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Antimicrob Agents Chemother. 2006 May; 50(5): 1696–1700.
PMCID: PMC1472197

Preclinical Safety and Efficacy Assessments of Dendrimer-Based (SPL7013) Microbicide Gel Formulations in a Nonhuman Primate Model

Abstract

Three gel formulations (1%, 3%, and 5% [wt/wt]) of SPL7013, a dendrimer known to have antiviral (anti-human immunodeficiency virus and anti-herpes simplex virus) activities, completed a range of preclinical tests in the pigtailed macaque models for vaginally and rectally applied topical microbicide safety assessments. The vaginal safety profile of the 3% SPL7013 gel formulation was equal to that of the 1% formulation but was superior to that of the 5% formulation. The 3% SPL7013 gel was further evaluated for rectal safety and for antichlamydial efficacy with cervical challenge with Chlamydia trachomatis. This first-generation dendrimer-based product was shown to be safe to the vaginal and rectal microenvironments with repeated daily use. However, a single intravaginal application of the 3% (wt/wt) SPL7013 gel did not provide protection from the acquisition of cervical chlamydial infection.

The use of topical microbicides is an emerging strategy for the prevention of transmission of human immunodeficiency virus (HIV) and other sexually transmitted infections. Topical microbicide products should optimally be safe and effective for repeated vaginal and/or rectal use.

Dendrimers are a novel class of macromolecules characterized by highly branched three-dimensional structures (15). Dendrimers have good antiviral activities against both HIV and herpesvirus and physical properties appropriate for microbicide applications (2, 3, 5, 7-9, 17). SPL7013, a dendrimer-based microbicide developed by Starpharma Pty Ltd., Melbourne, Australia, has emerged as a candidate for such a product (10, 15).

In this study, three gel formulations (1%, 3%, and 5% [wt/wt]) of SPL7013 were assessed in a range of preclinical safety tests performed with the established pigtailed macaque models of repeated vaginal and rectal application (11-13). The vaginal safety profile of the 3% SPL7013 gel formulation was equal to that of the 1% formulation but was superior to that of the 5% formulation. Unformulated SPL7013 has been shown in separate mouse models to protect against genital herpes simplex virus type 2 infection and significantly reduce the incidence of ascending genital tract infection caused by Chlamydia trachomatis (2, 3). Thus, the 3% SPL7013 gel formulation was further evaluated for rectal safety and antichlamydial efficacy with a cervical challenge of C. trachomatis.

MATERIALS AND METHODS

Test products.

Dendrimer gel formulations containing 1%, 3%, and 5% (wt/wt) SPL7013 and a placebo gel were provided by Starpharma Pty Ltd.

Animals.

Sexually mature, female Macaca nemestrina macaques were obtained from a colony of animals at the Washington National Primate Research Center. Prior approval for use of the monkeys in this protocol was obtained from the Institutional Animal Care and Use Committee at the University of Washington. The animals were handled humanely, and experiments were performed within the National Institutes of Health's animal use guidelines.

Experimental safety protocols. (i) Vaginal safety.

Three concentrations of SPL7013 in a water-based gel formulation were assessed for safety with vaginal application, and their safety was compared to that of the placebo (i.e., base) formulation, which contained no SPL7013. Thirty animals were prescreened by reviewing their medical and research histories and were monitored for 1 month for general health and to document normal menstrual cycles. One baseline vaginal biopsy specimen and one cervical biopsy specimen were obtained during this month. Six animals were randomly assigned to each of three test arms. A total of 12 animals completed the placebo arm of the study, which was run in two separate phases. Initially, the 1% and 5% dendrimer formulations, controlled by a placebo arm, were assessed for safety. A second series of experiments assessed the 3% dendrimer formulation controlled by a placebo arm. The same protocol was followed for each experiment. On study days 1 to 4, baseline colposcopy assessments were performed and swab specimen collections for vaginal pH and microflora determinations were obtained. Immediately following specimen collection, 1.5 ml of test or placebo gel was administered to each animal intravaginally. Thirty minutes after gel application, 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 determinations, and one cervical biopsy specimen and one vaginal biopsy specimen were obtained. No gel was applied. On day 8, colposcopy and vaginal swabs for pH and microflora determination were obtained to assess recovery.

Standardized colposcopic assessments were conducted by a team of three cross-trained individuals. Colposcopy took place immediately after speculum placement, before any swab collections. Vaginal pH was determined by rolling a swab onto a pH indicator strip with a resolution of 0.5 pH unit. A second swab specimen was collected and immersed in a transport tube (Port-a-Cul; Becton Dickinson Microbiology Systems, Cockeysville, MD) for quantitative microbiologic characterization. Histologic assessment of hematoxylin-eosin-stained biopsy samples was conducted by quantification of polymorphonuclear cells, lymphocytes, and plasma cells in five nonadjacent high-power fields.

