Contraception 72 (2005) 447 453

Original research article

Spermicidal activity of dermaseptins

Amira Zairi, Afifa BelaRd, Amina Gahbiche, Khaled HaniT
Laboratory of Biochemistry ( UR/08-45), Faculty of Medicine, 4002 Sousse, Tunisia
Received 16 July 2004; revised 6 June 2005; accepted 6 June 2005

Abstract
Objective: The present study was undertaken to elucidate the spermicidal efficacy of two synthetic antimicrobial peptides, dermaseptin
(DS1 and DS4).
Methods: Twenty samples of fresh semen were obtained from patients aged between 23 and 35 years. The ability of DS to kill sperm was
evaluated by the SanderCramer test under in vitro conditions.
Results: The data showed that sperm motility was inhibited with various concentrations of DS at different intervals ranging from 2 to
240 min. The effective 100% inhibitory concentration (EC100) of DS4 in 2 min during the sperm immobilization assay was 100 Ag/mL
whereas the sperm immobilization of EC100 of DS1 was 200 Ag/mL. The presence of 0.1% chelating agent ethylenediaminetetraacetic acid
(EDTA) reduced the EC100 of DS4 to 10 Ag/mL whereas less than a two-time enhancement in DS1 activity was observed upon combination
with EDTA. The action of DSs on sperm motility was observed to be dose dependent. Supplementation with pentoxifylline and that with
calcium are known to enhance the motility of sperm but they did not prevent the spermicidal action of DSs.
Conclusion: This present study indicates that DS is an effective agent to kill sperm. In view of this fact, it is suggested that DS4 has
antibacterial, antiviral, antifungal and potentially spermicidal activities and could be a potent vaginal contraceptive.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Dermaseptins; Antimicrobial; Spermicidal; Sperm motility; Contraceptive

1. Introduction
Safe and effective vaginal contraceptives are urgently
needed. Ideally, such products should have antimicrobial
and antiviral properties; as such, they could also play a role
in the reduction of sexually transmitted diseases (STDs),
particularly the human immunodeficiency virus (HIV). At
the present time, hundreds of spermicidal products have
been marketed. Most of them contain a detergent as an
active ingredient [1], such as nonoxynol-9 (N-9) and
benzalchonium chlorides; some of these detergents (e.g.,
N-9) have also exhibited microbicidal activity in vitro [2].
The spermicidal activities of these surfactants are associated with their structural affinity to the lipid membrane [35].
However, numerous laboratory studies have now shown that
detergent spermicides do not provide any protection against
STDs and that their effect in preventing HIV transmission
remains controversial [6]. The major drawback of using N-9
T Corresponding author. Laboratory of Biochemistry, Faculty of
Medicine, 4002 Sousse, Tunisia. Tel.: +216 73 21 96 32; fax: +216 73
22 49 98.
E-mail address: hani.k@planet.tn (K. Hani).
0010-7824/$ see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.contraception.2005.06.055

and other currently used surfactants is their detergent-type

effect on epithelial cells and normal vaginal flora [711]. The
repeated use of a surfactant as a vaginal contraceptive/
microbicide has been associated with an increased risk of
vaginal or cervical infection and irritation or ulceration
[1215]. Detergent-type spermicides alter vaginal bacterial
flora, and such disturbance of the vaginal microbial milieu
can lead to opportunistic infections [16 18], which in turn
increase the chances of HIV/STD transmission [7,19].
Because of this effect, it is important to identify and evaluate
a new generation of contraceptive antimicrobial agents that
act by an entirely different mechanism of action and can be
used vaginally in effective doses without causing overt
vaginal irritation or other toxicity.
Antimicrobial peptides are produced by both prokaryotic
and eukaryotic cells and have been the subject of many
studies in recent years because of their involvement in host
defense mechanisms and their potential use as therapeutic
agents [20 27]. These antimicrobial peptides include
dermaseptins (DSs), a family of five structurally and
functionally related peptides that were originally isolated
from the skin of a tree-dwelling South American frog

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A. Zairi et al. / Contraception 72 (2005) 447 453

