Contraception 64 (2001) 113123

Review article

Gel-microemulsions as vaginal spermicides and intravaginal drug

delivery vehicles
Osmond J. DCruza,*, Fatih M. Uckunb
a

Drug Discovery Program, Department of Reproductive Biology, Parker Hughes Institute, St. Paul, MN 55113, USA
b
Drug Discovery Program, Department of Virology, Parker Hughes Institute, St. Paul, MN 55113, USA

Abstract
There is a need for novel formulations to improve the bioavailability through the vaginal/rectal mucosa of microbicidal drug substances
against sexually transmitted diseases. In addition, there is a need for more effective and less toxic vaginal spermicides. Here we review our
recent discovery of novel gel-microemulsions (GM) as nontoxic, dual-function intravaginal spermicides, which can be used as delivery
vehicles for lipophilic drug substances targeting sexually transmitted pathogens. We describe the formulation and biologic properties of 2
novel, submicron-particle-size GMs, GM-4 and GM-144, which were prepared from commonly available pharmaceutical excipients. These
GMs comprising oil-in-water microemulsion and polymeric hydrogels were designed to solubilize lipophilic antiviral/antimicrobial agents
and exhibited rapid spermicidal activity in human semen. Preclinical studies comparing the in vivo contraceptive efficacy of GM-4 and
GM-144 versus nonoxynol-9-based detergent spermicide (Gynol II) in the rigorous rabbit model confirmed the potent contraceptive activity
of these GMs. Unlike nonoxynol-9, repeated intravaginal applications of GM-4 and GM-144 in the rabbit vaginal irritation test were not
associated with local inflammation or damage of the vaginal mucosa or epithelium. Furthermore, in short-term toxicity studies performed
in mice, repetitive intravaginal application of spermicidal GM-4 and GM-144 for up to 13 weeks was not associated with any local, systemic,
or reproductive toxicity. Spermicidal GMs have unprecedented potential as dual function microbicidal contraceptives to improve vaginal
bioavailability of poorly soluble antimicrobial agents without causing significant vaginal damage. 2001 Elsevier Science Inc. All rights
reserved.
Keywords: Microbicides; Microemulsion; Nonoxynol-9; Sexually transmitted diseases; Vaginal spermicides

1. Introduction
The vaginal vault is subject to conditions that render it a
target for disease and infection during sexual intercourse.
Treatment of the female reproductive system for the prevention of conception and sexually transmitted diseases
(STDs) usually involves the delivery of active agents to the
vaginal vault and its environs. Systems to effect the delivery
of such spermicidal or nonspermicidal microbicidal agents
are usually in the form of gels, foams, creams, suppositories, and quick dissolving tablets. At present, such over-thecounter dual function microbicides include the neutral surfactants isononyl-phenyl-polyoxyethylene-(9)-ether or
nonoxynol-9 (N-9), p-menthanyl-phenyl-polyoxyethylene(8,8)-ether or menfegol, and isooctyl-phenyl-polyoxyethylene-(9)-ether or octoxynol-9 (O-9) [1,2]. N-9 is the most
* Corresponding author. Tel.: 1-651-697-0041; fax: 1-651-6971057.
E-mail address: odcruz@ih.org (O.J. DCruz).

commonly used spermicidal contraceptive in the UK and

USA [3,4]. Worldwide, the cationic surfactant benzalkonium chloride and the anionic detergent sodium docusate
(dioctyl sodium sulphosuccinate) are also used as vaginal
spermicides [5]. N-9, sodium oxychlorosene, and benzalkonium chloride have been used as creams, gels, foams, suppositories, ovules, sponges, or film.
The spermicidal and microbicidal activities of these surfactants are associated with their structural affinity to the
membrane lipids [6,7]. Therefore, the major drawback of
using N-9 or other currently used surfactants is their detergent-type effect on epithelial cells and normal vaginal flora.
N-9 displays antiviral/antibacterial and spermicidal activities only at cytotoxic doses [8 11]. Frequent use of N-9 as
a vaginal contraceptive/microbicide has been associated
with an increased risk of vaginal or cervical infection, irritation, or ulceration [1215]. Detergent-type spermicides
alter vaginal bacteria or flora and lead to an increased risk of
opportunistic infections [16 19]. Chemical irritation that
disrupts the vaginal mucosa can enhance the risk of vaginal

0010-7824/01/$ see front matter 2001 Elsevier Science Inc. All rights reserved.
PII: S 0 0 1 0 - 7 8 2 4 ( 0 1 ) 0 0 2 3 3 - 5

