in totalWBC counts. It has been reported thatashwagandha increased the haemoglobin,total red blood cells and white blood cellsIII mice with chemically suppressedimmunity (5). Methanolic extract ofashwagandha significantly increased totalWBC counts in normal Balb/c mice andreduced leucopenia induced by sublethaldose of gamma radiation (5). Recently it hasbeen suggested that ashwagandha

Indian J Physial Pharmacal 2001; 45(2)

significantly increases bone marrowcellularity (8). It has ability to enhance stemcell proliferation and also stimulate spleenand thymus cells and thus enhance theimmune system. Ashwagandha inhibitedtumour growth and increased tumour freesurvival in a dose dependent manner in mice(5). Ashwagandha treatment beforeirradiation synergistically increasedsurvival time to 120 days even in advancedtumour (9). Withaferin A, a steroidal lactonefrom the roots of Withania sominifera alsohas antitumor activity against Ehrlichascites carcinoma cells (10, 11) andradiosenstizing effect on experimentalmouse tumours (12, 13). Ashwagandhaprevented myelosuppression inducedby cyclophosphamide, azathioperine orprednisolone (7). In the present study,W. s o m n ifer a prevented paclitaxelinduced leucopenia and neutropenia.Though the exact mechanism of actionof W. somnifera is still unknown, theenhanced production of growth factors suchas GM-CSF by W. somnifera has beenpostulated (8).

Protective effect of W. somniferaagainst paclitaxel induced neutropeniaindicates that it is a potential agent tobe evaluated clinically for its effecton anticancer treatment inducedmyelosuppression. The presently usedagents GM-CSF and G-CSF though effective,have a major limitation due to theirexhorbitant cost. W. somnifera could be andigenous and a economic alternative toreduce side effects of anticancer drugs.Additionally its immunostimulant propertywill help in improving cancer treatmentstrategies.

Indian J Physiol Pharmacol 2001; 45(2)

REFERENCESWithania Reversed Paclitaxel Neutropenia 257

1. Casper ES, Waltzman RJ, Schwartz GK, SugarmanA, Pfister D, Ilson D, Woodruff J, Leung D, BertinoJR. Phase II trial of paclitaxel in patients withsoft tissue carcinoma. Cancer Investigation 1998;16: 442-446.

2. Thatte UM, Dhanukar SA. Ayurveda andcotemporary scientific thoughts. Trends inPharmacol Sci 1986; 7: 247.

3. Thatte UM, Dhanukar SA. Immunotherapeuticmodifications of experimental infections byIndian medicinal plants. Phytother Res 1989; 3:43-49.

4. Sethi PD, Thiagrajan AR, Subramanian SA.Studies on the antiinflammatory and antiarthriticactivity of withaferin A. Ind J Pharmacol 1970; 2:165-167.

5. Kuttan G. Use of Witahnia somnifera Dunnal asan adjuvant during radiation therapy. Ind J ExpBiol 1996; 34: 854-864.

6. Thatte UM, Chliabria S, Karandikar SM.Dhanukar SA. Protective effects of Indianmedicinal plants against cyclophosphamideneutropenia. J Postgraduate Med 1987; 33:185-188.

7. Zlauddin M, Phansalkar N, Patki P, Diwanay S,Patwardhan B. Studies on immunomodulatory

effects of Ashwagandha. J Ethnopharmacol 1996;50: 69-72.

8. Davis L, Kuttan G. Suppressive effect ofcyclophosphamide induced toxicity by Withaniasomnifera extract in mice. J Ethnopharmacol 1998;62: 209-214.

9. Devi UP, Sharada AC, Solomon FE. In vivo growthinhibitory and radiosenstizing effects of WithaferinA on mouse Ehrlich ascites carcinoma. CancerLetters 1995; 95: 189-194.

10. Devi UP, Sharada AC, Solomon FE, Kamath MS.In vivo growth inhibitory effect of Withaniasomnifera (Ashwagandha) on a transplantablemouse tumour, Sarcoma 180. Ind J Exp Bioi 1992;30: 169-172.

u. Sharad AC, Solomon FE, Devi PU, Udupa N,Srinivasan KK. Antitumour and radiosenstizingeffects of Withaferin A on mouse Ehrlich ascitescarcinoma in vivo. Acta Oncology 1996; 35: 95-100.

12. Ganasoundari A, Zare SM, Devis PU. Modificationof bone marrow radiosensitivity by medicinal plantextracts. Br J Radiol 1997; 70: 599-602.

13. Devi UP, Akagi K, Ostapenko V, Tanaka Y,Sugahara T. Withaferin A: A new radiosensitizerfrom Indian Medical plant Withania Somnifera. IntJ Rad Bioi 1996; 69: 19-17.