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Safety, Tolerability, and Pharmacokinetics of SPL7013 Gel (VivaGel®): A Dose Ranging, Phase I Study

O'Loughlin, John MBBS, MS*; Millwood, Iona Y. PhD; McDonald, Helen M. PhD; Price, Clare F. BPharm§; Kaldor, John M. PhD; Paull, Jeremy R. A. PhD§

doi: 10.1097/OLQ.0b013e3181bc0aac
Original Study
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Objectives: To evaluate safety, tolerability and systemic pharmacokinetics of escalating doses of SPL7013 Gel in healthy women.

Design: Randomized, double-blind, placebo-controlled dose-escalation trial.

Methods: Thirty-seven healthy women were randomized to receive 3.5 g of 0.5% (N = 8), 1% (N = 8), or 3% (N = 9) SPL7013 Gel or placebo gel (N = 12), applied vaginally once daily for 7 consecutive days. Genital toxicity was determined by interview, physical examination, assessment of vaginal microflora and colposcopy. Systemic toxicity was determined by nongenital adverse events (AEs) and laboratory assessments. Plasma was collected for pharmacokinetic analysis.

Results: Genital AEs considered potentially product-related were all mild and reported by 5 (20%) women receiving SPL7013 Gel and 2 (17%) women receiving placebo gel. The most common were abdominal pain or discomfort, with no reports of vaginal burning or malodour, or genital-tract pain. There were no clinically significant colposcopic findings, including of genital inflammation or epithelial disruption. Lower concentrations of normal lactobacillary flora occurred during SPL7013 Gel and placebo gel use, with a decrease in anaerobes in the SPL7013 Gel groups. There were no reported cases of bacterial vaginosis, and lactobacilli returned to predose levels in most women after treatment. All nongenital AEs were of mild or moderate severity, expect for a severe tension headache in a woman receiving placebo. There was no absorption of SPL7013 into the systemic circulation.

Conclusions: SPL7013 Gel applied vaginally once daily for 7 days at concentrations of 0.5% to 3% was safe and well tolerated in healthy, sexually abstinent women, with no evidence of systemic toxicity or absorption.

A safety, tolerability and pharmacokinetics vaginal microbicide study found mostly transient microbiologic changes. Genital irritation was low with no evidence of epithelial disruption. Overall no safety concerns were detected.

From the *Royal Adelaide Hospital, Adelaide, Australia; †National Centre in HIV Epidemiology and Clinical Research, University of New South Wales, New South Wales, Australia; ‡Microbiology and Infectious Diseases Department, Women and Children's Hospital, Adelaide, Australia; and §Starpharma Pty Ltd, Melbourne, Australia

The authors would like to thank all the women who participated in the study; Yvonne Lungershausen and the clinical research staff at CMAX; the laboratory staff at the IMVS; Ena Ribic and Amanda Liebelt, Women's and Children's Hospital, Adelaide for vaginal microflora analysis; and Sepehr Shakib, the medical monitor for the study.

Supported by Starpharma Pty Ltd.

Correspondence: Jeremy R. A. Paull, PhD, PO Box 6535, St Kilda Rd Central, Victoria 8008, Australia. E-mail: jeremy.paull@starpharma.com.

Received for publication April 8, 2009, and accepted August 8, 2009.

The global HIV epidemic continues, with an estimated 2.5 million people newly infected in 2007,1 mainly by heterosexual transmission. Vaginal microbicides offer a potential strategy for the prevention of sexual transmission of HIV that can be initiated by women.

SPL7013 Gel (VivaGel®) is a vaginal microbicide being developed by Starpharma Pty Ltd (Melbourne, Australia) for the prevention of HIV and HSV-2. The active ingredient, SPL7013, is a polyanion macromolecule in a class of compounds called dendrimers.2 Agents of this kind have the ability to inhibit virus: cell interactions, making them candidates for topical microbicides.2,3

SPL7013 is active against HIV-1 in vitro,2,4,5 and in colorectal and cervical explant systems.6,7 In a macaque model, SPL7013 Gel was protective against vaginal simian humanized immunodeficiency virus challenge, in a dose-related manner, at concentrations of 1%, 3%, and 5%.5 Activity against HSV-1 and -2 has been demonstrated in vitro,8,9 and against HSV-2 infection in mice and guinea pigs.9 A contraceptive effect of SPL7013 Gel has also been observed in rabbits.10

This article reports on the first clinical study of SPL7013 Gel. The aim of the study was to assess the safety, tolerability and pharmacokinetics of 7 consecutive daily vaginal applications of SPL7013 Gel at 3 escalating dose levels in healthy, sexually abstinent, HIV-uninfected women.

