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From the TOPCAD Program and * Department of
Obstetrics and Gynecology, Rush Medical Center, Chicago, Illinois;
Department of Medical Microbiology and
Immunology, Southern Illinois University School of Medicine, Springfield,
Illinois; Department of Pediatric Infectious
Diseases, Mount Sinai School of Medicine, New York, New York;
Department of Obstetrics and Gynecology, Eastern
Virginia Medical School, Norfolk, Virginia;
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Department of Pharmaceutics and Pharmacodynamics,
University of Illinois at Chicago, Chicago, Illinois; and
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Contraceptive Research and Development Program,
Arlington, Virginia.
| Correspondence to: Robert A. Anderson Jr, Ob/Gyn Research, Rush Medical Center, Chicago, IL 60612 (e-mail: randerso{at}rush.edu ). |
| Received for publication July 19, 2001; accepted for publication January 9, 2002. |
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Abstract |
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Key words: Antifertility, microbicide, HIV, HSV, chlamydia, N. gonorrhoeae
Ideally, prevention of pregnancy and STIs would be controlled by women. It should not interfere with sexual pleasure for the woman or her partner, and should be accomplished through the self-administration of a single drug or drug combination. Vaginal application of the drug is reasonable, because this route permits use only when needed (i.e., during sexual intercourse). Therapeutic concentrations of the drug could be applied locally, thus reducing systemic levels and potential systemic side effects.
Methods for preventing STIs are limited. Until recently, interest groups for women's reproductive health issues advocated the use of the spermicide, nonoxynol-9 (N-9) for preventing HIV transmission (Uckun and D'Cruz, 1999). This was based upon in vitro efficacy of N-9 (Malkovsky et al, 1988; Polsky et al, 1988) and other surfactants (Krebs et al, 1999) against HIV and other microbes responsible for STIs (Judson et al, 1989; Jennings and Clegg, 1993), and preliminary clinical findings (Zekeng et al, 1993; Cook and Rosenberg, 1998). Additional clinical studies showed that N-9 is ineffective in preventing the transmission of HIV (Roddy et al, 1998). N-9 may actually increase its transmission under certain conditions (Kreiss et al, 1992; Piot, 2000; Stephenson, 2000). The only proven methods of preventing the transmission of HIV are so-called "safe-sex" techniques. These include use of male or female condoms (e.g., Johnson et al, 1999). However, personal preferences and cultural practices have limited the use of condoms (Zekeng, et al, 1993; Coleman, 1999; Johnson, et al, 1999; Schlumberger et al, 1999; Smits et al, 1999; Cornelius et al, 2000).
Safe, novel, active ingredients and acceptable barrier devices (or both) are needed that will allow women to control their reproductive health. This is illustrated by several conferences and interest from private and public sectors on this subject (Gabelnick and Harper, 1999). We established the Program for the Topical Prevention of Conception and Disease (TOPCAD) in 1993 as a collaboration among university research scientists. TOPCAD works closely with companies in the pharmaceutical industry; its primary mission is to discover or design, evaluate, and develop new vaginal methodologies to prevent conception and STIs.
Heparin-like compounds and other sulfated polysac-charides are active against several sperm-related functions (Lalich et al, 1989; Oehninger et al, 1991; Lee et al, 1994). These compounds are also active against STI-causing viruses, such as HIV (Mohan, 1992; Pearce-Pratt and Phillips, 1996; Witvrouw and De Clercq, 1997) and HSV (Shieh and Spear, 1994; Witvrouw and De Clercq, 1997; Zeitlin et al, 1997). The purpose of this study was to evaluate the contraceptive and anti-STI potential of a well-defined, high-molecular-weight form of cellulose sulfate (Ushercell).
Our data show that Ushercell has activity against mammalian spermatozoa. It is also active against HIV, HSV, Chlamydia trachomatis and Neisseria gonorrhoeae. Ushercell is contraceptive in the rabbit. It has no overt cytotoxic properties, and can be readily formulated. These results support the clinical evaluation of Ushercell for the prevention of conception and STIs. A patent for this use has been awarded (Anderson et al, 2000b).
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Materials and Methods |
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Unless specifically indicated, reagents in the present study were the
highest quality commercially available. Sheep testicular hyaluronidase (Type
III), hyaluronic acid from bovine vitreous humor,
N-acetylglucosamine, p-dimethylaminobenzaldehyde,
N
-benzoyl-L-arginine ethyl ester, pregnant mare serum (PMS),
human chorionic gonadotropin (hCG), Bismarck Brown, and Rose Bengal were
products of Sigma Chemical Company (St Louis, Mo). Lactobacillus MRS
broth and Gas Pak anaerobic pouches were purchased from Fisher Scientific
(Itasca, Ill). The calcium ionophore A23187 (free acid) was obtained from
Calbiochem (San Diego, Calif). N-9 was obtained from Ortho Pharmaceutical
Corp. (North Brunswick, NJ), and a suspension of penicillin (10 000 units/mL)
and streptomycin (10 mg/mL) was from Gibco BRL (Grand Island, NY).
