Preliminary results of identification and quantification of paclitaxel and its metabolites in human meconium from newborns with gestational chemotherapeutic exposure
Preliminary results of identification and quantification of paclitaxel and its metabolites in human meconium from newborns with gestational chemotherapeutic exposure
Elyce Cardonick 0 1
Robert Broadrup 1
Peining Xu 1
Mary T. Doan 1
Helen Jiang 1
Nathaniel W. SnyderID 1
0 Cooper Medical School, Rowan University , Camden , New Jersey, United States of America, 2 AJ Drexel Autism Institute, Drexel University , Philadelphia, Pennsylvania , United States of America
1 Editor: Eduardo Ortiz-Panozo, National Institute of Public Health , MEXICO
Cancer diagnosis during pregnancy occurs in 1 out of 1000 pregnancies with common malignancies including breast and hematological cancers. Fetal exposure to currently utilized agents is poorly described. We directly assessed fetal exposure by screening meconium from 23 newborns whose mothers had undergone treatment for cancer during pregnancy.
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This work was supported by National
Institute of Health Grants R21HD087866,
K22ES026235, and R03CA211820 to NWS. The
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
Meconium was collected from newborns whose mothers were diagnosed with cancer during
pregnancy and underwent chemotherapy in the second or third trimester as part of the
Cancer and Pregnancy Registry. We conducted screening of 23 meconium samples for
chemotherapeutics and known metabolites of chemotherapeutics by liquid chromatography-high
resolution mass spectrometry (LC-HRMS). Putative identification of paclitaxel and/or its
metabolites was made in 8 screened samples. In positively screened samples, we
quantified paclitaxel, 3?-p-hydroxypaclitaxel, and 6?-hydroxypaclitaxel by stable isotope
Mean (standard deviation) levels of paclitaxel in positively screened samples were 399.9
(248.6) pg/mg in meconium samples from newborn born to mothers that underwent
chemotherapy during pregnancy. 3?-p-hydroxypaclitaxel and 6?-hydroxypaclitaxel mean levels
were 105.2 (54.6) and 113.4 (48.9) pg/mg meconium, respectively.
Intact paclitaxel, 3?-p-hydroxypaclitaxel, and 6?-hydroxypaclitaxel were detected in
meconium, providing unambiguous confirmation of human fetal exposure. Variability in meconium
Competing interests: The authors have declared
that no competing interests exist.
levels between individuals may indicate a potential for reducing fetal exposure based on
timing, dosing, and individual characteristics. This preliminary study may provide an approach
for examining the effects of cancer diagnosis during pregnancy on other outcomes by
providing a measure of direct fetal exposure.
Cancer diagnosis occurs in 1 in 1000 pregnancies. The most common malignancies
complicating pregnancy are breast cancer, Hodgkin?s and Non-Hodgkin?s lymphoma, and melanoma
. The most common agents used during pregnancy include doxorubicin,
cyclophosphamide, epirubicin, 5-fluouracil and more recently paclitaxel. Studies of effects of chemotherapy
exposure during the second and third trimester have been reassuring, where the majority detail
the physical appearance at birth and general health during the first year of life [
]. A limited
number of studies provide longer follow up with developmental and physical evaluation of
children exposed in utero to chemotherapy up until the late teen years and young adulthood
]. In all existing studies, study design has used inclusion by maternal chemotherapy
during pregnancy correlated with post-natal growth and development of the children without a
direct measure of fetal exposure and internalized dose to the fetus.
Pharmacokinetics of drugs in an obstetric population is complicated by changes in the
mothers? pharmacokinetic parameters and in the unique placental/fetal compartments.
