Patterson, Kristine B. MD*; Dumond, Julie B. PharmD, MS†; Prince, Heather A. PA-C*; Jenkins, Amanda J. PharmD†; Scarsi, Kimberly K. PharmD‡; Wang, Ruili MD†; Malone, Stephanie MS†; Hudgens, Michael G. PhD*; Kashuba, Angela D. M. PharmD†
HIV-infected pregnant women receive antiretroviral therapy for their own health and to prevent mother-to-child transmission of HIV. The US Department of Health and Human Services guidelines have recommended lopinavir/ritonavir (LPV/RTV; Kaletra; Abbott Laboratories, Abbott, IL) as the first-line protease inhibitor for either goal.1 It has been previously demonstrated that standard dosing (400 mg/100 mg) using 3 LPV/RTV soft gel capsules twice daily results in the third trimester lopinavir area under the concentration–time curve (AUC) and trough concentrations below a target of 52 μg/·h/mL and 1 μg/mL, respectively.2–5 This decrease in exposure was not overcome with dose increases to 533 mg/133 mg (4 capsules) twice daily.6 Exposure between the new Meltrex tablet (200 mg/50 mg) formulation and standard dose LPV/RTV soft gel capsules is not significantly different, although over 17% of women receiving either formulation have lopinavir trough concentrations below 3 μg/mL.7 Dose increases to 600 mg/150 mg twice daily results in the third trimester concentrations similar to nonpregnant exposures at the standard 400 mg/100 mg dose.8 Based on these data, the US Department of Health and Human Services guidelines recommends increasing LPV/RTV dose to 600 mg/150 mg twice daily in the third trimester. However, the increased bioavailability of the tablet formulation has the potential to decrease tolerability and increase toxicity at this higher dose.
Lopinavir and ritonavir are 98%–99% bound to the blood plasma protein albumin and alpha-1-acid-glycoprotein (AAG), and only protein-unbound drugs are available to traverse cell membranes and exert a pharmacologic effect.9 Due to technical complexity and the requirement for advanced analytical instrumentation, pharmacokinetic (PK) studies generally measure total drug concentrations (protein bound plus unbound) rather than unbound drug. Additionally, because plasma protein binding in most populations is consistent, measuring unbound drug is often unnecessary to assess efficacy.10 Pregnancy, however, is associated with alterations in plasma proteins,11–13 which may affect unbound drug concentrations. Consequently, total lopinavir and ritonavir exposures may not reflect drug available for antiviral activity; in this situation, protein-unbound drug concentrations may be most relevant.
Few studies have longitudinally and comprehensively measured unbound lopinavir and ritonavir concentrations throughout pregnancy. Investigations with sparse sampling have had mixed results: either an 18% increase in the fraction of unbound lopinavir in the third trimester compared with postpartum14 or no significant changes.15
To address the hypothesis that the physiological changes associated with pregnancy will decrease the total (protein bound plus unbound) but not the protein-unbound (active) lopinavir and ritonavir concentrations, this study longitudinally evaluates total and unbound lopinavir and ritonavir exposures in HIV-infected pregnant women in the second and third trimesters of pregnancy and postpartum. The pediatric formulation of LPV/RTV (100 mg/25 mg twice daily), rather than an additional adult tablet (200 mg/50 mg), is empirically added to standard dosing (400 mg/100 mg twice daily) in the third trimester. The study is designed to investigate whether LPV/RTV 500 mg/125 mg is able to overcome any pregnancy-associated changes in total and free drug exposure while minimizing toxicity and maximizing tolerability.
Study Design and Subject Selection
HIV-infected pregnant women were enrolled in this open-label, longitudinal PK study to assess the total (protein bound plus unbound) and protein-unbound exposure of fixed-dose combination lopinavir/ritonavir tablets (LPV/RTV, Kaletra; Abbott Laboratories). Subjects were recruited from the Infectious Diseases Clinics at the University of North Carolina at Chapel Hill, NC, and Northwestern University, Chicago, IL The Biomedical Review Boards from each individual institution approved the study, and all subjects provided written informed consent before study procedures. Subjects were enrolled from October 2007 to September 2009 (National Clinical Trial Registry No. 00766818).
Subjects were eligible to participate if they were less than 20 weeks of gestation and receiving or planning to initiate LPV/RTV tablets (400 mg/100 mg twice daily). Subjects were required to be ≥18 years of age with a current singleton and uncomplicated pregnancy. Subjects were excluded if they were being treated for an opportunistic infection; had prior obstetrical complications or preterm delivery; had a hemoglobin <9.0 g/dL; were unable to complete a dose record card at home; were <80% adherent to the current antiretroviral regimen as determined by dosing card, provider, or self-report; or were receiving other protease inhibitors or concurrent medications known to interact with LPV/RTV. Subjects with documented virologic failure of an LPV/RTV regimen or who had a genotype that would predict failure to LPV/RTV were excluded.