Lactobacillus gasseri (ATC 9857) originated from a vaginal isolate
and was obtained from the American Type Culture Collection (Manassas, Va).
Sperm Function Tests
Sperm function tests were chosen on the basis of our current understanding
of sperm processes or properties required for successful fertilization. These
include adequate enzymic activities of hyaluronidase and acrosin, the ability
to release these and other critical acrosomal factors at the appropriate time
and place, and migration through cervical mucus. These tests are presented in
detail elsewhere (Anderson et al, 2000a), and are briefly described below.
Hyaluronidase and acrosin inhibition— Hyaluronidase (EC 3.2.1.35) and acrosin (EC 3.4.21.10) activities were estimated by spectrophotometric determination of their reaction products. The natural substrate (hyaluronic acid) was used for hyaluronidase assays. The enzyme was incubated with Ushercell for 5 minutes at 37°C, and the reaction was started by adding the substrate. Concentrations of Ushercell ranged from 0.5 to 4.0 mg/mL. Reversibility of inhibition was approximated by the method of Ackermann and Potter (1949), in which the level of inhibited enzyme activity was determined in the presence of different amounts of the enzyme. In this and other dose-response experiments, the data were best fit to curves with TableCurve software (version 5.0, SPSS Statistical Software, Chicago, Ill). These curves were used to calculate constants of inhibition (e.g., IC50).
The esterase activity of acrosin was measured spectrophotometrically with a
synthetic substrate (N-benzoyl-L-arginine ethyl ester, or
BAEE), as described by Anderson et al (1981). Concentrations of Ushercell
ranged from 0.05 mg/mL to 0.3mg/mL. Acrosin was preincubated with Ushercell
for 10 minutes at ambient temperature. Reactions were started with the
approximate Km concentration (0.05 mM) of BAEE. Data were expressed
as percentage of inhibition compared with control activity determined in the
absence of Ushercell.
Stimulation of acrosomal loss— Acrosomal loss was determined by direct visualization of the acrosomal region of the sperm head after staining (Anderson et al, 1992, 2000a). After equilibration in Biggers Whitten Whittingham medium at 37°C, washed spermatozoa were treated with different concentrations of Ushercell, ranging from 0.05 µg/mL to 0.1 mg/mL. Fifteen minutes after addition of Ushercell, acrosomes from treated spermatozoa were visualized after staining with Rose Bengal and Bismark Brown. Data are expressed as the percentage of total spermatozoa counted that lack acrosomes. Values were compared with the acrosomal loss induced by a maximally stimulating concentration of the calcium ionophore, A23187.
Cervical mucus penetration inhibition— Cervical mucus penetration was determined by measuring the distance through which the most progressive spermatozoa migrated through bovine cervical mucus in the presence of Ushercell. One end of a Penetrak (BioChem ImmunoSystems, Allentown, Pa) tube (contains bovine cervical mucus) was immersed for 30 minutes into a solution that contained either 1 mg/mL Ushercell, 16.7 mg/mL of the 6% Ushercell gel, or vehicle (phosphate-buffered saline). Each tube was removed from the test solution. Donor human semen (50 µL), was diluted with Bakers buffer (27.4 mM Na2HPO4, 0.6 mM KH2PO4, and 167 mM glucose, pH 8.1) to a final concentration of 60 x 106 motile sperm/mL (initial percentage of motile spermatozoa >50%). This suspension was added to each solution. The Penetrak tubes were reimmersed into the solutions and incubated at 37°C for 1 hour. The tubes were removed from the solutions, and were examined microscopically to find the length of the tube traversed by the most advanced motile spermatozoon. Migration by the spermatozoa contained in the test solution is reported as the percentage of the migration by spermatozoa treated with the vehicle.
Rabbit Contraception Assays
Pretreatment of spermatozoa with Ushercell before artificial
insemination—
Spermatozoa were pretreated with Ushercell. Conception
was measured in the rabbit as described by Joyce et al,
(1979) and Anderson et al,
(2000a). Female New Zealand white rabbits (age 8-12 months, 4.4 kg) were
injected (i.m.) with 200 IU PMS. After 96 hours, 200 IU hCG was given by i.v.
injection. Freshly ejaculated washed spermatozoa were pooled from 2 proven
breeders. The suspension was adjusted to a final sperm count of 25-31 x
106/mL, and was treated with either 1 mg/mL Ushercell or a vehicle
used to dissolve the Ushercell (modified Tyrode albumin lactate pyruvate).
After 15 minutes, rabbits were artificially inseminated (Anderson et al,
2000a). For comparative purposes, sperm were also pretreated with heparin
(either 100 µg/mL or 1 mg/mL) and with dextran sulfate (10 mg/mL) before
insemination.