(ii) Rectal safety.

Based on the results from the vaginal safety study, only the 3% SPL7013 formulation was assessed in the rectal application model. Eight animals were randomly selected for inclusion in the rectal safety protocol to evaluate the 3% SPL7013 gel formulation, placebo gel, and no product. Each animal underwent the experimental protocol three times, crossing test arms for each repetition. Each three-arm experiment lasted 4 days. One arm tested three daily intrarectal applications of 2.5 ml 3% SPL7013 formulation, one tested three daily applications of 2.5 ml placebo gel, and another tested no product application.

Prior to each gel application (days 1 to 3), rectal swab specimens were obtained for evaluations of rectal pH and microflora, followed by collection of a rectal lavage specimen. These sample collections were followed by an application of 2.5 ml gel product past the anal sphincter into the rectum. Fifteen minutes later, repeat samples were collected. Animals assigned to the no-product group underwent sample collection 15 min after completion of the “preapplication” sample collections. On the fourth day, a final rectal pH swab, microbiology swab, and lavage specimen were collected from all animals.

Rectal pH and microbiology procedures were performed as described above for the vaginal safety studies. However, rectal microbiology was characterized by semiquantitative measures. The rectal lavage was performed with a 10-ml syringe attached to an infant feeding tube. The tube was carefully inserted 3 to 5 cm beyond the anal sphincter (into the rectum), and 8 ml of saline wash was expelled. Twenty seconds later, the saline wash was recovered and deposited into a vial for characterization.

Safety measures. (i) Vaginal and rectal microbiologic characterization.

Each swab collected for microbiologic assessment was individually placed in Port-A-Cul tube, designed for transport of anaerobic, facultative anaerobic, and aerobic specimens on swabs. This transport device has been shown to preserve viability of aerobic and anaerobic bacteria in clinical specimens (1). Within 24 h of collection, samples were delivered to the Reproductive Infectious Disease Laboratory at Magee-Womens Research Institute (Pittsburgh, PA).

Swab specimens from all animals were evaluated for aerobic and anaerobic microorganisms by microbiologic assays described previously (11, 13). Quantitative analyses were performed with all vaginal specimens. Semiquantitative analyses were performed for each organism detected in the rectal swab specimens, and the findings were graded on a scale of 0 to 4 (where 0 is not detected and 4 is growth on all quadrants of a plate). For ease of presentation, species belonging to the genera Bacteroides, Porphyromonas, and Prevotella were grouped together as either anaerobic gram-negative rods (nonpigmented) or black gram-negative rods.

(ii) Colposcopic assessment.

Colposcopy was conducted following the CONRAD, IWGM, UNAIDS Manual for the Standardization of Colposcopy for the Evaluation of Vaginal Products: Update 2000 (9a). The mucosal tissues of the cervix and vagina were assessed by low-power magnification under white light and a green filter to accentuate the vasculature. Vaginal and ectocervical tissues were evaluated for erythema, vasculature pattern, epithelial integrity, and any exceptional findings. Tissue erythema was described as general erythema (reddening) or was further detailed by the vasculature noted. The term “intact vasculature” refers to a clearly demarcated vessel pattern noted below the epithelial surface. “Disrupted vasculature” refers to findings of diffuse vessels, petechiae, and/or petechial hemorrhage noted below the epithelial surface. Findings of intact and disrupted vasculature are commonly noted in baseline, healthy tissues. Indicators of tissue injury included areas of friability, abrasion, or ulceration, which are noted as “disrupted epithelium.” Colposcopic observations were noted on the daily examination records and were documented by digital photography.

(iii) Rectal lavage.

Two to four hours after sample collections, the rectal washes were individually examined under a dissecting microscope at ×7 magnification. Under low-power examination, each rectal lavage was evaluated for evidence of fecal matter, cellular debris, epithelial desquamation (epithelial sheets are defined as measuring >3 mm in at least one dimension), and stroma and/or blood associated with the epithelial sheets. After gross examination, the epithelial sheets were measured and lavage samples were photographed for documentation.

Statistical analyses.

