Indian Journal of Experimental Biology Vol. 40, December 2002, pp. 1373- 1377

Potentiation of spermicidal activity of 2',4'-dichlorobenzamil by lidocaine

P Moudgil , A Gupta, A Sharma, S Gupta, & A K Ti wary*

Department of Pharm aceuti cal Sciences and Drug Resea rch, Punjab i University, Pat iala, 147002, India

Received 16 May 2002: revised 12 Sep/ell/ber 2002

The present in vesti gation was des igned to study the spermi cida l acti vity or lidocaine. a membrane stabili zer, and its combinat ion with 2' ,4 '-dichlorobcnzamil hydroc hloride, a ~t-Ca2+ exc hange inhi bitor, on human semen and spermatozoa separated from semen. Both drugs per se produccd dose- and time-dependent reduc tion in motilit y of ejac ul ated human sperm. Lidoca ine was found to potenti ate the spe rmicidal activity of benwmil resulting in signi ficant decrease in time for producing complete loss of ejacu lated sperm moti lity . Sperm revival test revealed irreversible loss of sperm viability indicat­ing a sperilli cida l rather than spenni ostati c ac ti on by both the drugs . Furthermore, both benz<1mi I (10-40 mM) per .I'e and benzam il -li doca ine combinati on (0.5 and 16 mM) prod uced contraception in rabbit mode l.

A Na+-Ca2+ exchange inhibitor, 2',4'-dichlorobenzamil hydrochloride (DBZ), has been show n to prod uce ir­reversibl e loss of sperm viability in ejacul ated human semen and spermatozoa separated from semen. DB Z is 1.62-fold more potent than widely used spermicide, l1 onoxynol-9. Thi s action of DBZ has been found to he due to elevation of intrasperm calc ium ' and has been demonstrated to be potentiated by combining it with propranolol2. Potentiation of act ion produced by combinati on of DBZ and propranolol resulted in sig­ni ficantly reduced ti me and dose of respecti ve drugs for prod ucing complete loss of ejaculated human sperm motility.

It is important to note that spermi cida l ac ti vity of proprano lol is due to its membrane stabili zing prop­erty and not due to ~-b l oc king property3. However, propranolol has been reported to produce a fa ll in sys­to lic blood pressure, heart rate and forced expirato ry volume after insertion of vagin al tab lets sugges ting its systemic absorpti on from the vaginal mucosa" . Thi s indicates that propranolol may not be safely used as a spermicide alone or in combinati on with DBZ and there is a need to study the potenti ati on of spermicidal activity ofDI3Z wi th another safe membrane stabili zer.

Lidocaine hydrochloride, a membrane stabili zer, has high water solubility and ex hibits a pKa of 7.9 (Ref. 5). Hence, lidocaine hyd rochl oride is ex pected to be 1.2% uni onized at pH 3.5-6.0 of vagina l fluid which is normally in the range of pH 3.5-6.0. Due to lower amount present in the litJid solubl e (uni oni zed) form , it is envi saged that systemi c absorption of lido­caine across the vaginal mucosa would be very low.

*Correspondent author

Therefore, the present in vestigation aimed at study­ing the spermicidal act ivity of lidocaine hydrochloride and its combination with DBZ in samples containing ejacu lated hu man semen and spermatozoa separated fro m semen. Furthermore, contracepti ve efficacy of DBZ per se and that of its comb inat ion with lidocaine was studi ed in rabbit model.

Materials and Methods 2',4' -di chl orobenzami I hydrochloride, lidocaine

hydrochl oride and carbopol 934P were obtained as gift samples from SRI (USA), Astra- IDL (India) and Ranbaxy Research Laboratories (Indi a), respectively. Quin-2AM was purchased from Sigma Chemicals (USA) . All oth er chemicals were of AR grade and were pu rchased from S.D. Fine Chemica ls Limited (I ndia) .

