Nucleoside/tide reverse transcriptase inhibitors
Figure 1a depicts extracellular ZDV concentrations over a 12 h dosing interval. After a first dose, ZDV concentrations were detectable in both BP and GT secretions 2 h after a dose. Although BP concentrations steadily declined from that time point, GT concentrations continued to increase and reached a maximum concentration at 6 h post-dose. At steady state, concentrations were detectable in the GT, but not in BP, prior to dosing. The GT and BP concentrations were similar 4 h postdose; after this point, GT concentrations declined minimally, while BP concentrations declined significantly. An average extracellular BP concentration (Cav) previously suggested for efficacy [20,21], is indicated in the figure as a black horizontal line. GT concentrations were at or near this target for a longer period of time than BP concentrations.
Lamivudine exposure after once-daily dosing can be seen in Fig. 1b. Similar results were seen for subjects receiving twice daily dosing (not shown). GT concentrations were approximately 1-log higher than BP concentrations throughout the dosing interval after a single dose and under steady-state conditions. The GT concentrations exceeded the suggested average extracellular target concentration (Cav) previously suggested for efficacy [20,21]. Similarly, GT concentrations of FTC were higher than BP concentrations from 6–24 h after a single dose, and remained higher than BP concentrations during the entire dosing interval under steady-state conditions (Fig. 1c). For TDF (Fig. 1d), the GT concentrations were at or above BP concentrations 4 h after a dose, and remained higher throughout the dosing interval.
Pharmacokinetic profiles for ATV and LPV are shown in Fig. 1e and f. In contrast to the NRTIs, ATV GT concentrations (when given as 300 mg ATV/100 mg RTV once daily) were lower than BP throughout the dosing interval both after first dose and at steady-state. These concentrations still, however, exceeded a suggested BP trough concentration of 150 ng/ml for efficacy against wild-type virus : the mean (±SD) ATV trough concentration in the GT after a first dose was 417 ± 818 ng/ml, and at steady state was 169 ± 148 ng/ml. The GT concentrations for LPV were also lower than BP at both first dose and steady-state. A suggested LPV trough concentration for efficacy of 1000 ng/ml is depicted in Fig. 1f . The GT concentrations approached this target over a dosing interval, and briefly exceed this concentration at the 4-h time point (mean ± SD = 1522 ± 1659 ng/ml). At the end of the dosing interval, mean ± SD GT concentrations were 437 ± 631 ng/ml after a single dose and 354 ± 384 ng/ml at steady-state.
Nonnucleoside reverse transcriptase inhibitors
The summary EFV pharmacokinetic profiles are shown in Fig. 1g. The BP and GT exposures are clearly distinct. Whereas EFV was detected in the GT 2 h after a single dose, concentrations were approximately 10 ng/ml: 2 logs lower than BP. The suggested target trough concentration for EFV is 1000 ng/ml .
Table 2 lists steady-state pharmacokinetic parameter estimates in BP and GT for each drug. BP concentrations at the end of the dosing interval (Cτ) and the AUC over 24 h (AUC0–24h) for each drug were within the expected range. On average, GT time to maximal concentration (Tmax) was delayed in comparison with BP. All drugs were, however, detectable in GT secretions within 4 h of antiretroviral drug ingestion.
Table 3 lists median (IQR) GT: BP AUC ratios in rank order from the highest exposure to lowest exposure after single and multiple doses of antiretroviral drugs. A ratio equal to 100% indicates drug exposure in the GT that is equivalent to BP. Therefore, ratios greater than 100% indicate greater GT drug exposures than BP, and those less than 100% indicate lower GT exposures than BP.
ZDV, 3TC, and FTC achieved greater GT exposures than BP both after a single dose and under steady-state conditions. The median GT exposures for these drugs ranged from 111 to 371% that of BP after a single dose and 235 to 411% after multiple doses. TDF GT exposure at first dose achieved a median of 135%, and 75% of BP at steady state. In contrast, ATV, stavudine (d4T), EFV, and RTV achieved lower GT exposures than BP, ranging from 0.5 to 21% that of BP after a first dose, and 0.4 to 26% after multiple doses.
This is the first study to comprehensively evaluate antiretroviral drug exposure in the GT after single and multiple dosing. In this study of 27 HIV-infected women, GT concentrations of all 11 antiretroviral drugs reported here were detected rapidly after single doses. Differing GT exposures between the drugs relative to BP, however, suggest that only certain drugs may be preferable for PrEP or PEP regimens.
Of the nucleoside analogs evaluated, 3TC, ZDV, FTC, and TDF achieved GT concentrations similar to, or higher than, BP, and therefore would be excellent PEP and PrEP candidates for further study. Mean 3TC and ZDV concentrations in the GT were near or above extracellular concentrations suggested for clinical efficacy [20,21]. The median GT AUC of FTC at steady state was approximately three times higher than the suggested BP AUC of 10 ug*h/ml for clinical efficacy .
Of the other compounds studied, LPV and ATV achieved low to moderate GT concentrations. Since these may be similar to the recommended BP trough concentrations necessary for efficacy in patients with wild-type HIV-1 virus , further study of these agents is warranted for use in PrEP and PEP either given alone or in combination with NRTIs. EFV had very low GT concentrations relative to BP, and is unlikely to be useful in PEP/PrEP regimens.
