evaluation of the substantivity of chlorhexidine in associa~1

Pesqui Odontol Bras2003;17(1):78-81

Evaluation of the substantivity of chlorhexidine in association with sodiumfluoride in vitro

Avaliação da substantividade da clorexidina na associação com fluoreto desódio in vi troCarolina Saliba de Freitas*Henrique França Oliveira Diniz*Jânderson Breder Gomes*Rubén Dário Sinisterra** María Esperanza Cortés***

AB STRACT: The ef fi cacy of the flu o ride-chlorhexidine as so ci a tion in the pre ven tion of gin gi vi tis and car ies has been ad -vo cated for a num ber of years5,7,14. The ob jec tive of the as so ci a tion of these ther a peu tic agents is a syn er gis tic ac tion.The aim of the pres ent study was to de ter mine the substantivity of chlorhexidine as so ci ated or not to so dium flu o rideat dif fer ent in ter vals of time, in vi tro. Bo vine enamel sur faces were treated with 0.12% chlorhexidine gluconate(Periogard® – Colgate®) or 0.05% so dium flu o ride with 0.12% chlorhexidine (Duplak® – Dentsply®) so lu tions for onemin ute. Frag ments were placed in dis tilled wa ter and sam ples were col lected at in ter vals of 5, 30 and 360 min utes andan a lyzed by spectrophotometry in the vis i ble ul tra vi o let re gion. Substantivity was eval u ated by the mea sure ment ofchlorhexidine desorption from the treated slabs. The mean val ues ob tained were sta tis ti cally an a lyzed by Stu dent’st-test. The re sults showed that the con cen tra tion of chlorhexidine de creased when it was used in as so ci a tion with so -dium flu o ride. The dif fer ence be tween the groups was sta tis ti cally sig nif i cant (p < 0.05). These in vi tro re sults sug gestthat the as so ci a tion be tween so dium flu o ride and chlorhexidine in the stud ied con cen tra tions ap pears to have no ben -e fi cial ef fect be cause of the de crease in the substantivity of chlorhexidine.DESCRIPTORS: Chlor he xi di ne; So di um flu o ri de; Subs tan ti vity.

RESUMO: A efe ti vi da de da as so ci a ção flu o re to-clo re xi di na na pre ven ção da gen gi vi te e da cá rie vem sen do de fen di da háal guns anos5,7,14. O pro pó si to des sa as so ci a ção é ob ter um de sem pe nho si nér gi co. O ob je ti vo do pre sen te es tu do foiava li ar a subs tan ti vi da de da clo re xi di na em as so ci a ção ou não ao flu o re to de só dio em di fe ren tes in ter va los de tem po,in vi tro. Fo ram tra ta das su per fí ci es de es mal te bo vi no com so lu ções de di glu co na to de clo re xi di na 0,12% (Pe ri o gard® –Col ga te®) ou flu o re to de só dio 0,05% com clo re xi di na 0,12% (Du plak® – Dentsply®), du ran te um mi nu to. A subs tan ti vi -da de foi me di da pela des sor ção da clo re xi di na a par tir dos frag men tos pre vi a men te imer sos nas so lu ções tes te e co lo -ca dos em água des ti la da. Alí quo tas fo ram re mo vi das em in ter va los de 5 mi nu tos, 30 mi nu tos e 6 ho ras e ana li sa dasatra vés de es pec tros co pia na re gião do ul tra vi o le ta vi sí vel. Os va lo res ob ti dos fo ram ana li sa dos es ta tis ti ca men te pelotes te t de Stu dent. Os re sul ta dos mos tra ram que a con cen tra ção da clo re xi di na foi sig ni fi ca ti va men te re du zi da quan do em as so ci a ção com flu o re to de só dio (p < 0,05). Os re sul ta dos in vi tro su ge rem que a as so ci a ção da clo re xi di na ao flu o -re to de só dio nas con cen tra ções es tu da das pa re ce não ter efe i to be né fi co, de vi do à re du ção da subs tan ti vi da de da clo -re xi di na.DESCRITORES: Clorexidina; Fluoreto de sódio; Substantividade.