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O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

transmission of STDs including HIV-1, the causative agent

of AIDS, by mucosal erosion and local inflammation [15,
20 23]. Therefore, vaginal spermicidal microbicides lacking detergent-type membrane toxicity may offer a significant clinical advantage over the currently available
detergent-type spermicides.
Because vaginal microbicides would likely be used repeatedly over decades, an ideal spermicidal microbicide
should have an established safety record and lack genital
epithelial toxicity. Moreover, it should be inexpensive, be
produced from commonly available resources, and should
have a broad specificity for solubilizing the drugs for prevention of sexual transmission of several STDs including
HIV-1. The precise incidence of STDs is not known, but it
is estimated that each year there are 12 million new infections in the US [24]. This figure includes an estimated 4
million cases of Chlamydia trachomatis infection, 800,000
cases of gonorrhea, over 110,000 cases of syphilis, and
several million cases of Trichomonas vaginitis and nonspecific urethritis. Each year there are 0.51 million cases of
human papillomavirus (HPV) infection, 200,000 300,000
cases of hepatitis B virus (HBV) infection, and 40,000
80,000 new infections with HIV [24 26]. An estimated 1
million Americans are currently infected with HIV, 31 million infected with herpes simplex viruses (HSV), 24 million
infected with HPV, and over 1 million are chronic HBV
carriers.
Of the more than 7000 new HIV infections occurring in
the world each day, about 90% are the result of heterosexual
transmission. The emergence of HIV/AIDS as a disease
spread through sexual intercourse, combined with growing
public awareness about the problems associated with other
viral STDs, has prompted the search for new, effective,
acceptable, and safe vaginal microbicides for curbing mucosal and perinatal viral transmission [11,27]. Microbicides
would provide protection by inactivating microbes or by
preventing microbes from replicating either in semen or in
the infected host cells that line the vaginal wall.
In a systematic effort to develop a spermicidal microbicide with good solubility for lipophilic drugs, we evaluated
several components of microemulsion formulations for
spermicidal activity, drug solubility, particle size, stability,
and responses to in vivo and in vitro biologic models. We
found that two novel, submicron (30 80 nm) particle size
gel-microemulsion (GM) formulations (viz., GM-4 and
GM-144) prepared from pharmaceutical excipients commonly used in topical, oral, and injectable medications exhibit rapid spermicidal activity in human semen, although
these excipients by themselves exhibit little or no spermicidal activity [28]. In this review, we discuss the utility of
these spermicidal GMs as vehicles for microbicides for the
purpose of preventing transmission of pathogens to mucosal
membranes, particularly sexually transmitted viruses, such
as HIV-1 and HSV, and bacteria, such as Neisseria gonorrheae and C. trachomatis. In addition, the spermicidal properties of these GMs can be exploited to achieve in vivo

contraceptive effect without the toxic side effects characteristic of currently available detergent-type spermicides.
The effectiveness of two novel GM formulations was tested
for in vivo contraceptive efficacy, local, systemic, and reproductive toxicity in test animal species. Our main strategy
was to design fast-acting GMs that kill sperm in semen
on contact without exhibiting cytotoxicity to mucosal
epithelium.

2. Gel-microemulsion formulation
Microemulsions are thermodynamically stable, isotropically clear dispersions of two immiscible liquids, such as oil
and water, stabilized by an interfacial film of surfactant
molecules [29]. The surfactant may be pure, a mixture, or
combined with other additives. The role of surfactant is
stabilization of the microemulsion, for instance, by decreasing the interfacial tension. The microemulsion has an oilin-water (o/w), a water-in-oil (w/o), or a bicontinuous structure. In this kind of microemulsion, (a) the hydrophilic
component is dispersed as colloidal droplets in the lipophilic component, (b) the lipophilic component is dispersed as colloidal droplets in the hydrophilic component,
or (c) the hydrophilic and the lipophilic component form a
microemulsion with bicontinuous structure wherein said
components form elongated adjacent channels. The droplet
size is typically in the range of 1100 nm.
Microemulsions are superior to simple micellar solutions
in terms of solubilization potential, and their thermodynamic stability offers advantages over unstable dispersions,
such as emulsions and suspensions, and has a long shelf-life
[30]. GMs are suitable as carriers for both water-soluble and
lipo-soluble drugs. Oil-soluble drugs can be formulated in
o/w microemulsions, whereas, water-soluble drugs are better suited for w/o systems. If the microemulsion has a
bicontinuous structure, the composition is suitable as carrier
for both water-soluble and oil-soluble drugs.
An increasing number of reports in the literature suggest
that lipid-based microemulsions (o/w and w/o) can be used
to enhance the bioavailability of lipophilic antimicrobial
drugs [3136]. Drug delivery advantages offered by microemulsions include improved drug solubilization and protection against enzymatic hydrolysis, as well as the potential
for enhanced absorption afforded by surfactant-induced
membrane fluidity and thus permeability changes. The challenge for the pharmaceutical formulator is to predict which
oil(s) and surfactant(s) to select for a particular application,
taking into consideration their acceptability because of potential toxicity. The formulation of w/o and o/w microemulsions usually involves a combination of three to five basic
components, namely, oil, water, surfactant, cosurfactant,
and electrolyte. The tendency toward a w/o or an o/w
microemulsion is dependent on the properties of both the oil
and surfactant and the oil-to-water ratios. The nonionic or
zwitterionic surfactants are often considered for pharmaceu-