Differential toxicokinetics and timing of exposure impacts the potential effects from maternal
exposure, placental exposure, and direct fetal exposure [
]. Lipophilicity, affinity for
transporters, molecular size of the agent, and variations in biotransformation lead to
differential disposition across the maternal/fetal compartments and thus differential exposure for
mother versus child. Although maternal exposure is often quantified by urine and blood
analysis, direct fetal exposure is more difficult to ascertain and most would involve invasive
measures such as amniocentesis or cordocentesis each of which carries risk to the ongoing
pregnancy. Studies in non-human primate pregnant baboon model transplacental disposition
of chemotherapeutics was possible and fetal exposure to taxanes (paclitaxel and docetaxel)
occurred hours after infusion . Longer timepoints with docetaxel indicated a fetal
reservoir, with higher fetal than maternal liver concentrations 72 hours after infusion. However, it
is unknown whether fetal exposure occurs in human pregnancy and for which
Meconium, the first stool of a newborn that passes in the first few days after birth, begins
accumulation around the 13th week of gestation. For this reason, it provides a unique window
into gestational metabolism and exposures. Meconium also has the advantage of non-invasive
collection, making it a useful biosample in epidemiologic studies of humans[
]. This has been
used to quantify maternal use of drugs of abuse and environmental exposures during
], fetal exposure to anti-retrovirals [
], metabolomics during gestational diabetes
], and fetal sex steroid metabolism [
]. To fill in the gap of the knowledge of human
exposure in utero to chemotherapeutic agents we employed liquid chromatography-high resolution
mass spectrometry (LC-HRMS) to analyze meconium sampled from newborns whose mothers
underwent chemotherapy during pregnancy. Since no previous information was available on
human meconium analysis for chemotherapeutics to facilitate a directly targeted approach, we
used a two-step approach where we screened for the presence of major chemotherapeutics in
the meconium, detected paclitaxel, and then quantified paclitaxel and its major metabolites in
the positive samples.
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Materials and methods
This study was approved under the Cooper Health System IRB #1-028EX maternal cancer
diagnosis and treatment during pregnancy: Longitudinal follow-up of child development and
maternal psychological well being. Since the Cancer and Pregnancy Registry was first created,
382 women have voluntarily enrolled and provided diagnostic and treatment information
regarding their cancer during and after pregnancy. Of these women, 268 have received
chemotherapy during pregnancy. Since starting the current project, 30 women have enrolled and
received chemotherapy, and 23 have agreed to collect meconium at the time of delivering their
child. The majority of women in the study (20) were treated for breast cancer, 1 for colon
cancer and 2 for Hodgkin?s Lymphoma. Mean gestational age at the first chemotherapy treatment
was 20 +/- 4.8 weeks, and mean number of days between last treatment during pregnancy and
day of birth was 36 +/- 20.6 days. Other than parental education, sociodemographic
information was not collected. Plans for follow up in this study include annual tracking through
pediatricians of height and weight percentile, developmental milestones, and treatment for any
illness. Standardized developmental testing will be administered based on future funding and
Patients treated with chemotherapy during pregnancy who consented to participate in this
study were instructed to collect a single sample from the first 3 stools on day one of life.
Meconium was collected on day 1 of life in all cases. Collection was performed by the mother herself,
sent frozen in provided pre-labeled packaging and when received, were stored at -80?C until
batched and shipped to the laboratory for analysis. Meconium was also collected from healthy
newborns from the Hospital of the University of Pennsylvania in collaboration with the March
of Dimes prematurity research center, and then transmitted as de-identified samples falling
under exempt human subjects research as determined by the Drexel IRB. Samples were
collected by nurses in the hospital, stored at 4?C for under 24 hours, and then at -80?C until use.
These samples were used for analytical method development, as negative control meconium,
and as a matrix for spiking in analytes to generate quality control samples.
Water, methanol, ethyl acetate, methyl tert-butyl ether (MTBE), acetonitrile, and acetic acid
were Optima LC-MS grade solvents from Fisher Scientific (Pittsburgh, PA). Sodium chloride
and paclitaxel were from Sigma-Aldrich (St. Louis, MO) and 13C6-paclitaxel,
3?-p-hydroxypaclitaxel and 6?-hydroxypaclitaxel were from Toronto Research Chemicals (Toronto, Canada).
