Examining the Safety, Pharmacokinetics, and Pharmacodynamics of a Rectally Administered IQP-0528 Gel for HIV Pre-Exposure Prophylaxis: A First-In-Human Study

Abstract

A lubricating microbicide gel designed for rectal and vaginal use would provide a behaviorally congruent strategy to enhance pre-exposure prophylaxis adherence and reduce HIV infection risk. In this study, we report the first-in-human evaluation of such a gel containing 1% IQP-0528, an investigational antiretroviral. Seven HIV-1-negative participants received one 10 mL rectal dose of radiolabeled 1% IQP-0528 gel. We assessed safety; IQP-0528 pharmacokinetics in plasma, and rectal and vaginal tissue; ex vivo local pharmacodynamics (PD); and colorectal distribution. The 1% gel was determined to be safe with one mild event attributed to study product and no effects on rectal tissue histology. All concentrations measured in plasma and vaginal tissue were below the limit of quantitation. Median IQP-0528 concentrations in rectal tissue exceeded the in vitro EC₉₅ against HIV-1 (0.07 ng/mg) by 3–5 h of dosing and remained above this concentration for at least 24 h, despite a 3-log reduction in concentration over this duration of time. Rectal tissue PD—assessed by ex vivo HIV challenge—demonstrated significant p24 antigen reduction 3–5 h postdose compared with baseline (p = .05), but not 24–26 h postdose (p = .75). Single-photon emission computed tomography/computed tomography imaging revealed that product distribution was localized to the rectosigmoid. The IQP-0528 gel possesses desirable features for a topical microbicide including: local safety with no systemic absorption, delivery of locally high IQP-0528 concentrations, and significant reductions in ex vivo HIV infectivity. However, the gel is limited by its rapid clearance and inability to penetrate vaginal tissues following rectal dosing.

Clinical Trial Registration number: NCT03082690.

Introduction

The World Health Organization (WHO) estimated that 38 million people globally were living with HIV at the end of 2018.¹ The advent of antiretroviral (ARV) therapy has transformed HIV into a manageable chronic disease. In addition, tenofovir/emtricitabine as daily oral pre-exposure prophylaxis (PrEP) proved effective in 2012 in reducing HIV transmission. Despite this progress, HIV remains a threat to public health. In 2018, there were ∼1.7 million new infections, with 770,000 HIV-related deaths.¹ Thus, there is a gap in HIV prevention and treatment strategies that must urgently be addressed.

A primary contributor to this gap is low adherence to oral PrEP, which directly correlates with low efficacy.^2,3 Adherence is critical to achieve and maintain suppression of viral load, prevent emergence of drug-resistant virus, and curb new infections.^4,5 However, adherence is negatively impacted by a myriad of behavioral, psychological, and social factors.⁶ Two of the most common barriers to drug uptake and persistence for PrEP are the potential for short-term side effects and/or long-term toxicity,^6,7 and the burden of discrimination and stigma.⁶

One strategy to address these issues is topical microbicide PrEP. Topical microbicides encompass gels, films, and intravaginal rings that deliver high local concentrations of ARVs to rectal and/or vaginal mucosal tissues, the primary sites of viral exposure.⁸ Such microbicides have two key advantages: (1) they can be used discreetly by at-risk individuals for on-demand protection and (2) systemic exposure is decreased relative to traditional oral PrEP (because the microbicides are administered topically), thus reducing both the perceived stigma and the risk of systemic toxicity associated with traditional daily oral PrEP.⁸

Various topical microbicides have been explored clinically. A vaginal tenofovir gel^2,9,10 and a dapivirine vaginal ring^11,12 have proven effective in randomized clinical trials. In addition, a rectal tenofovir gel has been tested in early phase studies.^13–15 However, to date, no single product is suitable for use in both compartments. This limitation is a concern for at-risk women who engage in both vaginal and anal intercourse. Furthermore, inappropriate application of a hyperosmolar vaginal gel (e.g., tenofovir vaginal gel) in the rectum may increase the risk of HIV infection from anal intercourse because of local cytotoxicity and epithelial sloughing.¹⁶