O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

tical applications and microemulsion formulation because

they are less toxic and less affected by pH and ionic strength
changes. So far, microemulsions have been used mainly for
the oral delivery of peptide drugs [37,38], but microemulsions have also been reported as drug carriers for topical,
dermal, transdermal, and pulmonary administration of drugs
[32,33,36,39,40].
Microemulsions have great potential as intravaginal/rectal drug delivery vehicles for lipophilic drugs, such as microbicides, steroids, and hormones, because of their high
drug solubilization capacity, increased absorption, and improved clinical potency. However, the use of microemulsions for intravaginal or intrarectal administration imposes
rigorous demands on the nontoxicity of the formulation and
its bioavailability. In a systematic search for microemulsion
formulations suitable for intravaginal application, we developed lipophilic submicron (30 80 nm) particle-sized o/w
microemulsions using the common pharmaceutical excipients through systemic mapping of ternary phase diagrams.
Several microemulsion compositions were screened for particle size, stability, lipophilic drug solubility, and responses
to in vitro and in vivo biologic models [28]. The ingredients
tested included: medium-chain triglycerides (Captex 300),
purified soya phospholipid (Phospholipon 90G), Pluronic
poloxamers, ethoxylated castor oil (Cremophor EL), propylene glycol, and polyethelene glycol. The ingredients
selected included drug solubilizers and stabilizers (Captex
300, Cremophor EL, phospholipon 90G, Pluronic F-68,
propylene glycol, and polyethelene glycol 200) as well as a
preservative (sodium benzoate).
Various polymeric gels were screened to produce a gel
with desirable viscosity (range of 200-5000 centipoises).
Polymer suspensions of seaspan and viscarin carrageenan
and Xanthan gum (Rhodigel) were selected as additives to
the microemulsion-based system. These polymeric hydrogels yielded a gel with desirable viscosity (1000 centipoise) as well as a high thickening capability and compatibility with vaginal mucosa. The components of two GMs,
GM-4 and GM-144, are listed in Table 1. Long-term toxicity studies and clinical trials suggest that these pharmacological excipients are safe for human use. A brief description of the categories of permeation enhancers (surfactants,
fatty acids, medium chain glycerides), humectants, and hydrogels used for the formulation of GM-4 and GM-144 are
given below.

115

Table 1
Components of spermicidal gel-microemulsions*
Ingredients

Captex 300
Cremophor EL
Pluronic F-68
Phospholipon 90G
Propylene Glycol
PEG-200
Seaspan carrageenan
Viscarin carrageenan
Rhodigel
Sodium benzoate
Water

Type

Lipid
Surfactant
Surfactant
Phospholipid
Humectant
Humectant
Natural polymer
Natural polymer
Natural polymer
Preservative
Diluent

Final concentration
(%, by wt)
GM-4

GM-144

10.8
7.6
0
5.1
4.2
4.2
0.9
0.5
0
0.2
66.5

4.5
3.8
0.4
3.0
17.0
0
0
0
1.0
0.2
70.1

* Captex 300 (C8/C10 triglycerides from coconut oil), Cremophor EL

(polyethoxylated castor oil), Rhodigel (xanthan gum), Pluronic F-68
(polyoxyethylene and polyoxypropylene polymer), Phospholipon 90G
(modified saturated soybean phosphatidylcholine).

of a microemulsion. Microemulsion existence fields can be

identified from mapping of ternary phase diagrams of o/w
microemulsion [29,30]. Fig. 1 shows o/w microemulsion
existence field in the ternary phase diagram of a microemulsion system used for the formulation of GM-4, containing
Captex 300, a surfactant blend, and an aqueous phase. The
surfactant blend is a mixture of Cremophor EL, Phospholipon 90G, propylene glycol, PEG 200. In addition to a
one-phase region (the nongrid area) in which the microemulsions appear, the phase diagram also exhibits twophase and three-phase regions.

3. Components of GM-4 and GM-144

Microemulsions are usually four-component mixtures
that include a surfactant, a cosurfactant, an oil, and water.
When a mixture of surfactant and cosurfactant is added to a
biphasic water-oil system, a thermodynamically stable, optically transparent or translucent, isotropic mixture spontaneously forms. The nature and structure of the surfactant,
cosurfactant, and oil are essential features in the formulation

Fig. 1. Ternary phase diagram of an oil/surfactant/water system with

emphasis on microemulsion phase. Within the phase diagram, the nongrid
area represents the single-phase microemulsion region. The arrow points to
the o/w microemulsion region that was used for the formulation GM-4
listed in Table 1.

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O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

3.1. Surfactants/cosurfactants
Pharmaceutically acceptable surfactants may be ionic or
nonionic. Particularly suitable are naturally occurring surfactants such as phospholipids and triglycerides. The monomeric solubility of the surfactant both in water and in oil
must be low so that the surfactant could form a microemulsion with the highest possible stability. If the surfactant is
ionic, it must have two hydrocarbon chains to form a microemulsion. If the ionic surfactant does not have two hydrocarbon chains, a neutral inorganic salt and a cosurfactant
is added.
3.1.1. Captex 300
Medium chain triglycerides (MCTs) derived from coconut oil have been particularly attractive for formulating
orally active microemulsions because they are stable food
grade products and generally recognized as safe by the Food
and Drug Administration (FDA). MCTs are widely used in
foods, drugs, and cosmetics [40]. MCTs are essentially
nontoxic in acute toxicity tests conducted in several species
of animals [41]. Microemulsions incorporating these excipients can be formulated at ambient temperature over a wide
range of compositions. Medium-chain glycerides (mono, di,
and triglycerides) are reported to improve the mucosal absorption of co-formulated drugs [42].
3.1.2. Cremophor EL (polyethoxylated castor oil)
A naturally occurring surfactant known under the trade
name Cremophor is an ethylene oxide derivative of castor
oil. As a nonionic surfactant, Cremophor has been used
widely as an emulsifier, solubilizer, and formulation adjunct
in pharmaceuticals [43]. The main component of Cremophor EL is glycerol-polyethylene glycol (PEG) ricinoleate,
which, together with fatty acid esters of PEG, represents the
hydrophobic part of the product. The smaller part consists of
PEGs and ethoxylated glycerol. Cremophor EL, when used
up to 10% w/v, was found to cause no apparent membrane
damage to Caco-2 cell monolayers, nor did it cause lysis of
human leukemic cells [44].
3.1.3. Phospholipon 90G (modified saturated
soybean phosphatidylcholine)
Lecithin, a naturally occurring biologic surfactant
known under the trade name Phospholipon 90G is a
major component of membrane lipids. Lecithin is highly
lipophilic because of two hydrocarbon chains but at the
same time, it is hydrophobic because of polar zwitterionic head group. As a natural surfactant, Phospholipon
has rapid spreadability on the air-water interphase. Microemulsion compositions containing Phospholipon have
been shown to ameliorate the gastric lesions induced by
oral nonsteroidal anti-inflammatory drugs [45].

