Women account for an increasing proportion of new HIV infections worldwide. In sub-Saharan Africa, for example, 61% of HIV-infected adults are female . Thus, there is an urgent need for new female-initiated methods of HIV prevention. Vaginal microbicides for the prevention of HIV transmission may be an important option for protecting women from infection.
Vaginal gels that can be self-administered are one such microbicide delivery approach. The results of clinical trials have shown that microbicide gels have acceptability rates of more than 90% [2,3], and most study participants (92–100%) have found them easy to use [4–7].
The nonnucleoside reverse transcriptase inhibitor (NNRTI) dapivirine (TMC120) is a promising microbicide candidate currently in development in vaginal gel and vaginal ring formulations [8–11]. Dapivirine exhibits potent antiviral activity against both wild-type virus and strains harboring various resistance-inducing mutations [12,13].
This article describes two independent phase I/II studies designed to evaluate local and systemic safety of dapivirine gel vs. placebo gel in healthy, HIV-negative women. The systemic absorption of dapivirine was also measured.
Clinical trial design
IPM003 and IPM005B were randomized, double-blind, placebo-controlled phase I/II studies in healthy, HIV-negative women 18–50 years of age. IPM003 was conducted at four research centers in Africa (Kigali, Rwanda; Johannesburg, South Africa; Moshi, Tanzania; and Bloemfontein, South Africa). IPM005B was conducted at a single research center in Antwerpen, Belgium. Study dates were the periods of October 2005–October 2006 for IPM003 and December 2005–March 2006 for IPM005B.
The primary objectives in both studies were to assess local and systemic safety and tolerability of dapivirine vaginal gel vs. placebo gel when used twice daily for 42 days. Matching placebo gel was used in IPM003, the first trial of dapivirine gel, to identify any drug-related safety observations. Universal placebo gel was used in IPM005B, the expanded safety study, to gain safety and acceptability data on the universal placebo gel vs. dapivirine gel prior to phase III trials with this placebo.
Secondary objectives in both studies were to assess the acceptability of the study gels and compliance with the dosing regimen. In addition, dapivirine concentrations were measured in plasma. The occurrence of bacterial vaginosis was evaluated in IPM003.
Study protocols were reviewed and approved by the responsible regulatory authorities and ethical review committees prior to study start. All participants signed an informed consent form (or provided a thumbprint if illiterate) during screening. Screening visits were conducted up to 21 days (IPM003) or 28 days (IPM005B) prior to study entry on day 0.
In addition to their usual contraceptive method [oral contraceptive, combined injectable, depot-medroxyprogesterone acetate, levonorgestrel implant, combined hormonal patch (intrauterine device) IUD, or surgical sterilization], women were advised to use condoms. Participants were required to have normal Pap smears within 90 days prior to randomization. Women enrolled in IPM003 agreed to be sexually abstinent for the first week of the study.
In IPM003, 112 women were randomized to receive one of three concentrations of dapivirine gel (0.001, 0.002, or 0.005%) or matching placebo gel in a 2: 2: 2: 1 ratio. In IPM005B, 36 women were randomized to receive dapivirine gel (0.02%) or hydroxyethyl cellulose (HEC)-based universal placebo gel (universal placebo gel) in a 2: 1 ratio. In both studies, the gel was used twice daily for 42 days (Table 1).
Safety and tolerability were assessed by clinical evaluations (pelvic/colposcopic examinations, physical examinations, and vital signs), clinical laboratory tests (hematology, liver, and renal function tests), genital infection laboratory tests, pregnancy tests, and monitoring of treatment-emergent adverse events (TEAEs). Dapivirine concentrations were measured in plasma in both studies on days 0, 7, 28, and 42 and on day 56 in IPM003 only.
