Therapeutic drug Monitoring of Darunavir/Ritonavir in Pregnancy

Abstract

Background

Physiological changes during pregnancy can have a significant impact on antiretroviral pharmacokinetics (PK), which may result in reduced drug efficacy. Here we describe the PK of darunavir/ritonavir (DRV/r) 800/100 once daily in a cohort of pregnant women undergoing routine therapeutic drug monitoring (TDM) as well as transplacental passage of DRV by measuring and comparing cord blood and maternal blood samples at delivery.

Methods

Pregnant HIV-positive women received DRV/r as part of routine pre-natal care. Demographic and clinical data were collected. DRV plasma concentrations [DRV] were determined in the first (T1), second (T2) and third (T3) trimester and at postpartum (PP). The target concentration was 550 ng/ml. Where possible, paired maternal and cord blood samples were taken at delivery.

Results

A total of 33 women were enrolled. Samples were taken 14–20 h post-dose and measured concentrations were extrapolated to 24 h post-dose. At the time nearest to delivery, all but four had undetectable plasma viral loads (pVL). [DRV] were determined in 1 (T1); 14 (T2); 32 (T3) and 29 (PP). 1 sample was <550 ng/ml at T2, 6 at T3 and 3 at PP. [DRV] were significantly lower at T2/T3 relative to PP.

Conclusions

[DRV] in T2 and T3 were 36–55% when compared with PP. However, DRV PK in pregnancy were not associated with a lack of virological suppression at delivery as of the 33 patients enrolled in this study, 31 had no HIV transmission from mother to child. Data regarding two candidates were not available as they delivered in a separate health-care facility.

Introduction

Pregnancy is a state associated with significant alteration to drug absorption, distribution, metabolism and elimination, which can affect the efficacy of antiretroviral therapy for both mother and child [1,2]. Studies to better understand the pharmacokinetics of cART in pregnancy are an ongoing need in order to optimize therapy in this population.

The aim of this paper was to study the plasma concentrations, antiviral activity and safety of darunavir/ritonavir (DRV/r) 800 mg/100 mg once daily in HIV-infected pregnant women.

Methods

Subjects

HIV-infected pregnant women in receipt of DRV/r 800 mg/100 mg once daily attending the Rotunda Hospital were recruited and consented to be part of this study.

Study design

Blood sampling was performed in the first trimester (T1), second trimester (T2), third trimester (T3) and postpartum (PP). Maternal and cord blood samples were taken at labour and delivery. Demographic and clinical parameters were collected. Plasma viral load (pVL) of HIV and CD4⁺ T-cell counts were measured at time of TDM sampling and at delivery.

Analytical and pharmacokinetic methods

Blood samples were taken by venepuncture the morning after the previous dose of DRV/r (approximately 14–20 h post-dose). DRV/r was taken with food, as per recommended dosing guidelines [3]. Time of drug intake was recorded.

Blood was collected in heparin tubes and centrifuged at 1,000 g, 10 min; 4°C and the plasma removed and stored at −30°C. Prior to analysis the plasma was heat inactivated (58°C, 40 min). Total plasma DRV concentrations were determined using a validated HPLC-MS/MS methodology (Lab21, Cambridge, UK) [4]. The assay lower limit of quantification (LLQ) for DRV was 78.1 ng/ml.

Data analysis

Measured DRV plasma concentrations were expressed as the geometric mean (95% CI). The measured concentrations were then extrapolated to 24 h post-dose (C₂₄; ng/ml) using the established half-life (15 h) for DRV in the presence of RTV [3]. This enabled normalization of the effect of variable sampling times post drug intake. Values with missing drug intake data were excluded. Inter-subject variation in plasma concentrations was estimated using a coefficient of variation, expressed as a percentage (CV%) [%CV = (standard deviation/mean)*100]. DRV plasma concentrations at antepartum and PP were related to the drug's 50% effective concentration (EC₅₀; 550 ng/ml) for protease inhibitor resistant HIV-1 strains [3]. Placental transfer of DRV was determined by calculation of a cord blood-to-maternal plasma ratio (C/M ratio) from maternal and cord blood samples obtained at delivery.