3.1.4. Pluronic F-68 [poly(oxyethylene)poly(oxypropylene)]

Pluronics are water-soluble, nonionic detergents. The
compounds are formed from condensing ethylene oxide
with a hydrophobic base formed by the condensation of
propylene oxide with propylene glycol. The addition of
polyoxyethylene radicals to the hydrophobic portion tends
to increase the solubility of the molecule as a whole to make
the surfactant water soluble. Pluronic poloxamers are
widely used parenteral vehicles as nontoxic solubilizers for
lipophilic drugs and vitamins as well as to enhance absorption of drugs through the mucus membranes. Pluronic F-68
is a nonionic polyol that does not have any intrinsic antibacterial activity. It is commonly used to protect cultured
animal cells from the detrimental effects of sparging [46].
3.1.5. Polyethylene glycol
PEG is commonly used as a base for cream, gel, and
ointment preparations because of its physical characteristics
and the versatile consistencies that can be obtained by
mixing different proportions of its liquid and waxy forms.
PEG chain lengths in combination with vegetable oils have
been used to solubilize poorly water-soluble drugs and improve their bioavailability.
3.1.6. Propylene glycol (cosurfactant)
Propylene glycol is a common solvent used in industry,
food, and in consumer goods. It is commonly used as a drug
solubilizer in topical, oral, and injectable medications. In
intraperitoneal and oral toxicity studies performed in mice
and rats given up to 10% propylene glycol, no evidence of
adverse effects were noted on neuropsychopharmacological
activity, fertility, and reproduction [47].
3.2. Gelling agents
Suitable gel polymers and water are added to the
microemulsion, thereby bringing the microemulsion into
a gel form. The gelling agent includes conventional
gelling agents, those materials which on contact with
water, imbibe the water and thereby form hydrogels or
pseudoplastics. It is essential that for intravaginal application by women, a viscous GM is preferred so the
product does not seep from the body cavity. This was
achieved by the addition of mucoadhesive polysaccharide
gelling agents, carrageenan, and xanthan gum, for the
preparation of spermicidal GMs, GM-4, and GM-144,
respectively. The gelling agents used were 0.9% and
0.5% seaspan and viscarin carrageenan for GM-4 and 1%
xanthan for GM-144. The utility GM as a vaginal spermicide was based on the principle of bioadhesion, rather
than the viscosity, as the retentive mechanism. The viscosity of GMs used for the in vivo contraceptive efficacy
study was 100-fold greater than that of seminal viscosity
(8 9 centipoise). For intravaginal use, a desirable viscosity range (1,000 10,000 centipoise) can be achieved

O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

by increasing the percentage of mucoadhesive polymers

in the GM.
3.2.1. Carrageenan
Carrageenan is a naturally occurring family of polysaccharides derived from red seaweed and is made up of
sodium, potassium, magnesium, and calcium sulfate esters
of galactose and 3,6-anhydrogalactose units. It is used as a
stabilizer and gelling agent by the food industry. Viscarin
and seaspan carrageenans were selected as gel base for
GM-4 because of their excellent water binding capabilities,
which are useful in topical dosage forms.
3.2.2. Rhodigel (xanthan gum)
Rhodigel xanthan gum is a high molecular weight anionic polysaccharide from the bacterium Xanthomonas
campestris. The chemical structure of the xanthan gum
consists of a cellulosic backbone substituted on alternate
glucose residues with a trisaccharide sidechain. It is used as
a rheology control agent in aqueous systems and as a stabilizer for emulsions and suspensions. Its numerous areas of
applications cover a broad spectrum and range from the
food industry to oil recovery. Xanthan gum was preferred as
a gel base for GM-144 because of its safety, bioadhesive
properties, and wide acceptability as a pharmacological
excipient for topical application. Xanthan gum exhibits
pseudoplastic (shear thinning) behavior in solution (i.e. high
viscosity at rest, viscosity decreases with increasing shear
rates, and instantly returns as shear is reduced or removed)
that is higher than other commercial hydrocolloids. Xanthan
gum is extremely stable in both acidic and alkaline solutions
over a broad pH range of 2 to 12 and over a wide range of
temperatures (up to 60C).
3.3. Preservative
3.3.1. Sodium benzoate
Sodium benzoate is an FDA-approved preservative that
has been used by food manufacturers for over 80 years to
inhibit microbial growth. Sodium benzoate has been the
subject of extensive experimentation: it has been tested in
longitudinal and short-term feeding experiments in humans,
dogs, and rats. All evidence points to sodium benzoate as a
safe preservative.
3.4. Water
The final essential ingredient in the GM composition
having improved interfacial tension properties is water. The
proportion of water in the compositions generally is in the
range of 60 70% by weight. In the final form, the GMs are
clear compositions and exhibit stability at reduced and increased temperature. Such compositions remain clear and
stable in the range of 4 40C, and the compositions exhibit
a pH in the neutral to the acidic range.