IN TRO DUC TIONChemotherapeutic agents are used as ad juncts

to the me chan i cal con trol of plaque15. In 1890,Miller10 sug gested an ap pro pri ate and in tel li gentuse of an ti sep tic to de stroy bac te ria, or limit bac te -rial ac tiv ity, as a way of in hib it ing the de struc tioncaused by den tal de cay. The main chal lenge inpre ven tive den tistry con tin ues to be the re duc tionor con trol of the ac quired film in or der to pre vent

car ies and periodontal dis ease. Sev eral re searcheshave been car ried out and the in dus try con tin u ally launches new for mu la tions ca pa ble of in ter fer ingei ther in the met a bolic ac tiv ity or the bac te rial ad -he sion to the den tal plaque.

Flu o ride is the most prom i nent drug used as anaux il iary in the pre ven tion by enamel re mi ne ra -lization. Flu o ride has been used in car ies pre ven -tion since 1940. Mod ern con cepts of the mech a -

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Prevenção

* Under gra du a te stu dents, Scho ol of Den tistry; **Assis tant Pro fes sor, De part ment of Che mistry, Insti tut of Exact Sci en ce;***Assis tant Pro fes sor, De part ment of Res to ra ti ve Den tistry, Scho ol of Den tistry – Fe de ral Uni ver sity of Mi nas Ge ra is.

nism of ac tion of flu o ride rec om mend daily flu o ride sup ply to es tab lish and main tain a sig nif i cant con -cen tra tion in sa liva and in plaque flu ids, thus pre -vent ing and con trol ling enamel dissolution15. Fur -ther more, the ba sis of its ap pli ca tion is that af terap pli ca tion, ei ther as top i cal so lu tions or as tooth -paste, the free flu o ride is in cor po rated into theenamel in the form of cal cium flu o ride on the toothsur face and serves as a flu o ride res er voir when ever it is nec es sary in the de min er al iza tion/re minera -lization pro cess. The use of this agent as chemo -pro phylaxis or chemo therapeutics in den tal de cayis still stim u lated by the lack of ev i dence of harm -ful ef fects.

Al though flu o ride acts in the de min er al iza -tion/remineralization equi lib rium, this func tion isnot ex er cised in pH lower than 4.5, be cause at thislev els the den tal plaque is not sat u rated byfluorapatite. Fur ther more, there are no ev i dencesto show the di rect ben e fits of flu o ride on in di vid u -als with gin gi vi tis alone17. At low con cen tra tions,chlorhexidine can re duce plaque acid pro duc tion.Based on these prin ci ples, the fluoride- chlorhe -xidine as so ci a tion would be quite beneficial11,prob a bly be cause chlorhexidine re duces plaqueacid for ma tion for sev eral hours, pre vent ing thede crease in pH, and ad di tion ally, this drug hasgreat ef fec tive ness in the re duc tion of gin gi vi tis.

Chlorhexidine, a cationic agent, has been usedfor more than fif teen years in Eu rope, and its hy -dro pho bic and hy dro philic prop er ties are re spon -si ble for its ef fi cacy. Chlorhexidine is a very po tentchemoprophylactic agent. It has a broad-spec trum ac tion, and is more ef fec tive against Gram-pos i tive than Gram-neg a tive bac te ria. Strep to coc cus of themutans group is es pe cially sus cep ti ble to chlorhe -xidine, which acts by bind ing to the bac te rial cel lu -lar wall and af fect ing its functions15. Chlorhexidine binds readily to neg a tively charged bac te rial cellwalls and can thereby dis rupt mem brane in teg rity. In high con cen tra tions, chlor hexidine is bac te ri -cidal and acts as a de ter gent by dam ag ing cellmem brane. One pos i tive prop erty of chlorhexidineis its in trin sic abil ity to be re tained by oral sur -faces, and grad u ally re leased into oral flu ids overmany hours, which is de fined as substantivity15.

The flu o ride-chlorhexidine as so ci a tion was pro -posed three de cades ago based on re sults ob tained firstly by in vi tro and later by in vivo stud ies.Luoma et al.7 (1978) and Emilson et al.3 (1976) jus -ti fied this as so ci a tion as the search for a syn er gis -tic ef fect of the two drugs to con trol car ies and gin -

gi vi tis. Ullfoss et al15 (1994) re ported that a com bi -na tion of a 2.2 millimolar chlorhexidine mouthrinse used twice a day with sin gle daily rinses of11.9 millimolar NaF re sulted in min eral loss onlyslightly larger than that ob served in soundenamel.

