Pharmacodynamic correlations using fresh and cryopreserved tissue following use of vaginal rings containing dapivirine and/or maraviroc in a randomized, placebo controlled trial

Abstract

Background: The ex vivo challenge assay is a bio-indicator of drug efficacy and was utilized in this randomized, placebo controlled trial as one of the exploratory endpoints. Fresh and cryopreserved tissues were evaluated for human immunodeficiency virus (HIV) infection and pharmacokinetic (PK)/pharmacodynamic (PD) relationships.

Methods: HIV-negative women used vaginal rings containing 25 mg dapivirine (DPV)/100 mg maraviroc (MVC) (n = 12), DPV only (n = 12), MVC only (n = 12), or placebo (n = 12) for 28 days. Blood plasma, cervicovaginal fluid (CVF), and cervical biopsies were collected for drug quantification and the ex vivo challenge assay; half (fresh) were exposed immediately to HIV while the other half were cryopreserved, thawed, then exposed to HIV. HIV replication was monitored by p24 enzyme-linked immunosorbent assay from culture supernatant. Data were log-transformed and analyzed by linear least squared regression, nonlinear Emax dose-response model and Satterthwaite t test.

Results: HIV replication was greater in fresh compared to cryopreserved tissue (P = 0.04). DPV was detected in all compartments, while MVC was consistently detected only in CVF. Significant negative correlations between p24 and DPV levels were observed in fresh cervical tissue (P = 0.01) and CVF (P = 0.03), but not plasma. CVF MVC levels showed a significant negative correlation with p24 levels (P = 0.03); drug levels in plasma and tissue were not correlated with HIV suppression. p24 levels from cryopreserved tissue did not correlate to either drug from any compartment.

Conclusion: Fresh tissue replicated HIV to greater levels and defined PK/PD relationships while cryopreserved tissue did not. The ex vivo challenge assay using fresh tissue could prioritize drugs being considered for HIV prevention.

Figure 1

Capacity of fresh and cryopreserved cervical tissue to replicate human immunodeficiency virus (HIV). Cervical biopsies collected from women using the placebo vaginal ring were assessed for their capacity to replicate HIV. Cumulative p24 through day 11 of culture was determined and compared between fresh and cryopreserved cervical tissue. N = 6 for fresh tissue. N = 4 for cryopreserved tissue; 2 women did not have biopsies collected.

Figure 2

DPV and MVC concentration and fresh cervical tissue cumulative p24 dose–response relationships. DPV and MVC were quantified from paired cervical tissue (A), CVF (B), and plasma (C) on day 28 of vaginal ring use. The ex vivo challenge assay was performed on tissue collected on day 28 of VR use and cumulative p24 from day 11 are shown. The data shown are for 12 participants (n = 6 DPV only VR [red circle], n = 6 DPV/MVC VR [blue circle], left panels; and n = 6 MVC only VR [green circle], n = 6 DPV/MVC VR [blue circle], right panels). The vertical dotted line represents the limit of quantification for each matrix. CVF = cervicovaginal fluid, DPV = dapivirine, MVC = maraviroc, VR = vaginal ring.

Figure 3

Dose–response relationship between DPV tissue levels and cumulative p24 levels in fresh cervical tissue. Virus growth in the ex vivo challenge model, following both dapivirine and dapivirine/maraviroc vaginal ring use, was calculated as a proportion of the upper 95% confidence limit of the placebo fresh cumulative p24 levels (i.e., % placebo p24) and fitted with a nonlinear E_max model (gray line). The EC₅₀ for the E_max model is indicated with a dotted vertical line. DPV = dapivirine, EC₅₀ = 50% effective concentration.

Figure 4

DPV and MVC concentration and cryopreserved cervical tissue cumulative p24 dose–response relationships. DPV and MVC were quantified from paired cervical tissue (A), CVF (B), and plasma (C) on day 28 of VR use. The ex vivo challenge assay was performed on tissue collected on day 28 of VR use and cumulative p24 from day 11 are shown. The data shown are for 12 participants (n = 6 DPV only VR [red circle], n = 6 DPV/MVC VR [blue circle], left panels; and n = 6 MVC only VR [green circle], n = 6 DPV/MVC VR [blue circle], right panels). The vertical dotted line represents the limit of quantification for each matrix. CVF = cervicovaginal fluid, DPV = dapivirine, MVC = maraviroc, VR = vaginal ring.