Patton, Dorothy L. PhD; Cosgrove Sweeney, Yvonne T. BA; Paul, Kathleen J. MPH
THE DEVELOPMENT OF TOPICAL MICROBICIDES represents an important opportunity and a major source of hope in the global fight against HIV/AIDS and other sexually transmitted infections (STIs). A microbicide product that is safe, effective, and inexpensive has the potential to significantly reduce global transmission of STIs, with the added benefit that it would give women the ability to take an active role in protecting themselves.
In 1999, the Division of Microbiology and Infectious Diseases (DMID), of the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) began support for the Sexually Transmitted Disease Prevention-Primate Unit. This contract was established as a means to comparatively assess the safety of topical microbicide products to cervical and vaginal tissues after repeated use and to evaluate their efficacy in preventing cervical chlamydial infection. The US Food and Drug Administration (FDA) requires that safety and, whenever possible, in vivo activity of candidate topical microbicide products be evaluated in animals before their use in humans.
The pigtailed macaque (Macaca nemestrina) is a well-established primate model that has been used to further our understanding of the pathogenesis of experimentally induced Chlamydia trachomatis cervicitis and salpingitis since the mid-1980s,1–5 and more recently, we have extended the model to include studies of the lower reproductive tract as well.6–9 In this capacity, we have developed a clinically relevant animal model that has been used to evaluate the impact of formulated microbicidal compounds on vaginal microflora and cervicovaginal tissue and to investigate their chlamydiacidal activity.10–13 M. nemestrina is an ideal model for studying the female reproductive tract in that the menstrual cycle is similar to that of human females in length and pattern, and the species is naturally susceptible to many human STIs including C. trachomatis and Trichomonas vaginalis without the need for exogenous hormone treatments.14
Preclinical animal studies are particularly useful in that we have the unique ability to evaluate relatively immediate and cumulative effects of product exposures (pre- and postexposure exams for 4 consecutive days with follow-up on days 5 and 8), and to a certain extent, we can control the behavior of our test subjects (e.g., timed product administration, abstinence from intercourse). The standardized protocol allows for findings from any single test product to be compared to those of another. The main purpose of these studies is to provide evidence that helps to move promising products forward in development and, perhaps more importantly, to recommend reformulation for products that are observed to cause significant, deleterious changes or toxicity to the cervicovaginal environment.
This article summarizes the preclinical vaginal microbicide candidate safety and chlamydial efficacy studies completed in the pigtailed macaque model over the past 7 years of NIH contract support.
Materials and Methods
Candidate topical microbicide formulations and matched placebo gels were provided by suppliers or manufacturers via the NIH. The topical microbicide candidates may be gels, creams, foams, or films for intravaginal use with or without barrier devices. Before product enrollment, developers were requested to submit in vitro data showing the formulation’s activity against numerous STI pathogens, effect on vaginal flora, and cell toxicity observed in tissue/cell culture assays and toxicity data from rabbit vaginal irritation studies. Suppliers also ensured shelf life stability of at least 6 months and agreed to supply 500 mL each of formulated product and placebo prepared under good laboratory practices (GLP) conditions. Material transfer agreement and/or screening agreement and/or confidentiality agreements were signed by the developer and the Division Director, DMID.
Sexually mature female M. nemestrina were obtained from a colony of animals at the Washington National Primate Research Center (WaNPRC). Prior approval for use of monkeys in this protocol was obtained from the Institutional Animal Care and Use Committee at the University of Washington. Animals were handled humanely, and experiments were performed within the National Institutes of Health’s animal use guidelines.
A pool of macaques was kept available for enrollment in specific contract studies. For each safety evaluation, 6 animals were randomly selected for each study arm. All animals were allowed at least 2 weeks recovery time between studies, and a single animal was allowed to complete a maximum of 6 safety evaluations before being returned to the WaNPRC colony. Animals were not synchronized nor otherwise altered to control for menstrual or hormone status. If an animal was noted to have colposcopically abnormal tissues (e.g., friable tissue) on the first day of study, she was replaced by an alternate macaque. In the event of adverse product-related findings, repeat experiments could be conducted with additional macaques to increase the sample size.
Midway through the contract period, we refined the protocol to reduce our total demand for female macaques. We transitioned to a crossover study design, wherein each animal controlled for herself by completing both arms of the safety study in random order, with 2 to 3 weeks of recovery time in between. Given the small number of animals available and the expectation that individual animals would show correlation in their results over time, a crossover design is not only practical but also statistically efficient. In total, 16 studies were conducted with the original independent design, and 8 were conducted using the revised crossover design.