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MATERIALS AND METHODS

Study Design

The study was a single centre, randomized, double-blind, placebo-controlled, dose-escalation trial, conducted between January and October 2004 at CMAX, Royal Adelaide Hospital, South Australia.

Within each of 3 consecutively enrolled groups of 12, participants were randomized to receive either SPL7013 Gel (0.5%, 1% or 3%) or placebo gel in a 2:1 ratio.

Ethical approval was granted by the Royal Adelaide Hospital Research Ethics Committee.

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Study Participants

Female volunteers aged between 18 and 45 years were recruited by advertisement, and gave written informed consent. Participants were eligible if they: were nonpregnant, had a regular menstrual cycle of 25 to 35 days duration, were in good general health with no abnormal findings from physical or genital examination, including colposcopy, Pap smear and evaluation of vaginal microflora, and did not have any STI or vaginal infections.

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Study Products

Study products were provided in prefilled single-use applicators containing 3.5 g of 0.5%, 1%, or 3% w/w SPL7013 Gel, or 3.5 g of the vehicle gel as a placebo. The gels are Carbopol-based aqueous gels that have the same formulation except for the addition of SPL7013, and are similar in appearance.11

A randomization schedule was prepared with 4 participant numbers within each group of 12 randomly assigned to placebo gel. Participants were consecutively assigned a participant number at enrollment. Replacements were assigned the same treatment as the participant they had replaced. Participants and study staff remained masked to treatment allocation for the duration of the study.

Participants administered study products in the morning, in the presence of a member of study staff.

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Study Procedures

After consent was obtained a screening visit was conducted up to 28 days before enrollment. This involved medical history, physical examination (including assessment of vital signs and ECG), and colposcopy. A Pap smear was taken, and samples collected for a screen of sexually transmitted infections (STI) and reproductive tract infections including: Endocervical swab samples for Neisseria gonorrhoeae and Chlamydia trachomatis; blood sample for syphilis; and vaginal swab samples for trichomonas, bacterial vaginosis, candidiasis, Haemophilus ducreyi, and vaginal microflora. Blood samples were collected for evaluation of antibodies to HIV-1, HIV-2, HBV, HCV, and HBsAg. Blood and urine samples were taken for a standard laboratory evaluation (i.e., serum chemistry, haematology, coagulation, urinalysis) pregnancy, and drug and alcohol screen.

Participants were required to abstain from sexual activity for 5 days before screening, and for the period beginning 14 days before baseline and concluding at day 14. Participants were required to fast before blood chemistry and pharmacokinetic samples.

Eligible women who provided consent were enrolled in the trial. At baseline (day 1), participants were admitted to the clinic where they remained for 7 days of dosing (days 1–7) and 1 day of follow-up (day 8), with a further follow-up visit at day 14. Screening and baseline visits were scheduled for midmenstrual cycle.

At baseline (day 1), participants had a brief physical examination, colposcopy was performed, and samples collected for vaginal microflora evaluation, pregnancy testing, and standard laboratory evaluation.

On day 1, eligible subjects received the first dose of gel. Colposcopy was performed and vital signs were measured 4, 12, and 24 hours postdose. Samples were collected for pharmacokinetic analysis and standard laboratory evaluation.

On days 2 to 6, vital signs were measured twice daily, colposcopy was performed and samples collected for vaginal microflora, and standard laboratory evaluations. Pharmacokinetic samples were taken on days 3 and 5. Day 1 procedures were repeated on day 7 except for colposcopy.

Before leaving the clinic on day 8, women underwent a physical examination, (including vital signs and ECG). Colposcopy was performed and samples collected for STI and reproductive tract infections screen, pregnancy testing and standard laboratory evaluation. At the day 14 visit, colposcopy was performed and samples collected for vaginal microflora evaluation.

