To the Editors:
Research examining the pharmacokinetic profile of antiretrovirals in the male genital tract is important for both the purposes of characterizing the disposition of these drugs within this compartment and informing subsequent research aimed at ascertaining their effectiveness for interrupting the sexual transmission of human immunodeficiency virus (HIV).1 We therefore undertook a study to characterize the pharmacokinetic disposition of maraviroc, raltegravir, darunavir, and etravirine over a 12-hour dosing interval in the semen of HIV-infected men.
We prospectively recruited 10 HIV-infected men aged ≥18 years who had been receiving maraviroc- and raltegravir-based combination antiretroviral therapy (cART) for a minimum of 90 days, of whom 8 were receiving concomitant therapy with darunavir and etravirine. Additional eligibility criteria included having an undetectable viral load (<50 copies per milliliter) for a minimum of 1 month, ability to provide written informed consent, and no active illness or comorbidity, including acute renal or hepatic disease. We excluded patients who were nonadherent to their prescribed cART regimen, were receiving concomitant therapy with a non-antiretroviral inducer of CYP3A4 and who were expected to have difficulty adhering to the study protocol for any reason. We obtained written informed consent from all the participants of the study.
We used a staggered sampling approach in which semen samples were produced by participants over several days at different sampling times relative to the morning dose of antiretrovirals. Specifically, semen samples were collected 30 minutes to 1 hour before the morning dose of medication (day 1), and then at hours 1, 2, 4, 8, and 12 postdrug ingestion on days 2–6. We collected corresponding blood samples within 1 hour of the semen sample.
Semen and blood samples were centrifuged without delay, and isolated seminal and blood plasma were aliquoted and stored at −80°C until analyzed. Antiretroviral drug concentrations were determined by a validated high-performance liquid chromatography (HPLC) coupled to the tandem mass spectrometry (LC–MS/MS) method. Specifically, before analysis, protein was removed from the samples by precipitation with 0.1% acetic acid in methanol followed by centrifugation at 2000g for 5 minutes. One hundred microliters of a 2000 ng/mL of internal standard, 6,7-dimethyl-2,3-di (2-pyridyl)-quinoxaline (Aldrich, Milwaukee, WI), were added to 250 μL of supernatant, and the concentrations of maraviroc, darunavir, raltegravir, and etravirine were determined by LC–MS/MS. The gradient mobile phase system with a run time of 8 minutes consisted of 3 mobile phases: 5 mM ammonium acetate, pH 4.15 with 5% methanol; pure methanol; 0.1% acetic acid. A Hewlett Packard-1100 HPLC system (Agilent Technologies, Wilmington, DE) consisting of a Supelco (Supelcosil ABZ1Plus) 15 cm × 4.6 mm, 3 μm C18 column (Supelco, Bellefonte, PA) coupled to a PE Sciex API-2000 LC–MS/MS triple quadruple mass spectrometer (AB/MDS/Sciex, Concord, ON, Canada) equipped with a turbo ion spray source was used. Analyst software version 4.11 (AB/MDS/Sciex) was used as the system controller and integrator. All the solvents and chemicals were obtained from Sigma (Oakville, ON, Canada) and were of HPLC grade.
We used noncompartmental analyses to determine the pharmacokinetic parameters of maraviroc, raltegravir, darunavir, and etravirine in seminal and blood plasma. Specifically, for each drug, we used the exact sample collection and data observation times to determine the maximum concentration (C max), minimum concentration (C min), time to maximum concentration (T max), and area under the curve by using the trapezoidal rule from 0 to 12 hours (AUC0–12 h) in both compartments. We used published estimates of antiretroviral protein binding in seminal plasma and in vitro unbound EC90 for HIV-1 to facilitate interpretation of our results.2–6 We also determined the coefficient of variation (CV) of SP:BP ratios for each drug. Finally, we summarized baseline characteristics of the participants using medians and interquartile range (IQR) for continuous variables and proportions for categorical variables (Sigmastat 3.5). We obtained ethics approval for this study from Institutional Review Board Services and the Ottawa Hospital Research Ethics Board.