Safety evaluations are designed to assess the effects of the active component in a product formulation. Therefore product-specific placebos, defined as the same formulation as the test product less the active component(s), were supplied for each safety evaluation. Most studies were conducted with 2 arms (test product vs. product-specific placebo). In the case of a test product that was formulated with varying concentrations of the active ingredient, several formulations were compared to a single placebo arm.
Experimental Safety Protocol
A standardized vaginal safety protocol was followed for each experiment (Table 1). On study days 1 to 4, baseline (T0) colposcopy assessments and swab specimen collections for vaginal pH and microflora were obtained. Immediately after daily T0 specimen collections, intravaginal application of 1.5 mL of test or placebo gel was administered to each animal. Thirty minutes after each gel application (T30), vaginal swabs were again collected to assess pH and any acute fluctuations in the vaginal microflora. On day 5, colposcopy, vaginal swabs for pH, and microflora were collected. For the majority of test products were assessed, 1 cervical and 1 vaginal biopsy were obtained. On day 8, colposcopy and vaginal swabs for pH and microflora were obtained to document recovery.
Colposcopy took place immediately after speculum placement, before any swab collections. Standardized assessments were conducted by a team of 3 cross-trained individuals following World Health Organization guidelines.15–17 Vaginal and ectocervical mucosal surfaces were evaluated for erythema, vasculature pattern, epithelial integrity, and any exceptional findings. Observations were noted on daily examination records, and documented by digital photography.
Vaginal pH was determined at T0 and T30 by rolling a swab onto a pH indicator strip (resolution 0.5 pH units) to document transient, product-induced pH shifts. Recovery to baseline pH was documented at follow-up visits. Based on our observations of more than 100 animals examined at baseline, we have established the normal range of vaginal pH in the Macaca nemestrina as 5.0 to 8.5 (unpublished data); this range is inclusive of mensing animals, which tend to have higher pH values.
Each vaginal swab collected for microbiologic assessment was individually placed in Port-A-Cul tube, a transport device shown to preserve viability of aerobic and anaerobic bacteria in clinical specimens.18 Within 24 hours of collection, samples were delivered to the Reproductive Infectious Disease Laboratory at Magee-Womens Research Institute (Pittsburgh, PA), where they were quantitatively evaluated for aerobic and anaerobic microorganisms with microbiologic assays previously described.6 Species belonging to the genera Bacteroides, Porphyromonas, and Prevotella were grouped together as anaerobic Gram-negative rods (nonpigmented or black) for ease of presentation. Prior studies have observed that the normal vaginal microflora in pig-tailed macaques is remarkably similar to that of human females, although macaques tend to have slightly lower levels of H2O2-producing lactobacillus and higher levels of anaerobic bacteria.6
For safety studies conducted through May 2004 (n = 16), vaginal and cervical biopsies were collected before study enrollment and on study day 5. Histologic assessment of H&E-stained biopsy samples was conducted by quantification of polymorphonuclear cells, lymphocytes, and plasma cells in 5 nonadjacent high-powered fields as previously described.19,20 In 2004, interim analysis indicated that a single biopsy, representing a small area of a relatively large anatomical site, collected at a single time, did not reliably support colposcopic evaluation, particularly when an adverse finding was noted. Additionally, histologic evaluations of serial tissue sections indicated potentially significant variability in identification of inflammatory cell foci.21 Given the obvious undesirable effect of breaking the protective epithelial layer to collect the sample, this analysis suggested that the risk of biopsy collection outweighed the benefit of its potential as an indicator of microbicide toxicity. Thus, a protocol revision was established eliminating biopsy collections and histology from future contract studies (n = 8 studies conducted without biopsies).
Product Development Criteria
Product development guidelines for vaginal safety studies were established to allow for recommendations of product advancement or reformulation; these guidelines are summarized in Table 2. Although all test compounds that did not show deleterious effects in safety assessments were eligible for efficacy testing, some product developers did not wish for their products to undergo such tests when offered.
Experimental Efficacy Protocol
Efficacy trials are designed to assess a formulated product controlled by a no product arm. Rather than specifically assessing the ability of the active component(s) to prevent chlamydial infection, this experiment is designed to determine whether the formulated product provides protection from infection when compared with no product use; the mode of action is not explored. Typically, it was most feasible and economical to assess 2 to 3 test products compared with a single positive control (no product) arm. After an initial infection with C. trachomatis, detection of subsequent infections becomes problematic, and so each animal was allowed to complete only 1 efficacy assessment.