Dapivirine gel, matching placebo gel (IPM003), and HEC-based universal placebo gel (IPM005B) were manufactured and packaged by the International Partnership for Microbicides (Bethlehem, Pennsylvania, USA) as individual doses of 2.65 g (2.5 ml) in polyethylene vaginal applicators (HTI Plastics, Lincoln, Nebraska, USA). Dapivirine gel contained (w/w) 35.0% polyethylene glycol, 2.0% HEC, 0.65% polycarbophil, 0.60% carbopol 1342, 0.20% methylparaben, 0.05% propylparaben, 0.09% sodium hydroxide, dapivirine (0.001, 0.002, 0.005, or 0.02%), and purified water (quantity sufficient to 100%). The matching placebo gel used in IPM003 contained the same vehicle without dapivirine. The universal placebo gel used in IPM005B consisted of (w/w) 96.3% water, 2.7% HEC, 0.85% sodium chloride, 0.1% sorbic acid, and 0–0.02% caramel color (as needed). The pH was adjusted to 4.4 with sodium hydroxide. The safety and acceptability of universal placebo gel has been previously described . Applicators containing dapivirine or placebo gel were labeled in an identical manner to maintain blinding. The dapivirine and placebo gels were translucent and similar in appearance.
Safety laboratories and adverse events
Blood samples for hematology were collected at screening and on days 0 (IPM003 only) and 42. Liver and renal function tests and urine pregnancy tests were performed at screening and on days 0, 7, 28, and 42. Pap smears and urine dipsticks were done at screening. Laboratory tests for genital infections were performed prior to the first application of study gel and on day 42. In IPM003, Gram stain Nugent scores were calculated on days 0 and 42. HIV testing was performed at screening and on days 0, 28, and 42. Specimen collection, handling, and processing were performed according to the standard operating procedures of the local or central laboratory.
Participants were monitored for TEAEs at each study visit. Adverse event data were coded using MedDRA (version 8.1) System Organ Classes and Preferred Terms. Severity of TEAEs and laboratory abnormalities were graded by the investigator according to the National Institutes of Health (NIH) Division of AIDS (DAIDS) Table for Grading the Severity of Adverse Experiences. Causality of TEAEs was assessed by the investigator as related to study gel (definitely related, probably related, possibly related) or not related to study gel (probably not related, not related).
Macroscopic and microscopic changes to vulva, vagina, and cervix
A primary endpoint of both studies was macroscopic or microscopic evidence of change (not due to pathogens or mechanical trauma) to the vulval or vaginal epithelium or both or to the cervical mucosa including ulceration and other lesions, abrasion, severe erythema, and/or severe edema. Macroscopic change was reported as an adverse event and was assessed by pelvic examination at screening, preentry or day 0, and on days 7, 28, and 42. Microscopic change was noted by colposcopy performed on days 0, 7, and 42 in IPM003 and at preentry and on day 42 in IPM005B.
Dapivirine concentrations in plasma
Blood samples were collected by venipuncture for determination of dapivirine concentration in plasma on days 0, 7, 28, and 42 in both studies and on day 56 in IPM003 only. Blood samples were centrifuged within 2 h of collection for 10–15 min to separate plasma, which was then stored frozen at or below −18°C until shipment to the bioanalytical laboratory. Plasma samples were shipped on dry ice to the biochemical laboratory where they were stored frozen until analysis. Dapivirine concentrations in plasma were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS) at FARMOVS-PAREXEL Bioanalytical Services Division of the Campus of the University of the Free State, Bloemfontein, South Africa for IPM003 and at Analytisch Biochemisch Laboratorium BV (ABL), Assen, The Netherlands for IPM005B. The lower limit of quantification of the assay for both studies was 5 pg/ml.
Statistical analyses for IPM003 and IPM005B were performed by Quintiles South Africa (Bloemfontein). The randomization code was generated by the statistician at Quintiles South Africa (IPM003) or Siris Pharmaceuticals (Bloomsbury, New Jersey, USA) (IPM005B) and was provided to the manufacturing facility for clinical labeling. At the research center, each new volunteer entering the study was assigned a number in sequence. Any person directly participating in the study was blinded to the randomization assignment.
All participants who received dapivirine gel and who had pharmacokinetic data available were included in the pharmacokinetic population. The safety population included all participants who received at least one application of study gel and who had postbaseline safety assessment data. Baseline was defined as the last measurement before the first application of the study gel. Missing data were not imputed. The endpoint observation was defined as the last observed postbaseline data (last observation carried forward).