117

4. Spermicidal activity of GMs

We systematically evaluated the effect of individual excipients formulated in GM-4 and GM-144 on the motility of
sperm in human semen, which was quantitated by computer-assisted sperm analysis (CASA) [710,28]. A timecourse study of sperm motility impairment by each of the
individual components of GM-4 formulation revealed that
none of the eight excipients tested, including Captex 300,
phospholipon 90G, Cremophor EL, propylene glycol, PEG
200, viscarin carrageenan, seaspan carrageenan, and sodium
benzoate, was spermicidal in human semen (t1/2 60
min; n 3; Fig. 2A). By contrast, the submicron particle
size GM-4 formulation completely immobilized sperm in
human semen in less than 2 min (mean 1.2 0.3 min; n
6).
Similarly, the ingredients used for GM-144, Captex 300,
Cremophor EL, Phospholipon 90G, Pluronic F-68, and sodium benzoate demonstrated little or no inhibitory effects
on human sperm motility (t1/2 60 min) at the concentration used for GM-144 formulation (Fig. 2B). Furthermore, sperm motion kinematics using CASA confirmed that
these excipients did not significantly alter the sperm motion
parameters, such as the progressive velocity, straightness of
the swimming pattern, linearity of the sperm tracks, beatcross frequency, and the amplitude of lateral sperm head
displacement. Whereas treatment of human semen with
propylene glycol and Rhodigel at the concentration used for
GM-144 formulation induced only partial spermicidal activity with slow kinetics (t1/2 24 min). Progressive
sperm motility (30%) was evident even after 60 min of
exposure to these components. Similarly, the microemulsion without the Rhodigel polymer was partially spermicidal with slow kinetics (t1/2 15 min). By contrast, the
submicron particle size GM, GM-144, containing Rhodigel
as the polymer completely immobilized sperm in human
semen in less than 30 sec (mean 27 4 sec; n 9). The
oleaginous nonisotropic mixture of microemulsion components lacked spermicidal activity in semen. Thus, the combination of commonly used pharmaceutical excipients, as a
GM formulation, was a rapid spermicidal agent in human
semen. The kinetics of spermicidal activity of GM can be
enhanced by varying the GM components. We have tested
a panel of GMs with viscosities ranging from 300 to 2000
centipoise. We found that the kinetics of spermicidal activity in human semen was unrelated to viscosity but was
dependent on the type of hydrogel used for the formulation
of GM.

5. Contraceptive efficacy of GM-4 and GM-144

Because of the rapid spermicidal activity of the combination of these pharmaceutical excipients as a GM formulation, we performed in vivo contraceptive efficacy studies
of GM-4 and GM-144. Gynol II, a commercial contracep-

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O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

Fig. 2. In vitro spermicidal activity of individual excipients of microemulsion and GMs GM-4 and GM-144. Aliquots of liquefied semen were mixed with
an equal volume of GM excipients or an assay medium containing the final concentrations of GM-4 or GM-144 components. At timed intervals, sperm
motility was evaluated. GM-4 and GM-144 immobilized sperm in human semen in less than 2 min and 30 sec, respectively, whereas components of GM-4
and GM-144 demonstrated variable or no inhibition of sperm motility in human semen.

tive gel containing 2% N-9, was tested in the same way for
comparison. Because the rabbit provides a standard animal
model for testing vaginal agents for antifertility activity
[48,49], we tested the ability of intravaginally applied GM-4
and GM-144 to prevent pregnancy in ovulated rabbits following artificial insemination.
For the contraceptive efficacy studies of GM-4, 48 ovulated NZW rabbits in subgroups of 16 were given intravaginal application of GM-4 formulation or Gynol II immediately prior (2 min) to artificial insemination with fresh

pooled semen (30 107/mL) and the females were allowed to complete their pregnancy. The efficacy of GM-4
formulation versus Gynol II for preventing pregnancy in the
rabbit model is summarized in Table 2. In the control group,
15 out of 16 (93.7%) rabbits artificially inseminated became
pregnant and delivered 123 newborn rabbits. In contrast,
none of the 16 rabbits given GM-4 formulation before
artificial insemination became pregnant (p 0.0001, Fishers exact test). By comparison, 5 out of 16 (31.2%) rabbits
given Glycol II became pregnant (p 0.0006) and deliv-

O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

119

Table 2
Comparison of the contraceptive activity of gel-microemulsions versus N-9 formulation (Gynol II)
Treatment
Group I
None
GM-4
Gynol II (2%-N-9)
Group II
None
GM-144
Gynol II (2%-N-9)