Adverse events (AEs) and solicited clinical symptoms of genital irritation (specifically: appearance, viscosity and quantity of vaginal discharge/gel leakage; vaginal burning; genital malodour; genital tract pain; abdominal pain; and dysuria) were monitored from baseline until the study end.

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Colposcopy

Colposcopy was performed at screening, days 1 (4 hours postdose), 4 (before dosing), 8 and 14 in accordance with the procedures outlined in the 2000 Update of the CONRAD/WHO Manual for the Standardization of Colposcopy for the Evaluation of Vaginal Products.12 All colposcopic examinations were performed by the Investigator (JO), an experienced colposcopist. Colposcopy was directed to the examination of the vulva and the vaginal and cervical epithelia, and a comprehensive photographic record made of each examination.

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Microflora Evaluation

Vaginal specimens were obtained on days 1, 4, 8, and 14. A smear made on a glass slide, and 2 sterile cotton swabs placed in an anaerobic transport system (BBL Port-A-Cul Specimen Collection System). A gram stain was performed on the vaginal smear and the Nugent score determined to microscopically assess inflammation and vaginal flora.13

Within a biologic safety cabinet, the Port-A-Cul swabs were placed in 1.5 mL of buffered salt solution and vortexed. Serial 1:10 dilutions of the resultant suspension were made.14 Quantitative culture was performed following the procedures used by Clarke et al15 to determine frequencies and concentrations of the following microorganisms after gel use: Lactobacillus species (H2O2-producing and nonproducing), Gardnerella vaginalis, Escherichia coli and other gram-negative rods, anaerobic gram-positive cocci, Propionibacterium species, anaerobic gram-negative rods, aerobic gram-positive cocci including group B streptococcus, and yeasts.

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Pharmacokinetic Evaluation

Plasma samples were collected for pharmacokinetic analysis before and at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration of the first dose on day 1, and before dose administration on days 3 and 5. Plasma SPL7013 concentration was determined using a validated bio-analytical capillary electrophoresis method with a lower limit of quantification of 0.5 μg/mL (30 nM).

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Interim Safety Evaluation

A full masked review of safety variables from each dose level was performed before initiation of the next higher dose.

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Statistical Methods

Sample size was consistent with other phase I studies of topical microbicide products and it was accepted that the study had limited statistical power, as is typical of phase I studies in general.

AEs were coded using MedDRA version 7.0 and all participants receiving study products were included in safety analyses. Most analyses compared women on SPL7013 Gel, combined across the 3 dose levels, with placebo. Given the limited power, no statistical tests of significance were conducted to compare treatment groups.

Pharmacokinetic analysis and quantitative analysis of vaginal microflora was based on participants who received all 7 doses of study product.

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RESULTS

Enrollment

Thirty-seven women were enrolled into the study from February to October 2004 (Table 1). Eight women received SPL7013 Gel at 0.5% (group 1), 8 at 1.0% (group 2), and 9 at 3.0% (group 3). Four women in each group received placebo gel. Subject 028 in group 3 was withdrawn from treatment after receiving 3 doses of 3% SPL7013 Gel, due to detection of Clostridium botulinum on day 3 in a sample taken at baseline (deemed likely due to laboratory contamination), and was replaced. Thirty-six women completed the study, each receiving all 7 doses of study product.

TABLE 1

TABLE 1

Participants were aged between 18 and 43 years old (mean 24 years old). Twenty-four women were using hormonal contraception at baseline, and continued throughout the study. Baseline characteristics were similar between treatment groups.

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Urogenital Adverse Events

The incidence of solicited symptoms of genital irritation was low, and they were all reported as mild in intensity. Symptoms considered potentially related to study product use were reported by 3 (38%) women in the 0.5% SPL7013 Gel group (4 events), 1 (13%) in the 1% group (1 event), 1 (11%) in the 3% group (1 event), and 2 (25%) women in the placebo group (6 events) (Table 1). The most common genital symptom considered to have a potential relationship to study product was abdominal discomfort or pain, which was reported by 3 (38%) women in the 0.5% group (4 events), 1 (11%) in the 3% group (1 event), and 1 (8%) in the placebo group (1 event). Other potentially related symptoms were: a report of dysuria in 1 (13%) woman in the 1% group, and 5 reports of genital pruritis in 1 woman (8%) in the placebo group. Single reports of abdominal discomfort or pain by 1 (13%) woman in each of the 0.5% and 1% groups, and 2 (17%) women in the placebo group were considered unrelated to study product. There were no reports of vaginal burning, vaginal malodour, or genital tract pain.