Within 30 minutes of hCG treatment, the rabbits were inseminated with 0.75 mL of the treated or the control sperm suspension. After 28 hours, rabbits were killed with Sleepaway (pentobarbital-based; Fort Dodge Laboratories, Fort Dodge, Iowa), and oocytes were examined microscopically for fertilization. Data were reported as the percentage of recovered oocytes fertilized for each rabbit.
Vaginal application of Ushercell before artificial insemination— The study used 10 female rabbits over a total of 3 experiments. Each experiment included 1-2 controls (placebo gel applied), and 1-3 rabbits treated with formulated Ushercell. The formulation (6% Ushercell, w/w) was vaginally applied to sexually mature New Zealand White rabbits (8-9 months of age, approximately 4.4 kg in body weight). After 15 minutes, rabbits were artificially inseminated with washed spermatozoa (approximately 30 x 106) obtained from proven breeders. Fertilization was evaluated essentially as described above ("Pretreatment of spermatozoa with Ushercell before artificial insemination").
Antimicrobial Tests
Antimicrobial assays were divided into viral and bacterial assays. HSV and
HIV were selected as representative sexually transmitted viruses. N
gonorrhoeae and C trachomatis were selected as representative
sexually transmitted bacteria. Excluding N gonorrhoeae, tests were
designed to measure microbial infectivity. Microbes were exposed to Ushercell,
and the mixture was inoculated onto target cells. Ushercell was removed by
centrifugation or dilution before the microbe was allowed to replicate within
the target cells. Therefore, inhibition in Ushercell-treated samples was due
to its effect on viral/bacterial binding, entry into the target cells, or
both.
HSV inhibition— Infectivity of CaSki cells by HSV-2 (strain 333) and HSV-1 (strain 17) was measured after treating the virus with Ushercell. Different concentrations were used, ranging from 0.1 to 100 µg/mL (Herold et al, 1996). The viral inoculum was chosen to produce approximately 500 pfu per assay in control incubations (no added inhibitor). Two hours after adding the Ushercell-treated virus to CaSki cells, the culture was washed to remove the unbound virus and Ushercell. After approximately three days of culture, the CaSki cells were examined for viral plaques. Data are expressed as viral titer (pfu/mL).
HIV inhibition— HIV-1 (strain IIIB) infectivity in the presence of Ushercell was evaluated with a viral binding inhibition assay. Serial dilutions of test compound (from 3.2 µg/mL to 0.1 mg/mL) were added to MT-2 cells (Resnick et al, 1990). The virus (inoculum adjusted to produce 70-100 syncytia per well in untreated MT-2 cells) was added to the wells containing the test compound, or only control medium. The virus/cell cocultures were incubated at 37°C (5% CO2) for 48-72 hours, and cultures were scored for syncytia formation. Data are expressed as a percentage of control value (number of syncytia induced in wells with no compound added).
C trachomatis inhibition— Infection of HeLa cells by C trachomatis (serotype E/UW-5/CX) in the presence and absence of Ushercell was evaluated essentially as described by Cooper et al (1990). Each of 3 serial 1:10 dilutions of elementary bodies (104 to 106 IFU/mL) were added to different concentrations of Ushercell (1 µg/mL to 1 mg/mL). The mixtures were incubated at 37°C for 4 hours, after which they were inoculated onto HeLa cell monolayers. One hours after inoculation, free microbes and Ushercell were removed by washing, and the HeLa cell cultures were incubated for an additional 48 hours. Chlamydia-induced inclusions were measured by immunofluorescence after reacting the cultures with a Kallsted Chlamydia culture confirmation fluorescein-conjugated antibody (monoclonal). Data are expressed as bacterial titer (IFU/mL) at each concentration of Ushercell.
N gonorrhoeae inhibition— Inhibition of N. gonorrhoeae growth on agar by Ushercell was measured as described previously (Anderson et al, 2000a). Ushercell (1 µg/mL to 1 mg/mL) was incorporated directly into gonococcal agar, and the mixture was poured into culture dishes. The dishes (with and without Ushercell) were inoculated with serial dilutions of N. gonorrhoeae ranging from 102 to 106 CFU/mL. Colonies were enumerated after overnight incubation at 37°C in 5% CO2. Data are presented as bacterial titer (CFU/mL) at each concentration of Ushercell.
Safety Assessment
Sperm immobilization and growth of normal vaginal flora (e.g.,
Lactobacillus) in the presence of Ushercell were chosen for safety
assessment. In addition, general cytotoxic properties of this polymer were
evaluated by measurement of its effects on the target cells used for the
microbial assays.
The effect of an agent on the percentage of motile sperm in a sample can be used as an indirect measure of the cytotoxic effect of that agent on the spermatozoa. The spermicidal effect of N-9 and other detergents is due to a nonspecific cytotoxic disruption of plasma and other membranes of spermatozoa. This nonspecific property also gives N-9 its unwanted side effects, such as vaginal lesions (Roddy, et al, 1998; Johnson, et al, 1999; Uckun and D'Cruz, 1999). An agent may immobilize, without killing, the spermatozoa. However, spermatozoa are not killed without being immobilized. In this context, failure of the test agent to reduce the fraction of motile sperm in a sample (while showing contraceptive activity) is a desirable outcome.