Differences between antepartum and PP drug concentrations (both paired and independent data) were assessed using a one-way ANOVA, with a Bonferroni correction to test for multiple comparisons. Data were log transformed prior to statistical analysis. All statistics were performed and analysed using Statsdirect [5]. P-values were two-sided at the 0.05 significance level.

Ethical review

Ethical approval was granted by the Rotunda Hospital's research ethics committee.

Results

A total of 33 women were enrolled in the study. 11/33 were newly diagnosed at the antenatal clinic. Patient baseline characteristics are summarized in Table 1. 15 women were receiving ART prior to pregnancy. 18 women (11 treatment-naive, 7 experienced) initiated DRV/r therapy in pregnancy. Median (range) gestational age at time of DRV/r treatment initiation was 19 weeks (13–24).

Table 1

Baseline demographics and clinical characteristics

	Value
Age, years^a	35 (20–44; 33)
Ethnicity^b
Black African	22 (67)
Caucasian	11 (33)
Patients initiating ART in pregnancy^b	18 (55)
Naive to ART at baseline^{b, c}	Yes 11 (61); no 7 (38)
Gestational age at ART initiation, weeks^{^{a, c}}	19 (13.0–24.0; 18)
Gestational age at first TDM, weeks^a	26 (13.0–38; 33)
Co-ART^b
Combivir	2 (6)
Kivexa	1 (3)
Raltegravir	2 (6)
Truvada	28 (85)

Total n=33.

Values given as median (range; n/33).

Values given as n (%).

In patients initiating antiretroviral therapy (ART) during pregnancy. TDM, therapeutic drug monitoring.

DRV concentrations were determined in one patient in T1, 14 in T2 and 32 in T3. Median (range) gestational age at time of pharmacokinetic sampling was 21.5 weeks (20–24) in T2 and 30 weeks (25–39) in T3, respectively. 29 patients had measurements PP with a median follow up time after delivery of 8 weeks (2–57).

Measured concentrations of DRV in T1, T2, T3 and PP extrapolated to 24 h post-dose (C₂₄) are presented in Table 2.

Table 2

Darunavir plasma concentrations (observed and extrapolated C₂₄) and virological/immunological responses in 33 HIV-infected pregnant women receiving DRV/r 800/100 mg once daily

	T1 (n=1)	T2 (n=14)	T3 (n=32)	PP (n=29)	P-value
Measured [DRV], ng/ml^a	3,790	1,401 (1,041, 1,760)	1,201 (910, 1,493)	2,588 (1,655, 3,521)	P<0.0001 (all comparisons)
					P=0.003 (T2 versus PP)
					P<0.0001 (T3 versus PP)
CV, %	–	49	69	94	–
<550 ng/ml^b	0 (0)	0 (0)	6 (19)	3 (10)	–
Time post-dose, h^c	14.5	18.0 (14.5–24.0)	19.7 (11.5–25.4)	18.6 (10.0–29.3)	P=0.510 (all comparisons)
Extrapolated [DRV] C₂₄, ng/ml^a	2,280	1,069 (770, 1,368)	961 (683, 1,239)	2,111 (942, 3,281)	P<0.0001 (all comparisons) P=0.003 (T2 versus PP)
CV, %	–	53	82	144	P<0.0001 (T3 versus PP)
C₂₄ <550 ng/ml^b	0 (0)	1 (7)	6 (19)	3 (10)	–
Gestation/PP, weeks^c at time of TDM	13.0	21.5 (20–24)	30 (25–39)	8.0 (2–57)	–
pVL <50 copies/ml^b	1 (100)	11 (79)	23 (72)	24 (86)	–
CD4⁺ T-cell count, cells/μl^c	346 (1)	453 (209–723; 14)	462 (62–1,330; 31)	562 (111–1,697; 29)	–
Weight, kg^c	115	73.8 (65.0–118; 14)	74.3 (53.4–124; 30)	70.1 (43.6–119)	–

Values given as geometric mean (95% CI).