A dual-use product formulated for vaginal and rectal protection would be a convenient alternative to two application-site-specific microbicides. Furthermore, if this product possessed lubricant-like properties, its use would be behaviorally congruent among populations, such as men who have sex with men, who use lubricants during sexual activity, thereby potentially increasing adherence.¹⁷

We have previously described the development of DuoGel™, an isotonic product formulated for vaginal and rectal use containing 1% (wt/wt) IQP-0528, a pyrimidinedione non-nucleoside reverse transcriptase inhibitor (NNRTI),^18,19 with a second inhibitory mechanism of preventing viral entry into target cells.²⁰ IQP-0528 inhibited HIV-1_IIIB with subnanomolar potency (0.2 nM) in vitro and a wide therapeutic index (>2,500).²⁰ DuoGel yielded a 2-log reduction in HIV-1 p24 antigen following ex vivo application to both cervicovaginal and colonic tissue explants collected from premenopausal women with no associated reduction in tissue viability.¹⁹

Recently, DuoGel has been evaluated in vivo in simian–human immunodeficiency virus-positive Rhesus macaques, which resulted in delivery of IQP-0528 concentrations to the vagina several logs greater than the in vitro 50% effective concentration (EC₅₀) against clinical HIV-1 (0.93 ng/mL), 4 h after dosing.²¹ Unexpectedly, vaginal dosing also yielded concentrations in rectal tissue 4 h postdosing that exceeded the in vitro EC₅₀.²¹ This latter observation implies the possibility of obtaining dual compartment protection following single-compartment dosing. Collectively, these data suggest that DuoGel warrants further examination as a PrEP strategy.

Given these highly promising preclinical results, we evaluated DuoGel clinically via rectal administration in HIV-negative adults. The objectives of this first-in-human study were to assess local and systemic safety, colonic distribution, and single-dose pharmacokinetics (PK) and pharmacodynamics (PD) in plasma, and rectal and vaginal tissue.

Materials and Methods

Design, setting, and participants

Our phase 1 open-label study was conducted in an outpatient research clinic within the Johns Hopkins Hospital in Baltimore, MD. Eight healthy, HIV-1-negative adults free of any anorectal and/or vaginal abnormalities were recruited between October 2017 and May 2019. Full inclusion and exclusion criteria can be found at ClinicalTrials.gov. Informed consent was obtained from all participants before eligibility screening that involved a physical examination, medical history, and laboratory measurements. The protocol and informed consent document were approved by the Johns Hopkins Medicine Institutional Review Board.

Overall schema and study endpoints

At least 1 week before receiving study product, all participants underwent flexible sigmoidoscopy for baseline rectal biopsy collection, and women underwent vaginal biopsy collection.

Participants were given a single 10 mL rectal dose of radiolabeled 1% IQP-0528. Plasma samples, single-photon emission computed tomography/computed tomography (SPECT/CT) images, and rectal and vaginal biopsies were collected for histological, PK, and PD assessments.

The two safety endpoints were: (1) clinical or laboratory adverse events (AEs) Grade 2 or higher, based on clinician judgment at the time of any clinical events, as defined by the National Institute of Health Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, Version 2.0 (November 2014), and Addendum 3 (Rectal Grading Tables for Use in Microbicide Studies)^22,23; and (2) extent of tissue damage, as determined by quantitative scoring of rectal biopsy histology. The three PK endpoints were: (1) IQP-0528 PK in plasma, (2) drug concentrations in biopsy homogenates (rectum and vagina), and (3) luminal distribution of radiolabel in the rectum and vagina. The PD endpoint was an ex vivo HIV challenge of biopsy explants (rectum and vagina).

