The primary objective of the study was to assess the safety of VivaGel on the vulvar and cervicovaginal mucosa of healthy sexually active HIV-negative women aged 18–24 years. Secondary objectives included assessment of product adherence and acceptability as well as the impact of product administration on vaginal microflora. Exploratory objectives included (i) assessment of product administration on a range of immunological parameters including cervicovaginal levels of cytokines, chemokines, and innate immune factors, (ii) systemic absorption of SPL7013, and (iii) the effects of VivaGel on colposcopic findings of the cervix and vagina.
MTN-004 began as a Phase 1, double blind, randomized, placebo controlled comparison of twice daily exposure to VivaGel and VivaGel placebo (1:1) at two clinical sites (San Juan, PR and Tampa, FL). Enrollment began in August 2007 under Version 2.0 of the protocol. The protocol was paused in October 2007 as five of the seven women enrolled in the study had experienced a low grade genital AE. Following an interim data review the protocol was amended to include a third comparison arm (the HEC placebo gel). Enrollment recommenced in October 2008 under Version 3.0 of the protocol. 61 participants were randomized 1:1:1 to the three study arms. In May 2009, a third clinical site (Pittsburgh, PA) was added to accelerate recruitment to the study. Sample size was based on similar Phase I studies of topical microbicide studies. A blinded statistician from the Statistical and Data Management Center (SCHARP) created lists containing randomly generated unique three-digit codes in blocks of six (two per arm) for each clinical site. Prescriptions containing the randomly assigned codes were contained in sealed envelopes, numbered sequentially and assigned by the clinic staff to study participants in sequential order. Site pharmacists dispensed three boxes of product labeled with the same unique three-digit randomization codes and containing ten applicators to each participant. Pharmacists removed tear-off labels from the boxes of product and placed them on pharmacy dispensing records that were contained in concealed envelopes and pre-printed with the randomization codes. Participants, study staff, pharmacists, clinicians and statisticians were blinded to study assignments.
The study population consisted of healthy, non-pregnant, sexually active, HIV-negative women of the ages 18 through 24 years inclusive with a normal genital tract (anatomically normal pelvic exam without evidence of genital infection or deep disruption of the genital epithelium). Participants were required to have regular menstrual cycles with >/= 21 days between menses, to be using adequate contraception (hormonal method, intrauterine device, sterilization, or sexual activity with a vasectomized partner), and to have had a normal pap smear within one year prior to enrollment.
The VivaGel formulation used in the study comprised SPL7013 formulated in a water-based gel (methyparaben (0.18%), propylparaben (0.02%), EDTA (0.1%), Carbopol 971P (5.0%), propylene glycol (1.0%), glycerin (1.0%)), with purified water and sodium hydroxide added to adjust the gel pH to approximately 5.0. The VivaGel placebo for this study is the base formulation without SPL7013. The HEC gel contains hydroxyethylcellulose as the gel thickener (2.7%), sodium chloride (0.85%), sorbic acid (0.1%), with purified water and sodium hydroxide added (18). The gel is isotonic and formulated at a pH of 4.4 to avoid disrupting the normal vaginal pH and has minimal buffering capacity in order to avoid the inactivation of sexually transmitted pathogens. For all gels, the dose volume of 3.5 g (equivalent to 3.5 mL) has been selected as the dose that is intended to provide optimum vaginal and cervical coverage while minimizing leakage of product from the vagina . Participants were asked to insert the product in the morning and in the evening, or approximately every 12 hours for fourteen days. All study products had a similar appearance and were provided in identical HTI polypropylene pre-filled applicators (HTI Plastics, Lincoln, NE) ().
The HTI vaginal applicator used to deliver the microbicide gel in MTN-004
A total of five scheduled clinic visits were performed. After obtaining informed consent all participants underwent a thorough medical history, a targeted physical examination, and a complete pelvic examination including vaginal swabs for pH, wet prep and Gram stain. A pap smear was obtained when necessary.