The University of Washington Chlamydia Reference Laboratory provided the clinical cervical isolate of C. trachomatis serovar E for efficacy studies. The isolate was prepared in McCoy cell culture and purified by renografin methylglucamine diatrizonate linear gradient column. The stock inocula were titered (1.0–1.3 × 107 IFU/mL), in sucrose-phosphate-glutamate (SPG) buffer and frozen (−70°C) until dilutions were prepared for experimental inoculations. For inoculation, a frozen aliquot of chlamydiae was thawed and diluted to 5 × 105 IFU/mL.
Six animals were randomly assigned to each study arm (n = 6 per test product and n = 6 positive control) to populate each efficacy trial. Baseline samples, including cervical swabs for chlamydia detection by culture and nucleic acid amplification (NAAT) and 5-mL blood for serum antibody detection, were collected from each animal. A single intravaginal application of test microbicide (1.5 mL) was then administered to each animal in the test product group. Thirty minutes later, all (test and positive control) animals underwent inoculation by expelling 1-mL C. trachomatis from a 1-mL tuberculin syringe into the vaginal fornix, thereby exposing the cervix to the organism. Follow-up samples to document chlamydial infection by culture, NAAT, and serology were collected on days 2 and 7 and weekly thereafter for 5 weeks. Culture and NAAT samples were collected with Dacron-tipped swabs making direct contact with the cervical os. For these studies, animals were considered infected if they tested positive by culture beyond study day 2, or positive by NAAT on more than 1 study day, or developed serum antibody.
Chlamydia Trachomatis Detection Assays
Cervical swab specimens were cultured on cycloheximide-treated McCoy cells in 96-well microtiter plates and stained from 40 to 72 hours later with a monoclonal antibody specific for C. trachomatis detection.22
Ligase Chain Reaction.
Cervical swab specimens were vortexed, then incubated at 95 to 100°C for 15 minutes followed by cooling to 20°C. Before use in LCR assays, specimens were either frozen for batched runs or refrigerated to be processed within 48 hours. Specimens were subsequently added to the chlamydia LCR Unit Dose Tubes (Abbott Laboratories, Abbott Park, IL) and then amplified according to the manufacturer’s instructions.23 In late 2001, Abbott’s LCR detection system was discovered to have both specificity and reproducibility problems and was subsequently replaced in these studies with an alternate NAAT system (GenProbe Aptima2).
GenProbe APTIMA Combo 2.
Cervical swab specimens were collected, stored, and transported to the laboratory for assessment according to the manufacturer’s instructions (Gen-Probe, San Diego, CA).24 The APTIMA assay employs transcription-mediated amplification, in which the RNA target molecule from C. trachomatis (23S rRNA) is isolated and specific regions are amplified by using a separate capture oligomer and a unique set of primers for the target.
Serum IgG and IgM chlamydial antibody titers were measured using the microimmunofluorescence technique.25
A total number of 28 test products were evaluated for vaginal safety and 9 for efficacy under this contract.
Overall safety profiles were acceptable in 24 products (Table 3). Microbiologic findings common to most products included stable populations of H2O2-producing lactobacilli and Viridans streptococci and transient decreases in anaerobic Gram-negative rods. The majority of products induced a transient decrease in vaginal pH 30 minutes after product exposure, although pH typically returned to baseline by follow-up on day 8. In studies that included cervical and vaginal biopsies, no product-induced inflammatory response was observed. Common colposcopic observations included notations of vasculature, petechiae, and mild color changes (blanched to slightly reddened tissues), all of which are considered typical findings in nonstudy animals.
Four candidate products were identified as causing cervicovaginal irritation. Four percent Pro-2000 led to abnormal colposcopic findings in 8 of 14 animals; tissue abnormalities included epithelial friability, abrasion, and disruption. Four of the 8 animals with abnormal colposcopic findings developed cervical epithelial disruptions, 2 developed friable cervical tissues, 1 developed cervical epithelial abrasion, and 1 progressed only to elevated white bumps. After treatment with 2% Pro-2000, abnormal colposcopic findings were noted in 5 of 6 animals, but were less pronounced than those noted during study assessing 4% Pro-2000. Three of the 5 animals had cervical epithelial abrasions noted at a single time. Two animals presented with friable tissue on day 5, whereas the other had epithelial abrasions noted on day 2, which improved daily thereafter. Presence and quantity of H2O2-producing lactobacilli remained stable after 4% and 2% Pro-2000 treatments. The frequency of anaerobic Gram-negative rods decreased after exposure to both formulations, but the decrease was more pronounced after exposure to the 4% Pro-2000. Vaginal pH decreased slightly after treatments with both formulations but returned to baseline levels at the day 8 follow-up visit.