Dose preparation and administration

DuoGel containing 1% (wt/wt) IQP-0528 (ImQuest BioSciences, Frederick, MD) was stored at ambient temperature (20–25°C) and atmospheric humidity. Doses were radiolabeled by mixing 10 mL DuoGel with 1 mCi ^99mTc-diethylenetriaminepentaacetic acid (DTPA) (Cardinal Health, Beltsville, MD). The study clinician administered the 10 mL dose to participants in the anorectum via syringe attached through Luer lock to a rectal applicator (no. 001-921-6670; Northern Pharmacy and Medical Equipment). All dosing materials, including the emptied syringe, were assessed for residual radiolabel, decay corrected, and used to assess percent retention of the radiolabeled gel dose.

Collection of plasma samples

Blood (4 mL) was collected in K₂EDTA vacutainer tubes (BD, Franklin Lakes, NJ). For the first 4 participants, 12 blood samples were collected: predose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 24, and 72 h postdose. Based on preliminary plasma PK results from the first four participants, the sampling scheme was simplified for the remaining participants to seven blood samples collected predose and 1, 2, 4, 8, 24, and 48 h postdose.

Collection and distribution of rectal and vaginal tissue biopsies

A gastroenterologist collected rectal tissue biopsies via flexible sigmoidoscopy. Five rectal tissue biopsies were collected during the baseline visit. Fourteen rectal biopsies were collected each at 3–5 h and 24–26 h after dosing. Rectal biopsies were obtained 10–20 cm from the anal verge, using Radial Jaw 4 Jumbo forceps (2.8 mm jaw opening distance; Boston Scientific, Marlborough, MA). Four and one of the baseline rectal biopsies were allocated for ex vivo HIV challenge assays and histology, respectively. For each postdosing collection of 14 rectal biopsies, the allocations were: ex vivo challenge assays (6), measurements of IQP-0528 tissue concentrations (6), and histology (2).

Vaginal tissue biopsies were obtained by a gynecologist. Two vaginal biopsies were collected during the baseline visit, and four were collected 24 h postdosing. All biopsies were collected from the posterior wall of the vagina, using Tischler-Morgan biopsy punch forceps (7 × 3 mm bite). Both vaginal biopsies collected at baseline, and two collected 24 h postdosing were allocated for ex vivo challenge assays. The remaining two biopsies collected 24 h postdosing were assessed for IQP-0528 concentrations.

Ex vivo histology of rectal biopsies

Rectal biopsies were individually fixed with 1 mL of 10% neutral buffered formalin, stained with hematoxylin–eosin, and evaluated by a pathologist. Epithelial surface denudation and lamina propria hemorrhage were assessed as previously described¹⁶ using a categorical scale from 0 to 3 representing the amount of denudation or hemorrhage: 0, none; 1, less than one-third of the epithelial surface; 2, one-third to two-thirds; and 3, more than two-thirds. The apoptotic body average was assessed as the decimal equivalent of the number of bodies per × 20 field.

Analysis of IQP-0528 concentrations

IQP-0528 measurements in plasma and tissue biopsies were performed via liquid chromatographic-tandem mass spectrometric analysis by the Clinical Pharmacology Analytical Laboratory at the Johns Hopkins University School of Medicine. Plasma and tissue assays were validated in accordance with the Food and Drug Administration Bioanalytical Method Validation, Guidance for Industry, recommendations, and assay validation reports were externally reviewed by the Division of AIDS (DAIDS)-sponsored Clinical Pharmacology Quality Assurance program.^24,25

For IQP-0528 quantitation, the structural analog IQP-0410 was used as an internal standard. For isolation of IQP-0528 from plasma, 0.1 mL plasma was combined with internal standard and subjected to solid-phase extraction via an Oasis MCX, 30 μm solid-phase extraction plate (Waters Corporation, Milford, MA). For drug quantitation in tissue, biopsies were homogenized in 0.5 mL 70% cold methanol; 0.05 mL was then combined with internal standard and subjected to protein precipitation using a Captiva 0.45 μm protein precipitation plate (Agilent Technologies, Wilmington, DE).

