Metabolism of Long-Acting Rilpivirine After Intramuscular Injection: HIV Prevention Trials Network Study 076 (HPTN 076)

Abstract

A long-acting injectable formulation of rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor, is currently under investigation for use in human immunodeficiency virus (HIV) maintenance therapy. We previously characterized RPV metabolism after oral dosing and identified seven metabolites: four metabolites resulting from mono- or dioxygenation of the 2,6-dimethylphenyl ring itself or either of the two methyl groups located on that ring, one N-linked RPV glucuronide conjugate, and two O-linked RPV glucuronides produced via glucuronidation of mono- and dihydroxymethyl metabolites. However, as is true for most drugs, the metabolism of RPV after injection has yet to be reported. The phase II clinical trial HPTN 076 enrolled 136 HIV-uninfected women and investigated the safety and acceptability of long-acting injectable RPV for use in HIV pre-exposure prophylaxis. Through the analysis of plasma samples from 80 of these participants in the active product arm of the study, we were able to detect 2 metabolites after intramuscular injection of long-acting RPV, 2-hydroxymethyl-RPV, and RPV N-glucuronide. Of the total of 80 individuals, 72 participants exhibited detectable levels of 2-hydroxymethyl-RPV in plasma samples whereas RPV N-glucuronide was detectable in plasma samples of 78 participants. In addition, RPV N-glucuronide was detectable in rectal fluid, cervicovaginal fluid, and vaginal tissue. To investigate potential genetic variation in genes encoding enzymes relevant to RPV metabolism, we isolated genomic DNA and performed next-generation sequencing of CYP3A4, CYP3A5, UGT1A1 and UGT1A4. From these analyses, four missense variants were detected for CYP3A4 whereas one missense variant and one frameshift variant were detected for CYP3A5. A total of eight missense variants of UGT1A4 were detected, whereas two variants were detected for UGT1A1; however, these variants did not appear to account for the observed interindividual variability in metabolite levels. These findings provide insight into the metabolism of long-acting RPV and contribute to an overall understanding of metabolism after oral dosing versus injection. ClinicalTrials.gov Identifier: NCT02165202.

Keywords: HIV prevention; Rilpivirine; intramuscular injection; long-acting; metabolism.

Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.

Detection of 2-hydroxymethyl-RPV and RPV N-glucuronide in plasma samples of HTPN 076 research participants after oral dosing of RPV (25 mg, once-daily) for 4 weeks. (A) 2-hydroxymethyl-RPV and (B) RPV N-glucuronide in plasma samples of HPTN 076 study participants were detected by using an ultra-high-performance liquid chromatography-tandem mass spectrometry assay, as previously published. The 2-hydroxymethyl-RPV metabolite was quantified by using a synthetic standard, and the levels of 2-hydroxymethyl-RPV are represented as ng/mL. Due to the lack of a synthetic standard for RPV N-glucuronide, data are represented as a peak area ratio to the IS, RPV-d6. A total of 83 plasma samples collected from study sites, Bronx/Newark, USA n = 23, Cape Town, South Africa n = 25, Harare, Zimbabwe n = 35 were analyzed. Statistical significance was denoted as follows: **p ≤ .01; ***p ≤ .001. IS, internal standard; RPV, rilpivirine.

FIG. 2.

Detection of 2-hydroxymethyl-RPV and RPV N-glucuronide in plasma samples of HTPN 076 research participants after an intramuscular injection containing RPV (1,200 mg of RPV was delivered in two 2 mL injections at 8-week intervals). (A) 2-hydroxymethyl-RPV and (B) RPV N-glucuronide in plasma samples of HPTN 076 study participants were detected by using an ultra-high-performance liquid chromatography-tandem mass spectrometry assay, as previously published. The 2-hydroxymethyl-RPV metabolite was quantified by using a synthetic standard, and the levels of 2-hydroxymethyl-RPV are represented as ng/mL. Due to the lack of a synthetic standard for RPV N-glucuronide, data are represented as a peak area ratio to the IS, RPV-d6. A total of 80 plasma samples collected from study sites, Bronx/Newark, USA n = 22, Cape Town, South Africa n = 24, Harare, Zimbabwe n = 34 were analyzed.

FIG. 3.

Variation of the plasma levels of RPV metabolites, 2-hydroxymethyl-RPV, and RPV N-glucuronide in study participants at post-oral dose versus post-injection. Levels of 2-hydroxymethyl-RPV in plasma samples of HPTN 076 participants from (A) Bronx/Newark, USA, (B) Cape Town, South Africa, and (C) Harare, Zimbabwe after oral dosing versus injection. RPV N-glucuronide levels in plasma samples collected from (D) Bronx/Newark, USA, (E) Cape Town, South Africa, and (F) Harare, Zimbabwe after oral dosing versus injection. Eighty-three plasma samples collected from study sites, Bronx/Newark, USA n = 23, Cape Town, South Africa n = 25, Harare, Zimbabwe n = 35 were analyzed for post-oral dose whereas 80 plasma samples collected from study sites, Bronx/Newark, USA n = 22, Cape Town, South Africa n = 24, Harare, Zimbabwe n = 34 were analyzed for post-injection.

FIG. 4.

Detection of RPV metabolites, 2-hydroxymethyl-RPV, and RPV N-glucuronide in rectal fluid, cervicovaginal fluid, and vaginal tissue samples of HTPN 076 research participants after RPV delivery via an intramuscular injection. (A) Detection of 2-hydroxymethyl-RPV in rectal fluid samples. For this, 79 rectal fluid samples from study sites Bronx/Newark, USA n = 21, Cape Town, South Africa n = 23, Harare, Zimbabwe n = 35 were analyzed. The 2-hydroxymethyl-RPV metabolite was quantified by using a synthetic standard, and the levels of 2-hydroxymethyl-RPV are represented as ng/mg of sample. Detection of RPV N-glucuronide in (B) rectal fluid (n = 79), (C) cervicovaginal fluid (80 samples: Bronx/Newark, USA n = 21, Cape Town, South Africa n = 24, Harare, Zimbabwe n = 35), and (D) vaginal tissue (22 samples from Bronx/Newark, USA), samples using an ultra-high-performance liquid chromatography-tandem mass spectrometry assay as previously published. RPV N-glucuronide data are represented as a peak area ratio to the IS, RPV-d6. Statistical significance was denoted as follows: *p ≤ .05; **p ≤ .01.