MA TE RIALS AND METH ODS

Bo vine teeth were sec tioned with a wa ter-cooled di a mond saw to ob tain the crowns. The 40 frag -ments ob tained were em bed ded in poly sty reneresin and ves tib u lar sur faces of 4 x 8 mm were leftex posed. Af ter that, the resin sur faces were cov -ered with nail pol ish and ran domly di vided to eval -u ate the substantivity in Group 1, by one-min uteim mer sion in 0.12% chlorhexidine gluconate so lu -tion (Periogard®), or the as so ci a tion of 0.05% flu o -ride so dium and 0.12% chlorhexidine (Duplak®) inGroup 2. The sam ples were im mersed in 1 ml ofdis tilled and ster il ized wa ter and kept in glasstubes. Af ter 5, 30 and 360 min utes, an aliquot wastaken from the tubes and the same vol ume was im -me di ately re placed and an a lyzed by ul tra vi o let in a spectrophotometer at 260 nm. The val ues ob -tained were an a lyzed with Stu dent’s t-test.

RE SULTS

Graph 1 shows the chlorhexidine re lease fromteeth im preg nated with 0.12% chlorhexidinegluconate and those im preg nated with 0.05% so -dium flu o ride and 0.12% chlorhexidine. The peakdesorption of chlorhexidine in the first 5 min utes(Ta ble 1) was lower for the so dium flu o -ride-chlorhexidine as so ci a tion (12.14 µg/ml) thanthat of the chlorhexidine gluconate so lu tion(16.68 µg/ml). Af ter 30 min utes, there was a sharp fall in the con cen tra tion of the two groups as fol -lows, Group 1, 45% (9.16 µg/ml), and Group 2,82% (2.10 µg/ml). Chlorhexidine con cen tra tioncor re spond ing to 360 min utes was 4 µg/ml forchlorhexidine gluconate, whereas so dium flu o -ride-chlorhexidine had a con cen tra tion of only1.51 µg/ml. How ever, one of the most im por tantprop er ties of chlorhexidine is its substantivity,which was shown to be re duced in all time in ter -vals an a lyzed for the as so ci a tion.

DIS CUS SION

Prog resses in sci ence and phar ma col ogy fa cil i -tate the ac cess of den tists to a larger num ber ofdrugs ev ery day. How ever, the pro fes sional knows

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Fre i tas CS de, Di niz HFO, Go mes JB, Si nis ter ra RD, Cor tés ME. Eva lu a ti on of the subs tan ti vity of chlor he xi di ne in as so ci a ti on withso di um flu o ri de in vi tro. Pes qui Odon tol Bras 2003;17(1):78-81.

that for the use of new agents to be ben e fi cial forthe pa tient, more com par a tive stud ies should becare fully per formed to es tab lish the ap pro pri atether apy strat egy.

As pre vi ous papers5-7 fo cused on the ben e fi cialef fect of the flu o ride-chlorhexidine ther apy to ar -rest car ies and to remineralize in cip i ent le sion, thesubstantivity was as sumed to be sat is fac tory in re -la tion to the cur rent dose. How ever, Melo et al.9

(1999) ad vised that the flu o ride-chlorhexidine as -so ci a tion was not ben e fi cial, be cause it re ducedthe agent con cen tra tion.

Brambilla et al.1 (1998) con cluded that this as -so cia tive mouth rinse might be used in car ies pre -ven tion pro grams for preg nant women, be cause itre duces Strep to coc cus mutans scores in the sa livaand be cause it re tards col o ni za tion in chil dren upto four months.

Di ver se stu di es jus ti fi ed the va lue of the flu o ri -de-chlor he xi di ne as so ci a ti on on the syner gis tic ef -fect of the two drugs3,7. Due to the low mo le cu larwe ight of flu o ri de mo le cu les, they could link to pla -ces of dif fi cult ac cess for chlor he xi di ne and re achStrep to coc cus mu tans re ten ti on ni ches and in ci pi -

ent de cay le si ons, whe re the pH is low, thus inhi bi -ting acid pro duc ti on with a con co mi tant pro lon ged pre ven ti ve ef fect8. No net he less, the flu o ri de-chlor -he xi di ne as so ci a ti on could ge ne ra te low chlor he xi -di ne con cen tra ti on in cer ta in re ten ti on pla ces, and the re fo re sup press the po ten ti al re ser vo irs of thedrug, such as ton gue, mu co sa, ton sils and sa li va,which is fa vo ra ble to inhi bit the re co lo ni za ti on oftre a ted le si ons.