Breakthrough bleeding or spotting occurred in 3 (38%) women receiving 0.5% SPL7013 Gel and 2 (25%) women receiving 1% SPL7013 Gel, of whom all except 1 were using contraceptive tablets, and in 2 (17%) women receiving placebo, both of whom were using subcutaneous contraceptive implants.

Subject 029 receiving 3% SPL7013 Gel had pyuria, determined by urinalysis, but this event was not considered related to study product use.

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Other Adverse Events

Overall, a total of 90 AEs were reported by 31 (84%) of the women. At least 1 AE occurred in 8 (100%) women receiving 0.5% SPL7013 Gel, 7 (88%) receiving 1% SPL7013 Gel, 6 (67%) receiving 3% SPL7013 Gel, and 10 (83%) receiving placebo gel. The most common nongenital AEs were headache (28% SPL7013 Gel, 33% placebo) and venipuncture site bruise (16% SPL7013 Gel, 17% placebo). The only nongenital AE considered possibly related to study product was a jawline rash of moderate severity in 1 woman in the placebo group. All AEs were of mild or moderate severity, except for a tension headache classified as severe in a woman from the placebo group.

There were no clinically significant changes in systemic laboratory safety parameters as determined by the standard laboratory evaluations during the study. No serious AEs, deaths or pregnancy were reported during the study. There was no apparent relationship between colposcopy findings or changes in vaginal flora and hormonal contraceptive use.

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Colposcopy Findings

Colposcopic examination identified no evidence of vulval, vaginal or cervical inflammation or disruption in any of the active or the placebo groups. A total of 33 new colposcopy findings were reported postproduct use, with 6 findings in 4 (50%) women receiving 0.5% SPL7013 Gel, 5 findings in 3 (38%) women receiving 1% SPL7013 Gel, 13 findings in 5 (56%) women receiving 3% SPL7013 Gel, and 9 findings in 4 (33%) women receiving placebo gel. Findings at both baseline and follow-up were of a minor nature, and of no clinical significance and comprised small localized areas of erythema, ecchymosis, petechiae, and petechial hemorrhage without epithelial disruption. The majority of these findings were considered to be related only to the study procedures.

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Vaginal pH

There was an apparent marginal increase in vaginal pH at day 4 (no greater than 1.0 except in subject 022), which was similar in all treatment groups including placebo, with measurements returning to predose levels by day 14. The only pH value above 5.0 was reported by a woman in the placebo group (subject 022) on day 4 (pH 5.8), who was experiencing breakthrough bleeding at the time.

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Microbiology Findings

A moderate (2–3 log difference) or major decrease (≥4 log difference) in lactobacilli concentrations was observed during gel use in 6 (75%) women receiving 0.5% SPL7013 Gel, 6 (75%) women receiving 1% SPL7013 Gel, 4 (50%) women receiving 3% SPL7013 Gel and 7 (58%) women receiving placebo gel (Table 2).

TABLE 2

TABLE 2

Lactobacilli returned to baseline levels by day 14 in all but 6 of the women. Three of the women had begun menstrual bleeding before the day 14 assessment, and among these a 3 log10 decrease in lactobacilli remained in 1 woman receiving 0.5% SPL7013 Gel and 1 receiving placebo gel, and a 4 log10 decrease remained in 1 woman receiving 3% SPL7013 Gel. Of the 3 nonmenstruating women, a 3 log10 decrease in lactobacilli concentration remained at day 14 in 2 women receiving 0.5% and 3% SPL013 Gel, respectively, and a 4 log10 decrease remained in 1 women given 1% SPL7013 Gel.