Sell/en collection and separation of sperl71atozoa ­Semen was collected by masturbation from fi ve non­drinkers and non-smoking male vo lunteers hav ing a mean spermatozoal count of > 20 x 106 spennatozoa/ml (permi ss ible level according to WHO manual ) with > 76% normal sperm morphology. An abs tinence period of not less th an 48 hI' and not more than 5 days was all owed between two collect ion periods6

. Care was taken to avoid cold shock to ejacul ated sperm by coll ec ting the sample in a warm sterili zed beaker. Fresh samples were all owed to liquefy and subjec ted to further in vest igat ions at room temperature (35°C). For separation of spermatozoa, liquefied semen was diluted with an equal volume of Biggers-Whitten­Whittingham (BWW) med ium7 and centrifuged at 1000 rpm for lO min . The supernatant was di scarded

1374 INDIAN J EXP BIOL, DECEMBER 2002

and the procedure was repeated. The pellet so ob­tained was finally resuspended in BWW medium so as to yield a final spermatozoal co unt of> 20 x 106

.

TOlal speml cUlInt-Liquefied semen was diluted ( I :20) with formalin-bicarbonate so lution (sodium bicarbonate 5g; formalin I ml ; dist illed water qs 1000 ml ). Spermatozoa were counted microscopically (40 x magnification) using a Neubauer chamber as per the procedure laid down in the World Health Organization Manuals.

Sperl1l viabiliry - Eosi n stai ns the dead spermato­zoa, whereas the plasma membrane of viable sperm rema ins un stained~ . Stock so lu tion of DBZ and lido­caine hydrochl oride were prepared in BWW medium. The drug so lution was mi xed in eq ual proportion with I iquefi ed semen or spermatozoa separated from semen and incubated at 35°-37°C. ]n the studi es employ ing combination of drugs, the solution of each drug was added to a sample of semen (0.5 :0.5: 1.0) and the mix­ture was incubated. Incubated samples were with­drawn at vari ous time intervals, mi xed with 0.5 ml eosin so lution (0.5% w/v in normal saline) and observed microscopicall y. Fract ional motility was calculated by the formula : % motile sperm in treated sample 7 % motil e sperm in control sample.

Sperlll revival test-Glucose so lution was added to the sample of immotile sperm so as to obtain a fina l concentration of 250mg/m l. The mi xture was incu­bated at 35°-37°C for 60 min and then observed fo r rev ival of sperm motilit/.

MeaslIrement of intracellllla r Ca2+-Effcct of li­docaine and its combination with DBZ on intracellu­lar ci + was studi ed in spermatozoa separated from human semen by using fluorescent dye, Quin-2AM (acctoxy methyl es ter of Quin-2) according to the method outlined by White et ali a.

Contracepti ve efficacy testing in rabbit 1Il0del­The drugs were incorporated ina carbopol gel fo rmu­lation (carbopol 934P, 1.25; tri eth anol amine, 0.81; EDTA , 0.008; methy l paraben , 0. 18; propyl paraben, 0.02; propylene glyco l, 5. 18; water 92.55 %w/w ) fo r delivering them into the vagina of rabbits. Parabens and EDTA were di sso lved in 1/41h quantity of water by warming and allowed to cool. Carbopo l was sepa­ratel y added to the remain ing quantity of water in small increments with stirring. To thi s carbopol di s­persion, DBZ (l0, 20 or 40 mM) or a combinati on of DBZ (0 .5 mM) and lidoca ine hydrochl oride (16 mM) was added. J\dding tri ethanolamine (drop wise) CO I11-

pleted ge l formation. Finally, paraben so lution and propy lene glyco l were added.

A cervicovaginal cannula was prepared using a sy­ringe (5 ml) and a polyethylene tube (15 cm long, in­ner diam 2.75 mm; outer diam 3.25 mm) for deli ver­ing the gel into vag ina of doe. A plastic bulb (0.75 inch long) having a hole in the center was attached to the free end of the pol yethylene tube that served as a guide for the tube during its tra vel to the cervico­vagin a.