No. of does
inseminated

No. of does fertile

(%)

Contraceptive efficacy
(%)

Mean litter size

(median)

Total litter
size

16
16
16

15 (93.7)
0 (0)*
5 (31.2)

100
68.7

8.2 3.1 (8)

0
6.8 1.1 (7)

123
0
34

28
28
28

24 (85.7)
6 (21.4)*
7 (25.0)*

75.0
70.8

7.7 3.3 (7)

5.6 3.1 (6)
6.7 1.0 (6)

185
34
47

Aliquots (0.5 mL) of fresh, pooled semen obtained from fertile bucks (n 12) were used to artificially inseminate the does within 1-2 min following
intravaginal application of 2 mL of GM-4, GM-144, or N-9 formulation. Does were induced to ovulate by an intravenous injection of 100 IU HCG and
allowed to complete term pregnancy.
* Significantly different from control by Fishers exact test (p 0.0001).
Significantly different from control by Fishers exact test (p 0.0006).

ered 34 newborn rabbits. Thus, the GM-4 formulation was

far more effective than Glycol II as a vaginal spermicidal
contraceptive (100 vs. 68.7%, p 0.05, Fishers exact test).
For the contraceptive efficacy studies of GM-144, 84
ovulated NZW rabbits in subgroups of 28 were artificially
inseminated with fresh pooled semen (30 107/mL) with
and without intravaginal application of GM-144 formulation or Gynol II and allowed to complete term pregnancy.
The efficacy of GM-144 formulation versus Gynol II for
preventing pregnancy in the rabbit model is summarized in
Table 2. In the control group, 24 out of 28 (85.7%) rabbits
artificially inseminated became pregnant and delivered a
total of 185 newborn rabbits. By contrast, only 6 out of 28
(21.4%) rabbits given GM-144 formulation prior to artificial
insemination became pregnant (p 0.0001, Fishers exact
test) with a total of 34 newborn pups. Similarly, only 7 out
of 28 (25%) rabbits given Gynol II became pregnant (p
0.0001, Fishers exact test) and delivered a total of 47
newborn rabbits. Thus, the GM-144 formulation was as
effective as Gynol II as a vaginal spermicidal contraceptive
(75 vs. 70.8%).
We confirmed that intravaginal application of GM-4 or
GM-144 formulation prior to artificial insemination drastically prevents pregnancy in the rabbit model. Our in vivo
contraceptive efficacy studies included term pregnancy as
well as the analysis of normalcy of the resulting pregnancies. Rabbits that delivered litters following single intravaginal application of GM-144 or Gynol II prior to artificial
insemination had healthy offspring with no peri or postnatal
repercussions. A 100% and 75% contraceptive effect was
obtained with GM-4 and GM-144, respectively, despite the
fact that the rabbit ejaculate used contained 1000-fold
larger inseminating doses than in humans [48]. Under identical conditions, Gynol II showed 68% to 71% inhibition of
fertility. Because of the semen dose used (108/mL), a
partial contraceptive activity in rabbits can be considered
essentially 100% contraceptive in humans [48,49]. To our
knowledge, these experiments are the first to demonstrate
the in vivo contraceptive efficacy of GM formulations that

were prepared from commonly used pharmaceutical excipients. The potent contraceptive activity obtained with GM-4
and GM-144 is most likely due to their rapid spreadability
across the vaginal mucosa as well as to their rapid spermicidal activity. In addition to their spermicidal property, the
microemulsions known ability to alter membrane potentials
or oxidation-reduction properties may in part account for
the potent in vivo contraceptive effect. These properties are
important in sperm-egg interactions.
Despite the rapid in vitro spermicidal activity of N-9containing spermicides, their in vivo contraceptive effect
has been shown to be highly dependent on the time interval
between delivering the agent to the vagina and coitus or
artificial insemination. It takes several minutes for commercial formulations of N-9 to distribute in the rabbit vagina
[49]. Thus, remarkably viscous N-9 gels (48,000-120,000
centipoise) may be slower to mix with vaginal secretions
than the bioadhesive GMs (1000 centipoise). Therefore, a
large excess of N-9 (400-fold greater dose) is required to
achieve in vivo contraceptive activity. In fact, in over-thecounter formulations, N-9 is being used at concentrations of
2 6% in creams and gels, 12% in foams, and as high as
18% in condom lubricants. The partial (68.7%) contraceptive effect of a commercial 2% N-9 gel (120,000 centipoise)
observed in our study when compared with 100% efficacy
of GM-4 is in agreement with the high contraceptive failure
rates reported for users of N-9 [50,51]. In several large
studies for users of N-9, the average 6-month pregnancy rate
is 26%, and the first-year pregnancy rates range from 11 to
31%. Our studies suggest that this is most likely due to
incomplete mixing of semen with traditional N-9 gels or to
inadequate distribution of the agent in the vagina.

6. Lack of intravaginal toxicity of GMs

The second objective of our study was to determine

the toxic effects, if any, resulting from repeated intravag-

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O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

Table 3
Comparison of histological changes in the rabbit vaginal tissue after 10
days of intravaginal application of gel-microemulsions versus N-9
formulation

Epithelial ulceration
Lamina propria thickness
Leukocyte infiltration
Vascular congestion
Total score

GM-4
(n 6)

GM-144
(n 6)

4% N-9
(n 4)

0*
11
11
0
2 1

0
11
21
21
51

3 2
21
32
11
92

* Rabbits were administered intravaginally with 1 mL of GM-4, GM144, or 4% N-9 formulation.

