300 mg

1. Guidance algorithm for Protocol Steering Committee (PSC): female subjects If 300mg GT levels ≥ "adequate": Complete enrolment to total 20 women in 300mg cohort Complete enrolment to total 20 women in 600mg cohort Enrol 20 women in 150 mg cohort 300 mg 300mg GT levels "not adequate": abandon recruitment to 300 mg cohort PSC review: 10 female participants per cohort with data up to day 14 PHASE 2 PHASE 1 Randomised allocation to dose in PHASE 1 600mg GT levels "not adequate": Abandon the 300mg cohort Complete enrolment to total 20 women in 600mg cohort Enrol 20 women in 1200mg cohort 600 mg If 600mg GT levels ≥ "adequate": Complete enrolment of 20 women total in 600mg and 300mg cohort Do not initiate the 1200mg cohort

2. Outcomes of PSC meetings following phases 1 & 2 The above figure was stated as guidance in the original protocol, approved by UK regulatory and ethics authorities, for the first Protocol Steering Committee (PSC) meeting. • As approved, the protocol allowed the inclusion of 6 male volunteers, and 60 female volunteers total; in three dose groups of 20 each. PHASE 1 • The first phase of study explored the plasma pharmacokinetics of 300mg (n=10) and 600mg (n=10), and the PSC was convened to compare the genital tract (GT) and plasma exposure to day 14, comparing this to the concentration of 50 ng/ml* seen in phase 3 treatment studies with oral rilpivirine25 mg od. Given that the data showed that 300mg was below this reference, with mean exposure for 600mg approximately matching the reference range with no safety concerns, the PSC deemed that the second phase should proceed to enroll 10 volunteers to receive 1200mg and to complete enrolment to the 600mg dose. PHASE 2. • The second PSC meeting reviewed plasma data from the phase 2 - 1200mg and 600mg cohorts - in addition to complete plasma exposure from the first phase and GT compartment exposure in CV fluid. There were no further concerns regarding safety data with 600mg or 1200mg and plasma exposure at 600mg matched the reference at day 28 whilst the 1200mg dose exceeded this concentration at days 28 and 56. The ratio of CV:plasma concentrations was greater than 1.0 at all timepoints. Viral inhibition studies in phases 1 & 2 were however unsuccessful, with directly aspirated vaginal fluid; the viscidity of the fluid causing technical failure of the assay. • The decision of the second PSC therefore was to complete enrolment to the 1200mg dose, with an amendment to the collection method for CV fluid at days 28 and 56 using a lavage method rather than direct aspiration, in order to facilitate the viral inhibition assay. • Given that there would be limited data available on viral inhibition at lower concentrations than those achieved with 1200mg, the decision was taken to also complete enrolment to the 300mg cohort with this amended collection method. The ultimate outcome of the three phases is outlined in the manuscript in Table 1. * This concentration was based on the upper limit of the lowest quartile of exposure, in which group the virologic response was the lowest. This reference was termed ‘adequate’ for the purpose of the PSC.