Two to 4 weeks before enrollment, subjects underwent screening in which informed consent, physical examination, and routine safety laboratory testing (complete blood count, serum chemistries, liver function tests, urinalysis, and absolute CD4+ cell count and HIV RNA) were performed, and medical and obstetrical histories were obtained. Subjects agreed to allow investigators to capture any laboratory results, clinical events, or sonograph results that occurred between study visits. HIV DNA results for the infants were obtained at 0–2 days, 2–4 weeks, 4–6 weeks, and 4–6 months of life as part of routine infant care. All infants received oral zidovudine 2 mg/kg every 6 hours for 6 weeks postpartum in addition to intravenous zidovudine at delivery.
Subjects underwent 4 intensive PK visits with observed dosing within 15 minutes of a standard meal in the clinical research units at the following time points: 20–24 weeks (PK1), 30 weeks (PK2), 32 weeks (PK3) of gestation, and 8 weeks postpartum (PK4). Subjects were contacted 1 week before each PK visit to reinforce compliance, to record the time of each dose on provided cards, and to bring cards to study visit. Adherence was also assessed by pill counts at each study visit. Safety labs, including hemoglobin and chemistries, were obtained at all visits before blood samples for PK analysis were initiated. At each PK visit, blood samples were obtained 30 minutes before dosing and 2, 4, 6, 8, 10, and 12 hours postdose. The LPV/RTV dose was 400 mg/100 mg twice daily at PK1 and PK2. Immediately after completion of PK2, the dose was increased to 500 mg/125 mg twice daily by adding a 100 mg/25 mg tablet (pediatric formulation) to standard dose to determine possible effects on drug exposure using a smaller dose as an alternate dosing strategy. PK3 occurred 2 weeks later. The 500 mg/125 mg dose was continued for the remainder of pregnancy through 2 weeks postpartum, at which time, the dose was decreased to 400 mg/100 mg twice daily for the duration of the study.
Whole blood was obtained using K2-EDTA–containing collection tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged at 3000g for 15 minutes at 4°C within 30 minutes of collection. The resulting plasma was divided into labeled cryovials and stored at −80°C until analysis. Total drug concentrations in plasma for lopinavir and ritonavir were measured using validated and Division of AIDS CPQA-approved high-performance liquid chromatography method with ultraviolet detection. Intraday and interday variability was <10%, and the dynamic range was 10–10,000 ng/mL.16
Protein binding quantification was performed by rapid equilibrium dialysis as previously described.17 Briefly, plasma was incubated at 37°C for 18 hours in rapid equilibrium dialysis cartridges (Thermo Scientific, Pittsburgh, PA) followed by liquid–liquid extraction with methyl tert-butyl ether (MTBE) (Fisher Scientific, Norcross, GA). Darunavir (in 50/50 methanol:water) was used as the internal standard. An Agilent 1200 series HPLC System and an Agilent 1100 MSD (Agilent Technologies, New Castle, DE) in positive electrospray ionization mode were used. Analytes were separated on an Agilent Zorbax Eclipse XDB-C8 (3.0 × 50 mm, 1.8 m) column. For both lopinavir and ritonavir, assay sensitivity was 2 ng/mL, precision was within 15%, and accuracy was 95%–110% for lopinavir and 90%–107% for ritonavir. Recovery for both drugs was >90%.
PK parameters were estimated using noncompartmental methods (Phoenix WinNonlin Pro 5.2; Certara, L.P., St Louis, MO). Maximum concentration (Cmax), time at maximum concentration (Tmax), and concentration 12 hours after dosing (C12h) were determined by visual inspection of the subject profiles, and used the log-linear trapezoidal method to calculate the area under the time–concentration curve over the 12-hour dosing interval (AUC0–12h). Total drug apparent oral clearance was calculated as dose/AUC0–12h. Detectable concentrations that were below the limit of quantification were imputed as 50% of the lower limit of quantification. Descriptive statistics using Phoenix WinNonlin Pro 5.2 were performed. PK parameters are presented in median [interquartile range (IQR)] unless otherwise noted. Differences in PK parameters at different time points were assessed using the Wilcoxon signed-rank test. No adjustments were made for multiple comparisons.
The percent protein unbound of lopinavir was calculated by subtracting percent protein bound from 100%. Correlation between albumin or AAG and unbound drug concentrations were assessed using Spearman rank correlation coefficients.