Analysts were blinded to all sample identities during processing, including untargeted and
targeted analysis. 3 aliquots of meconium were analyzed per newborn. Meconium analysis was
conducted by liquid-liquid extraction with either ethyl acetate or MTBE. Briefly, 50 mg
(average 50.6 mg) of meconium was weighed into 10 mL screw cap glass tubes. Wet weight was
recorded to 0.1 mg on a balance with tolerance to +/- 0.01 mg. Samples were spiked with an
internal standard to adjust for variation in extraction and analysis. For screening, 500 pg
diclofenac (50 ?L of 10 pg/ ?L stock in methanol) was used as an internal standard to adjust for
consistency of extraction and system suitability. In quantitative analysis, 500 pg of 13C6-paclitaxel
(50 ?L of 10 pg/ ?L stock in methanol) was used as the internal standard. During screening
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experiments, ethylacetate and MTBE extraction were tested since a variety of extractions have
been reported from other matricies for the chemotherapeutics studied here. Briefly,
ethylacetate extraction was conducted by adding 100 ?L of saturated NaCl solution in water, 900 ?L of
water, sonicating in a bath sonicator for 10 minutes then adding 8 mL of ethyl acetate and
vortexting for 30 minutes. MTBE extraction was similar except that 8 mL of MTBE was used. The
organic layer was then removed and evaporated to dryness under nitrogen gas. Dried residue
was re-suspended in 100 ?L of 80:20 water: methanol.
LC-HRMS analysis was conducted on an Ultimate 3000 quaternary UPLC equipped with a
refrigerated autosampler (6? C) and a column heater (60? C) coupled to a Thermo QExactive
Plus. LC separations were conducted on a Waters XBridge C18 (3.5 ?m, 100 ?, 2.1 x 100 mm).
A multi-step gradient at 0.3 mL/min flow with solvent A (water 1% acetic acid) and solvent B
(acetonitrile 1% acetic acid) was as follows: 10% B from 0 to 1 minute, increasing to 100% B
from 1 to 5 minutes, holding 100% B until 12 minutes, then the column was returned to
starting conditions and re-equilibrated at 20% B from 12 to 15 minutes. This gradient was
sufficient to resolve an interfering peak (retention time of 5.5 min) in meconium samples that
interfered with the detection of paclitaxel by HRMS. Column effluent was diverted to waste
from 0 to 0.5 minutes and from 12.5 to 15 minutes. The mass spectrometer was operated in
positive ion mode with a second-generation heated electrospray ionization source (HESI-II)
alternating between full scan (200?900 m/z) at a resolution of 70,000 and data independent
analysis (MS/MS) at 17,500 resolution with a precursor isolation window of 0.7 m/z. In the
targeted experiments MS/MS was conducted on the M+H of paclitaxel, 13C6-paclitaxel,
3?-phydroxypaclitaxel and 6?-hydroxypaclitaxel. Source parameters were as follows: capillary
temperature, 425?C, probe temperature, 425?C, sheath gas, 45 arb units, aux gas, 15 arbitrary
units, sweep gas, 2 arbitrary units, spray voltage 4.0 kV, S-lens, 50. Targeted data analysis was
performed in Xcalibur and TraceFinder software (Thermo Fisher, San Jose, CA).
Assay precision and accuracy was calculated from independently prepared triplicates
(n = 3) on the same day and then across 3 separate days at each indicated QC level of 100, 200,
and 500 pg/mg meconium using blank meconium from healthy newborns. Precision was
calculated as coefficient of variation (%) defined as (standard deviation/mean) 100. Accuracy (%)
was calculated as [1-(expected?observed)/expected] 100.
Data was analyzed and summary statistics were calculated in Tracefinder, Excel 2016
(Microsoft), and/or with Graph Pad Prism (v7).
