Phase 1 randomized pharmacokinetic and safety study of a 90-day tenofovir vaginal ring in the United States.

INTRODUCTION: Tenofovir-based oral pre-exposure prophylaxis is currently approved for HIV prevention; however, adherence in women has been low. A vaginal gel containing tenofovir (TFV) demonstrated partial protection to HIV but protection was not confirmed in additional studies. Vaginal rings offer user-controlled long-acting HIV prevention that could overcome adherence and protection challenges. TFV may also help prevent herpes simplex virus type 2 acquisition when delivered intravaginally. We evaluated the pharmacokinetics, safety, adherence and acceptability of a 90-day TFV ring. METHODS: Between January and June 2019, Microbicide Trials Network (MTN)-038 enrolled 49 HIV-negative participants into a phase 1, randomized (2:1) trial comparing a 90-day ring containing 1.4 grams (g) TFV to a placebo ring. TFV concentrations were quantified in plasma, cervicovaginal fluid (CVF), rectal fluid and cervical tissue, and TFV-diphosphate (TFV-DP) in cervical tissue. Used rings were analysed for residual TFV. Safety was assessed by adverse events (AEs); acceptability and adherence by self-report. RESULTS: Mean age was 29.5; 46 identified as cisgender-female and three gender non-conforming. There were no differences in the proportion of participants with grade ≥2 genitourinary AEs in the TFV versus placebo arms (p = 0.41); no grade ≥3 AEs were reported. Geometric mean TFV concentrations increased through day 34 in CVF/rectal fluid and day 59 in plasma, but declined across compartments by day 91. Geometric mean TFV-DP tissue concentrations exceeded the 1000 fmol/mg target through day 56, but fell to 456 fmol/mg at day 91. Among 32 rings returned at the end of the study, 13 had no or low (<0.1 g) residual TFV. Residual TFV did not differ by socio-demographics, sexual activity, Nugent Score or vaginal microbiota. Most participants reported being fully adherent to ring use: 85% and 81% in the TFV and placebo arms, respectively (p = 1.00). A majority of participants reported liking the ring (median 8 on a 10-point Likert scale) and reported a high likelihood of using the ring in the future, if effective (median 9). CONCLUSIONS: The 90-day TFV ring was well-tolerated, acceptable and exceeded target cervical tissue concentrations through day 56, but declined thereafter. Additional studies are needed to characterize the higher release from TFV rings in some participants and the optimal duration of use.

Amygdalobacter indicium gen. nov., sp. nov., and Amygdalobacter nucleatus sp. nov., gen. nov.: novel bacteria from the family Oscillospiraceae isolated from the female genital tract.

Four obligately anaerobic Gram-positive bacteria representing one novel genus and two novel species were isolated from the female genital tract. Both novel species, designated UPII 610-JT and KA00274T, and an additional isolate of each species were characterized utilizing biochemical, genotypic and phylogenetic analyses. All strains were non-motile and non-spore forming, asaccharolytic, non-cellulolytic and indole-negative coccobacilli. Fatty acid methyl ester analysis for UPII 610-JT and KA00274T and additional isolates revealed C16 : 0, C18 : 0, C18:1ω9c and C18:2ω6,9c to be the major fatty acids for both species. UPII 610-JT had a 16S rRNA gene sequence similarity of 99.4 % to an uncultured clone sequence (AY724740) designated as Bacterial Vaginosis Associated Bacterium 2 (BVAB2). KA00274T had a 16S rRNA gene sequence similarity of 96.5 % to UPII 610-JT. Whole genomic DNA mol% G+C content was 42.2 and 39.3 % for UPII 610-JT and KA00274T, respectively. Phylogenetic analyses indicate these isolates represent a novel genus and two novel species within the Oscillospiraceae family. We propose the names Amygdalobacter indicium gen. nov., sp. nov., for UPII 610-JT representing the type strain of this species (=DSM 112989T, =ATCC TSD-274T) and Amygdalobacter nucleatus gen. nov., sp. nov., for KA00274T representing the type strain of this species (=DSM 112988T, =ATCC TSD-275T).