A total of 148 healthy, HIV-negative female participants were enrolled in two phase I/II safety studies: 112 in IPM003 and 36 in IPM005B (Table 1). The mean age for participants in each study group in IPM003 and IPM005B was 30–31 years. In IPM003, most participants were black and married; in IPM005B, most participants were white and not married. In each study, approximately 60% of participants had had sexual intercourse within 1 week prior to baseline. In IPM003, most participants (61%) had not used a condom when they last had sexual intercourse. Baseline characteristics for participants were similar across study groups in each study.
Exposure and disposition
In IPM003, 90 out of 96 (94%) participants who used dapivirine gel and all 16 (100%) participants who used matching placebo gel completed the study according to protocol. Reasons for discontinuation were ‘withdrew consent’ (n = 4), ‘lost to follow-up’ (n = 1), and ‘stopped using her contraception’ (n = 1). One participant randomized to 0.005% dapivirine gel withdrew consent prior to the first administration of study gel. In IPM005B, one participant in the dapivirine group prematurely discontinued study gel because of a serious adverse event (SAE), abdominal abscess, which occurred on day 10 and was assessed by the investigator as probably not related to the study gel. All 12 participants who used universal placebo gel completed the study.
Adverse events and safety laboratories
A total of five SAEs occurred in the two studies, and none was considered to be related to study gel. SAEs in IPM003 were severe headache (0.002% gel group on days 5–7), gastritis (0.001% gel group on days 22–24), and thrombotic thrombocytopenic purpura (0.002% gel group at 6 months after the last application of study gel). Two SAEs were reported for a single participant in the dapivirine gel group in IPM005B, abdominal abscess on days 10–18, and uterine leiomyoma on days 14–44.
In both studies, similar percentages of placebo (25–31%) and dapivirine (17–28%) gel users had TEAEs that were considered by the investigator to be related to study gel (Table 2). Among dapivirine gel users, the percentages were independent of dose. Five dapivirine gel users in IPM003 (5%) had neutropenia considered to be related to the gel. Vaginal candidiasis, increased alanine aminotransferase (ALT), increased aspartate aminotransferase (AST), increased bilirubin, and uterine spasm considered to be study gel-related occurred only in dapivirine gel users in IPM003 (2–3%). Other gel-related events occurred in a similar percentage of dapivirine and placebo gel users or in a greater percentage of placebo gel users.
A total of six participants in IPM003 had grade 3 TEAEs during the study. In addition to the three participants with SAEs, three participants had grade 3 neutropenia: one in the 0.002% gel group on day 0 (not related), one in the 0.002% gel group on day 56 (possibly related), and one in the 0.005% gel group on day 42 (possibly related). One participant in the matching placebo group had grade 3 total bilirubin (high) on day 28 that was not reported as a TEAE. All TEAEs and clinical laboratory values in IPM005B were of grades 1 or 2.
In IPM003, at baseline, 12% of dapivirine gel users and 6% of matching placebo gel users were positive for bacterial vaginosis (Nugent score 7–10). On day 42, the incidence of bacterial vaginosis was 3% of dapivirine gel users and 0% of matching placebo gel users.
Macroscopic changes to vulval, vaginal, and cervical tissues
In IPM003, four participants (two in the 0.001% gel group, one in the 0.002% gel group, and one in the matching placebo group) had macroscopic changes that were detected during pelvic examination and reported as TEAEs. Vaginal discharge was reported in three participants: two out of 32 (6%) in the 0.001% gel group and one out of 16 (6%) in the matching placebo group. All other macroscopic changes were reported in two participants, one in the 0.001% gel group and one in the 0.002% gel group (cervix disorder, cervix hemorrhage uterine, erythema, petechiae, and edema). Only the cervix disorder (cervical ectopy), which occurred on day 42, was classified as grade 2. All other macroscopic changes were classified as grade 1. All macroscopic changes were considered by the investigator to be related to the study gel.