(Phyllomedusa sauvagei). These compounds are linear

polycationic peptides composed of 28 34 amino acids that
are structured in amphipathic a-helices in apolar solvents
[28,29]. The antimicrobial action of DS is thought to be
mediated by interaction of the amphipathic a-helix with the
membrane phospholipids, resulting in lethal cell permeation
[20,26,30 33]. In this respect, these agents are similar to
other families of amphipathic helical peptides such as bombinins and magainins, which are potentially active against a
large spectrum of microorganisms [20,26,34]. Although the
precise mechanism of action of antimicrobial peptides is not
fully understood, they are believed to permeate the target
cell by destabilizing the plasma membrane via either a
bbarrel staveQ mechanism or a bnonpore carpet-likeQ
mechanism [35].
Yet, many of these peptides have no activity upon normal
eukaryotic cells, but there are some exceptions. It has been
previously reported that hamster [36] and human [37]
spermatozoa are sensitive to magainins and that their
motility is reduced.
DSs show a remarkable ability to inhibit microbial
cells efficiently, rapidly and irreversibly without a toxic
effect on mammalian cells. DSs display cytolytic activity
in vitro, generally against a broad spectrum of host-free
microorganisms, including bacteria (Gram positive and
negative) [29,30,3841], protozoa (Leishmania mexicana
and Plasmodium falciparum) [42 45], yeasts and filamentous fungi (Aspergillus fumigatus and Aspergillus
niger) [26,30,46]. In addition, it has been demonstrated
that DSs are able to inhibit herpes simplex virus-1
replication through interference with viruscell surface
interactions [47].
Despite DSs possessing a full range of desired microbicide activity against STDs, they have never been assessed
against spermatozoa. It would be interesting to develop a
vaginal contraceptive with these compounds that have many
biologic properties. Antimicrobial agents are of particular
clinical interest to act as contraceptives because consumer
preference studies suggest that most women worldwide
prefer a vaginal contraceptive agent to have contraceptive
and antimicrobial activity [7].
We therefore focused on the analysis of the spermicidal
effects of this new family of antimicrobial peptides to
explore their potential as vaginal contraceptive agents. We
also looked for a possible synergism between DSs and
ethylenediaminetetraacetic acid (EDTA).
Calcium, magnesium and potassium play important roles
in regulating vital sperm functions such as motility,
capacitation and acrosome reaction [48]. EDTA is a CaMg
chelating agent and was found to also have a relatively mild
spermicidal activity (EC100) at concentrations N5000 Ag/mL
[49,50]. Our study suggests that the potential of DSs as a
vaginal contraceptive might be enhanced further in the
presence of EDTA.
The present study has been carried out to determine the
effectiveness of DSs for inhibiting sperm motility. In

addition, the mechanism of their spermicidal action was

investigated and possible synergistic spermicidal effects
exerted by EDTA were evaluated.
2. Materials and methods
2.1. Peptide synthesis
Peptides (DS1 and DS4) were prepared by stepwise
solid-phase synthesis using Fmoc polyamide-active ester
chemistry on a Milligen 9050 pepsynthesizer. All Fmoc
amino acids were from Milligen-Waters (France).
4-(hydroxymethyl) Phenoacetic acid-linked polyamide/kieselguhr resin (pepsin kA), Fmoc amino acid pentaflurophenyl and 3-hydroxy-2, 3-dehydro-4-oxo-benzotriazine
esters were from Milligen/Bioresearch. Cleavage of
peptidyl resin and side chain deprotection were carried
out at concentrations of 5 mg of peptidyl resin in a 1-mL
mixture composed of trifluoroacetic acid, para-cresol,
thioanisol, water and ethyl methyl sulfide (82.5%, 5.5%
and 2.5% vol/vol) for 2 h at room temperature. After
filtering to remove the resin and ether extraction, the crude
peptides were purified by a combination of Sephadex gel
filtration, ion exchange chromatography and preparative
high-performance liquid chromatography (HPLC). Homogeneity of synthetic peptides was assessed by analytic
HPLC, amino acid analysis, solid-phase sequence analysis and mass spectrometry [29]. All peptides were stored
frozen as stock solutions at 1 mg/mL in double-distilled
water at 208C.
2.2. Reagents
The nonionic detergent N-9 (Igepal CO630) and EDTA
were purchased from Sigma (St. Louis, MO, USA).
Roswell Park Memorial Institute medium was obtained
from GIBCO, Ferticult IVF/FIV from Fertipro (Beernem,
Belgium), Sil-select stock from Fertipro 09/13RO1A.4,
pentoxifylline from Sigma P-1784, eosin Y (10 g/L; i.e.,
1% in distilled water) and nigrosin (50 g/L; i.e., 5% in
distilled water).
2.3. Collection and treatment of spermatozoa
Twenty fresh human semen samples collected by
masturbation were obtained from patients aged between
23 and 35 years undergoing routine semen analysis at the
Biological Laboratory of Reproduction and Cytogenetics
Farhat Hached (Sousse, Tunisia). The samples were allowed
to liquefy at 378C for 30 min. The semen characteristics,
volume, pH, viscosity and morphology were determined as
per World Health Organization guidelines [51].
Spermatozoa were separated by centrifugation through a
discontinuous Sil-select gradient. One milliliter of semen
was carefully layered over 50%/70%/90% gradient of Silselect and centrifuged for 20 min at 1500 rpm and
centrifuged again for 10 min at 2000 rpm, resuspended in
an appropriate volume (5 7 mL) of Ferticult and then used