The median age and baseline CD4+ cell count of the 10 participants were 51 years (IQR 46–57 years) and 335 cells per cubic millimeter (IQR 218–348 cells per cubic millimeter). Virologic suppression to <50 copies per milliliter had been maintained for a median of 10 months (IQR 2.5–10.5 months) at the time of study enrolment. A total of 60 semen samples and 60 blood samples were obtained from all the participants during the study period. Concentrations of maraviroc and raltegravir were determined in all the samples, whereas pharmacokinetic parameters of darunavir and etravirine could be determined from 48 matched semen and blood plasma specimens.
Pharmacokinetic parameters of maraviroc, raltegravir, darunavir, and etravirine in blood plasma and seminal plasma are summarized in Table 1. Both maraviroc and raltegravir were observed to accumulate in seminal plasma, with median SP:BP ratios of 0.92 (IQR 0.59–1.91) and 4.32 (IQR 4.1–5.0), respectively. Similarly, the median AUC0–12 h SP:BP ratios for maraviroc and raltegravir were 0.93 (IQR 0.71–3.89) and 3.63 (IQR 2.22–4.22). In contrast, median SP:BP and AUC0–12 h SP:BP ratios for darunavir were 0.20 (IQR 0.14–0.24) and 0.20 (0.15–0.34), whereas those for etravirine were 0.16 (IQR 0.15–0.23) and 0.17 (IQR 0.14–0.24), respectively. As reflected by the coefficients of variation, a greater degree of interindividual variability in both the AUC0–12 h (123%) and SP:PB (120%) ratios was observed over the dosing interval for maraviroc relative to the other antiretrovirals examined (Table 1).
The results of our study demonstrate that the distribution of maraviroc and raltegravir into the male genital tract exceeds that of both etravirine and darunavir when expressed as ratios of trough concentrations and total drug exposure in the seminal compartment relative to the blood plasma compartment. Although concentrations of etravirine and darunavir in seminal plasma are approximately 16% and 20% of those attained in blood plasma over a 12-hour dosing interval, these findings are likely a reflection of the greater affinity for blood plasma proteins exhibited by these drugs relative to maraviroc and raltegravir. Specifically, blood plasma protein binding of darunavir and etravirine is reported to be 95% and 99.9%, respectively,7 compared with 76% for maraviroc8 and 83% for raltegravir.9 Consequently, it is possible that darunavir and etravirine exhibit lower accumulation ratios relative to maraviroc and raltegravir because less unbound drug is available to access the male genital tract.
However, the lower accumulation ratios attained for darunavir and etravirine may not necessarily be indicative or predictive of poor antiviral effectiveness activity within the male genital tract. That is, when interpreting drug concentrations within body compartments, it is important to consider both absolute measures of exposure and ratios. In this context, because the concentration of albumin in seminal plasma (0.1 g/dL) is far lower than that of blood plasma (3.4–5.4 g/dL), unbound concentrations of antiretrovirals in seminal plasma may still exceed the level required to inhibit replication of HIV within this compartment.10 For example, the seminal plasma protein binding of darunavir and etravirine has been recently reported as being 14% and 96.7%, respectively.3 Applying these estimates to our data, the median unbound trough concentrations of etravirine and darunavir observed among our participants are estimated as 0.00033 and 0.34 mg/L, respectively. Therefore, unbound trough concentrations of darunavir in the seminal plasma of our participants are >200-fold greater than the unbound EC90 of the drug for HIV-1 (0.0015 mg/mL).5 In contrast, trough concentrations of etravirine are below the reported unbound EC90 of this drug for HIV-1 (0.0013 mg/mL).4 Using a similar approach, the unbound trough concentration of maraviroc was calculated to be 0.07 mg/L, which exceeds the unbound EC90 of the drug for HIV-1 (0.00057 mg/L) by >100-fold.6 We could not estimate the unbound trough concentration of raltegravir because the degree of protein binding of this drug in seminal plasma is unknown. However, because the semen plasma trough concentration exceeds the raltegravir IC95 for wild-type HIV-1 (0.0146 mg/L) almost 12-fold,11 it would seem reasonable to infer that the observed levels are adequate for viral suppression within this compartment.