A healthy vagina contains a normal complement of microbial flora, the most prevalent of which are lactobacilli (Mardh, 1991). These beneficial bacteria help protect the vagina from pathogenic microbes, including those causing STIs such as HIV (Klebanoff and Coombs, 1991). Agents with activity against STI-causing microbes but not Lactobacillus would likely cause less risk for secondary infections. Failure of an active antimicrobial agent to inhibit growth of Lactobacillus is a favorable outcome.
Sperm immobilization— Sperm immobilization by Ushercell was evaluated by a modification (Anderson, et al, 2000a) of the method of Sander and Cramer (1941). Thirty seconds after adding different concentrations of Ushercell, ranging from 2.5 mg/mL to 50 mg/mL, the fraction of motile spermatozoa was determined with brightfield microscopy (400x). Data are presented as the percentage of motile spermatozoa.
Lactobacillus inhibition— L gasseri growth was estimated turbidometrically, as previously described (Anderson et al, 1998). Ushercell (5 mg/mL) was added to an active culture of the microbe contained in a stoppered flask under anaerobic conditions (nitrogen headspace). Absorbency (550 nm) of the suspensions, with and without Ushercell, was measured beginning at 120 minutes of incubation at 37°C and at 20-minute intervals for a total of 260 minutes. Doubling times for bacterial growth were calculated from plots of Ln (absorbency) vs. time.
Cytotoxicity assessment of host cells used for antimicrobial testing— Host cells (CaSki for HSV, MT-2 for HIV, HeLa for C. trachomatis), were examined at the end of each experiment for signs of Ushercell-induced damage (confluency and general condition). More-sensitive assays for cytotoxicity of Ushercell were performed with CaSki cells by quantifying cellular DNA synthesis, and with HeLa cells by measuring the uptake of propidium iodide.
CaSki cells were grown to half-confluence in glass scintillation vials. They were cultured overnight in the presence of four serial dilutions of Ushercell (in triplicate) and 3H-thymidine. This was followed by extensive washing of the cells and determining incorporated thymidine by liquid scintillation spectrometry.
HeLa cells were grown to confluency in 24-well plates. They were cultured in the presence of 1 mg/mL Ushercell for either 4 or 24 hours. The cells were removed from the wells with trypsin, and treated with 5 µg/mL propidium iodide for 5 minutes. Red fluorescence emission of propidium iodide was detected with flow cytometry, with a dichroic bandpass filter (585 ± 42 nm). Cells that had high propidium iodide fluorescence were considered membrane-damaged and nonviable. Data from samples that contained Ushercell were compared with a positive control (HeLa cells fixed and permeabilized with a cell permeabilization kit from Caltag Labs, Burlingame, Calif). HeLa cells that were not exposed to Ushercell served as a negative control.
Statistical Analyses
Hyaluronidase and acrosin activities are expressed as averages, with the
standard errors of the mean. Data for hyaluronidase were best fit to curves
generated with TableCurve software (version 5; SPSS, Inc), from which were
calculated IC50 values of enzyme inhibition and coefficients of
determination of the fitted curves. Percentage data collected for the
acrosomal loss, and rabbit contraception and sperm immobilization tests were
subjected to arcsine transformation (Sokal
and Rohlf, 1981) before further analysis; data were
back-transformed for presentation. They are presented as either average
percent maximal acrosomal loss, percent fertilization, or percent motile
sperm, with 90% confidence limits. Differences in acrosomal loss and rabbit
conception in the presence and absence of Ushercell were compared with the
Student t-test on the transformed data.
Data for the antimicrobial activity of Ushercell against HSV, N gonorrhoeae, and C trachomatis were subjected to logarithmic transformation (Sokal and Rohlf, 1981) before further analysis. Data are presented as the back-transforms (average titers) with 90% confidence limits. IC50 values and concentrations of Ushercell yielding 3-log reductions in infectivity (99.9% inhibition of microbial titer) were calculated from curves fit to the data with TableCurve software. Lactobacillus growth was expressed as a change in absorbency at 550 nm over time. This was measured in the presence and absence of Ushercell. Slopes of growth curves generated from the data were compared, from which doubling times and their 90% confidence limits were calculated. Slopes were considered not different if the 90% confidence limits overlapped.
All differences were considered significant at the 0.05 level of confidence. Values were considered not different at the 0.1 level of confidence.
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Results |
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Acrosin Inhibition
Ushercell appears to inhibit human acrosin, as measured
spectrophotometrically by the method of Anderson et al (1980). Approximately
20% inhibition occurs with 50 µg/mL of the polymer. However, inhibition is
not dose-dependent. Ushercell concentration and the apparent percentage of
inhibition observed is as follows: 50 µg/mL, 20%; 100 µg/mL, 24%; 200
µg/mL, 25%; 300 µg/mL, 24%. No correlation exists between concentration
of polymer and apparent acrosin inhibition (Spearman coefficient of rank
correlation [rs] = 0.435; P >.1). We conclude that
Ushercell is not an effective acrosin inhibitor.