The doe was held in left arm with its back flat and straight. The tip of the plastic bu b attached to poly­ethylene tube was slowl y in serted into vaginal open­ing and gentl y pressed inside. Care was taken in ma­neuvering the bulb so that it does not enter the bladder by rupturing it. Beyond urovagina i sph incter, the bulb was gently inserted up to the base of the cervices. Then the gel was passed through the cervicovaginal cannu la by appli cation of steady press ure on the sy­ringe pi ston. After thi s the can nul a was gentl y re­moved from the vagina and the doe was kept in su­pine position fo r 5 min before allowing it to return to the normal position.

After allowing 10 min for even di stribution of ge l in the doe vagina, the doe was placed in a cage along wi th a buck (known fertility) for mating. The doe was separated immediately after one mating and qu aran­tined till 40 days for observa ti on of deli ve ry of litter. The doe used for this study had been quarantined for 40 days in order to ensure no carry over of pregnancy to the study peri od and were ensured for their being in the receptive phase after examination of red colour of their vulval I. Five doe were used for each group.

Results and Discussion Both DBZ and lidocai ne prod uced time- and dose­

dependent red uction of sperm motility in ejacul ated human sperm and spermatozoa se parated from semen samples . Complete loss of sperm viability immed i­ately on additi on to ejaculated human semen was pro­duced by DBZ at a concentration of 4 mM (Fig. I ). The same effect in samples conta ining spermatozoa separated from semen was obtained by adding DBZ at a concentration of 0.5 mM (Fig. 2). However, lido­ca ine required higher concentrati on of 28 mM in se­men samples and 24 mM in samples containing sper­matozoa separated from semen for immediately pro­ducing co mplete loss of sperm viab ility (Fig. 3) . Eo­sin staining revealed dead sperm to be stained red af­ter treatment with both DBZ and lidocaine. A combi­nation of lidocaine (16 111M) and DBZ (0.5 IllM) pro­duced co mplete loss of sperm viability immediately afte r additi on to semen samples sugges ting potentiation

MOUDGIL el al.: SPERMICIDAL ACTIVITY POTENTIATION OF BENZAM IL 1375

of spermicidal acti vity of OBZ (Table t ). A negati ve result for sperm rev ival at the end of the ex periments suggested spermicidal rather th an sperm iostat ic act ion by OBZ, lidocai ne and their combination . Carbopol gel fo rmul ations containing OBZ ( 10-40 mM) and a combination of OBZ (0.5 mM) with lidocaine ( 16 mM) were found to be effective contraceptives in fe­male rabbits (Tab le 2).

Results showed that OBZ was more potent spenni­cide than lidocaine in both semen and spermatozoa separated from semen sam pl es. However, the differ­ence in spermi cidal potency of lidoca ine in presence or absence of seminal fluid was less pronounced th an OSZ. Although no exact reaso n for the red uced po­tency of spermicidal agents in semen samples cou ld be suggested , the ro le of hi gher viscos ity and panial neutrali zation of the agents by constituents of seminal flu id cannot be rul ed out9-

12.

Red ucti on of sperm moti lity by OBZ has been re­ported to be accompanied with an elevati on of in-

0.8

0.7 -0-0.25 mM

-+- 0.5 mM

-tr-- 1.0 mM

0.6 --"- 2.0 mM ~4.0mM

>. ~

~ 0.5 0

::i I1l c: 0 ~ 0.4 v I1l ... u..

0.3

o 200 400 600

Time (min)

Fig. I - Inrl ucn cc or variq,~l s doses o r DBZ Oil 1ll0lilil Y or sperlll in cj,lculalccI human sC lllcn.

traspenn calcium l. Furthermore, propranolol that ex ­

hibits spermicida l ac tivity due to its membrane stabi­li zin g property has been shown to elevate intrasperm ca lcium 'o and potentiate the spermicidal act ivity of OBZ2. It is noteworthy that neither lidocaine per se nor its combination with OBZ was found to elevate the intrasperm Ca2