Mean SD values representing the upper (cervico-vagina), middle,

and lower (uro-vagina) regions of vagina from each rabbit.

Semiquantitative scoring based on Eckstein et al. [52]. Individual

score: 0 none, 1 minimal, 2 mild, 3 moderate, 4 intense. The
scoring system correlates to human irritation potential as follows: scores of
0 to 8 are acceptable, scores of 9 to 11 indicate borderline irritation
potential, and scores greater than 12 are potentially irritating.

inal application of spermicidal gel microemulsions. Because of the potent in vitro and in vivo spermicidal
activity of GM-4 and GM-144 formulation, it was necessary to evaluate the toxicity to vaginal mucosa particularly in the rabbit vaginal irritation test. Rabbits have a
simple cuboidal or columnar epithelium that is highly
sensitive to mucosal irritants when compared to the stratified squamous epithelium of human vagina. In intravaginal toxicity studies in rabbits, tissue irritation is usually
evaluated by gross examination of the entire vaginal area
as well as complete histopathological evaluation of the
cervix-vaginal junction, mid-vagina, and uro-vagina for
epithelial ulceration, edema, leukocyte infiltration, and
vascular congestion, after daily intravaginal application
of the test agent for 10 consecutive days [52]. Absence of
any lesions is, in general, indicative of no tissue damage.
We tested the effect of GM-4 and GM-144 on vaginal
irritation in rabbits. Sixteen adult NZW female rabbits in
subgroups of four or six were treated intravaginally with
1 mL of GM-4, GM-144, or 4% N-9 for 10 consecutive
days. The animals were killed on Day 11, and the reproductive tract of each rabbit was examined grossly and
microscopically after completion of the study. Histologic
examination was made to assess acute local toxicity and
mucosal damage as described by Eckstein et al. [52].
When compared with N-9 gel, both GM-4 and GM-144
were well tolerated by the animals, and no local or
systemic abnormalities were detected (total scores of 2
and 5, respectively; Table 3). In contrast, vaginal tissues
from rabbits treated with 4% N-9 as a positive control
revealed mild to moderate irritation. These conditions
were characterized by epithelial ulceration, edema, leukocyte influx, and vascular congestion characteristic of
inflammation (total score 9). These results clearly demonstrated that both GM-4 and GM-144 are not damaging
to vaginal mucosa of the rabbit despite being potent

spermicidal agents when added to human or rabbit semen. This was expected because the ingredients used for
GM-4 and GM-144 are nontoxic solubilizers for lipophilic drugs used in the preparation of a variety of
topical, oral, and injectable medications. Therefore, unlike the currently used nonionic and cationic detergent
spermicides, the submicron particle-based GM-4 and
GM-144 formulations are not likely to cause harmful side
effects following repetitive intravaginal application.

7. Short-term toxicity studies

We performed 13-week intravaginal toxicity studies
on GM-4 and GM-144 formulations in mice to support
their further development as dual-function spermicides
and intravaginal drug delivery system for lipophilic
drugs. Female B6C3F1 mice (10 or 20/subgroup) were
treated with intravaginal application of GM-4 or GM144, 5 days per week, for 13 consecutive weeks. The
endpoints that were used for evaluation included survival, body weight, hematologic and clinical chemistry
profiles, absolute and relative organ weights, and histopathology [28]. No effects related to GM-4 or GM-144
treatments were observed on survival, mean body weight,
and mean body weight gain. Repeated intravaginal exposure of mice to GM-4 and GM-144 for 13 weeks had no
toxicologically significant effect on organ weights and
did not cause any adverse changes in hematology parameters or blood chemistry profiles [28]. Extensive histopathologic examination of tissues showed no lesions of
pathologic significance. Thus, intravaginal application of
GM-4 and GM-144, for up to 13 weeks, does not cause
systemic toxicity. Experiments to test the effect of daily
intravaginal administration of spermicidal GMs on longterm (2-year) toxicity and carcinogenesis studies in
B6C3F1 mice are currently in progress.
We also tested whether repetitive intravaginal application of GM-4 and GM-144 has any adverse effects on
subsequent fertility. For these studies, female CD-1 mice
(20/subgroup) were given daily intravaginal application of
GM-4 or GM-144 for 13 weeks, and then they were mated
with untreated CD-1 males and allowed to complete a pregnancy. Thirteen-week treatment with GM-4 or GM-144 had
no significant effect on subsequent fertility, median litter
size, neonatal survival, pup morphology, or development.
On the basis of these studies, we are hopeful that repetitive
intravaginal application of spermicidal GMs, GM-4 and
GM-144, will have no significant adverse systemic side
effects in clinical settings. GM-4 and GM-144 show unique
clinical potential as safe, dual-function spermicidal and intravaginal drug delivery systems for curbing mucosal transmission of STDs.