A. Zairi et al. / Contraception 72 (2005) 447 453

449

spermatozoa were considered to be alive and stained

spermatozoa were considered to be dead [53].
2.6. Sperm morphology
Sperm morphology of treated sperm was studied microscopically using the eosinnigrosin staining technique
previously described. A drop of seminal fluid with DS1 and
DS4 was examined separately at 400 under a phase-contrast
microscope to detect any morphological change of sperm.
2.7. Calcium supplementation
This study was conducted by adding 12 mM of calcium
diluent to human sperm suspended in Hams F-10 medium
containing DS (at EC100) in a microfuge tube that was
incubated at 378C in a CO2 incubator (5%) for 1 h. Various
motility parameters were then recorded.
2.8. Pentoxifylline supplementation
This study was conducted by adding 1.5 mg/mL of
pentoxifylline diluent to the medium containing DS (at
EC100). Incubations were carried out in a CO2 incubator
(5%) at 378C for 1 h, and the motility patterns were
recorded every 30 min.
Fig. 1. Effect of different concentrations of DS1 on spermatozoal motility.

3. Results
for further procedures at a concentration of 20106 sperm
per milliliter of Ferticult.
2.4. In vitro spermicidal test and EC100 determination

3.1. Motility analysis

The inhibitory effect of DS1 and DS4 on sperm motility
is shown in Figs. 1 and 2. Although all of these compounds

The spermicidal action was determined by studying

various concentrations of DS1 and DS4 (0, 5, 25, 50, 100,
125 and 200 Ag/mL) and EDTA alone or combined with
DS1 and DS4 on spermatozoa separated from seminal
plasma and was evaluated by the SanderCramer test [52].
Briefly, 1 mL of spermicidal compound was mixed with
0.2 mL of liquefied semen by shaking and incubating at
378C. Samples were taken at various intervals and
analyzed for percentage and grade of sperm motility
microscopically. These measurements were performed at
20-s and 30-min intervals up to 240 min of incubation.
The weakest dilution that completely immobilized all the
spermatozoa in 20 s was recorded as EC100. Killing of
sperm was confirmed by supravital staining (eosin
nigrosin). Commercial N-9 (100 Ag/mL) was used as
positive control.
2.5. Sperm viability test
Sperm were incubated with a spermicidal solution or
control. At specific times, sperm viability was determined
using an eosinnigrosin staining technique: one drop of the
treated semen was mixed with one drop of 1% eosin. After
30 s, two drops of 5% nigrosin solution were added and
mixed. A drop of the semeneosinnigrosin mixture was
placed on a clear microscope slide, which was allowed to
dry and then observed under a microscope. Unstained

Fig. 2. Effect of different concentrations of DS4 on spermatozoal motility.

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A. Zairi et al. / Contraception 72 (2005) 447 453

Table 1
EC100 of test compounds on sperm motility after 20 s of exposure
Compounds
DS4
DS1
EDTA
N-9

EC100 (Ag/mL)
0%

0.1% EDTA

100
200
5000
100

10
100

alone immobilized sperm, their effective doses diverged by

orders of magnitude. Whereas DS4 abolished sperm motility
within 20 s (EC100) at a concentration of 100 Ag/mL, DS1
had no effect at this dose. At least twice the concentration of
DS1 was required to achieve the same effect as that of DS4.
It was also observed that with an increase in concentration
or with an increase in time at a specific concentration, there
was significant suppression of motility. It was found that the
effect of DS1 and DS4 on sperm motility and viability was
dose dependent; also, inhibition of human spermatozoa
activity was more sensitive to DS4 than to DS1.
The potency of DS4 was significantly greater than DS1
based on the concentration required to inhibit sperm motility.
Hence, only DS4 at a concentration of 100 Ag/mL was
selected for further experiments.
3.2. Sperm viability
To confirm that DS1 and DS4 are toxic to human
spermatozoa, we performed an eosinnigrosin stain. The
treatment of human sperm with DSs at their EC100 had
a potent spermicidal effect. The pink stain permeated the
membranes of the dead sperm. In contrast, untrea-