Induction of acrosomal loss— Acrosomal loss is induced by Ushercell (Figure 2). It produces a 50% maximal loss of acrosomes at 52 ng/mL. In this context, maximal acrosomal loss is the response to a maximally stimulating concentration of the calcium ionophore, A23187 (Anderson et al, 1992). Nearly maximal (99.9%) acrosomal loss in response to Ushercell is estimated at 1.7 µg/mL. The highest concentration used in these experiments (0.6 µg/mL) does not affect sperm motility (57%; 90% confidence limits = 44.4%-69.5%; average control motility = 58%; 90% confidence interval = 46.4%-69.6%, n = 3), suggesting that the induction of acrosomal loss is not secondary to dead or dying spermatozoa.
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Inhibition of cervical mucus penetration by spermatozoa— Ushercell inhibits the migration of human spermatozoa through bovine cervical mucus (Table 1). At 1 mg/mL, it inhibits migration by approximately 70% (t = 26.7, df = 12; P <.001). Similar results were obtained in the presence of 16.7 mg/mL of the 6% Ushercell gel formulation (equivalent to 1 mg/mL of the polymer). The effect seems independent of whether Ushercell is introduced from the bulk powder, or from the gel (t = 1.455, df = 21; P >.10).
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Contraception in the rabbit— Ushercell prevents conception in rabbits. Fertilization is inhibited by nearly 95% when freshly ejaculated rabbit spermatozoa are treated with 1 mg/mL (final concentration) of the polymer for 15 minutes before artificial insemination. In contrast, neither heparin (up to 1 mg/mL) nor dextran sulfate (10 mg/mL) inhibited conception when added to spermatozoa 15 minutes before insemination (Table 2). Higher concentrations of heparin were not tested because of the risk of vaginal bleeding.
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Similar efficacy is seen for Ushercell in formulation. The 6% (w/w) gel completely blocks conception when it is vaginally applied 15 minutes before insemination of untreated spermatozoa. Out of 187 oocytes examined from the Ushercell-treated rabbits, none were fertilized. In contrast, nearly 80% of oocytes harvested from rabbits treated with an equal volume of placebo gel without Ushercell were fertilized (Table 2).
Antimicrobial Activity
HIV and HSV inhibition—
Ushercell is highly effective against
the enveloped viruses, HIV and HSV. The IC50 for
HIV-1IIIB infection is 32 µg/mL. The calculated 3-log reduction
in activity occurs at 206 µg/mL (Figure
3). It should be noted that all values reported for 3-log
reductions in viral or bacterial titers are based upon regression analysis.
Occasionally, they are greater than concentrations of Ushercell that produced
complete inhibition. Other strains of HIV inhibited by Ushercell (and the
corresponding IC50 values; data not shown) include the clinical
isolates ROJO (3.0 µg/mL) and TEKI (3.9 µg/mL), and the monocytotropic
strains ADA (1.3 µg/mL) and BaL (78 µg/mL).
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Ushercell is more active against HSV-1 and HSV-2 (Figure 4). Constants of inhibition are similar for both types; IC50 and 3-log reduction values are 59 ng/mL and 6.2 µg/mL, respectively, for HSV-1; and 24 ng/mL and 13.9 µg/mL, respectively, for HSV-2.
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C trachomatis inhibition— Ushercell inhibits infection of HeLa cells by C. trachomatis. Inhibition is dose-dependent (Figure 5). Higher concentrations are required to inhibit infectivity, compared with that required to inhibit viral infectivity (see above). The calculated IC50 for chlamydial inhibition is approximately 78 µg/mL. The highest concentration tested against Chlamydia (1 mg/mL) inhibits infectivity by 75%. The concentration of Ushercell required for 95% inhibition (90% confidence limits for inhibition = 93.4% -96.6%) is estimated from the dose-response curve at approximately 7 mg/mL.
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N gonorrhoeae inhibition— Ushercell inhibits the growth of N. gonorrhoeae on agar. Inhibition is dose-dependent (Figure 6) at concentrations of the polymer ranging from 1.0 µg/mL to 1.0 mg/mL. The calculated IC50 for gonococcal inhibition is approximately 2 µg/mL. Ushercell causes a 3-log reduction in growth at 1.4 mg/mL.
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Safety Assessment
Sperm immobilization—
In contrast to its effects on other
sperm function variables, Ushercell minimally immobilizes human spermatozoa.
At 50 mg/mL it inhibits the fraction of motile spermatozoa by only
approximately 30% (Figure 7).
