potentiation of spermicidal activity of 2',4...
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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_
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