For both specimen sources, reconstituted samples were analyzed using an API 5500 mass analyzer (SCIEX, Redwood City, CA) interfaced with a Waters Acquity UPLC System. The mass spectrometer was operated in positive ionization and selected reaction monitoring modes. Chromatographic separation was achieved using a Waters Acquity BEH C8, 1.7 μm (2.1 × 50 mm) column; IQP-0528 and IQP-0410 were eluted at 0.61 min. The analytical run time was 4.25 min. Ion transitions for IQP-0528 and IQP-0410 were monitored at m/z 341.2→287.0 and m/z 353.3→273.2, respectively.

Assay lower limits of quantitation (LLOQ) for plasma and tissue homogenates were 3 ng/mL and 0.25 ng/sample, respectively. For tissue homogenates, results were normalized to the weight of tissue homogenate tested and are reported as ng/mg. When normalized to biopsy weights, the median LLOQs for rectal and vaginal homogenates were 0.021 and 0.014 ng/mg, respectively.

SPECT/CT imaging to determine IQP-0528 distribution in the colorectum

The luminal distribution of ^99mTc-DTPA was determined via SPECT/CT 1–2 h after dosing. Participants were imaged using a Discovery NM/CT 670 SPECT/CT (GE Healthcare, Waukesha, WI). The CT images provided an anatomical reference and were used for attenuation correction of SPECT images. SPECT images were reconstructed using an ordered subset expectation maximization algorithm and subsequently fused using the Xeleris Functional Imaging Workstation, software version 3.1 (GE Healthcare). Images were fused into a 256 × 256 × 256 matrix with 1.724 mm³ voxels.

Curve fitting and concentration-by-distance calculations were performed using principal curves with a length penalty.^26–28 Centerlines were translated into concentration–distance curves. Quantitative concentration–distance parameters were estimated using noncompartmental analysis in Phoenix WinNonlin, 8.2 (Certara USA, Princeton, NJ) by replacing time with distance as follows: (1) D_min, the minimum distance where radiolabel was detected (the centerline origin), corrected for distance from the anorectal junction; (2) D_max, the distance to the most proximal radiolabel signal; (3) DC_max, the distance to maximum radiolabel concentration; and (4) D_ave, the mean residence distance.

Because the flexible sigmoidoscope distances are relative to the external opening of the anus and the SPECT/CT scans are relative to the anal verge at the internal opening into the rectum, there is roughly a 4 cm difference in these assessments.

Ex vivo HIV challenge of rectal and vaginal tissue biopsy explants

Rectal and vaginal biopsies allocated for ex vivo challenge assays were collected in a solution of 0.5 mg/mL Zosyn^® in cRPMI (20 mL). Ex vivo challenge of rectal and vaginal biopsy explants were performed using a common stock of HIV-1_BaL [3.43 × 10⁶ tissue culture infectious dose (TCID₅₀)/mL], as previously described.^29–31 The final virus solution was either 1 × 10⁵ (rectal) or 5 × 10⁴ (vaginal) TCID₅₀/mL. At days 4, 7, 10, and 14 after HIV challenge, 500 μL (rectal) or 700 μL (vaginal) culture supernatant was collected and replaced with an equal amount of fresh medium.

Enzyme-linked immunosorbent assay was used to quantify p24 antigen in collected media. The LLOQ was 8 pg/mL. The pharmacodynamic unit of analysis was the cumulative, biopsy weight-adjusted p24 values over 14 days. Values below the LLOQ were imputed as the median biopsy weight-adjusted LLOQ/2 (range: 0.09–0.19 pg/mg; dependent on individual biopsy weights).

Statistical analyses

Drug concentrations and histology were summarized using descriptive statistics. Differences among histology scores (epithelial surface denudation, lamina propria hemorrhage, and average apoptosis) and explant readouts (median weight-adjusted cumulative p24 antigen) collected at different sampling times were assessed separately in rectal and vaginal tissue using the Friedman analysis of variance (ANOVA). Individual pairwise comparisons between sampling times were tested using the Wilcoxon signed rank test. All statistical analyses were performed using Stata (StataCorp, College Station, TX), and a p value <.05 was considered statistically significant.