As ve ri fi ed in this study, the ma xi mum con cen -tra ti on of chlor he xi di ne ob ser ved af ter 5 mi nu teswas lar ger for chlor he xi di ne glu co na te than for theas so ci a ti on (Graph 1). The ini ti al lo wer con cen tra -ti on of chlor he xi di ne in re la ti on to the as so ci a ti onwas due to the com pe ti ti on bet we en the flu o ri deions and the chlor he xi di ne mo le cu les for the ad -sorp ti on sur fa ce. Other aut hors3,7 sug gest that there are io nic re ac ti ons bet we en flu o ri de andchlor he xi di ne, ge ne ra ting a de cre a se in the con -cen tra ti on of chlor he xi di ne, and inhi bi ting thebac te ri ci dal ef fect of the as so ci a ti on. Ho we ver, thefact that chlor he xi di ne has se ve ral po si ti ve char -ges and that it is in lar ger con cen tra ti on in the as -so ci a ti on would ne u tra li ze the ef fect of the drug byflu o ri de9.

In agre e ment with the re la ted li te ra tu re7, in thepre sent study it was ob ser ved that the subs tan ti -vity of chlor he xi di ne was re du ced. A stra tegy to in -cre a se the subs tan ti vity of flu o ri de on ena mel sur -fa ce is its as so ci a ti on with ami ne groups, whichwould link to the ne ga ti ve char ges ava i la ble in te -eth and ena mel film. This would be in te res ting be -ca u se it would pro long flu o ri de re a di ness on theden tal sur fa ce. Ho we ver, Ete mad za deh et al.4

(1985) sho wed that the ami ne flu o ri de-chlor he xi -di ne as so ci a ti on does not inhi bit pla que growth.Ho we ver, the po ten ti al to inhi bit ca ri es throughthe use of the flu o ri de-chlor he xi di ne as so ci a ti onwas ve ri fi ed by long-term stu di es2,6,7,13,14, even in the tre at ment of in ci pi ent root le si ons, with which theas so ci a ti on was used6.

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GRAPH 1 - Chlor he xi di ne con cen tra ti on (µg/ml) re le a -sed from bo vi ne ena mel frag ments im preg na ted withchlor he xi di ne glu co na te 0.12%, 0.05% so di um flu o ri deand 0.12% chlor he xi di ne glu co na te.

TA BLE 1 - Chlorhexidine re leased (µg/ml) from teeth im preg nated with 0.12% chlorhexidine gluconate and from teethim preg nated with 0.05% so dium flu o ride and 0.12% chlorhexidine. The val ues were ob tained by Stu dent’s t-test(p < 0.05).

0.12% chlorhexidine gluconate (Periogard®) 0.05% sodium fluoride with 0.12%chlorhexidine (Duplak®)

Time (mi nu tes) 5 30 360 5 30 360

Mean 16.68 9.16 4.00 12.14 2.10 1.51

Stan dard de vi a ti on ± 9.43 ± 7.94 ± 4.58 ± 7.08 ± 2.29 ± 1.56

Va ri an ce 88.93 63.18 20.98 50.24 5.25 2.44

The change in the con cen tra tion pro file ofchlorhexidine shown in this study raises new con -cerns about its in ter ac tion with so dium flu o ride,and con se quently about the ben e fit of the as so ci a -tion of these two drugs.

CON CLU SIONBased on in vi tro ex per i men tal con di tions of this

work and the re sults ob tained, we can con clude

that the flu o ride-chlorhexidine as so ci a tion is un -fa vor able, due to the sig nif i cant de crease in thesubstantivity of chlorhexidine.

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doy F, Stroh men ger L. Ca ri es pre ven ti on du ring preg -nancy: re sults of 30-month study. J Am Dent Assoc1998;129:871-3.

2. Dol les O, Gjer mo P. Ca ri es in cre ment and gin gi val sta tusdu ring 2 ye ars’ use of chlor he xi di ne- and flu o ri de-con ta i -ning den ti fri ces. Scand J Dent Res 1980;88:22-7.

3. Emil son CG, Kras se B, Wes ter gren G. Effect of a flu o ri -de-con ta i ning chlor he xi di ne gel on bac te ria in hu man pla -que. Scand J Dent Res 1976;84:56-62.