Fig. 4. Effect of calcium on the spermicidal action of DS4.

ted spermatozoa remained vital and impermeable to the

pink dye.
3.3. Sperm morphology
The morphological study of sperm was performed using
the eosinnigrosin stain. It was observed that no morphological changes were found in the sperm head, midpiece or
tail when compared with untreated sperm.
3.4. Synergism between spermicidal agents
The synergistic spermicidal activity of DSs and EDTA is
shown in Table 1. EDTA at a concentration of 0.1% showed
mild spermicidal activity. The EC100 of DS4 decreased to
10 Ag/mL as the added dose of EDTA was increased up to
0.1%. This procedure demonstrated the presence of a

Fig. 3. The effect of 0.1% EDTA on human sperm motility by DS4.

Fig. 5. Effect of pentoxifylline on the spermicidal action of DS4.

A. Zairi et al. / Contraception 72 (2005) 447 453

synergistic effect between DS4 and EDTA on sperm

motility as shown in Fig. 3. However, incubation of DS1
in the presence of EDTA at a concentration of 0.1% caused
no significant effect on motility after 30 min of exposure
and was reduced by only 50% within 4 h of incubation.
3.5. Calcium supplementation
This study was done to determine whether DS4 kills
sperm by depleting intracellular calcium. It was found that
calcium supplementation, even at the 2-mM concentration,
did not affect motility in the presence of DS4 (Fig. 4).
3.6. Pentoxifylline supplementation
The effect of pentoxifylline on motility of sperm in the
presence of DS4 is shown in Fig. 5. It is evident that the
presence of pentoxifylline did not delay the spermicidal
action of DS4 on sperm.

4. Discussion
DSs that were purified from the skin of tree-dwelling
South American frogs (P. sauvagei) have been reported to
have varied biologic activities against bacteria, fungi,
protozoa and viruses.
Our in vitro studies have demonstrated that DS1 and DS4
were effective spermicidal agents, and their effects were
found to be dose and time dependent. DS possessed
spermicidal activity to a certain extent at much lower doses
(Fig. 1) than other known spermicidal agents [37,48].
Between the two DSs used, DS4 was found to be more
potent than DS1 as far as sperm immobilization is
concerned. This observation may be correlated to their
differences in amino acid sequence, charge disruption and
hydrophobicity [30]. The present study also suggests that the
spermicidal effect of DS4 is significantly higher than that of
some other vaginal contraceptive preparations that are
available in the market, including magainins [37], gramicidins [48,49] and gossypol polyvinylpyrrolidone [54,55].
For example, the concentration of magainin A required to
immobilize spermatozoa within 20 s is 23 mg/mL [37], but
the concentration of DS4 was sufficient to achieve the same
efficacy, indicating that DSs have some advantages over
other known spermicidal agents. Moreover, the contraceptive use of these other compounds has limitations for a
number of reasons such as vaginal irritation and no
suppressive activity against STDs [12,13,17]. Although
both DS4 and N-9 immobilize all sperm with the same
EC100, a number of studies have shown that detergent
spermicides do not provide any protection against STDs
[13,17]. Besides, repeated use of a surfactant as a vaginal
contraceptive may cause lesions in the vaginal mucosa,
resulting in an increased risk of STD transmission [48].
Thus, DSs with their antibacterial, antiviral, antifungal and
spermicidal activities could be highly desirable vaginal
contraceptive agents in the future.