Microflora were analyzed for differences across treatment arms (the Mann-Whitney test was applied to vaginal data; Fisher's exact test was applied to rectal data), for acute fluctuations in microorganism prevalence after product application (Wilcoxon signed-rank and McNemar's tests), and for sustained alterations to microorganism populations (Friedman's and Wilcoxon signed-rank tests). Vaginal and rectal pH measures from the test groups were compared at each time point to those of the placebo and the no-product groups (Mann-Whitney test). The effects of product application on vaginal or rectal pH were compared within each treatment group by using the Wilcoxon signed-rank test. Finally, McNemar's test was used to examine changes in the prevalence of epithelial sheets in rectal lavages pre- and postapplication of the product, and pairwise comparisons of 3% SPL7013 gel and placebo gel samples were conducted.

Experimental efficacy protocol: cervical challenge.

Twelve animals were randomly assigned to the cervical efficacy study to evaluate protection from chlamydial infection. Six animals served as test animals and received the 3% SPL7013 gel product. Six animals received no gel prior to challenge (chlamydia control group). Baseline samples, including cervical swabs for chlamydia detection by culture and a nucleic acid amplification test (NAAT; Aptima Combo 2) and 5 ml blood for serum antibody detection, were collected from each animal. A single intravaginal application of 3% SPL7013 gel (1.5 ml) was then administered to each test animal. Thirty minutes later, all 12 animals were inoculated with chlamydia by expelling 1 ml C. trachomatis E to the vaginal fornix, thereby exposing the cervix to the organism.

Follow-up sample collections for documentation of chlamydial infection by culture, NAAT, and serology were conducted on days 2 and 7 and then weekly thereafter for 5 weeks. All samples for chlamydia culture and NAAT were collected with Dacron-tipped swabs by making direct contact with the cervical os.

Chlamydia trachomatis.

The University of Washington Chlamydia Reference Laboratory provided the clinical cervical isolate of C. trachomatis strain E (UW/11027) for these studies. The chlamydial isolate was prepared in McCoy cell culture and purified on a Renografin methylglucamine diatrizonate linear gradient column. The titers of the stock inocula were determined (1.0 × 107 to 1.3 × 107 inclusion-forming units/ml) in sucrose-phosphate-glutamate buffer and were frozen (−70°C) until dilutions were prepared for experimental inoculations. For inoculation, a frozen aliquot of chlamydia was diluted to 5 × 105 inclusion-forming units per milliliter. A disposable 1-ml tuberculin syringe was used to draw 1 ml of inoculum, which was then inserted into the fornix of the vagina and expelled toward the cervix.

Chlamydia trachomatis detection assays. (i) Cell culture.

Cervical swab specimens were cultured on cycloheximide-treated McCoy cells in 96-well microtiter plates (14) and stained with a monoclonal antibody specific for C. trachomatis detection.

(ii) Nucleic acid amplification test.

For the NAAT (Gen Probe APTIMA Combo 2), cervical swab specimens were collected, stored, and transported to the laboratory according to the manufacturer's instructions (Gen-Probe, Inc., San Diego, CA). The APTIMA Combo 2 assay (6) employs the amplification technology of transcription-mediated amplification, in which the RNA target molecule from C. trachomatis is isolated and specific regions are amplified by using a separate capture oligomer and a unique set of primers for the target. This test uses transcription-mediated amplification to detect a specific C. trachomatis 23S rRNA target.

(iii) Serology.

Serum immunoglobulin G (IgG) and IgM chlamydial antibody titers were measured by the microimmunofluorescence technique (4, 16).

RESULTS

Vaginal safety of 1%, 3%, and 5% SPL7013 formulations. (i) Colposcopy.

Four consecutive daily applications of the test product containing 5% SPL7013 in a 1.5-ml gel volume led to cervicovaginal tissue disruption and/or friability in four of six animals assessed in this study (Table (Table1).1). Adverse effects (friability and epithelial disruption) were noted by colposcopy in three animals 24 h after the first product application. These findings remained through day 5 and resolved by day 8 (after 3 days of no product use). Friable tissues were noted in the fourth animal on days 4 and 5 of the experiment. None of the animals treated with the 1% SPL7013 or the 3% SPL7013 formulation demonstrated cervicovaginal irritation. Observations of subepithelial vasculature were noted in the majority of animals from each arm of the study. No significance is drawn from these observations. Mild erythema was noted in 2 of 12 placebo-treated animals.

TABLE 1.
Colposcopic findings after vaginal applications of SPL7013 (1%, 3%, 5%) and placeboa

(ii) Vaginal pH.