Participants who met the inclusion and exclusion criteria during screening proceeded to an enrollment visit. Enrollment was scheduled within 1–2 days of the end of menses to avoid menstrual bleeding during product use and during evaluation of the cervix and vagina. At the enrollment visit participants were randomized, safety labs were repeated and a blood sample was collected for measurement of SPL7013. Pelvic samples were collected for vaginal pH, Gram stain, wet prep, quantitative vaginal cultures and immunological assays (cytokines, chemokines, and innate factors). Colposcopic assessment was performed and photographed. Participants received the first vaginal application of study product in clinic and were provided with 20 applicators for use at home. One and two weeks later participants returned to the clinic for repeat assessment including clinical assessment of AEs, pregnancy testing, safety labs, and gynecologic exam with vaginal pH, wet prep, vaginal microflora, and immunological assays. Used applicators were collected and counted at each visit. Ten additional applicators of study product was distributed at the Week One visit. A colposcopy was performed at the Week Two visit. A final visit was conducted one week after completion of product administration with clinical assessment, pregnancy testing, and collection of safety labs, and vaginal samples. All study visits were timed to avoid menses and included the option to conduct additional laboratory assessment and examination as clinically indicated. Interim contact and unscheduled visits were held as necessary for reported AEs or by request of the participant or investigator.
Clinical Safety and Laboratory Assessment
Emergent AEs were graded using the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, Version 1.0, December 2004 as well as Addendum 1 (Female Genital Grading Table for Use in Microbicide Studies
). Asymptomatic bacterial vaginosis/candidiasis and colposcopic findings were not reportable AEs. In cases where an AE was covered in both tables, the Female Genital Grading Table for Use in Microbicide Studies
was the grading scale utilized. The extent of SPL7013 absorption was determined via assessment of SPL7013 levels in serum samples taken on Day 0 and Day 14, using a validated capillary electrophoresis bioanalytical method conducted at Starpharma Pty Ltd.
Overall product like (or dislike) and likelihood of gel use in the future were assessed using an internet based computer assisted self interview.
Analysis of study outcomes
For analyses comparing VivaGel to the VivaGel placebo, data from 43 women were included (4 and 3 participants, respectively, enrolled under Version 2.0 of the protocol, and 18 participants in each of the 2 arms enrolled under Version 3.0 of the protocol) whereas for analyses comparing VivaGel to the HEC gel, data from 36 women were included (18 participants per arm enrolled under Version 3.0).
The primary endpoint of abnormal genital symptoms was defined as those judged by the study investigator to be possibly, probably, or definitely related to product use. Genital symptoms included were as follows: Genital tract pain (pelvic pain, vaginal & vulvar pain); tenderness; dyspareunia; dysmenorrhoea; vulva (itching, edema, erythema, lesions, abrasions, rash); vagina (itching, edema, erythema, dryness, discharge, abrasions, lesions, masses; ruptured cyst); cervix (edema, erythema, discharge, lesions); perianal (erythema, laceration, irritation); urinary tract (urinary frequency, dysuria, hematuria, infection); vulvovaginitis; cervicitis; pelvic inflammatory disease; genital herpes; candida; bacterial vaginosis; menorrhagia; metrorrhagia; unexplained infrequent bleeding; post-coital bleeding. Comparisons were made across the arms of the rates of at least one related abnormal genital symptom using the global Chi-square test for independence. Additionally, to assess the incidence of related abnormal genital symptoms, the number of symptoms was summed for each woman during her follow-up. Due to the short follow-up time each type of symptom was only counted once per woman. A global test for homogeneity of the incidence rates across the arms was conducted based on the Poisson distribution. Similarly, pairwise comparisons of the rates were also made between arms.
The secondary endpoint of product adherence was calculated in two ways, including and excluding the time participants spent on product holds. Product acceptability was defined as the proportion of participants who at their two-week follow-up visit reported via the acceptability questionnaire that they would very likely use the candidate microbicide during sexual intercourse in the future.
Vaginal flora was assessed from Nugent scores defined as follows: Normal, 0 to 3; Intermediate, 4 to 6; Bacterial Vaginosis, 7 to 10. Quantitative measures of >= 1 log change in the dominant members of the microflora included the following:
- Lactobacillus (H2O2 positive and negative strains)
- anaerobic gram negative rods
- Gardnerella vaginalis
- Escherichia coli
- Staphylococcus aureus
- Candida species
- Group B Streptococcus, and Enterococcus species
Odds ratios comparing VivaGel placebo or HEC gel versus VivaGel (reference group) for a >= 1 log change in microflora levels were estimated using Generalized Estimating Equations. A logit link function with the exchangeable correlation structure and model-based standard errors were used. All analyses were performed using SAS Version 9.1.3 (SAS Institute, Cary, North Carolina, USA).