Values given as n (%).

Values given as median (range; n). CV, coefficient of variation; C₂₄, trough concentration; 550 ng/ml = darunavir (DRV) minimum effective concentration; PP, postpartum; pVL, plasma viral load; TDM, therapeutic drug monitoring; T1, first trimester; T2, second trimester; T3, third trimester. Bonferroni critical P for 3 comparisons (T2/T3/PP) =0.017. Assay limit of quantification (LLQ) =78.1 ng/ml; T3 = one patient had [DRV] <LLQ; PP = three patients had [DRV] <LLQ.

The difference in the total number of patients who had TDM undertaken in T2 (n=14), T3 (n=32) and PP (n=29), indicate the possibility of inaccuracy due to different cohort sample sizes. Considering only the plasma concentrations of patients who had TDM sampling in T2, T3 and PP inclusive (n=10), we see a difference of 12% between T2 (1,463 ng/ml, 982–1,945) and T3 (1,289 ng/ml, 842–1,736; P=0.585), and a 48–53% difference between antepartum and PP (2,802 ng/ml, 1,847–3,756; P<0.009). Following normalization there was a 45–53% decrease in 24 h trough concentrations antepartum compared with PP (P<0.015), and a 15% difference between T2 and T3, respectively (P=0.474).

Inter-subject variation in DRV plasma concentrations was moderately high throughout pregnancy (49–69%) and increased at PP (94%). Median time of sampling (post-dose) was consistent during T2, T3 and PP, at approximately 18–20 h (P=0.510).

5/33 (15%) patients had DRV concentrations below the proposed DRV MEC (<550 ng/ml) in T3, with one undetectable reading. Only one of the five patients had her DRV concentration checked during T2, and it was noted to be above 550 ng/ml then. One of the five patients developed swine flu during T3 testing, which may have affected medication compliance. All five achieved undetectable pVL based on the VL closest to delivery. At PP, one patient had DRV TDM <550 ng/ml, two had undetectable readings and two were unavailable. One of the patients with undetectable readings admitted to reduced medication compliance due to forgetfulness. The one patient with a DRV TDM <550 ng/ml had a TDM of 346 during T3.

18 patients had paired maternal and umbilical cord blood samples taken at delivery to ascertain DRV placental transfer. 15 of them were taken before delivery (median time of 5.13 h, range: 1.00–17.45 h), 2 after delivery (median time of 3.37 h, range: 1.65–5.1 h), 1 at delivery. Cord blood samples were obtained minutes after delivery, and DRV plasma concentrations are depicted in Figure 1. The median (range) time from last DRV dose and collection of maternal and cord blood was 11 h (1.9–36) and 15 h (1.9–44), respectively. DRV was detectable in all maternal plasma samples, and in 17/18 cord plasma samples. One subject with undetectable DRV in cord plasma (<78 ng/ml), had corresponding maternal plasma concentration of 607 ng/ml. Geometric mean (95% CI) DRV concentrations were higher in maternal plasma (1,878 ng/ml, 1,172–2,584) than cord plasma (309 ng/ml, 0.3–618) in 16/18 patients with paired samples. Median C/M ratio was 0.11 (0.06–2.46).

Figure 1

DRV maternal and cord plasma concentrations DRV, darunavir; LLQ, lower limit of quantification.

At the time nearest to delivery 28 patients had undetectable pVL, 4 patients had detectable pVL (77, 91, 114, 176 copies/ml) with 1 unavailable. Although TDM sampling in these subjects was limited, two/four patients had therapeutic concentrations in T2 with the other two not having a TDM. In T3, three of four patients had therapeutic concentrations and one had subtherapeutic levels. One subject had undetectable plasma concentrations in T3, suggesting non-adherence. Of 33 livebirths, one death occurred due to Trisomy 13. Two patients (6%) delivered in another maternity unit, so mode of delivery was unavailable. Of the remaining 31, 14 (45%) had spontaneous vaginal delivery and 17 (55%) were by caesarean section (8 elective; 9 emergency). Median gestational age at delivery was 39.6 weeks (30.0–41.6 weeks) and median birthweight 3.16 kg (1.76–3.89 kg). Of the 31 neonates for whom data were available, all were HIV-negative.