In the SPL7013 Gel groups, the number of women with anaerobic gram-negative rods detected by culture, decreased during gel use, from 13 (54%) at baseline, to 6 (25%) at day 4, and 4 (17%) at day 8, returning to 13 (54%) at day 14. In the placebo group, the proportion of women with anaerobic gram-negative rods remained at 33% throughout. Conversely, the proportion of women with facultative gram-negative rods increased between days 1 and 8, in both the SPL7013 Gel (13%–71%) and placebo (17%–50%) groups, returning to predose levels at day 14 in all but the 3% SPL7013 Gel group, where colonization continued in 50% of women. The balance of lactobacilli and anaerobes as measured by the Nugent Gram stain score did not change during gel use, and there were no women with a Nugent score of 7 or more.

Three women (placebo group) had a light growth of Candida species at screening or day 8. In all cases this growth was asymptomatic and not considered to be candidiasis.

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Vaginal Discharge

Vaginal discharge was reported in 35 of the 37 subjects (24 [96%] in the SPL7013 Gel groups and 11 [92%] in the placebo group). Most reports of discharge took place during the treatment period and descriptions were consistent with leakage of the gel. Discharge in all instances was mild, transient and well tolerated, and was not associated with colposcopic findings, patterns of change in vaginal microflora, vaginal burning, pruritis, or malodour.

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Pharmacokinetic Findings

SPL7013 was not detected above the lower limit of quantification of the assay in any plasma samples from participants receiving 3% SPL7013 Gel. As SPL7013 was not detectable at highest dose, samples from the lower dose groups were not analyzed.

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DISCUSSION

It is important that microbicide candidates demonstrate a low propensity to cause genital irritation and systemic toxicity to be considered appropriate for development. It is also important that microbicide candidates do not disrupt the vaginal microenvironment, which may also lead to additional irritation.

In this double-blind study of healthy, HIV-uninfected, sexually abstinent women, SPL7013 Gel was safe and well tolerated when administered vaginally at doses up to 3% w/w, once daily for 7 days. Except for 1 woman who was withdrawn and replaced, all women completed the study.

The levels of potential symptoms of genital irritation reported during the study were low in the 3 SPL7013 treatment and placebo groups, and at levels comparable with those reported from other phase I studies of microbicide candidates.16–18 The incidence of systemic toxicities reported was also low and unremarkable. There were no dose-related trends within the active treatment groups or apparent differences between active or placebo groups. Nearly all AEs in the study were mild or moderate, and none resulted in discontinuation of gel use.

Nearly all women experienced vaginal discharge during the study, and these reports were consistent with leakage of the study gels. Vaginal discharge of this type attributable to gel leakage is commonly reported in microbicide studies.19,20

On colposcopic assessment, there were no clinically significant findings of vulval, vaginal or cervical inflammation or other pathology that was determined to be related to the study gels. All findings in the study were minor, and the majority were classified as being due to aspects of the study procedures. It must be considered that use of colposcopy will often detect abnormalities independent of an effect of the microbicide under assessment.21

Quantitative analysis of vaginal microflora identified a decrease in lactobacilli, accompanied by an increase in colonization of facultative gram-negative rods, in both the treatment groups. In those using SPL7013 Gel, there was also a decrease in the proportion of women with anaerobic gram-negative rods, but the Nugent score did not change. In most cases, the microflora returned to predose levels a week after treatment had ended. The clinical significance of lower lactobacilli concentrations may not be important if anaerobes are also in low concentrations, and there were no cases of bacterial vaginosis. The clinical significance of increased facultative gram-negative rods is uncertain.

None of the women tested at the highest dose had detectable plasma levels of SPL7013, indicating that the active ingredient was not absorbed systemically. This is consistent with the lack of clinically significant systemic toxicity seen in the study, preclinical studies to date, and molecular structure of SPL7013.2

The low incidence of genital irritation and lack of systemic absorption are consistent with the recently published study investigating safety and tolerability in men.22

In conclusion, SPL7013 Gel was safe and well tolerated in sexually abstinent women at strengths up to 3%, with a low potential for systemic toxicity and no evidence of genital irritation by reported symptoms or colposcopy. SPL7013 is considered an appropriate candidate for further exploration as a microbicide.

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