Twelve HIV-infected pregnant women from 2 academic institutions (11 from University of North Carolina and 1 from Northwestern University) were enrolled, and all completed the study as designed. Demographic information is presented in Table 1. The median age of the participants was 28 (range, 18–35) years; median body mass index was 32 (range, 19–41) kg/m2 at enrollment. Nine (75%) were black. Eight subjects were antiretroviral naive and initiated therapy at a median of 127–18 weeks of gestation, consisting of lopinavir/ritonavir 400 mg/100 mg (LPV/RTV) twice daily in addition to the nucleoside reverse transcriptase inhibitor fixed-dose combinations of either zidovudine 300 mg/lamivudine 150 mg (ZDV/3TC) twice daily or tenofovir disoproxil fumarate 300 mg/emtricitabine 200 mg (TDF/FTC) once daily. The remaining 4 subjects had received 1 or 2 prior antiretroviral regimens. One subject was on ZDV/3TC plus an unboosted protease inhibitor before pregnancy and was switched to LPV/RTV after pregnancy was confirmed, and the other 3 subjects were not on antiretroviral therapy and initiated LPV/RTV (plus ZDV/3TC or TDF/FTC) after 12 weeks of gestation for fetal protection.
Study enrollment occurred between 16 and 20 weeks of gestation. At enrollment, 11 (92%) subjects were receiving ZDV/3TC and 1 (8%) TDF/FTC. One subject developed hemolytic anemia at 24 weeks of gestation, which had occurred in a pregnancy 12 years prior when the subject was HIV negative. As a result, ZDV/3TC was discontinued and the subject was changed to TDF/FTC for the remainder of the pregnancy. Therefore, 2 subjects received TDF/FTC for the majority of gestation. The median absolute CD4 count was 509 (116–880) cells per cubic millileters at 20–24 weeks. HIV RNA was <48 copies per milliliters in all 12 women at delivery. There were no perinatal transmissions.
Total lopinavir AUC0–12h did not change between the second and third trimesters (PK1 vs. PK2, P = 0.58) (Fig. 1A). The increased dose also did not result in a significant difference in total lopinavir AUC0–12h [PK2: median (IQR), 64.1 (51.3–69.7) vs. PK3: 69.1 (55.2–78.2) μg·h/mL, P = 0.27] (Table 2). Despite a 25% increase in dose, total lopinavir AUC0–12h increased by only 8% (P = 0.37). The median total lopinavir AUC0–12h, regardless of dose or gestation, was significantly lower than postpartum (PK1, PK2, or PK3 vs. PK4, P = 0.005). The apparent oral clearance of lopinavir was significantly higher during pregnancy than postpartum: 3.1 (2.2–4.2) L/h in the second trimester, 3.4 (3.1–4.2) in the third trimester, 3.6 (3.1–3.9) L/h after the dose increase, and 1.3 (0.6–1.8) L/h postpartum (PK1, PK2, or PK3 vs. PK4, respectively, P < 0.03).
The greatest changes in lopinavir C12h were between the third trimester (PK2) and postpartum (PK4) [4.0 (3.4–5.4) vs. 7.2 (6.1–9.3) μg/mL, respectively, P = 0.001] (Fig. 2A). Postpartum C12h was 38%–80% higher than at any point during pregnancy. The increased dose increase to 500 mg/125 mg resulted in a 23% increase (4.0 vs. 4.9 μg/mL, P = 0.03) in total lopinavir C12h as measured at 30 and 32 weeks, respectively.
Figure 1B highlights that neither dose nor gestation had an effect on unbound lopinavir median AUC0–12h. Table 2 more specifically outlines that lack of significant changes in median-unbound lopinavir AUC0–12h after the increased dose [PK2 vs. PK3, 1.6 (IQR, 1.3–1.9) vs. 1.8 (IQR, 1.3–2.1) μg·h/mL, respectively, P = 0.47]. Similarly, gestation period did not significantly alter unbound lopinavir exposure before the dose increase (PK1 vs. PK2, P = 0.41) or after the dose increase (PK1 vs. PK3, P = 0.76). Compared with the postpartum AUC0–12h, unbound lopinavir exposure was significantly lower throughout pregnancy regardless of dose (PK1, PK2, or PK3 vs. PK4, P = 0.05, P = 0.01, and P = 0.03, respectively).