Results

Subjects

Ten participants (six men and four women) provided written informed consent and underwent eligibility screening (Fig. 1). Eight participants (five men and three women) satisfied the inclusion criteria and were enrolled. At baseline, the median age was 34 years (range: 31–53 years). Per participant self-report, five identified as African American, two as White, and one as White/American Indian. The median baseline body mass index was 27.3 kg/m² (range: 22.8–41.2 kg/m²).

FIG. 1.

CONSORT diagram detailing movement of participants through study procedures. *This participant completed all other study procedures and was excluded only from pharmacokinetics, pharmacodynamics, and histological analysis of colorectal tissue.

One participant was withdrawn from the study before receiving any study product due to AEs. One participant withdrew consent for rectal biopsy collection but completed all other study procedures. Overall, seven participants contributed to the analysis of plasma PK and IQP-0528 distribution in the colorectum; six participants contributed to the PK, PD, and histological analysis in colorectal tissue; and three participants contributed to the PK and PD analysis in vaginal tissue.

Adverse events

Six AEs from four participants were reported throughout the study. Before dosing, one participant presented with low hemoglobin and subsequent weight loss, both deemed Grade 3 events and judged unrelated to study product. Due to concerns regarding the participant's safety on study, the decision was made to withdraw the participant. One AE was deemed related to study product: a Grade 1 headache occurring on the day of dosing that resolved shortly after onset. Of the remaining three AEs, two were Grade 2 (headache and abdominal cramps) and one was Grade 1 (high bilirubin) in severity. Two of these events, occurring in two participants, were reported before receipt of study product. The remaining event was reported ∼6 days after dosing. All three events were deemed unrelated to study product.

Histology

Comparing scores at baseline against those 3–5 h and 24–26 h after dosing, no significant increases in histological parameters were observed (p > .05 for global comparison by the Friedman ANOVA) (Table 1).

Table 1.

Histological Scoring of Rectal Biopsies

Parameter	Baseline	3–5 h postdose	24–26 h postdose
Surface denudationa	1 (0–2)	1 (0–3)	1 (0–3)
Lamina propria hemorrhagea	1 (1–1)	1 (0–1)	1 (0–2)
Apoptosis averageb	0.0 (0.0–0.8)	0.0 (0.0–1.3)	0.0 (0.0–0.3)

Values are reported as median (range).

No significant differences in parameters were observed across visits by the Friedman ANOVA (p > .05).

^aReported using semiquantitative scoring system: 0, no surface involved; 1, <1/3 surface involved; 2, 1/3–2/3 surface involved; 3, >2/3 surface involved.

^bReported as the decimal equivalent of the average number of bodies per multiple × 20 fields.

ANOVA, analysis of variance.

Pharmacokinetics

After accounting for residual radiolabel and decay correction, all participants retained at least 92% of the radiolabel gel dose. The only anatomic compartment with detectable IQP-0528 was rectal tissue, with values below the LLOQ at each time point across all seven participants in plasma (0.5–48 or 72 h) and across all three female participants in vaginal tissue 24–26 h postdose.

The median IQP-0528 rectal tissue concentrations 3–5 h postdose were 4,914 ng/mg [interquartile range (IQR): 1,682–5,135 ng/mg]; by 24–26 h postdose, the median concentrations decreased 3-logs to 5.4 ng/mg (IQR: 3.5–11.2 ng/mg). Despite this, for at least 24 h, rectal tissue concentrations remained above the in vitro EC₉₅ of 0.07 ng/mg (70.1 ng/mL, 206 nM), determined in the presence of simulated seminal fluid (Investigator's Brochure, data not shown). Based on these two measurements, the median crude half-life of IQP-0528 in rectal tissue was determined to be 2.2 h (2.2–2.3 h).

Distribution in the colorectum via SPECT/CT

SPECT/CT imaging 1–2 h after rectal dosing revealed that the distribution of DuoGel was primarily localized to the rectosigmoid (Fig. 2). After correcting for the location of the anorectal junction, the median D_min was 0 cm (IQR: −0.75 to 0.89 cm), the median D_max was 13.11 cm (IQR: 10.68–15.77 cm), the median DC_max was 8.60 cm (IQR: 6.70–9.61 cm), and the median D_ave was 7.77 cm (IQR: 6.55–8.41 cm).