Tenofovir vaginal film as a potential MPT product against HIV-1 and HSV-2 acquisition: formulation development and preclinical assessment in non-human primates.

Tenofovir (TFV) is an adenosine nucleotide analog with activity against HIV and HSV-2. Secondary analyses of clinical trials evaluating TFV gel as pre-exposure prophylaxis (PrEP) for HIV have shown that gel formulations of TFV provide significant protection against both HIV and HSV-2 acquisition in women who had evidence of use. An alternate quick-dissolving polymeric thin film, to deliver TFV (20 and 40 mg) has been developed as a potential multipurpose technology (MPT) platform. Film formulation was developed based on excipient compatibility, stability, and ability to incorporate TFV doses. Placebo, low dose (20 mg), and high dose (40 mg) films were utilized in these studies. The developed film platform efficiently incorporated the high dose of TFV (40 mg/film), released more than 50% of drug in 15 min with no in vitro toxicity. Pharmacological activity was confirmed in an ex vivo HIV-1 challenge study, which showed a reduction in HIV-1 infection with TFV films. Films were stable at both doses for at least 2 years. These films were found to be safe in macaques with repeated exposure for 2 weeks as evidenced by minimal perturbation to tissues, microbiome, neutrophil influx, and pH. Macaque sized TFV film (11.2 mg) evaluated in a pigtail macaque model showed higher vaginal tissue concentrations of TFV and active TFV diphosphate compared to a 15 mg TFV loaded gel. These studies confirm that TFV films are stable, safe and efficiently deliver the drug in cervicovaginal compartments supporting their further clinical development.

Phase 1 pharmacokinetics and safety study of extended duration dapivirine vaginal rings in the United States.

INTRODUCTION: Vaginal rings are a promising approach to provide a woman-centred, long-acting HIV prevention strategy. Prior trials of a 25 mg dapivirine (DPV) ring have shown a favourable safety profile and approximately 30% risk reduction of HIV-1 infection. Extended duration rings replaced every three months may encourage user adherence, improve health service efficiency and reduce cost overall. We evaluated safety, pharmacokinetics, adherence and acceptability of two three-month rings with different DPV dosages, compared with the monthly DPV ring. METHODS: From December 2017 to October 2018, MTN-036/IPM-047 enrolled 49 HIV-negative participant in Birmingham, Alabama and San Francisco, California into a phase 1, randomized trial comparing two extended duration (three-month) rings (100 or 200 mg DPV) to a monthly 25 mg DPV ring, each used over 13 weeks, with follow-up completed in January 2019. Safety was assessed by recording adverse events (AEs). DPV concentrations were quantified in plasma, cervicovaginal fluid (CVF) and cervical tissue, at nominal timepoints. Geometric mean ratios (GMRs) relative to the comparator ring were estimated from a regression model. RESULTS: There were no differences in the proportion of participants with grade ≥2 genitourinary AEs or grade ≥3 AEs in the extended duration versus monthly ring arms (p = 1.0). Plasma and CVF DPV concentrations were higher in the extended duration rings compared to the monthly ring. Plasma GMRs were 1.31 to 1.85 and 1.41 to 1.86 and CVF GMRs were 1.45 to 2.87 and 1.74 to 2.60 for the 100 and 200 mg ring respectively. Cervical tissue concentrations were consistently higher in the 200 mg ring (GMRs 2.36 to 3.97). The majority of participants (82%) were fully adherent (ring inserted at all times, with no product discontinuations/outages) with no differences between the monthly versus three-month rings. Most participants found the ring acceptable (median = 8 on 10-point Likert scale), with a greater proportion of participants reporting high acceptability (9 or 10) in the 25 mg arm (73%) compared with the 100 mg (25%) and 200 mg (44%) arms (p = 0.01 and p = 0.15 respectively). CONCLUSIONS: The extended duration DPV rings were well-tolerated and achieved higher DPV concentrations compared with the monthly DPV ring. These findings support further evaluation of three-month DPV rings for HIV prevention.