+ in the present study. However, lidoca ine was found to potentiate the

spermicidal ac ti vity of OSZ. OB Z per se produced complete loss of sperm viability in 550, 300, 230 and 11 0 min at a concentration of 0.25,0.5, 1.0 and 2.0 mM, respect ively. Lidocaine per se (16 mM) pro­duced complete loss of sperm viab ility in semen and spermatozoa separated frolll semen at 50 and 40 min, respecti vely. This concentration of lidoca ine was cho­sen for potentiation studies so that the red uction in spenn icidal time in combi nat ion with DSZ could be more apparent. It is ev ident from Table I that lido­caine ( 16 mM) produced co mplete loss of sperm vi­abi I ity in semen sam ples at 10, I , I and I min , when combined with 0.25, 0.5 , 1.0 and 2.0 mM concentra­ti ons of OBZ, respectively. Thi s indicated a red ucti on

0.8 -0- 0.05 mM -+- 0.1 mM

-0-0.15 mM --"- 0.2 mM

0.7 -0- 0.25 - 0.5mM

0.6

>. ."'::: ~

0.5 0 ::i t1l c: 0

'';:; 0.4 v t1l ... u..

0.3

O.L

0.1

0

0 50 100 150 Time (min)

Fig. 2- ln rlucllcc or va riou s doses or DBZ on mOlilil Y or spcr lll ill spe rlllalozoa separal t:d I'rolll hUlllall scmcll.

1376 INDI AN J EX P BIOL, DECEMB ER 2002

Tab le 1- Fractional motil ity of sperm in (A) human semen and (B) spermatozoa separated fro m semcn fo llowi ng combined treatmen t of lidoca ine ( 16 mM) and diffe rent concentrati ons (mM) of 2' , 4'-di cholorbenzam il hydrochl oride (DBZ)*

[Values are mean ± SO of five replications l

Time (min)

5

10

15

*0. I

0.45 ± 0.023 0.24 ± 0.025 0 .05 ± 0.0 17

0

A *0.25 *0.3 *0 .4

0 .4 8 ± 0.54 ± 0.28 ± 0 .026 (l. O33 0.03 1 0. 15 ± 0 0 0.0 19

0

B *0 .5 * 1.0 *2.0 *0. I *0.2 *0.3 *0.4 *0.5 * 1.0 *2.0

0 0 0 0.30 ± 0. 18 ± o o o o o 0 .0 17 0.0 12 0.03 ± 0 0 .0 I I

0

Blank rows ind icate that the expe ri ment was term inatcd after obscrving 100% illlillotilc sperm

1.2

0.8

>-.~ -0 ::i: !1l 0.6 t: 0 :z u !1l ... u..

0.4

0.2

o 20

---e--- 8 mM Semen - - • - - 8 mM Spenmatozoa --tr- 12 mM Semen - - • - - 12 mM Spermatozoa

---a-- 16 mM Semen - - • - - 16 mM Spermatozoa _____ 20 mM Semen

- - ')C- - - 20 mM Spermatozoa --+-- 24 mM Semen - - + - -24 mM Spermatozoa ~28 mMSemen

- - -x- - -28 mM Spermatozoa

40 60 80

Time (min)

100 120

Fig. 3- lnf'luencc of va rious doses of lidocai ne on motility of sperm in cjacul ated human semen (so li d lines) and spcrmatozoa scparated fro m scmen (broken lincs) samples.

of time fo r prod uci ng complete loss of sperm viability in semen samples by 300-fold by using a combination of lidoca ine and DBZ. Similarl y, the time for produc-

Table 2- Contraceptivc efficacy tcsting in i'c male rabbit s

DBZ (0.5 mM)+LD ( 16 mM)

COll trol DBZ conc. (m/v1J 40 20 10 0.5 + LON ( 16 mM)

+ * :;: * :::

* ::: * ::: * + * :I: * * + :(. * * * + ::: ::: ::: >.:

Note: (+) indicates concepti on in doc; (* ) indiC<ltes cOll tracept ion in doc

ing complete loss of sperm viability of spermatozoa separated from semen decreased from 60, 30 and 10 min (DB Z per se) to 10,5 and I min by combin ing lidocaine ( 16 m M) with DB Z at concentrations of 0. 1, 0.2 and 0.5 mM, respecti vely_

Although the data generated in the present st udy cou ld not suggest the exact mechanism of potentia­tion, the results nevertheless indi c8 ted a hi ghly sig­ni ficant potentiat ion of spermi cidal acti vity of DBZ in combination with lidoca ine in ejac dated human se­men and contraceptive acti vity in female rabbits. Thi s st rategy will help in red ucing the dose of respecti ve drugs in a contracepti ve fo rmulat ion.