451

However, the major downside of DS4 is reflected in its

toxicity, which is a major concern in its pharmaceutical
application [47]. In previous studies, monolayers of Hep-2
cells were cultured in the presence of DSs to evaluate their
cytotoxicity [47]. The data showed that the cytotoxic effect
of these peptides was concentration dependent and that
DSs did not have any effect on cell viability at 16 Ag/mL.
So, with an EC100 of 100 Ag/mL, DS4 is known to damage
Hep-2 cell viability; this activity can also damage
application sites such as the vaginal epithelium. In the
present study, a low nontoxic dose of DS4 (10 Ag/mL) was
tested in combination with EDTA to reduce its toxicity and
the synergistic effect of these two compounds on inhibition
of sperm motility was evaluated.
EDTA, at a concentration of 0.1%, showed mild
spermicidal activity; it is often used as a preservative
potentiator in nasal drops or sprays at the same concentration [50]. The recommended dose of EDTA in the food,
cosmetic and pharmaceutical industries ranges between
25 and 500 Ag [49]. Besides, humans infused with EDTA
over a period of 2 years showed no serious side effects and
the acute toxicity of EDTA is negligible [49]. Whereas the
combination of N-9 with EDTA has an additive effect [56],
DS4 and EDTA acted together in a synergistic fashion as
their combined activity on sperm motility was two times
more potent than a similar concentration of N-9/EDTA [50].
Our study showed that DS4 alone at a nontoxic
concentration (10 Ag/mL) caused approximately 40% inhibition of sperm motility whereas the addition of 0.1% EDTA
achieved 100% sperm motility inhibition. This finding
indicated the presence of a synergistic effect between DS4
and EDTA on sperm motility. The mean effects of DS4
alone and in combination with 0.1% EDTA on motility of
human sperm are shown in Table 1. Thus, we found that
EDTA enhances the spermicidal activity of DS4 and reduces
its EC100 to allow use of a nontoxic concentration so that
the desired inhibition occurs without toxicity. This indicates
that DSs show a different synergistic spermicidal effect
with EDTA.
As both DS4 and EDTA are used in low nontoxic
concentrations, further studies examining the combination
of these compounds are clearly warranted to evaluate their
safety and efficacy for use as a vaginal contraceptive. The in
vivo toxicity of DS4 at 10 Ag/mL has to be studied to
determine if this peptide could disrupt epithelial barriers at
such low doses, especially those forming the lining of the
reproductive tract.
When used at high concentrations, DSs are cytotoxic in
vitro for host cells [41,43]. However, modifications can be
achieved on the sequence of administration to reduce their
toxicity without altering activity. Previous reports showed
that native DS4 displayed aggregation in solution and high
binding affinity. These properties correlated with high
cytotoxicity [40]. However, a large body of experimental
data demonstrates that peptide aggregation in solution has
dramatic consequences on cytotoxic properties, including

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A. Zairi et al. / Contraception 72 (2005) 447 453

selectivity of the peptides [40]. Thus, tampering with the

composition of the peptide hydrophobic domains by
reducing hydrophobicity or by increasing the net positive
charge dramatically affected the peptides activity and
resulted in various analogs that display potent bioactivity
with low cytotoxicity [41]. Previous studies performed in
mice with K4-S4(116), a derivative of DS4, have
demonstrated antimicrobial activity in vivo of this short
peptide with no toxicity [38]. Recently, the same peptide
was evaluated on P4-CCR5 cells. The data showed that
this analog did not cause cell toxicity with concentrations
up to 100 AM [56]. The same data also showed that human
endometrial epithelial cells are less sensitive to the toxic
effect of K4-S4, another modified peptide of DS4, than
other cells. This finding is important because these cells
would be in contact with spermicides. These shorter
derivatives with high activity but lower toxicity should
be evaluated by both in vitro and in vivo experimental
tests to determine their efficacy against spermatozoa.
Considering the available information about safety and
contraceptive efficacy, the precise mechanism of DS (DS4)
action on sperm motility can only be inferred. Unlike N-9,
which acts by indiscriminately solubilizing membrane
lipids, DS does not act by lysing sperm but appears to
display highly specific inhibition of sperm motility that
may account for its ability to reduce irritation. The
decreased motility and viability of sperm observed in the
presence of DSs may be attributed to the loss of
permeability of the plasma membrane by forming cation
channels that cross the cell membranes, which leads to cell
death. In addition, DS does not significantly affected select
sperm viability parameters such as calcium.
Because calcium supplementation did not protect the
sperm from the spermicidal action of DSs, it was evident
that DSs did not cause any depletion of intracellular
calcium. Supplementation with pentoxifylline, which is
known to enhance the motility of sperm, also could not
prevent the spermicidal action of DS (Fig. 5).
It is likely that the mechanism of antimotility action of
DS4 is different from that of other more toxic compounds
such as N-9. This high spermicidal potency of DS may be
caused by its high partition to the hydrophobic region of
the sperm membrane and channel-forming property causing deregulation of cation exchange (Na+, K+), which leads
to cell death. This observation, along with the striking
phenomenon of synergistic enhancement with EDTA,
suggests that DS probably affects the motility of sperm
membrane by depleting cytosolic potassium and sodium
whereas EDTA chelates extracellular calcium. However, it
is not clear how the disturbance in ionic levels of K+, Na+
and Ca2+ can lead to rapid and irreversible sperm
immobilization. Further studies are necessary to elucidate
the mode of DS action.
Considering the available information about contraceptive efficacy, the different biologic properties of these
peptides should be studied as new candidates for human

use as a barrier contraceptive agent with additional protection against STDs.