The increased dose also did not significantly alter the median-unbound lopinavir C12h concentration [PK3 vs. PK2: 0.12 (0.10–0.15) vs. 0.10 (0.08–0.15) μg/mL, respectively, P = 0.09] (Fig. 2B). Unbound lopinavir C12h concentrations were highest in the second trimester [PK1: 0.15 (0.08–0.16) μg/mL] and did not significantly change after delivery [0.16 (0.14–0.27) μg/mL, P = 0.09]. Only the C12h at 30 weeks of gestation before dose escalation (PK2) was significantly different from the postpartum C12h [PK2: 0.10 (0.08–0.15) vs. PK4: 0.16 (0.14–0.27) μg/mL, P = 0.05]. Less than 2% of all concentrations were below the wild-type IC50 (50% inhibitory concentration) for unbound lopinavir of 0.00064 μg/mL.18
Total ritonavir median AUC0–12h was lower throughout pregnancy, regardless of dose increase, compared with postpartum AUC0–12h (Fig. 1C). The median total ritonavir AUC0–12h in the second trimester (PK1), the third trimester (PK2), and after the dose increase (PK3) were 2.5 (1.7–3.0), 2.3 (1.7–3.0), and 2.4 (1.9–3.3) μg·h/mL, respectively, compared with 5.2 (2.9–5.8) μg·h/mL postpartum (PK4) (P = 0.002, P = 0.002, and P = 0.002, respectively). Overall, total ritonavir exposure was not significantly changed during pregnancy. Apparent oral clearance of ritonavir (Table 2) was greater during pregnancy than the postpartum period (PK1, PK2, or PK3 vs. PK4, P < 0.01).
Total ritonavir C12h were similar between the second and third trimesters [0.12 (0.09–0.20) vs. 0.12 (0.08–0.14) μg/mL, respectively, P = 0.5] (Fig. 2C). The higher dose did result in a significant increase in median C12h to 0.15 (0.11–0.18) μg/mL, P = 0.02. Total ritonavir C12h was significantly lower during pregnancy, despite the additional dose, compared with postpartum C12h [0.28 (0.2–0.4) μg/mL; PK1, PK2, or PK3 vs. PK4, P ≤ 0.01].
Similar to lopinavir, the protein-unbound ritonavir median AUC0–12h did not change regardless of gestation or dose (Fig. 1D). The dose increase (PK2 vs. PK3, 0.3 vs. 0.3 μg·h/mL, P = 1.0) did not alter the unbound ritonavir exposure. Nor was there a difference in exposure in the second or third trimesters before (PK1 vs. PK2, P = 0.12) or after (PK1 vs. PK3, P = 0.97) the dose increase. Postpartum exposure was significantly higher than during pregnancy regardless of dose (PK1, PK2, or PK3 vs PK4, P = 0.01, P = 0.01, and P = 0.03, respectively).
The increased dose did not significantly alter the median-unbound ritonavir C12h (PK3 vs. PK2, P = 1.0). The unbound ritonavir C12h was highest in the second trimester (PK1) (0.02 μg/mL) and was the only time in which a significant difference in postpartum C12h was found (0.02 vs. 0.03 μg/mL, P = 0.01).
Less than 1% of all unbound ritonavir concentrations were under 1 mg/mL. A single subject had concentrations below this threshold at the beginning of her postpartum visit, suggesting a missed dose or a delay in dosage.
The median (IQR) albumin and AAG are shown in Table 1. There were no significant changes in albumin between the first 3 PK evaluations (PK1 vs. PK2: P = 0.2; PK1 vs. PK3: P = 0.8, and PK2 vs. PK3: P = 0.9). There were also no significant changes in AAG (PK1 vs. PK2: P = 0.4; PK1 vs. PK3: P = 0.2, and PK2 vs. PK3: P = 0.5). Both albumin and AAG were significantly greater at postpartum PK4 (P < 0.001). The drug-binding proteins albumin and AAG were modestly correlated (rs = 0.48, P = 0.0006), as were the PK parameters LPV AUC0–12h and C12h (rs = 0.47, P = 0.0009). However, in this data set, only albumin significantly correlated with LPV AUC0–12h fraction unbound (rs = 0.3, P = 0.03).
The additional 100 mg/25 mg LPV/RTV tablet was tolerated without any complaints of nausea or vomiting. There were no symptomatic or biochemical adverse events above grade 1 that were related to LPV/RTV. No subject required premature discontinuation or dosing alterations.
The widespread use of antiretroviral therapy has resulted in reduction of perinatal HIV transmission rates to <1%–2% in developed countries.19 However, the pregnancy-associated variations in drug-binding proteins may alter antiretroviral bioactivity,20,21 resulting in decreased efficacy, suboptimal maternal viral suppression, and ultimately perinatal transmission. Conversely, overdosing unnecessarily may increase maternal and fetal toxicity.22 Because of such risks, understanding the PKs of LPV/RTV is paramount to provide dosing guidance for LPV/RTV in HIV-infected pregnant women.