Zim CHIC: A cohort study of immune changes in the female genital tract associated with initiation and use of contraceptives.

PROBLEM: Contraceptive hormones are systemically active, potent, and likely to invoke biological responses other than known fertility regulation impacts. We hypothesized that initiation of depot medroxyprogesterone acetate (DMPA) would increase genital HIV-target-cells and soluble immune mediators compared with baseline and initiation of other contraceptive methods. METHOD OF STUDY: We collected cervical cytobrushes and cervicovaginal fluid from healthy Zimbabwean women aged 18-34 to assess immune cell populations, cytokines, and innate anti-HIV activity at baseline and after 30, 90, and 180 days use of DMPA (n = 38), norethisterone enanthate (n = 41), medroxyprogesterone acetate/estradiol cypionate (n = 36), levonorgestrel implant (n = 43), etonogestrel implant (n = 47), or copper intrauterine device (Cu-IUD) (n = 45). Cells were quantified by flow cytometry, cytokines were detected by multiplex assays, and innate anti-HIV activity was assessed by in vitro HIV challenge. RESULTS: Compared to baseline, the number of cervical HIV target cells (#CD4 cells P < .04 and #CD11c cells P < .04), the concentration of the inflammatory cytokine IL-1ß (P < .01), and the innate in vitro anti-HIV activity (P < .001) significantly decreased following DMPA initiation. In Cu-IUD users, genital HIV target cells increased (#CD4 cells P < .001, #CD4CCR5 cells P = .02, #CD4CD69 cells P < .001, #CD8CD69 P = .01, and #CD11c cells P = .003) at day 30 and resolved by day 180. IFN-γ (P < .001), IL-1ß (P < .001), IL-6 (P < .001), IL-8 (P < .001), IL-10 (P < .01), and RANTES (P < .001) were also significantly increased at day 30. Minimal alterations were observed following initiation of subdermal implantable contraceptives. CONCLUSIONS: This head-to-head study compared six contraceptives and found increased HIV target cells and cervical inflammation temporally associated with Cu-IUD initiation. Use of hormonal contraception, including DMPA, did not increase cervical HIV target cells or inflammation. Clinical Trial Number: NCT02038335.

Optimization of processing female genital tissue samples for lymphocyte analysis by flow cytometry.

PROBLEM: A variety of methods have been used to process cervical cytobrush and genital tissue for flow cytometric evaluation of immune cell populations. We sought to optimize genital tract specimen processing and to determine if blood could be used as a model for assessment of tissue processing methods. METHOD OF STUDY: Cervical cytobrushes, PBMCs, and genital tissue samples (cervical and endometrial biopsies) were subjected to varying processing conditions to characterize the effects on cell yields, lymphocyte viability, and surface receptors. We exposed PBMC and tissue specimens to varied collagenase types, concentrations, and exposure durations and cytobrushes to immediate vs delayed processing with/without vortexing. RESULTS: PBMCs and tissues exposed to varying enzymatic digestion conditions demonstrated stability of some cell surface receptors, including CD3+ , CD4+ , and CD8+ , while others, including CCR6+ , were cleaved when exposed to any concentration of collagenase B, or ≥0.25 mg/mL of collagenase D. We observed increased CD69 expression (marker of cell activation) after exposure to collagenase B. Neither a 2-hour delay in cytobrush processing nor vortexing at a setting of 50% for 30 seconds had significant impacts on viability or quantities of genital immune cells of interest. CONCLUSION: Although tissue digestion with collagenase D was sufficient to recover and analyze cells from endometrial biopsy specimens, cervical biopsy specimens required a limited exposure to collagenase B at 1 mg/mL to optimize cell yield and viability for cytometric analysis. PBMCs can be used as a model to assess the impact of tissue processing on co-receptor expression and to optimize methods prior to study implementation.