Several limitations of our work merit emphasis, including the small sample size and the inability to directly measure unbound antiretroviral drug concentrations in seminal plasma. However, these limitations are common to pharmacokinetic studies evaluating drug concentrations in semen.12 Furthermore, our study builds upon earlier research assessing the distribution of these drugs into the male genital tract, which utilized single semen samples from HIV-infected men or staggered sampling in healthy volunteers by providing the first evaluation of the seminal plasma pharmacokinetics of etravirine, maraviroc, and raltegravir over the entire dosing interval in HIV-infected men receiving cART with these drugs.2,3,13–15 In addition, our study corroborates the findings of Taylor et al,16 who described the disposition of darunavir in the seminal plasma of HIV-infected men. In conjunction with the results of these earlier studies, our work suggests that maraviroc, raltegravir, and darunavir may prevent the evolution of drug-resistant HIV in the genital tract and have a role in the primary and secondary prevention of HIV. Further research examining the potential of these agents in the latter regard is warranted.
1. Else LJ, Taylor S, Back DJ, et al.. Pharmacokinetics of antiretroviral drugs in anatomical sanctuary site: the male and female genital tract. Antivir Ther. 2011;16:1149–1167.
2. Brown KC, Patterson KB, Malone SA, et al.. Single and multiple dose pharmacokinetics of maraviroc in saliva, semen and rectal tissue of healthy HIV-negative men. J Infect Dis. 2011;203:1484–1490.
3. Brown KC, Patterson KB, Jennings SH, et al.. Single and multiple dose pharmacokinetics of darunavir plus ritonavir and etravirine in semen and rectal tissue of HIV-negative men. J Acquir Immune Defic Syndr. 2012;61:138–144.
4. DeJesus E, Lalezari JP, Osiyemi OO, et al.. Pharmacokinetics of once-daily etravirine without and without once-daily darunavir/ritonavir in antiretroviral naive HIV type-1-infected adults. Antivir Ther. 2010;15:711–720.
5. De Meyer S, Azijn H, Surleraux D, et al.. TMC114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitor-resistant virus, including a broad range of clinical isolates. Antimicrob Agents Chemother. 2005;49:2314–2321.
7. Hughes CA, Robinson L, Tseng A, et al.. New antiretroviral drugs: a review of the efficacy, safety, pharmacokinetics, and resistance profile of tipranavir, darunavir, etravirine, rilpivirine, maraviroc, and raltegravir. Expert Opin Pharmacother. 2009;10:2445–2466.
8. Kromdijk W, Huitema AD, Mulder JW. Treatment of HIV infection with the CCR5 antagonist maraviroc. Expert Opin Pharmacother. 2010;11:1215–1223.
9. Temesgen Z, Siraj DS. Raltegravir: first in class HIV integrase inhibitor. Ther Clin Risk Manag. 2008;4:493–500.
10. Cao YJ, Hendrix CW. Male genital tract pharmacology: developments in quantitative methods to better understand a complex peripheral compartment. Clin Pharmacol Ther. 2008;83:401–412.
11. Iwamoto M, Wenning LA, Petry AS, et al.. Safety, tolerability, and pharmacokinetics of raltegravir after single and multiple doses in healthy subjects. Clin Pharmacol Ther. 2008;83:293–299.
12. Avery LB, Rakshi RP, Cao YJ, et al.. The male genital tract in not a pharmacological sanctuary from efavirenz. Clin Pharmacol Ther. 2011;90:151–156.
13. Barau C, Delaugerre C, Braun J, et al.. High concentration of raltegravir in semen of HIV-infected men: results from a substudy of the EASIER-ANRS 138 trial. Antimicrob Agents Chemother. 2010;54:937–939.
14. Tiraboschi JM, Niubo J, Curto J, et al.. Maraviroc concentrations in seminal plasma in HIV-infected patients. J Acquir Immune Defic Syndr. 2010;55:e35–e36.
15. Calcagno A, Bonora S, D’Avolio A, et al.. Raltegravir penetration in seminal plasma of healthy volunteers. Antimicrob Agents Chemother. 2010;54:2744–2745.
16. Taylor S, Jayasuirya AN, Berry A, et al.. Darunavir concentrations exceed the protein-corrected EC50 for wild-type HIV in the semen of HIV-1 infected men. AIDS. 2010;24:2583–2587.