O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

8. Spermicidal GMs as intravaginal drug delivery

vehicles
The vaginal mucosa is, in fact, able to allow the diffusion
of considerable quantities of pharmacologically active substances from the application surface to the dermic stratum, which has a rich vascular area able to absorb and
drain in the systemic circle, besides the deep dermic
stratum. However, an important characteristic, which allows high absorption values in the vaginal area, is the
bioadhesion of the formulation and the bioavailability of
the drug. The vaginal cavity exhibits an aqueous environment containing glands whose secreted fluids create
an acidic pH in the range of 4.0 5.5.
Many pharmaceutically active compounds (intended as
antiviral/antimicrobial agents) are poorly soluble in water.
This hydrophobic property often makes it difficult to formulate a drug that exhibits a satisfactory bioavailability
profile in vivo. It has been found that gels, foams, creams,
suppositories, and tablets that are presently used as vaginal
delivery systems break down almost immediately following
insertion into the vaginal cavity and have minimal bioadherence to the vaginal walls. This is believed to be due to
their water miscibility and/or their lack of physical stability
at body temperature. Thus, they exhibit limited effectiveness. Poor bioavailability may lead to ineffective therapy,
the need for higher dosing, and/or undesirable side effects.
In this regard, some of the more conventional treatment
regimens for various vaginal diseases, such as HSV, chlamydial and trichomonas infections include oral and injectable drugs. Such treatment may induce some unwanted side
effects, and precautions include issues of carcinogenesis, mutagenesis, and impaired fertility. For HIV infection, there is no
treatment or preventive product available.
Microemulsions appear to have the ability to deliver larger
amounts of topically applied agents into the mucosa than
traditional vehicles (lotions, creams) because they provide a
better reservoir for a poorly soluble drug through their capacity
for enhanced solubilization. The drug, dissolved rather than
suspended in the vehicle, is in a form for immediate absorption
and is generally more rapidly and more effectively absorbed.
The microemulsion-based lipophilic and vaginal spermicides
GM-4 and GM-144 appear to offer several benefits for
vaginal delivery, including increased absorption, potent
contraceptive activity, and decreased toxicity. The spermicidal GM-4 and GM-144 formulations show unique clinical
potential to become clinically useful vaginal contraceptives and potential drug delivery vehicles for preventing
the sexual transmission of STDs while preventing unwanted pregnancies.
The use of GM-4 and GM-144 as vaginal drug delivery
vehicles opens new perspectives in the formulation of
poorly soluble drugs especially for preventing transmission
of pathogens to mucosal membranes, especially HIV-1 and
HSV. The GM can be used to treat a particular disease state
as well as to prevent sexual transmission of pathogens. For

121

example, the water insolubility of the anti-HIV drug of

choice, zidovudine (ZDV), makes the formulation for intravaginal use difficult. In addition, the anti-HIV efficacy of
ZDV as an inhibitor of HIV-1 reverse transcriptase is dependent on cell membrane permeability and cellular thymidine kinases [53]. Inasmuch as the thymidine kinase activity
is known to be low or lacking in monocyte/macrophage
cells, the main carriers of HIV in semen, ZDV by itself is
unlikely to have significant anti-HIV activity in genital tract
secretions.
In an effort to bypass the thymidine kinase-dependency
of ZDV activation as well as cell permeability, we have
rationally designed aryl phosphate derivatives of bromomethoxy ZDV as prodrugs of ZDV [54,55]. They exhibit
enhanced lipophilicity, superior pharmacokinetics, and retain full activity in thymidine kinase-deficient cells when
compared with ZDV. These ZDV analogues can inactivate
HIV-1 in cells that have low or deficient thymidine kinase
activity. We demonstrated that human female genital tract
vaginal and cervical epithelial cells as well as sperm efficiently convert these prodrugs to bioactive ZDV metabolites
despite their thymidine kinase deficiency [56]. However, the
lipophilic characteristics of these antiviral drugs limits their
solubility in aqueous buffers and, therefore, lipophilic delivery vehicles are needed. The development of lipophilic
GMs formulations GM-4 and GM-144 designed to enhance
the solubility of these drugs is expected to significantly
increase the therapeutic index of these antiretroviral agents
as dual-function microbicides. Using GM-4, we have been
successful in solubilizing 2000-fold higher concentrations
of the ZDV derivative for intravaginal/rectal administration
than would be available in aqueous formulations. In addition, these nontoxic lipophilic GMs may be useful for intravaginal application of antimicrobial agents to prevent
sexual transmission of such diseases as genital herpes, gonorrhea, and chlamydia.

9. Conclusion
In this review, we reported the discovery of novel pharmaceutical formulations in the form of GMs of various
compositions, which impart rapid spermicidal activity in
human semen. GM-4 and GM-144 were selected after a
systematic comparison of the solubility of lipophilic antiHIV drugs. These GMs are highly contraceptive in the
rigorous rabbit model. These formulations were more effective contraceptives than a commercially available N-9 gel.
Repeated intravaginal applications of spermicidal GMs to
rabbits and mice were found to be safe and did not cause
local, systemic, or reproductive toxicity. As potent contraceptive agents that are inexpensive and devoid of mucosal
toxicity, the lipophilic GMs meet the criteria for a vaginal
spermicide and warrant further in vivo evaluation in humans
as intravaginal/rectal drug delivery vehicles to prevent the

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O.J. DCruz, F.M. Uckun / Contraception 64 (2001) 113123

sexual transmission of such diseases as AIDS, genital herpes, gonorrhea, and chlamydia.

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