4. Ete mad za deh H, Ai na mo J, Mur to maa H. Pla quegrowth-inhi bi ting ef fects of an abra si ve flu o ri de-chlor he xi -di ne to oth pas te and a flu o ri de to oth pas te con ta i ning oxi -da ti ve enz ymes. J Clin Pe ri o don tol 1985;12:607-16.

5. Katz S. The use of flu o ri de and chlor he xi di ne for the pre ven -ti on of ra di a ti on ca ri es. J Am Dent Assoc 1982;104:164-70

6. Kelt jens H, Scha e ken M, Van der Ho e ven J, Hen driks JC.Ca ri es con trol in over den tu re pa ti ents: 18-month evalua -tion on flu o ri de and chlor he xi di ne the ra pi es. Ca ri es Res1990;24:371-5.

7. Lu o ma HH, Mur to maa H, Nu u ja T, Nyman A, Num mi kos kiP, Ai na mo J, et al. A si mul ta ne ous re duc ti on of ca ri es andgin gi vi tis in a group of scho ol chil dren re ce i ving chlor he xi -di ne-flu o ri de ap pli ca ti ons. Re sults af ter 2 ye ars. Ca ri esRes 1978;12:290-8.

8. Mcder mid AS, Marsh PD, Ke e vil CW, Ellwo od DC. Addi ti veinhi bi tory ef fects of com bi na ti ons of flu o ri de and chlor he xi -di ne on acid pro duc ti on by Strep to coc cus mu tans andStrep to coc cus san guis. Ca ri es Res 1985;19:64-71.

9. Melo GB, Ba tis ta de G, Pi nhe i ro CM, Osó rio CN, Zar di niFA. Po ten ci al de efi cá cia da as so ci a ção de clo re xi di na comflúor. Rev CROMG 1999;5:43-6.

10. Mil ler WD. The mi cro or ga nisms of the hu man mouth: thelo cal and ge ne ral di se a ses which are ca u sed by them. Phi -la delp hia: SS Whi te, 1890. Re print. Ba sel: Kar ger, 1973.

11. Opper mann RV, Röl la G. Effect of some poly va lent ca ti onson the aci do ge ni city of den tal pla que in vivo. Ca ri es Res1980;14:422-7.

12. Oste la I, Kar hu va a ra L, Te no vuo J. Com pa ra ti ve an ti bac te -ri al ef fects of chlo re xi di ne and stan nous flu o ri de-ami neflu o ri de con ta i ning den tal gels aga inst sa li vary mu tansstrep to coc ci. Scand J Dent Res 1991;99:378-83.

13. Spets-Hap po nen S, Lu o ma H, Forss H, Ken ta la J, Ala lu u -sua S, Lu o ma AR, et al. Effects of chlor he xi di ne-flu o ri -de-stron ti um rin sing pro gram on ca ri es, gin gi vi tis andsome sa li vary bac te ria among Fin nish scho ol chil dren.Scand J Dent Res 1991;99,130-8.

14. Te no vuo JJ, Häk ki nen P, Pa u nio P, Emil son C G. Effects ofchlo re xi di ne-flu o ri de gel tre at ments in mot hers on the es -ta blish ment of mu tans strep to coc ci in pri mary te eth andthe de ve lop ment of den tal ca ri es in chil dren. Ca ri es Res1992;26:275-80.

15. Ullsfoss BN, Öga ard B, Arends J, Ru ben J, Röl la G, AfsethJ. Effect of a com bi ned chlor he xi di ne and NaF mouth rin se: an in vivo hu man ca ri es mo del study. Scand J Dent Res1994;102:109-12.

16. Wann ma cher L, Fer re i ra MBC. Far ma co lo gia clí ni ca paraden tis tas. 2nd ed. Rio de Ja ne i ro: Gu a na ba ra Ko o gan S. A.;1999.

17. Wort hing ton HV, Da vi es RM, Blink horn AS, Man ko di S, Pe -tro ne M, De Vi zio W, et al. A six-month cli ni cal study of theef fect of pre-brush rin se on pla que re mo val and gin gi vi tis.Bri tish Dent J 1993;175,322-6.

Recebido para publicação em 24/04/02Enviado para reformulação em 29/11/02

Aceito para publicação em 07/02/03

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evaluation of the substantivity of chlorhexidine in associa~1

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