FIG. 2.

Spatial distribution of IQP-0528 gel in the rectal lumen estimated via SPECT/CT imaging of ^99mTc-labeled microbicide surrogate 1–2 h after rectal dosing. (A) SPECT image of radiolabel distribution in a male participant with distal and proximal labels for directional reference. (B) Data resampling (gray-shaded region) with estimated centerline in red following application of our curve-fitting algorithm. (C) Concentration–distance curve with labeled parameters estimated using noncompartmental analysis of the centerline. Distance set to 0 cm across SPECT/CT scans is consistent with anatomic location of the anal verge; the distance indicated on a flexible sigmoidoscope would be roughly 4 cm greater due to the full distance of the anal canal. Note: predicted concentration indicates relative changes in luminal signal intensity and not necessarily an absolute concentration value. CT, computed tomography; SPECT, single-photon emission computed tomography.

Pharmacokinetic–pharmacodynamic correlation

In colorectal tissue explants, the median (IQR) cumulative p24 antigen 3–5 h postdose, 0.2 pg/mg (0.1–0.6 pg/mg), dropped significantly relative to baseline, [38.5 pg/mg (22.1–63.7 pg/mg); p = .05], but increased back to levels similar to baseline values 24–26 h after dosing [53.2 pg/mg (6.7–144.9 pg/mg); p = .75 compared with baseline] (Fig. 3A).

FIG. 3.

Median (IQR) weight-adjusted cumulative p24 measured from ex vivo explant challenge assays versus time (A), and versus IQP-0528 concentration (B) in rectal tissue. Colored points represent median (IQR, vertical error bars) of cumulative biopsy weight-adjusted p24 for each biopsy of four (baseline) or six (postdosing) replicates at a given sample time for each individual participant. Cumulative p24 values below the LLOQ were set as the median biopsy weight-adjusted LLOQ/2 (0.09–0.19 pg/mg; range represented by the gray-shaded regions). (A) Values at 0 h represent baseline measurements. Black symbol with error bars represents the overall bidimensional median and IQR, respectively, for values at each nominal time. (B) Predose IQP-0528 concentrations are imputed as 0.01 ng/mg to enable graphic comparison to postdose samples on the log scale. *Indicates p < .05 by the Wilcoxon signed rank test. IQR, interquartile range; LLOQ, lower limits of quantitation.

This reduction in the 3–5 h postdose sample coincided with the greatest IQP-0528 concentrations; the concentration–response relationship is evident in Figure 3B. Data were too sparse to perform formal PK/PD modeling. In vaginal tissue biopsies, none of which had detectable drug concentrations, the median cumulative p24 was not significantly different at baseline [0.8 pg/mg (0.5–48,283 pg/mg)] versus 24–26 h postdose [53.7 pg/mg (31.5–4,286 pg/mg); p = 1.00] and was associated with much greater variability when compared with rectal tissue explants.

Discussion

Our study was a first-in-human evaluation of the safety, PK, and PD of rectally administered DuoGel, a gel designed for rectal and vaginal delivery containing the investigational NNRTI IQP-0528, for HIV PrEP. Our results demonstrated that a single rectal dose of DuoGel was safe and well tolerated, yielded rectal concentrations above the in vitro EC₉₅ against HIV-1 within 3–5 h of dosing and sustained for at least 24 h, and reduced HIV p24 antigen production in rectal tissue after ex vivo HIV challenge with higher IQP-0528 concentrations associated with greater p24 suppression.

Despite being designed for dual use, our phase 1 study focused on rectal delivery for two reasons: (1) condomless receptive anal intercourse typically carries the greatest risk of HIV transmission per sex act relative to unprotected oral and vaginal intercourse,^32–34 and (2) studying rectal versus vaginal or dual administration allowed us to recruit both men and women.