In IPM005B, cervix disorder (grade 1, possibly related) was reported as a TEAE on day 42 for three participants: 8% (1/12) in the universal placebo group and 8% (2/24) in the dapivirine group. The verbatims for the events were ‘aceto-white finding’ for the participant in the universal placebo group and ‘mosaicism’ for the two participants in the dapivirine group. Grade 1 vaginal hemorrhage assessed as probably not related to study gel occurred on days 2–8 in one participant (4%) in the dapivirine group.
Microscopic changes to vulval, vaginal, and cervical tissues
In each study, 13–17% of participants in each study group had microscopic changes to the vulva, vagina, or cervical tissues that either started after using the study gel or were present at baseline but worsened or recurred after using the study gel. Changes to the cervix were most common, occurring in 6–17% of participants in each study group. Petechiae occurred in 4% of dapivirine and 6% of matching placebo gel users in IPM003, ‘aceto-white finding’ occurred in 3% of dapivirine and 13% of universal placebo gel users in IPM005B, and erythema occurred in 4% of dapivirine gel users in IPM003. Other findings occurred in one or two participants.
Mean plasma concentrations of dapivirine were approximately dose proportional, and a plateau was reached on day 7 for all gel doses (Fig. 1). On day 56, 2 weeks after the final application of study gel in IPM003, mean plasma concentrations decreased to less than 5 pg/ml (Table 3). The range in values for participants within a study group was large.
Acceptability data were collected by questionnaire on days 7, 28, and 42. After use of the study gel for 42 days, the majority of questionnaire responders (88 out of 88 and 15 out of 23 dapivirine gel users and 15 out of 16 and 8 out of 12 placebo gel users in IPM003 and IPM005B, respectively) were ‘very willing’ or ‘willing’ to use the gel again in the future if it were proven to be effective for HIV prevention (Fig. 2). At the same time point, most responders (96% in IPM003 and 70% in IPM005B) reported that their male partners liked or had neutral feelings toward the study gel. Gel leakage during insertion was reported by 52% of questionnaire responders in IPM003; leakage after insertion was reported by all participants in IPM005B. Most responders (94% in IPM003 and 63% in IPM005B) felt that the leakage would not prevent future use of the gel. Furthermore, most reports of leakage (90% in IPM003 and 73% in IPM005B) were described as slight, that is, requiring the participant to wear a panty liner. The remaining reports described the leakage as moderate, that is, requiring the use of a regular menstrual pad.
Compliance was calculated by counting the number of gel applicators dispensed and the number of unused applicators returned at each study visit. Mean compliance with the gel administration regimen was over 90% in each study group in each clinical trial (92–97% in IPM003 and 96–99% in IPM005B).
Dapivirine vaginal gel at concentrations of 0.001–0.02% used twice daily for 42 days was found to be safe and well tolerated in healthy, HIV-negative women. None of the five SAEs was considered to be related to study gel. Safety data were similar for dapivirine gel and the placebo gels. Events that occurred in more than 5% of participants across both studies (headache, metrorrhagia, neutropenia, lower abdominal pain, influenza, and genital pruritus) occurred with similar frequencies in the dapivirine and placebo gel groups. Furthermore, similar percentages of placebo (25–31%) and dapivirine (17–28%) gel users had TEAEs that were considered by the investigator to be related to study gel.
In IPM003, neutropenia occurred in 14 participants (13% in each of the four study groups). As all but one case of neutropenia occurred at a single research center (Kigali, Rwanda), it is probably a laboratory artifact due to improper handling of laboratory specimens (testing was delayed for up to 5 days after specimen collection). Another possibility is that the hematological reference ranges used in this study did not adequately represent the Rwandan population . Lower and more relevant hematology ranges were recently established for reference populations in Rwanda, Uganda, Kenya, and Zambia . For 64% of participants with neutropenia, the event was recorded on day 0 prior to the first administration of study gel. Furthermore, for most participants with neutropenia (64%), the event was assessed by the investigator as ‘not related’ or ‘probably not related’ to study gel. All neutropenia events resolved before the end of the study. Neutropenia has not been observed in other studies of dapivirine topical microbicides, including IPM005B.