Low drug exposures in the GT may contribute to higher viral shedding in the GT and the development of viral resistance. Recently, receipt of NNRTI-containing therapy was found to be independently associated with HIV shedding at the cervix (odds ratio = 2.24 compared to a PI-based regimen) in 107 women (31 on NVP and 76 on EFV) . Additionally, data presented from the ACTG study A5077 suggested an increased rate of NNRTI mutations in the female GT  in individuals who were highly antiretroviral drug-experienced. Additionally, a recent study of 14 women found three with partial or complete compartmentalization of HIV in the GT compared to BP . In our study, the low exposures of EFV (0.5% GT: BP AUC) observed in this study suggest NNRTI drug resistance to be biologically plausible [27,28]. As drug concentrations in the GT are seldom measured, however, a direct relationship between drug exposure, viral burden, and viral resistance patterns in the GT has not been established.
It is currently unclear which specific mechanisms and physicochemical properties of drugs dictate passage into the female GT. However, drug penetration appears to be related to the protein-binding capacity of each drug. Highly protein-bound drugs such as LPV (98–99% protein bound)  and ATV (86% protein bound)  had lower GT exposures (8 and 18%, respectively, at steady state), whereas drugs with low protein-binding such as the nucleoside/tide analogs ZDV, 3TC, and FTC (< 4% to < 38% protein bound) [31–33], generally had higher GT exposures (ranging from 235–411%). d4T and ddI were exceptions to this trend, as GT exposures were low (5 and 21%, respectively) despite < 5% protein binding [34,35]. For d4T, peak plasma concentrations occur at approximately 2 h post-dose. Since our first sampling time was 2 h post-dose, it is possible that the true peak concentration was not captured, and complete exposure information was not obtained.
To further this initial pharmacokinetic evaluation, a number of investigations are desirable. Linking drug exposure to biologic response (e.g., decrease in HIV RNA) in the female GT is critical to confirm that higher extracellular drug concentrations are predictive of a more potent response and guide drug selection for PrEP and PEP. Pharmacokinetic/pharmacodynamic modeling analyses are ongoing in our laboratory to evaluate the correlation between differential GT drug exposure and virologic response. Additionally, our previous NRTI work in male GT mononuclear cells suggests similar or lower concentrations of intracellular triphosphate active metabolites compared to BP despite higher GT extracellular drug exposures . Therefore, research characterizing the intracellular active triphosphorylated metabolites for NRTIs in the female GT is necessary. Our study observed an extracellular half-life of TDF in the GT secretions twice as long as the extracellular half-life of TDF in BP, suggesting that TDF might be dosed at extended intervals for PEP or PrEP. Under first dose conditions, the median half-life was 14.5 h (95% CI: 3.6 - 29.2 h) in the GT, and 7.4 h (95% CI: 4.8–12.4 h) in BP (data not shown). However, characterizing intracellular concentrations of tenofovir diphosphate in GT mononuclear cells would be most important to move this application forward.
Although for most drugs, no differences in plasma drug exposure have been found between HIV-infected subjects and healthy volunteers [28–32], it may also be beneficial to evaluate the GT exposures of these drugs in healthy women, as they will be the primary recipients of PrEP and PEP. Additionally, no data exist on the influence of local infection and inflammation on drug concentrations in the female GT. As 130–1800% increases in drug concentrations have been documented with the inflammation present in meningitis and prostatitis, [37–39], GT infections may also increase antiretroviral drug concentrations in addition to increasing susceptibility to HIV infection [40–42].
Finally, understanding antiretroviral protein binding in the GT is necessary. This investigation measured total (protein unbound drug + protein bound drug) drug concentrations in both BP and GT. However, it has been suggested that concentrations of drug binding proteins (specifically albumin and alpha-1 acid glycoprotein) in the female GT may be lower than in BP . If this is true, despite differences in total drug concentrations between BP and GT, free drug concentrations in the GT may be similar to, or greater than, BP. Hence, free drug concentrations may be a more important predictor of efficacy. Although these types of investigations are technically difficult and lend themselves better to in-vitro study, this work will be required for a more thorough understanding of antiretroviral behavior in the GT.
In conclusion, this investigation is the first to comprehensively evaluate antiretroviral drug exposure in the female GT. Regardless of drug, GT concentrations were detected 2–4 h after a single antiretroviral dose. Additionally, GT exposures were similar after a first dose and at steady state. The results of this investigation support the use of 3TC, ZDV, TDF, and potentially FTC as excellent PrEP/PEP candidates. ATV and LPV/r may prove useful agents due to favorable GT concentrations in relation to HIV-1 wild-type susceptibility. We believe agents that achieve less than 10% of BP exposure, such as EFV and d4T, are less likely PrEP/PEP candidates.
We are indebted to the women volunteers, and the staff of the UNC Infectious Diseases Clinic, the Verne S. Caviness General Clinical Research Center, and the UNC CFAR Clinical Pharmacology and Analytical Chemistry Core.
Sponsorship: This work was supported by the National Institute of Allergy and Infectious Diseases (AI54980; A.D.M.K.), the UNC Center for AIDS Research/National Institute of Allergy and Infectious Disease (AI50410; N.L.R., A.S.B.), the UNC General Clinical Research Center/National Institutes of Health (RR00046), and the UNC Building Interdisciplinary Research Careers in Women's Health program (HD001441; A.D.M.K., K.B.P.).
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
antiretroviral therapy; genital tract; HIV; pharmacokinetics; prophylaxis; sexual transmission; women