Acknowledgement SRl (USA) is gratefully ack nowledged fo r prov id­

ing the sample of 2',4'-dichlorbenzam il hyd rochloride as part of Chemica l Synthesis Program of the a­tional Institu te of Mental Health , (Contract NOIMH30003 )for thi s study_

References I Rcddy P R, Patni A, Sharma A, Gupta S & Tiw:Jry A K, Ef­

fcc t of 2',4'-d ichlorobenzamil hydrochloride, a Na+-Ca2+ ex­

change inhi bitor, on human spermatozoa , £11 '- J Phanll(lcol, 4 18 (200 I) 153.

MOUDG IL et al.: SPERMI CIDAL ACT IVITY POTENTIAT IO OF BE IZAM IL 1377

2 Patn i A K. Gupta S. Sharma A. Tiwary A K & Garg S K. Role of" illlraeellula r calc ium in the spermicidal action of" 2'. 4'-d ichl orobenza mil , a nove l contact spermic ide. J IJlwJ"J1l

PIWI"/IWCIII, 53 (200 1) 1387. 3 Hong C Y. Chaput de Sa in lOnge D M & Turner P. The in­

hi bitory act ion o f" proca ine. (+ )- propranolol and (±)­pl"Opranoiol on hu man sperm motility: Ant agonism by caJ"­re ine. 81" J Ciill 1>lwI"lIIacol , 12 ( 198 1) 75 I.

~ Patel C G, \-Varrington S J & Pearso n R M. Propranolol con­centration in pl asma alkr in se ni on into vag ina . /11" Med J, 287 ( 1983) 1 2~ 7.

5 Lund W. The Plwnlla("elltiml C(ld('.\" ( The Pharmace uti ca l Press. London. U. K) 1 99~. 938.

6 Rey nolds T R & Na rang B S. in i\ll edicalla/)oJ"(l/o l"v /11111111111

.If!!' tropical coilil/I"ies, \'(II II. ed it ed hy M Cheesebol"Ough (BulICl"wonh and Co. Lid .. Kent ) 1 98~. 18(1.

7 l3i ggcrs J D. Whillen W K & Whillingham DG. The culture oj" mouse embryos ill I'it/"(}. in Methods ill IIwlllllw/iall 1'111-

/)I"rologv, ed it ed by J. C Danie l. (Freeman, San r'ranci sco. USA) 197 1. 86.

8 WHO IIII){)J"(I/o l"r IIwlllla l Jill" e.l"ll lII ill(l/iOIl oj" hlllllllli .1'(:'1111'11

alld spenll -cen 'iwl IIIIICIiS i llt cmClioll. (Ca mbridge Univer­sity Press, Ca mbridge. UK) 1999.

9 Redd y KV . Sahani K S & Meherj i K P. Spe rmi cida l ac ti vity or magainins: III l'ill"O IIIld ill l'i l'lJ slUdi es. COlltJ"(lCep ti(III,53

( 1996) 205. 10 White R D. Jane S C, Ratnasooriya W D & Aitken J. Co m­

pl ementary e J"rects o j" propranolol and l1 onoxy nol -9 upon human sperm motilit y. COlltJ"(lceptioll.52 ( 1995) 2~ I.

II Castl e P E. Hoell T E. Whaley K J & Cone R A, Contracep­tive tes ting oj" vagi nal agent s in rabbits. COlltmceptiol l. 58

( 1998) 5 I .

12 Ede lste in M C, Fulgha n D L. Gretly J fC. Alexa nder J. BauCl" T J & Archer D F. Studies on the i ll I'itm spenni cidal activ ity o J" sy nthet ic magainins. Feuit Staii. 55 ( 199 1) 6~7 .