Megasphaera lornae sp. nov., Megasphaera hutchinsoni sp. nov., and Megasphaera vaginalis sp. nov.: novel bacteria isolated from the female genital tract.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6T, KA00182T and BV3C16-1T. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus Megasphaera. UPII 199-6T and KA00182T had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as Megasphaera type 1 and Megasphaera type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6T exhibited sequence identities ranging from 92.9 to 93.6 % with validly named Megasphaera isolates and KA00182T had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1T was most closely related to Megasphaera cerevisiae with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6T, KA00182T and BV3C16-1T were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named Megasphaera species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C12 : 0, C16 : 0, C16 : 0 dimethyl acetal (DMA) and summed feature 5 (C15 : 0 DMA and/or C14 : 0 3-OH) in UPII 199-6T; C16 : 0 and C16 : 1 cis 9 in KA00182T; C12 : 0; C14 : 0 3-OH; and summed feature 5 in BV3C16-1T. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6T, KA00182T and BV3C16-1T represent novel species within the genus Megasphaera. We propose the following names: Megasphaera lornae sp. nov. for UPII 199-6T representing the type strain of this species (=DSM 111201T=ATCC TSD-205T), Megasphaera hutchinsoni sp. nov. for KA00182T representing the type strain of this species (=DSM 111202T=ATCC TSD-206T) and Megasphaera vaginalis sp. nov. for BV3C16-1T representing the type strain of this species (=DSM 111203T=ATCC TSD-207T).

Phase 1 Pharmacokinetic Trial of 2 Intravaginal Rings Containing Different Dose Strengths of Vicriviroc (MK-4176) and MK-2048.

BACKGROUND: Vaginal rings (VRs) are a promising approach for sustained delivery of antiretroviral (ARV) medication to prevent human immunodeficiency virus (HIV) infection in women. Combination ARV VRs could increase efficacy. METHODS: MTN-028, a phase 1 trial in 19 HIV-uninfected women, evaluated 2 VRs containing vicriviroc (VCV) and MK-2048. Participants were randomized 2:1 to a low-dose (VCV, 91 mg; MK-2048, 10 mg) or original-dose (VCV, 182 mg; MK-2048, 30 mg) ring used for 28 days. Safety was assessed by documenting adverse events (AEs). Drug concentrations were evaluated in plasma, cervicovaginal fluid (CVF), and cervical tissue samples. RESULTS: All AEs reported were grade 1 or 2, with no statistically significant differences in related genitourinary AEs or grade ≥2 AEs observed between arms (P = >.99). VCV/MK-2048 concentrations rose rapidly, with higher plasma area under the concentration-time curve (AUC) in the original-dose arm (geometric mean ratio, 3.29 for VCV and 1.49 for MK-2048) and similar AUCs across arms for CVF samples. Cervical tissue concentrations were higher in the original-dose arm (geometric mean ratio, 7.94 for VCV and 6.45 for MK-2048), with greater drug released based on residual drug levels. Plasma and CVF concentrations for both drugs fell rapidly after ring removal. CONCLUSIONS: In this first study evaluating 2 doses of a combination VCV/MK-2048 VR, both rings were found to be safe and well tolerated. VCV and MK-2048 were detectable in plasma, CVF, and cervical tissue samples, and drug release and plasma drug exposure were higher for the original-dose than for the low-dose ring.

Bacterial species colonizing the vagina of healthy women are not associated with race.

The vaginal microbiota of 36 white versus 25 black asymptomatic women were compared using both cultivation-dependent and -independent identification. Significant differences by race were found in colonization and density of bacterial species. However, exclusion of 12 women with bacterial vaginosis by Nugent criteria resulted in no significant differences by race.