For a contraceptive to be effective, it should demonstrate potent spermostatic effects as well as lack of
toxicity. Therefore, DS4 derivatives deserve to be further
evaluated in animal models to test their toxicity in
mucosal tissues and their ability to be a barrier contraceptive agent both alone or in combination with other
potential compounds.

Acknowledgments
We thank Dr. R. Khelifa (H. Thameur Hospital, Tunis,
Tunisia) for the first reading of this manuscript. We
acknowledge with gratitude the assistance of Dr. Saad
Ali and Ajina Mounir from the Laboratory of Reproduction
and Cytogenetics, CHU Farhat Hached, in providing
semen samples.
This study was supported by a grant from the Secretariat
of Scientific ResearchTunisia and performed at the
Laboratory of Biochemistry (UR/08-45), Faculty of Medicine, 4002 Sousse, Tunisia.

References
[1] DCruz OJ, Venkatachalam TK, Uckun FM. Structural requirements
for potent human spermicidal activity of dual-function aryl phosphate
derivate of bromo-methoxy zidovudine (compound WHI-07). Biol
Reprod 2000;62:37 44.
[2] Bird KD. The use of spermicide containing nonoxynol-9 in the
prevention of HIV infections. AIDS 1991;55:791 6.
[3] Schill WB, Wolf HH. Ultrastructure of human spermatozoa in the
presence of the spermicide nonoxynol-9. Andrologia 1981;13:42 9.
[4] Wilburn WH, Hahn DW, McGuire JJ. Scanning electron microscopy
of human spermatozoa after incubation with the spermicide nonoxynol-9. Fertil Steril 1983;39:717 9.
[5] DCruz OJ, Venkatachalam TK, Zhu Z, Shih M-J, Vekun FM. Bromomethoxy and aryl phosphate derivates of azidothymidine are dualfunction spermicides with a potent anti-HIV activity. Biol Reprod
1998;59:503 15.
[6] Roddy RE, Zekeng L, Rayan KA, Tamoufe U, Tweedy KG. Effect of
nonoxynol-9 gel in urogenital gonorrhea and chlamydial infection: a
randomised controlled trial. JAMA 2002;287:1117 22.
[7] DCruz OJ, Uckun FM. Gel-microemulsions as vaginal spermicides
and intravaginal drug delivery vehicles. Contraception 2001;
64:113 23.
[8] Krebs FC, Miller SR, Catalan BJ, et al. Sodium dodecyl sulfate and
C31G as microbicidal alternatives to nonoxynol-9 sensitivity of
primary human vaginal keratinocytes. Antimicrob Agents Chemother
2000;44:1954 60.
[9] DCruz OJ, Shih M-J, Yiv SH, Chen C-L, Uckun FM. Synthesis,
characterization and preclinical formulation of a dual-action phenyl
phosphate derivative of bromo-methoxy zidovudine (compound
WHI-07) with a potent anti HIV activity. Mol Hum Reprod 1999;
5:421 32.
[10] DCruz OJ, Uckun FM. Novel derivates of phenethyl-5-bromopyridylthiourea (PBT) and dihydroalkonybenzyloxopyrimidine (DABO)
are dual function spermicides with a potent anti-HIV activity.
Biol Reprod 1999;60:1419 28.
[11] Klebanoff SJ. Effects of the spermicidal agent nonoxynol-9 on
vaginal microbial flora. J Infect Dis 1992;165:19 25.