This small effect is far less than sperm immobilization caused by the
spermicide, N-9 (IC50 = 88 µg/ml; Anderson et al, 2000a). It is
likely due, at least in part, to the high viscosity of the polymer at this
concentration. Ushercell at 5% has a gel-like consistency, and may physically
prevent movement of spermatozoa. In support of this contention, approximately
90% of sperm immobilization caused by 50 mg/mL Ushercell was reversed when the
Ushercell was diluted 1:20 with phosphate-buffered saline (data not shown). We
conclude that Ushercell is not cytotoxic to spermatozoa at concentrations that
would likely be encountered if it were used vaginally as a contraceptive
antimicrobial agent.
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Host cell cytotoxicity— Ushercell produces no overt toxic effects on the host cells (MT-2 cells for HIV, CaSki cells for HSV) used in the antiviral assays. The highest concentrations tested were 100 µg/mL for HIV and 500 µg/mL for HSV. Similarly, no overt effect on the HeLa monolayer was observed when Ushercell was tested against C trachomatis at concentrations up to 1 mg/mL.
The calculated concentration of Ushercell required to reduce thymidine uptake in CaSki cells by 50% (CD50) is 80 µg/mL (dose-response data not shown). From this, the selectivity index (SI), defined as the CD50/IC50, for HSV-2 is equal to 3200. In similar experiments with N-9, SI is only 1.4 (CD50 = 35 µg/mL, IC50 = 25 µg/mL).
Based on propidium iodide uptake by HeLa cells, 98% (90% confidence limits = 96.7%-99.6%) and 95% (87.0%-99.6%) of the cells were classified as viable after 4 hours and 24 hours exposure to Ushercell (1 mg/mL), respectively. These data suggest no cytotoxicity to HeLa cells by this concentration of the polymer.
Lactobacillus inhibition— In contrast to the inhibitory effect of Ushercell against STI-causing organisms, it does not affect the growth of a commercial culture of the beneficial microbe, Lactobacillus gasseri, originating from a human vaginal isolate (Figure 8). The doubling time of Lactobacillus growth in the presence of 5 mg/mL Ushercell is 89 minutes (90% confidence limits = 78.7-102.5 minutes). This is essentially the same as the doubling time of 98 minutes seen for control cultures (90% confidence limits = 88.1-111.7 minutes). Similar negative data were obtained for other preparations of cellulose sulfate at concentrations as high as 18 mg/mL (highest tested; data not shown). This concentration is several times higher than concentrations of Ushercell required for contraception or for inhibition of the viral and bacterial STI-causing microbes.
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Discussion |
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Possible Common Penetration Pathways of Spermatozoa and Pathogenic
Microbes
Hyaluronidase and acrosin (a serine proteinase) are within and around the
sperm acrosome. They ease passage of spermatozoa through the cumulus oophorus
and possibly the zona pellucida, vestments that surround the unfertilized
oocyte (Rogers and Bentwood,
1982; Yanagimachi,
1988). Hyaluronidase inhibitors
(Pincus et al, 1948; Sieve, 1952;
Parkes, 1953;
Joyce et al, 1979;
Joyce and Zaneveld, 1985), and
acrosin inhibitors (Zaneveld et al,
1970; Joyce et al,
1979; Zaneveld,
1982; Kaminski et al,
1985) are contraceptive.
Different forms of hyaluronidase may help the penetration of target tissues by pathogenic microbes, including Treponema pallidum (Fitzgerald and Gannon, 1983; Fitzgerald and Repesh, 1987), cocci (Linker, 1966; Steiner and Cruce, 1992), and Trichomonas vaginalis (Rosso, 1975). Similarly, proteinases (including serine proteinases) are implicated in tissue invasions by microbes. Examples include N. gonorrhea (O'Reilly and Bhatti, 1986), Treponema pallidum (Poulsen et al, 1986), Trichomonas vaginalis (Arroyo and Alderete, 1989), and HIV (Hattori et al, 1989; Mohan, 1992; Bourinbaiar and Nagorny, 1994; Bourinbaiar and Lee-Huang, 1995). Hyaluronidase and proteinase activities may therefore support both mammalian fertilization and infection by certain STI-causing microbes.
Interaction of heparan sulfate and other glycosaminoglycans (GAGs) with their respective receptors may also be involved in tissue penetration by spermatozoa and infectious microbes. GAGs have a key role in viral and bacterial infectivity, phagocytosis and endocytosis, intercellular communication, and cell-cell interaction (Tumova et al, 2000). Complex interactions occur between heparin and related compounds and their receptors on spermatozoa, and possibly the oocyte. They modulate spermatozoa and their interaction with the oocyte (Jones, 1990). Similar interactions influence the infectivity of HSV, Chlamydia, N gonorrhoeae, and possibly HIV (Rostand and Esko, 1997).
Evidence for GAG-mediated infectivity is most compelling for HSV. Heparin and structurally related compounds bind to receptors on the surface of HSV (WuDunn and Spear, 1989; Spear, 1993). The first step in infection by both HSV-1 and HSV-2 infection may be the binding of the virus to target cell heparan sulfate (WuDunn and Spear, 1989; Shieh et al, 1992).