When pH measures were assessed within treatment groups, statistically significant decreases in vaginal pH were noted 30 min after the application of each SPL7013 gel formulation. The vaginal pH measurements remained lower than those at the baseline at 24 h after application but recovered to baseline levels at day 8. A less pronounced decrease in vaginal pH was noted after placebo gel application, although there were no statistically significant differences in pre- and postapplication pHs with the placebo gel. As expected, there were no statistically significant differences in pH values after application of the 1%, 3%, and 5% SPL7013 gels compared to that after application of the placebo gel at each time point (Table (Table22).

TABLE 2.
Vaginal pH pre- and postapplication of 1%, 3%, and 5% SPL7013 gels and placebo gel

(iii) Vaginal microbiology.

The microorganisms for which each vaginal swab specimen were evaluated included H2O2-producing lactobacilli and viridans group streptococci, as well as non-H2O2-producing species, Gardnerella vaginalis, Staphylococcus aureus, Enterococcus, Escherichia coli, and anaerobic gram-negative rods (GNRs; black and nonpigmented species). When the quantities of vaginal microflora between the placebo group and treatment groups were compared at each respective time point, there were few significant differences between the placebo group and the treatment arms in the quantity of vaginal microflora. Significant differences were found between the placebo-treated group and the 3% SPL7013-treated group concerning H2O2-producing lactobacillus on day 4 (for day 4 immediately before product application, P = 0.041; for day 4 30 min after product application, P = 0.041) and between the placebo-treated group and the 5% SPL7013-treated group concerning H2O2-producing lactobacilli on day 5 (for day 5 immediately before product application, P = 0.021). Due to the small sample size, these may not be true differences, and no pattern of product-induced suppression of these organisms emerged.

Product-induced shifts in microflora were detected by the detection of decreases in the organism population 30 min after product application compared to the size of its population immediately prior to product application. Transient shifts in anaerobic gram-negative rods in the groups treated with placebo and 1% and 5% dendrimer were noted. Additionally, a transient decrease in H2O2-producing viridans group streptococci was noted on days 3 and 4 with 5% SPL7013 use.

To look for sustained effects to the vaginal flora, we compared the quantity of microflora immediately before product application on each day for the individual treatment arms. There were no significant differences in the microflora over time for all vaginal microflora except anaerobic black GNRs and nonpigmented anaerobic GNRs. Both anaerobes decreased in quantity over time in all treatment arms, including the placebo group; however, they did rebound by day 8.

(iv) Biopsy specimens.

The profiles of the vaginal and cervical biopsy specimens collected 24 h after the fourth application of 3% SPL7013 were mostly similar to the baseline profiles assessed in these studies (layers of epithelial cells and the presence of polymorphonuclear cells, plasma cells, and lymphocytes). Biopsy specimens from animals that received the test gel had histologic profiles similar to those from animals that received placebo gel.

Rectal safety of 3% SPL7013. (i) Rectal pH.

The rectal pH in the animals in the 3% SPL7013 gel arm was compared to that in animals in the placebo gel arm and the no-treatment arm at each time point. The rectal pH measures in the 3% SPL7013 test group did not differ significantly from the pH values in the placebo gel group at each time point (Table (Table3).3). The rectal pH decreased significantly after application of both 3% SPL7013 and placebo gel on days 1, 2, and 3. For the group with no treatment, significant increases in rectal pH were observed on days 1 and 3. The rectal pH was shown to recover (day 1 compared to day 4) in each treatment group.

TABLE 3.
Rectal pH pre- and postapplication of 3% SPL7013 gel, placebo gel, and no product

(ii) Rectal microbiology.

The microorganisms for which the rectal swab specimens were evaluated included lactobacilli, viridans group streptococci, Enterococcus, E. coli, and anaerobic GNRs (black and nonpigmented). Semiquantitative assessment of the rectal microbiology revealed no significant shifts in the normal flora populations with 3% SPL7013 use. There were no significant differences in the prevalence of microflora before and after rectal application of 3% SPL7013 gel, placebo gel, and no product. In addition, there were no significant differences between the prevalence of rectal microflora on day 1 compared to that on day 4.

(iii) Rectal lavage specimen examinations.

There were no significant differences in the prevalence of epithelial sheets, blood, or stroma before and after application of 3% SPL7013 and the placebo gel product, nor was there a difference in the group that did not receive any product.

There were no significant differences in the number of epithelial sheets at each time point among the animals in each product group. When pairwise comparisons were conducted between 3% SPL7013 gel and no product, no associations were observed. When pairwise comparisons were conducted between 3% SPL7013 gel and placebo gel, there was an association between placebo gel use and the presence of epithelial sheets on day 1 after product application (for day 1 15 min after product application, P = 0.041). On the first day there were no significant differences between study arms before product application, yet seven of eight macaques had epithelial sheets after exposure to placebo gel, whereas only two of eight macaques had epithelial sheets after exposure to the 3% SPL7013 gel.

