Method
Study design
We performed a longitudinal pharmacokinetic study in pregnant women ≥ 18
years of age treated for RR-TB, and their infants, at King Dinuzulu
Hospital in Durban, Kwazulu-Natal - nested within a cohort, which has
been previously described. (13) KDH is a specialist provincial RR-TB
hospital where, until recently, all pregnant women with RR-TB were
referred for care. With some individual regimen variability, all
participants were treated with a minimum of five drugs including
bedaquiline. Other drugs included: pyrazinamide, isoniazid, clofazimine,
linezolid, moxifloxacin, and less commonly: ethambutol, terizidone,
levofloxacin, ethionamide and para-aminosalycylic acid. We performed
pharmacokinetic sampling pre-dose, and two, four and six hours post-dose
in the third trimester of pregnancy (≥28 weeks), and at the six week
postpartum visit. Dosing on both sampling days was observed after a
standard breakfast; the tablets/capsules were ingested with 250 mL of
water. Considering bedaquiline is dosed three times a week (after the
two week loading dose), it was not always logistically possible to
schedule pharmacokinetic sampling on a day when bedaquiline was
administered. We therefore recorded the last date and time when
bedaquiline was dosed to interpret the exposures with our modelling. All
concurrent medications, including antiretroviral therapy were recorded.
If available, human milk samples were taken from breastfeeding mothers
by manual expression at the same timepoints that blood was drawn at the
postpartum visit (i.e. pre-dose, and two, four and six hours post-dose);
samples were frozen within 30 minutes of sampling at minus 80° C. To
evaluate infant drug exposure, a single random plasma sample was taken
from infants at the postpartum visit. If applicable, the time of the
most recent breastfeeding prior to the infant blood draw was recorded.
Pharmacokinetics
Plasma and human milk samples were stored at minus 80°C and transported
to the University of Cape Town, Division of Clinical Pharmacology
laboratory where total plasma and human milk bedaquiline assays were
performed using liquid chromatography with tandem mass spectrometry. The
plasma assay for total bedaquiline has previously been described. (14)
Bedaquiline and its M2 metabolite in human milk were analysed with a
validated assay developed at the Division of Clinical Pharmacology
laboratory; the standards and quality checks were performed using blank
donated human milk. The extraction procedure consisted of protein
precipitation and solid phase extraction, followed by gradient liquid
chromatography on an Agilent Poroshell 120 SB-C18 (2.1 mm x 50 mm, 2.7
μm) analytical column with tandem mass spectrometry detection. An AB
Sciex API 3000 mass spectrometer at unit resolution in the multiple
reaction monitoring mode was used to monitor the transitions of the
protonated precursor ions m/z 555.1, m/z 561.1, m/z 541.1, and m/z 545.1
to the product ions m/z 58.2, m/z 64.1, m/z 480.3, and m/z 480.4 for
bedaquiline, TMC207-d6, M2, and M2-d3C13, respectively. Electro Spray
Ionisation was used for ion production. The calibration curves fitted
quadratic (weighted by 1/x) regressions based on peak area ratios over
the ranges 0.0780 – 5.00 µg/mL for bedaquiline and 0.0312 – 2.00 µg/mL
for M2. The combined accuracy (%Nom) and precision (%CV) statistics of
the lower limit of quantification, low, medium, and high quality
controls of bedaquiline and M2 during intra- and inter validations were
between 96.7% and 106.5%, and 3.4% and 7.5%, respectively.
Modelling
Bedaquiline concentrations were interpreted using population
pharmacokinetic modelling in NONMEM version 7.4.5 (15).
Perl-speaks-NONMEM version 5.2.6, Pirana version 3.0, and R with the
package xpose4 were used to facilitate the model development process,
data manipulation, and generation of model diagnostics (16). As a
starting point, we used a published population pharmacokinetic model of
bedaquiline in non-pregnant adults with HIV and drug-resistant
tuberculosis (14). Briefly, the published model consists of three
disposition compartments for bedaquiline and one disposition compartment
for M2. There was a correlation between bedaquiline and M2
between-subject-variability on clearance, and residual variabilities.
The effect of body weight on all disposition parameters was included
using allometric scaling, and albumin also affects the drug disposition
parameters. The co-administration of ritonavir-boosted lopinavir reduced
bedaquiline and M2 clearance by 65% and 42%, respectively. Molar
concentrations were used during model development to account for mass
balance between bedaquiline and its metabolite M2. Participant albumin
information were not captured in the current study, therefore we imputed
a reported albumin concentration from a previously study in South Africa
patients with RR-TB (17).
When analysing the data, we first fit the original model as published,
without re-estimating any of the population parameters, but using the
study covariate, doses and dosing regimen information. This is similar
to using the current data as an “external” validation of the model,
i.e. assessing how the previous model predicts the current data based
solely on covariate information and assuming no effect of pregnancy
(which was not part of the origina model). Afterwards, we attempted to
use the data to re-estimate parameter values, using the general
principles of model development including drops in NONMEM objective
function value (OFV) for assessment of statistical significance and
inspection of diagnostic plots.
Calculation of infant bedaquiline intake with human
milk
To estimate how much bedaquiline is ingested per day by a typical child
breastfed by a mother receiving bedaquiline, we assumed an average
infant milk ingestion of 0.15 L/kg/day (18). The following equation was
used to calculate the infant dose (19):
\begin{equation}
D_{\text{infant}}=\ C_{m}\bullet\ V_{m}\nonumber \\
\end{equation}where Vm is the volume of milk ingested by breastfeeding, andCm is the drug concentration in human milk. This
was calculated using the formula below:
\begin{equation}
C_{m}=M:P\ \bullet\ C_{\text{pss}}\nonumber \\
\end{equation}where Cpss is the average maternal plasma level
at a steady-state, and M:P is the human milk-to-plasma ratio.
Ethics
Ethics approval for the study was granted by the South African Medical
Research Council Ethics Committee (EC017-6/2016), and the University of
Cape Town Human Research Ethics Committee (HREC: 666/2018). Informed
consent was taken from all participants in a language of their choice
(either English or isiXhosa).