Spermatozoa bind heparin with high affinity (Delgado et al, 1982; Handrow et al, 1984; Marks and Ax, 1985; Miller et al, 1988; Lalich et al, 1989; Prins et al, 1989). Heparin and other GAGs induce acrosomal loss (Handrow et al, 1982; Lenz et al, 1983a,b; Meizel and Turner, 1986). GAG-induced acrosomal loss and heparin binding to spermatozoa are correlated with the fertility of spermatozoa (Marks and Ax, 1985; Parrish et al, 1988; Lalich et al, 1989; Prins et al, 1989; Vasquez et al, 1989). Heparin decreases the fertilization of homologous pig oocytes (Wang et al, 1991). This may be related to the ability of heparin to induce acrosomal loss.
We propose that the activities of Ushercell may be at least partly due to its interference with the interaction between sperm-associated and microbial-associated proteoglycan receptors and their target cell ligands. This suggestion is supported by the broad spectrum of antimicrobial activity of Ushercell, its ability to inhibit hyaluronidase (possibly by competing with the natural GAG substrate, hyaluronic acid [Astrup and Alkjaersig, 1950]), and its ability to stimulate acrosomal loss. However, its validity is subject to empirical verification.
Ushercell Effects on Sperm Function and Conception
Earlier work showed that cellulose sulfate inhibits hyaluronidase
(Astrup and Alkjaersig, 1950; Rogers and Spensley, 1954;
Spensley and Rogers, 1954), but not acrosin (Andolz et al,
1983). However, these studies were qualitative or semiquantitative
in nature. Also, neither the molecular weights nor the sources of the
cellulose sulfate were known. We extended the preliminary description of
hyaluronidase inhibition by determining constants of inhibition
(Figure 1) and reversibility of
inhibition for a defined, high molecular weight form of cellulose sulfate. Our
work shows a small apparent inhibitory effect of Ushercell on acrosin.
However, the effect is not dosedependent. We suggest this effect to be
secondary to interaction of Ushercell with the assay components, and that
Ushercell has little, if any, inhibitory action on enzyme activity.
Acrosomal exocytosis (acrosome reaction) is a prerequisite to successful oocyte penetration by spermatozoa. This process releases enzymes from the acrosome (including hyaluronidase and acrosin) that help the spermatozoon to pass through the outer vestments of the oocyte. It also exposes sperm-specific ligands that bind to the zona pellucida, a first step in the penetration process. It is important that this reaction occurs in capacitated spermatozoa near the oocyte, in response to the appropriate stimulus. Spermatozoa that have lost their acrosomes are shorter lived (Jones, 1990; Tarlatzia et al, 1993). Sperm are therefore less likely to fertilize if their acrosomes are lost prematurely. Agents that induce acrosomal loss in spermatozoa that are not near the oocyte are likely to be contraceptive. Ushercell induces acrosomal loss at low concentrations (Figure 2).
Ushercell inhibits the penetration of cervical mucus at 1 mg/mL (Table 1). This effect is relevant in view of the high concentrations (30-100 mg/mL) of this polymer that would be present in vaginal formulations. The consistency of cervical mucus changes as a function of the menstrual cycle. The molecular arrangement of its mucopolysaccharides provides a barrier to the migration of spermatozoa into the reproductive tract (Prins et al, 1979; Aitken et al, 1987). Agents or actions that impair sperm penetration of cervical mucus may be contraceptive, at least in humans.
Ushercell is contraceptive when tested in the rabbit (Table 2). A lower molecular weight form of cellulose sulfate was used clinically to prevent pregnancy. Its action was believed to be due to its inhibitory effect on hyaluronidase (Doring 1954; Andolz et al, 1983). However, the present study argues against this contention. Although hyaluronidase is inhibited, the IC50 for this effect (1.7 mg/mL) is higher than the concentration of Ushercell required for nearly 95% inhibition of conception in vivo (Table 2). Thus, although hyaluronidase inhibition may be supportive in the mechanism for contraception by Ushercell, other actions (e.g., a stimulus of acrosomal loss) may be more important.
Agents that impair sperm function in vitro are not necessarily contraceptive in vivo. Other polymers, such as heparin and dextran sulfate, have similar effects to those of Ushercell on sperm function, including hyaluronidase inhibition (Hadidian and Pirie, 1948; Astrup and Alkjaersig, 1950; Bernfeld et al, 1961; Mann, 1964; Mathews, 1966; Zimmermann et al, 1983) and induction of acrosomal loss (Delgado et al, 1982; Meizel and Turner, 1986; Parrish et al, 1989). However, these compounds are not contraceptive in vivo (Table 2). Factors such as its disposition after vaginal application, or its pharmacokinetics, may also be important. Ushercell is much less likely than either heparin or dextran sulfate to be metabolized by mammalian tissues, due to the ß,1-4 linkage of cellulose. Additional work is required to find the reasons for the differences between in vitro and in vivo efficacies among these polymers.