Discussion

Our study reflects a common trend where plasma concentrations of DRV progressively drop from T1–T3, then showing a rise PP, noted in both DRV/r 600 mg/100 mg twice daily and 800 mg/100 mg once daily [2,3,6–15]. Participants in our study exhibited median plasma DRV concentrations of 3,790 ng/ml, 1,401 ng/ml, 1,201 ng/ml and 2,588 ng/ml in T1, T2, T3 and the PP period, respectively. However, it should be noted that data from T1 is representative of a single patient. This is in line with previously reported data on DRV/r 800 mg/100 mg once daily by Colbers et al. [6] which observed a median C₂₄ of 1,140 ng/ml and 2,050 ng/ml at T3 and PP respectively. DRV EC₅₀ is 55 ng/ml for wild-type virus and 550 ng/ml for resistant virus strains [3]. A single patient (4%) had C₂₄ <550 ng/ml at T2, 6 (19%) at T3 and 3 (10%) at PP. One patient (3%) at T3 and 3 (10%) at PP had DRV concentrations <LLQ. In the majority of our patients, plasma C₂₄ remained above 550 ng/ml throughout pregnancy and in PP, supporting claims that DRV/r 800 mg/100 mg once daily is sufficient. This study is unfortunately limited by the lack of T1 data, but the critical time for transmission is late in pregnancy, and an undetectable VL at this time period is critical to prevent peri-partum transmission. However, there have been documented cases where perinatal transmission has occurred despite undetectable or low levels of HIV RNA in pregnant mothers [16–18], suggesting that maternal viral suppression alone is insufficient and that transplacental transfer of ART to provide peripartum prophylaxis is another important factor which may require monitoring.

Protease inhibitors such as DRV are documented to have a relatively low degree of transplacental transfer with considerable variation between individuals [2,3,7,18,19]. Foetal DRV concentrations are the result of multiple factors, primarily maternal plasma concentration, placental blood flow, placental permeability and the rate of foetal elimination of darunavir [19].

The Department of Health and Human Services (DHHS), the International Antiviral Society-USA, and French guidelines recommend a dose of 600/100 mg twice daily DRV/r in pregnancy [8,20,21]. Guidelines from the British HIV Association (BHIVA) recommend continuation of the 800/100 mg once daily dose in pregnant women who have plasma HIV RNA suppression [22]. According to prescribing information from Janssen, DRV/r 800/100 mg once daily should only be considered in certain pregnant patients who are already on a stable regimen of DRV/r 800/100 mg once daily prior to pregnancy and virologically supressed (HIV-1 RNA <50 copies/ml), and in whom a change to twice-daily DRV/r 600/100 mg might compromise tolerability or compliance [3].

Our study observes the ability of DRV/r 800 mg/100 mg once daily at providing peripartum prophylaxis by comparing cord levels obtained from our patients and comparing them to documented and published EC₅₀ levels. Janssen quotes an in vitro EC₅₀ of 1.2 to 8.5 nM (0.7 to 4.7 ng/ml) [3]. Cord blood levels were more than 10-fold of the quoted values, which correlates with good in vitro viral suppression.

In conclusion, we have shown adequate DRV levels and VL suppression with a once-daily regime of DRV/r 800 mg/100 mg. We suggest that once-daily dosing of DRV/r 800 mg/100 mg provides sufficient exposure with good efficacy, viral suppression and peripartum prophylaxis. Further studies should be conducted to confirm these findings.

Footnotes

Acknowledgements

This research was supported by a grant from Janssen-Cilag Ltd.

The authors have no conflicts of interest to disclose.

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