The 3% SPL7013 gel and cervical Chlamydia trachomatis infection.

Four of six animals that received the 3% SPL7013 gel intravaginally 30 min prior to cervical chlamydial inoculation tested positive for cervical chlamydial infection by culture and NAAT during the 5 weeks of follow-up (Table (Table4).4). Three of these animals also developed serologic IgG titers >1:8 during follow-up. Five of the six animals that received no intravaginal product before chlamydial inoculation developed cervical infection, as documented by the same assays. In the positive control arm of the study, a single animal tested positive by NAAT only on days 2 and 7 postinoculation. Similarly, one animal in the test arm of the study tested positive on days 2 and 7 postinoculation by NAAT only. Another tested positive by NAAT only on days 2 through 21. Because the results of NAAT were not confirmed by any other test in these instances, it is difficult to say that these animals were successfully infected with C. trachomatis.

TABLE 4.
Chlamydia trachomatis detection in pigtailed macaques treated with 3% (wt/wt) SPL7013 gel or no product

DISCUSSION

It is desirable that a topical microbicide product be effective at preventing HIV infections and sexually transmitted diseases while not irritating the mucosal surface or adversely affecting the normal flora of the vagina or rectum after repeated use. In order to protect oneself during each sexual encounter, men and women will require repeat applications of the microbicidal formulation. In this preclinical study, the nonhuman primate model was used to investigate the safety of SPL7013 (1%, 3%, and 5% [wt/wt]) dendrimer formulations after repeated vaginal applications. The safety profiles of the 1 and 3% formulations were excellent. After repeated applications of the 5% (wt/wt) SPL7013 gel, abnormal colposcopic findings were observed in four of six animals. Thus, studies with the 5% (wt/wt) SPL7013 gel formulation were not extended. However, the 3% (wt/wt) SPL7013 gel was further evaluated for rectal safety and efficacy against C. trachomatis.

After four daily applications of the SPL7013 gel formulations in the macaque, neither vaginal nor rectal populations of H2O2-producing lactobacilli were adversely affected. In a recently completed phase I clinical trial, the safety profile of VivaGel, which contains 0.5 to 3% (wt/wt) SPL7013, was shown to be comparable to that of the placebo gel following once-daily intravaginal dosing for 7 consecutive days (Starpharma, unpublished results). Lower concentrations of normal lactobacillary flora commonly occurred during gel use in both the placebo and the active gel test groups, and there was no statistically significant difference in the reduction between the placebo and the active gel test groups. In the subjects receiving active gels, this decrease in the concentration of beneficial lactobacilli was accompanied by a proportional decrease in potentially harmful anaerobic bacteria. The vaginal flora had generally returned to pre-gel treatment levels when the levels were assessed again 7 days after the end of dosing.

Antichlamydial activity was evaluated after a single application of 3% SPL7013 gel. Two of the test animals tested positive by NAAT only. One control animal was also positive by NAAT only. Because these results were not confirmed by any other test, it is difficult to be certain that the animals were infected, as opposed to shedding nonreplicating inoculum. These test animals may have been partially protected from C. trachomatis infection. However, the remaining four test animals were not protected from chlamydial infection after pretreatment with 3% SPL7013 gel.

Repeated daily vaginal use of 1% and 3% SPL7013 gels resulted in an acceptable safety profile, as evaluated by colposcopy, pH determination, microflora evaluation, and histology, compared to the profiles achieved with the placebo gel. The safety profile following the use of the 5% SPL7013 formulation did indicate deleterious effects on the cervicovaginal environment, as assessed by colposcopy, in this model, although the microflora and histology samples indicated no change compared to the profiles obtained with the placebo gel. The 3% SPL7013 gel was well tolerated by rectal tissues and microflora compared to the tolerance of the placebo gel, with which the level of epithelial desquamation was slightly increased.

From this study with macaques, we documented that 3% SPL 7013 is nonirritating after repeated vaginal and rectal applications and does not adversely affect the microbiology of either ecosystem. While this study showed that a single application of SPL7013 did not provide protection against C. trachomatis in the pigtailed macaque, efficacy against simian immunodeficiency virus has been reported in a separate study with this animal model (9). Continued development of a combination microbicide will expand efficacy against multiple sexually transmitted infections.

Acknowledgments

This work was supported by Public Health Service grants NO1-AI-95388 and PO1 AI39061 and WaNPRC RR00166.

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