Effects of Ushercell on STI-Causing Microbes
HIV-1 inhibition by Ushercell (Figure
3) agrees with earlier data obtained with low-molecular-weight
forms of this polymer (Mizumoto et al,
1988; Yamamoto et al,
1990). However, the efficacy of Ushercell is much greater. These
findings were extended to include several laboratory strains of HIV, and
clinical isolates. These data provide convincing evidence that in vitro,
Ushercell is highly effective against HIV.
Ushercell inhibits HSV-1 and HSV-2, with IC50 values in the submicrogram/mL range (Figure 4). These low inhibition constants argue against hyaluronidase inhibition as a primary determinant of its anti-HSV activity. Competition with proteoglycan-receptor interactions by Ushercell may at least partially explain the inhibition of HSV, HIV, and Chlamydia infectivity observed in the present study (see above, "Possible common penetration pathways of spermatozoa and pathogenic microbes").
Ushercell inhibits the growth of N gonorrhoeae on agar plates, an effect that does not likely require ligand receptor interaction. To our knowledge, this is the first report of inhibition of this microbe by any form of cellulose sulfate. This effect is probably a selective, rather than a nonspecific cytotoxic action of Ushercell. We observed a general lack of cytotoxic effects on several other cell types, including spermatozoa, MT-2 cells, CaSki cells, HeLa cells, and L gasseri. The mechanism for inhibition of gonococcal growth is likely distinct from that responsible for inhibition of infectivity by other microbes. Opa proteins and lipo-oligosaccharides found on N gonorrhoeae are required for microbial pathogenicity and are recognized by mammalian lectins (Mandrell et al, 1994). Ushercell may be interacting with these substances through ionic effects and hydrogen bonding to disrupt normal function of the microbial cell wall, and inhibit cell division. Additional work is required to characterize the inhibition of microbial replication by this polymer.
Initial Safety Assessment of Ushercell
A vaginally applied agent intended for contraception and prevention of STIs
must have a high safety index. Risk of ill side effects of its use should be
less than risks typically associated with therapeutic agents. A vaginally
applied product should have few effects on the normal vaginal flora (eg,
Lactobacillus). The low pH and the hydrogen peroxide produced by
Lactobacillus create a hostile environment for pathogenic
microorganisms, and help to maintain a healthy vaginal ecosystem
(Mardh, 1991).
The present data show that Ushercell should have a high margin of safety. No evidence exists that the antimicrobial activities of Ushercell are mediated through cytotoxic mechanisms. Our results show that host cells used in the viral and bacterial assays were not overtly damaged when Ushercell was present at concentrations several times higher than concentrations required to inhibit infectivity (Results). Although several STI-causing microbes are inhibited by Ushercell, the growth of Lactobacillus gasseri is unaffected (Figure 8). Similarly, sperm mobility (an index of cytotoxicity to spermatozoa) is only marginally affected at concentrations as high as 50 mg/mL (Figure 7). This concentration is at least 50 times higher than that required for approximately 95% inhibition of conception (Table 2) or for altered sperm function (Figures 1 and 2; Table 1). Different molecular forms of cellulose sulfate have been used for the prevention of pregnancy (Doring, 1954). It is also used as a food additive (Burke and Turbak, 1975) and as a component in the encapsulation of pancreatic islet cells (Merten et al, 1991; Lacik et al, 1998).
A high molecular weight form of cellulose sulfate, such as Ushercell, is not likely to be absorbed when applied vaginally (Wood et al, 1984), due to its high molecular weight and charge density. Poor systemic absorption reduces the expressions of any yet-to-be-determined side effects.
Initial phase I clinical trials with Ushercell suggest it to be as safe as K-Y Jelly (a marketed lubricant used as a control in those studies). Ushercell may cause less genital irritation than K-Y Jelly (C. Mauck, unpublished). These observations support an excellent safety profile for this polymer.
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) or HSV-2 (
) was carried out, as described in
"Materials and Methods," in the presence of different
concentrations of Ushercell, as indicated. Data were subjected to logarithmic
transformation before further analysis. Values are reported as average viral
titer (pfu/mL x 10-8), corrected to an average control titer
of 5 x 108 pfu/mL to permit direct comparison among
experiments. Error bars are upper 90% confidence limits. Data for HSV-1 were
fit (r2 = 1.000) to a curve described by the equation:
In(Titer) = a + b([Ushercell]0.5 + c(exp-[Ushercell]),
where a = 2.1399015, b = -2.994577, and c = -0.5304636. This curve was used to
estimate the IC50 (59 ng/mL) and amount of polymer required for
3-log reduction in infectivity (6.2 µg/mL). Data for HSV-2 were fit
(r2 = 0.999) to a curve described by the equation: Titer =
a + b(atan(([Ushercell]—c)/d) + pi/2)/pi, where a = 0.035518, b =
8.6916, c = 0.0055699, and d = -0.024508. This curve was used to determine
IC50 (24 ng/mL) and 3-log reduction (13.9 µg/mL) values. Data
for Ushercell concentrations ranging from 0 to 2 µg/mL are more easily
visualized in the figure inset.
/2)