One key advantage of topical microbicides over traditional oral PrEP is the potential to minimize systemic exposure and any associated toxicities. For topically delivered microbicides, it is also paramount to demonstrate epithelial tissue safety. Previous observations from our group showed that rectally administered hyperosmolar enemas, gels, and lubricants may increase gastrointestinal AEs and damage epithelial tissues.^15,16,35

In our study, DuoGel was found to be safe and well tolerated. Five of six AEs were deemed unrelated to study product, including all four that were judged Grade 2 or higher. A Grade 1 headache was the only AE judged related to study product based on evaluation at the time of the event. However, given that IQP-0528 was undetectable in systemic circulation, the relatedness of this AE is unlikely. Local toxicity assessed by histological scoring of colorectal tissue also revealed that DuoGel did not negatively impact the integrity of the colorectal epithelium.

Another theoretical advantage of topical PrEP over oral PrEP is the ability to deliver high concentrations of ARVs directly to mucosal tissues susceptible to HIV infection. No plasma absorption of IQP-0528 was observed; IQP-0528 was detected only in rectal tissue, the site of administration. Undetectable concentrations in plasma may be an advantageous feature as it would predict low potential for systemic side effects.

Unfortunately, concentrations were also undetectable in vaginal tissue. Although rectal-to-vaginal transfer has not been studied in Rhesus macaques, vaginal dosing studies of DuoGel in Rhesus macaques demonstrated vaginal-to-rectal transfer within 4 h of dosing.²¹ Numerous differences between humans and macaques could contribute to differences, including thickness of the vaginal submucosa, percent of vaginal surface area covered by stratified squamous epithelium, hormonal cycle impact on vaginal thickness, and microbiome, among other factors, and could result in cross species differences.

Vaginal-to-rectal (tenofovir, emtricitabine, and maraviroc) and rectal-to-vaginal (tenofovir) drug transfer has been reported in vaginal ring and gel studies; however, the concentration gradients are large, ranging from 1.5 to 4 log₁₀ depending on the drug and drug combination.^36,37

The timing of our vaginal biopsy sampling scheme, in which biopsies were only collected 24–26 h after dosing, may also have contributed to this observation. Sampling at similar times as rectal sampling, 3–5 h postdose, similar to the 4 h postdose Rhesus sampling, might have increased the ability to capture higher vaginal tissue IQP-0528 concentrations as has been carried out for other vaginal PrEP candidates. We cannot exclude the possibility that concentrations were detectable at earlier time points after dosing with substantial clearance occurring before the 24–26 h biopsy collection.

One day after dosing (24–26 h postdose), the smallest possible gradient from rectal tissue to vaginal tissue is estimated to be at least 215-fold based on assay LLOQ. Multicompartment PrEP coverage from single anatomic site of administration would be a desirable feature, particularly for women who engage in both unprotected vaginal and anal intercourse within the same sexual encounter. However, we demonstrated that rectal-to-vaginal transfer was very low at best and did not exceed EC₉₅ concentrations. Although not as simple as single-site dosing, women could apply DuoGel both vaginally and rectally, if needed, given that it is formulated for both rectal and vaginal use; however, vaginal safety studies remain to be performed to demonstrate vaginal safety.

The highest observed concentrations in rectal tissue occurred 3–5 h postdosing, with a median concentration more than 4-logs greater than the in vitro EC₉₅ of DuoGel against HIV-1 in the presence of simulated seminal fluid (206 nM) (Investigator's Brochure). Within 24–26 h of dosing, the median IQP-0528 concentration fell 3 × log₁₀, suggesting a very short tissue half-life, although concentrations remained above the in vitro EC₉₅. This characteristic contrasts substantially with some nucleoside analogs, for example, tenofovir and MK-8591, which have very long tissue half-lives due to being trapped in charged phosphorylated forms within tissue cells.

As a surrogate for in vivo efficacy, we performed HIV challenge assays ex vivo on rectal and vaginal biopsy tissue collected before and after IQP-0528 gel administration. At the time of the highest IQP-0528 rectal tissue concentrations observed (3–5 h postdose), explant p24 concentrations fell 3-log₁₀ units when compared with baseline, before returning to baseline values the next day. Relatively large interindividual variability, even at baseline, was observed in p24 values, consistent with prior reports¹⁵; with rectal tissue from at least one participant (006) proving difficult to infect at any time point.