A. Zairi et al. / Contraception 72 (2005) 447 453

[12] Niruthisard SR, Roddy E, Chutivongse S. The effect of frequent
nonoxynol-9 use on the vaginal and cervical mucosa. Sex Transm Dis
1991;18:176 9.
[13] Rekart Ml. The toxicity and local effect of the spermicide nonoxynol-9. J Acquir Immune Defic Syndr 1993;4:165 70.
[14] Weir SS, Roddy RE, Zekeng L, Feldblum PJ. Nonoxynol-9 use,
genital ulcers and HIV infection in a cohort of sex workers. Genitourin
Med 1995;71:78 81.
[15] Roddy RE, Cordero M, Cordero C, Fortney JA. A dosing of
nonoxynol-9 and genital irritation. Int J STD HIV 1993;4:165 70.
[16] Hooten TM, Hillier S, Johnson C, Roberts PL, Stamm WE.
Escherichia coli bacteriuria and contraceptive method. JAMA
1991;265:64 9.
[17] Stafford MK, Ward H, Flanagan A. A safety study of nonoxynol-9 as
a vaginal microbicide: evidence of adverse effects. J Acquir Immune
Defic Syndr Human Retrovirol 1998;17:327 31.
[18] Rosenstein IJ, Stafford MK, Kitchen VS, Ward H, Weber JN,
Robinson D. Effect on normal vaginal flora of three intravaginal
microbicidal agents potentially active against human immunodeficiency virus type 1. J Infect Dis 1998;177:1386 90.
[19] Augenbraun MH, McCormack WM. Sexually transmitted diseases in HIV-infected persons. Infect Dis Clin North Am 1994;8:
439 48.
[20] Zasloff M. Antimicrobial peptides of multicellular organisms. Nature
2000;415:389 95.
[21] Mor A, Nicolas P. Isolation and structure of novel defensive peptides
from frog skin. Eur J Biochem 1994;219:145 54.
[22] Ganz T. Defensins: antimicrobial peptides of innate immunity.
Nat Rev Immunol 2003;3:710 20.
[23] Hancock REW, Leher R. Cationic peptides: a new source of
antibiotics. Trends Biotechnol 1998;16:82 7.
[24] Hancock R, Chapple D. Peptide antibiotics. Antimicrob Agents
Chemother 1999;43:1317 23.
[25] Leher RI, Ganz T. Antimicrobial peptides in mammalian and insect
host defense. Curr Opin Immunol 1999;11:23 7.
[26] Nicolas P, Mor A. Peptides as weapons against microorganisms in the
chemical defense system of vertebrates. Annu Rev Microbiol
1995;49:277 304.
[27] Corzo P, Escoubas P, Villags E, Barnaham KJ, Norton R, Naka T.
Characterization of unique amphipathic antimicrobial peptides from
venom of scorpion Pandinus imperator. Biochem J 2001;359:35 45.
[28] Mor A, Nguyen VH, Delfour A, Migliore-Samour D, Nicolas P.
Isolation, amino acid sequence and synthesis of dermaseptin, a novel
antimicrobial peptide of amphibian skin. Biochemistry 1991;30:
8824 30.
[29] Mor A, Rouffaud MA, Montagne JJ, Nguyen VH, Nicolas P. Natural
and synthetic dermaseptins: in vitro large spectrum antimicrobial
peptides. J Mycol Med 1993;3:137 43.
[30] Mor A, Hani K, Nicolas P. The vertebrate peptide antibiotics
dermaseptins have overlapping structural features but target specific
microorganisms. J Biol Chem 1994;50:31635 41.
[31] Moll GN, Brul S, Konings WN, Dreissen JM. Comparison of the
membrane interaction and permeabilization by the designed peptide
AC-MB21-NH2 and truncated dermaseptins S3. Biochemistry
2000;39:11907 12.
[32] Feder R, Nehushtai R, Mor A. Affinity driven molecular transfer from
erythrocytes membrane to target cells. Peptides 2001;22:1683 90.
[33] Shephered CM, Vogel HJ, Tieleman DP. Indications of the designed
antimicrobial peptides MB21 and truncated dermaseptins S3 with
lipid bilayers: molecular dynamics stimulations. Biochemistry
2003;233 43.
[34] Cruciani RA, Barker JL, Zasloff M, Chen HC. Antibiotic magainins
exert cytolytic activity against transformed cell lines through channel
formation. Proc Natl Acad Sci USA 1991;88:3792 6.
[35] Cudic M, Otvos L. Intracellular targets of antimicrobial peptide. Curr
Drug Targets 2002;3:101 6.

453

[36] De Waal A, Gomes AV, Mensunk A, Grootegoed JA, Westerhoff HV.