Repeated daily use of 1.7% C31G produced mild abnormal colposcopic findings in 4 of 6 animals treated, and caused a marked reduction in frequency of microorganisms detected at T30, including the potentially advantageous H2O2-producing lactobacilli. Most microorganisms were again detected at 24 hours, but growth was diminished. Vaginal pH was unchanged after use of 1.7% C31G.
Five percent SPL7013 led to friability and epithelial disruption in 3 of 6 animals after the first product application. These findings persisted through day 5, but resolved by follow-up on day 8. A fourth animal showed friable tissues only on days 4 and 5. Vaginal microflora remained largely unchanged, with the exception of a transient decrease in anaerobic Gram-negative rods. Vaginal pH decreased at T30 but returned to baseline levels at follow-up on day 8.
All 4 products showing potentially deleterious cervicovaginal effects were reformulated and observed to have acceptable safety profiles when used at lower concentrations (0.5% Pro-2000, 1.0% C31G, and 1% and 3% SPL7013).
We have evaluated single applications of 9 topical microbicide candidate products (Table 4) to determine whether any of these microbicidal products showed efficacy against cervical chlamydial infection. As a positive control, 1 arm (n = 6) in each study received no product before cervical challenge with C. trachomatis. Unexpectedly, the positive control arm of the first efficacy trial only resulted in a 50% infection rate. Even at this low infectious challenge, significant protection against chlamydial infection was not noted with any of the test products. Similarly, chlamydial infection was not prevented by any products tested in subsequent trials. The majority of positive control animals (no microbicide treatment before inoculation) tested positive for infection by culture and/or NAAT in the second, third, and fourth trials.
We developed and tested a pig tailed macaque model designed to comprehensively evaluate the safety and efficacy of topical microbicide candidate products. In total, 28 products underwent safety evaluations and 9 were also tested for efficacy against cervical chlamydial infection. Our safety investigations identified 4 products that showed deleterious effects on the cervicovaginal environment, and these products were successfully reformulated by the manufacturers with improved safety profiles.
In light of recent concerns regarding the safety of topical microbicides in international clinical trials, the importance of preclinical safety investigations is especially clear. Female-controlled products like microbicides offer a critical opportunity in the fight against HIV and other STIs globally, but such products must first undergo rigorous safety and efficacy testing. Preclinical evaluations in an animal model such as ours can provide significant product comparison information obtained under standardized laboratory conditions, offering early warning of products that may have harmful effects and helping to usher promising products into clinical trials more quickly.
However, it is important to bear in mind that preclinical nonhuman primate testing is necessarily limited by the small numbers of animals involved; we cannot necessarily detect rare adverse events or safety problems, which may arise when larger clinical trails are conducted. Thus, our overall goal is not to definitively establish product safety, but rather to detect any major toxicities and recommend reformulation before the product enters clinical testing. When toxicity problems are observed, we may be able to conduct further testing to isolate the specific component of the product producing the deleterious effects, which is very useful information for the developer in terms of reformulation. This learning process is critical to future product development.
In summary, the NIH-contract mechanism provides a resource to further the development of topical microbicides aimed at the prevention and control of STIs through preclinical testing in nonhuman primates. Continuing safety evaluations and efficacy evaluations for the pathogens C. trachomatis and T. vaginalis are also supported through the recently awarded NIH/NIAID primate contract mechanism through 2014.
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29. Cosgrove Sweeney YT, Butler K, Peterson A, et al. Preclinical Safety Evaluation of Acidform in the Macaque Model. In: Microbicides 2004 Conference. 2004; London, England. Abstract 02343–3.
30. Cosgrove Sweeney YT, Patton DL. Tenofovir 1%: Vaginal safety evaluation in the macaque model. In: Microbicides 2006 Conference. 2006; Cape Town, South Africa. Abstract 56.
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32. Cosgrove Sweeney YT, Patton DL. Cellulose acetate phthalate (CAP): Vaginal safety evaluation in the macaque model. In: Microbicides 2006 Conference. 2006; Cape Town, South Africa. Abstract 57.
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model. In: Microbicides 2002 Conference. 2002; Antwerp, Belgium. Abstract A-184.
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