Microscopic changes to the vulva, vagina, and cervix were observed during colposcopy in 13–17% of participants in each study group, and the differences between the dapivirine and placebo groups were not statistically significant. Across both studies, changes to the cervix were most common, occurring in 7–13% of dapivirine gel users and 6–17% of placebo gel users. Erythema of the cervix, external genitalia, or vaginal wall was observed in two out of 16 (13%) of matching placebo gel users and five out of 95 (5%) of dapivirine gel users in IPM003. One case of erythema of the cervix was reported as a TEAE (0.002% dose group, grade 1, possibly related). Erythema was not reported in IPM005B. These findings support those reported previously in a 14-day safety and acceptability study of the universal placebo gel in which erythema was observed in one out of 14 (7%) of universal placebo gel users . Furthermore, cervical petechiae, reported here in a similar percentage of matching placebo (6%) and dapivirine (4%) gel users in IPM003, was previously reported in 7% of universal placebo gel users .
Systemic absorption of dapivirine was low and dose proportional. In IPM003, gel concentrations of 0.001–0.005% yielded mean values in the range of 17–84 pg/ml on days 7, 28, and 42. In IPM005B, 0.02% dapivirine gel yielded mean values in the range of 427–474 pg/ml during the same time frame. The maximum recorded value (964 pg/ml on day 28 in IPM005B) is over 2000-fold lower than the Cmax of 2.3 μg/ml observed at the maximum tolerated multiple dose with the oral formulation of dapivirine. The systemic effects of exposure to dapivirine gel beyond 42 days are not known. However, accumulation of drug in plasma was not observed in these studies. Two weeks after the final application of study gel, mean plasma concentrations were 5 pg/ml or less in each dose group. Furthermore, a pharmacokinetic study of dapivirine gel showed a plasma half-life for dapivirine of 65–88 h (unpublished data).
The risk for selection and transmission of HIV drug resistance through use of antiretroviral drug (ARV)-based microbicides is currently unknown. As drug resistance has important implications for the outcome of HIV treatment, the potential emergence of drug-resistant virus will be investigated in appropriately designed future clinical trials with ARV-based microbicides.
At the end of each study, most study participants who responded to an acceptability questionnaire found the dapivirine or placebo gel formulation to be acceptable and were willing to use it in the future (126 out of 139, 91%). This is supported by the finding that mean compliance with the gel administration regimen was over 90% in each study group in each clinical trial. The most common complaint was product leakage during/after gel application; however, the majority of responders reported that gel leakage was ‘slight’ and would not prevent them from using the product in the future. Leakage associated with application of vaginal microbicide gels is a potential issue that is being addressed in current clinical trials with newer formulations of dapivirine gel.
In conclusion, twice daily administration of dapivirine vaginal gel for 42 days was safe, well tolerated, and accepted in healthy, HIV-negative women with low systemic absorption. Dapivirine gel is currently being tested at a concentration of 0.05% in additional safety studies.
The two clinical trials in this article were funded by the International Partnership for Microbicides, Silver Spring, Maryland, USA. The study design was a collaborative effort among Z. Rosenberg (IPM), P. Coplan, and the principal investigators (S.H.K., H.V.R., J.V., and J.H.v.d.W.). Oversight of the study at the research centers was the responsibility of the principal investigators and subinvestigators (G.M., C.v.M., E.G., J.M., and I.K.). We acknowledge Dr Maria Ferreira at FARMOVS-PAREXEL, Campus of the University of the Free State, Bloemfontein (South Africa) for her contributions as Principal Investigator for IPM003 and Drs Eva Vets and Magali De Wulf for their contributions as Principal Investigator and Subinvestigator, respectively, at SGS Biopharma, Antwerpen (Belgium) for IPM005B. S.C.S. was responsible for study supervision, site coordination, and study monitoring. S.C.S., A.M.N., and W.H. (Quattro, San Francisco, California, USA) were responsible for the integrity of the data and for the accuracy of the data analyses. Data interpretation was supported by A.M.N. and J.R. All authors critically reviewed the manuscript for important intellectual content. We are grateful to the study teams in Rwanda, Tanzania, and South Africa and to Contract Laboratory Services and the CROs in South Africa (Triclinium and Quintiles). In addition, we wish to acknowledge P. Kaptur for preparation of the manuscript.
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