Magainins affect respiratory control, membrane potential of mobility
of hamster spermatozoa. FEBS Lett 1991;293:19 23.
[37] Reddy KV, Shahani SK, Meherji PK. Spermicidal activity of magainins: in vitro and in vivo studies. Contraception 1996;53:205 10.
[38] Navon-Venezia S, Feder R, Gaidukov L, Carmeli Y, Mor A.
Antimicrobial properties of dermaseptin S4 derivatives with in vivo
activity. Antimicrob Agents Chemother 2002;46:689 94.
[39] Jouenne T, Mor A, Bonato H, Junter GA. Antimicrobial activity of
synthetic dermaseptins against growing and non-growing Escherichia
coli cultures. J Antimicrob Chemother 1998;42:87 90.
[40] Feder R, Dagan A, Mor A. Structureactivity relationship study of
antimicrobial dermaseptin S4 showing the consequences of peptide
oligomerization on selective cytotoxicity. J Biol Chem 2000;275:
4230 8.
[41] Kustanovich I, Shallev DE, Mikhlin M, Gaidukov L, Mor A.
Structural requirements for potent versus selective cytotoxicity for
antimicrobial dermaseptin S4 derivates. J Biol Chem 2002;277:
16941 51.
[42] Hernandez C, Mor A, Dagger F, et al. Functional and structure
damage in Leishmania mexicana exposed to the cationic peptide
dermaseptin. Eur J Cell Biol 1999;59:414 24.
[43] Dagan A, Efron L, Gaidukov L, Mor A, Ginsburg H. In vitro
antiplasmodium effects of dermaseptins. Antimicrob Agents Chemother 2002;46:1059 66.
[44] Efron L, Dagan A, Gaidukov L, Ginsburg H, Mor A. Direct
interaction of dermaseptin S4 amino heptanoyl derivate with intraerythrocytic malaria parasite leading to increased specific antiparasitic
activity in culture. J Biol Chem 2002;277:24067 72.
[45] Krugliak M, Feder R, Zolotarev VY, et al. Antimicrobial activities of
dermaseptin S4 derivates. Antimicrob Agents Chemother 2000;44:
2442 51.
[46] Coote PJ, Holyoak CD, Bracey D, Ferdinando DP, Pearce JA.
Inhibitory action of truncated derivative of the amphibian skin peptide
dermaseptin S3 on Saccharomyces cerevisiae. Antimicrob Agents
Chemother 1998;42:2160 70.
[47] Belaid A, Aouni M, Khelifa R, Trabelsi A, Jemmali M, Hani K. In
vitro antiviral activity of dermaseptins against herpes simplex virus
type 1. J Med Virol 2002;66:229 34.
[48] Centola GM. Doseresponse effects of gramicidin-D, EDTA, and
nonoxynol-9 on sperm motion parameters and acrosome status.
Contraception 1998;58:35 8.
[49] Bourinbaiar AS, Lee CH. Synergistic effect of gramicidin and EDTA
in inhibiting sperm motility and cervical mucus penetration in vitro.
Contraception 1996;54:367 72.
[50] Lee CH, Bagdon R, Chien YW. Comparative in vitro spermicidal activity of chelating agents and synergistic effect with
nonoxynol-9 on human sperm functionality. J Pharm Sci 1996;85:
91 6.
[51] World Health Organization. Laboratory manual for the examination of
human semen and spermcervical mucus interaction. 4th ed. New
York7 Cambridge University Press; 1999.
[52] Sander FV, Cramer SD. A practical method for testing the spermicidal
action of chemical contraceptives. Hum Fertil 1941;6:134 7.
[53] Eliasson R. Supravital staining of human spermatozoa. Fertil Steril
1977;28:1257.
[54] Zavos PM, Correa JR, Nosek D, Mohammadi F, Digenis GA.
Comparative spermicide performance of iodinated and non-iodinated
contraceptive formulations of nonoxynol-9 co-precipitated with
polyvinylpyrrolidone. Contraception 1996;54:39 41.
[55] Zavos PM, Ed S, Correa JR, Zarmakoupis-Zavos PN. Assessment
of a tablet drug delivery system incorporating nonoxynol-9
coprecipitated with polyvinylpyrrolidone in preventing the onset
of pregnancy in rabbits. Fertil Steril 1998;69:768 73.
[56] Lorin C, Saidi H, Belaid A, et al. The antimicrobial peptide dermaseptin
S4 inhibits HIV-1 infectivity in vitro. Virology 2005;334:264 75.