Several study limitations restricted the IQP-0528 PK and PD inferences in this first-in-human study. Grant completion and study product expiration prevented completion of the 12 planned participants. We were also only able to collect rectal biopsies at two time points after dosing from six participants, precluding a meaningful sparse sampling analytic approach and tentative half-life estimation. Despite the limited sample size, more robust plasma sampling enabled our observation that there was undetectable systemic absorption of IQP-0528.

Another limitation was the decision to study product distribution without simulated unprotected receptive anal intercourse after product dosing, as has been performed previously.^14,38,39 Including simulated receptive anal intercourse and imaging the co-distribution of a virus surrogate intermixed with IQP-0528 gel would have provided additional pragmatic insights regarding the protective potential of DuoGel at sites of luminal viral distribution. We did, however, capture the localized distribution of radiolabeled gel in the rectosigmoid as would occur before sex.

In our first-in-human evaluation of IQP-0528, we have described the performance of DuoGel, containing 1% IQP-0528, across multiple domains, following rectal delivery in HIV-1 negative volunteers. We have demonstrated that DuoGel is safe, well tolerated, and not systemically absorbed when dosed rectally. In addition, IQP-0528 concentrations in rectal tissue exceeded the in vitro EC₉₅ within 3–5 h of dosing and lasted for at least 24 h and exhibited ex vivo antiviral activity following in vivo dosing. Aside from these strengths, our study also identified two key weaknesses. First, rectal tissue IQP-0528 concentrations were transient, with a short tissue half-life. It is, therefore, questionable whether DuoGel could provide protection from sexual exposure, although the temporal persistence of virus in the lumen and colon tissue has not been well established.

Second, there was no evidence of vaginal penetration after rectal dosing. Future studies of rectal dosing of DuoGel in women would benefit from vaginal sampling at earlier time points, such as 3–5 h after dosing, as was carried out with rectal biopsy collection. Evidence from this study suggests that women would need to separately dose in the rectum and vagina for protection at both sites of exposure during sex. These data suggest a potential role for DuoGel as an on-demand PrEP product, although additional formulation optimization may be needed depending on, as yet, uncertain mucosal viral dynamics and unknown mixing effects of virus and gel following ejaculation. In addition, it remains critical to characterize IQP-0528 safety, PK, and PD following vaginal delivery of DuoGel.

Authors' Contributions

E.S., E.J.F., A.S.H., K.W.B., R.W.B., Jr., and C.W.H. conceived the trial. A.A.-K., E.J.F., J.B., and E.D.W. interfaced with participants and collected samples for analysis. M.A.M., R.P.B., and P.H. performed method validation and analyzed all samples. H.C. and B.S.C. developed and implemented the curve-fitting algorithm for distribution analysis. A.A.-K. analyzed data and carried out statistical analyses. A.A.-K. wrote the article with support from C.W.H. All the authors provided critical feedback that helped to guide data analysis and the overall article.

Author Disclosure Statement

R.W.B., Jr., is an officer in ImQuest (licensee of IQP-0528). R.W.B., Jr., K.W.B., and A.S.H. are all shareholders in ImQuest. E.J.F. serves on the scientific advisory board for Lubrinovation, a subsidiary of ImQuest Life Sciences, Inc. C.W.H. serves on the scientific advisory boards for Gilead, Merck, and ViiV/GSK. A.A.-K., E.S., M.A.M., R.P.B., P.H., J.B., E.D.W., H.C., and B.S.C. have nothing to disclose.

Funding Information

The study was supported by the National Institutes of Health (NIH) Integrated Pre-Clinical/Clinical HIV Topical Microbicide Program (U19 AI101961). A.A.-K., E.S., and E.D.W. were supported by the Clinical Pharmacology Training Program (T32 GM066691). H.C. and B.S.C. were supported by the NIH grants NS060910-09A1 (NINDS) and 110156-0818 (NIBIB).