Unlike previous evaluations,5,6,8,23,24 this study used a unique design in which comprehensive PK measurements of both total (protein bound and unbound) and protein-unbound lopinavir and ritonavir were performed in the same 12 women at predetermined gestational ages. The intrasubject comparison strategy has not been previously used but is essential in assessing the impact of dose escalation on both the total drug and protein-unbound drug concentrations. Moreover, intensive PK assessments were performed before and after a 100 mg/25 mg (or 25% of standard dosing) pediatric tablet was empirically added to standard dosing (400 mg/100 mg) as an alternative to “recommended” third trimester dose increases (600 mg/150 mg or 50% dose increase). Our a priori hypothesis was that dose increases were not necessary as protein-unbound (or active) drug concentrations were not expected to significantly change despite previously reported changes in total drug concentrations.3,5,6,8,15 The ability to assess tolerability and potential toxicity of a 25% rather than a 50% dose increase would provide clinically useful information as an alternate strategy for women who may require a dose adjustment. Two weeks postpartum, all women return to standard 400 mg/100 mg dosing. PKs were subsequently performed 8 weeks postpartum consistent with normalization of plasma concentrations in other studies,15 although the precise timing of normalization of pregnancy-related changes has not been determined. These factors provide the most comprehensive PK analysis of LPV/RTV in HIV-infected pregnant women to date.
Our study found that a 25% increase in LPV/RTV dose results in <10% increase in total lopinavir exposure, which is not clinically relevant as concentrations remain above the minimum effective concentration (1 μg/mL for wild-type virus).25 One explanation for the decrease total lopinavir concentrations is an increase in total lopinavir clearance. Our estimate of lopinavir clearance during pregnancy is between 120%–216% higher than postpartum estimates, potentially due to changes in enzymatic processes.26 The relative consistency of Cmax would suggest that oral absorption remains stable.
Higher total lopinavir C12h (4 μg/mL)27,28 is targeted in protease inhibitor treatment-experienced patients. Of the 12 participants in this study, standard dose LPV/RTV results in total lopinavir C12h below this threshold in 33% (n = 4) of women in the second trimester and 50% (n = 6) in the third trimester. After the dose increase, 91% (n = 11) of women achieve C12h above 4 μg/mL with C12h of 2.9 μg/mL in a single-naive subject. All participants remain virologically suppressed before and after dose changes despite the decrease in C12h. However, our study population, by circumstance, had received 2 or less prior regimens. Our study suggests that evaluating C12h in the later part of the second trimester may be beneficial in women who are treatment experienced to determine whether predose concentrations are below target and therefore require a dose increase. This strategy would diminish the risk of potential toxicities when a dose increase is not necessary. The use of the pediatric formulation (100 mg/25 mg) provides an alternative dosing strategy when the C12h falls slightly below the targeted threshold, further minimizing the risk of toxicities.
The pharmacological effects of LPV/RTV are determined by the protein-unbound (or active) drug concentration, as only the unbound drug is able to transverse biological membranes and exert antiviral activity. In contrast to previous investigations, full PK profiles of unbound lopinavir and ritonavir were generated with our methodology. Using a protein-unbound IC50 (50% inhibitory concentration) for lopinavir of 0.00064–0.00077 μg/mL,18 unbound C12h exposures in all women were greater than 70-fold, this threshold in the third trimester before the dose increase. The increased dose resulted in <15% increase in protein-unbound AUC0–12h and <12% increase in C12h. The unbound fraction of lopinavir did not significantly change during the second or third trimesters and postpartum regardless of dose, and AUC0–12h modestly correlated with physiological changes in albumin. The addition of a standard LPV/RTV tablet (200 mg/50 mg) could possibly have resulted in higher protein-unbound C12h, which may have been unlikely to benefit our study participants and could have resulted in toxicity or intolerability.
In conclusion, this study demonstrates the PK effects of pregnancy on total and unbound lopinavir and ritonavir concentrations before and after empiric dose escalation. For many pregnant women, especially treatment naive or women with wild-type virus, LPV/RTV dose increases during pregnancy may not be necessary because the protein-unbound drug exposure is not altered. If drug exposure in women with resistance mutations is of concern, a dose increase of 100 mg/25 mg could be used to minimize toxicity. Future studies aimed toward providing accurate dosing recommendations in pregnancy of any antiretroviral agent will be best achieved through a longitudinal evaluation of total and unbound drug exposures throughout pregnancy in the same women.
The authors thank Certara for providing Phoenix WinNonlin Pro 5.2 to the Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, as a member of the Pharsight Academic Center of Excellence Program.
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