Putative detection of paclitaxel and its metabolites in human meconium by
Samples from 23 newborns born to mothers who underwent chemotherapy during pregnancy
were screened by LC-HRMS for the presence of chemotherapeutics and their major
metabolites. Meconium from 10 healthy newborns was used for assay development; as a negative
control to establish blank levels, and as a matrix to spike known quantities of analyte to serve as
positive controls for calibration curves and quality controls. A chromatographic peak
putatively corresponding to intact paclitaxel was detected in meconium from 7/23 meconium
samples, with putative detection of two paclitaxel metabolites but no parent drug in one other
sample retrospectively found after unblinding. The mothers of all positively screened samples
had been diagnosed with breast cancer. Detection frequency was markedly lower with MTBE
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extraction (4/23) from meconium despite this being a common liquid-liquid extraction solvent
for quantification of paclitaxel in other matricies[
Quantification of paclitaxel and its metabolites in positively screened
To confirm the screening results by LC-HRMS we utilized targeted stable isotope
dilutionLC-HRMS using commercially available 13C6-paclitaxel instead of the surrogate internal
standard diclofenac used in screening. Human metabolism of paclitaxel occurs via cytochrome
P450s to hydroxylated and epoxide metabolites, with high levels of excretion in the bile as well
as urinary excretion [
]. Major metabolites include 3?-p-hydroxypaclitaxel, and
6?-hydroxypaclitaxel produced by CYP3A4 and CYP2C8, respectively, thus we added these analytes to
our targeted assay [
]. 3?-p-hydroxypaclitaxel and 6?-hydroxypaclitaxel were well resolved on
our LC-HRMS method and were quantified using 13C6-paclitaxel as an internal standard (Fig
1A). Standard curves over the range of 4?4,000 pg/mg meconium were linear with R2 values of
0.9949, 0.9964 and 0.9971 for paclitaxel, 3?-p-hydroxypaclitaxel and 6?-hydroxypaclitaxel,
respectively. All positively detected samples were interpolated from these curves. Assay
performance was confirmed by inter- and intra-day accuracy and precision with bias and %
coefficient of variation less than 20% across quality controls of spiked meconium samples at 100,
200 and 500 pg/mg meconium (Table 1). The positive bias (accuracy) observed across quality
control points that was higher than the precision may reflect a slight overestimation of
paclitaxel and 3?-p-hydroxypaclitaxel in meconium. However, since no major interfering peaks
were detected in blank meconium samples or the negatively screened meconium samples, this
indicates that this bias is not likely to cause false positive identifications of meconium.
Quantification of paclitaxel across samples, taking an average of all three aliquots, revealed
a mean (standard deviation) of 399.9 (248.6) pg/mg in positively screened samples (Fig 1B).
3?p-hydroxypaclitaxel and 6?-hydroxypaclitaxel were quantified at 105.2 (54.6) pg/mg and 113.5
(48.9) pg/mg, respectively. Spearman rank correlation was used to analyze the correlations of
the averaged values of paclitaxel and its metabolites in the meconium samples corresponding
to the same individual. Correlation of paclitaxel with its metabolites was strong (Spearman r
(p-value) for each pair paclitaxel/3?-p-OH-Pac 0.952 (p = 0.001) paclitaxel/6?-OH-Pac 0.86
(p = 0.001)) and correlation between the two metabolites was strong at 0.95 (p = 0.001).
For 6 samples where data was available on treatment schedule, levels of paclitaxel and its
metabolites was plotted by days of chemotherapy received until delivery (Fig 1C). Correlation
Fig 1. Quantification of paclitaxel in meconium. (A) Representative chromatogram of paclitaxel and its metabolites by isotope dilution-LC-HRMS in
meconium. (B) Pg per mg wet weight of meconium for paclitaxel (Pac), 3?-p-hydroxypaclitaxel (3-OH-Pac), and 6?-hydroxypaclitaxel (6-OH-Pac) in 8
meconium samples. The mean of three averaged replicates per individual is shown with the standard deviation. (C) Pg per mg wet weight of meconium plotted
by days of chemotherapy received before delivery.
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between meconium levels and days of chemotherapy before delivery was negative, weak, but
not statistically significant (Spearman r (p-value) -0.14 (0.80), -0.086 (0.91), 0.086 (0.92) for
paclitaxel, 3?-p-hydroxypaclitaxel and 6?-hydroxypaclitaxel, respectively.
To our knowledge, this is the first measurement of chemotherapeutics in human meconium.
As a preliminary study, there are severe limitations to our findings. First, we were unable to
obtain any other maternal or fetal biospecimens to examine circulating maternal
chemotherapeutic concentrations. Combined with a lack of dosage information and a complete
accounting of covariates (other pharmaceuticals given, comorbidities, body weight, etc.) this prevents
informed pharmacokinetic analysis within this study design. Accounting for these factors in
larger future studies may explain some of the variability in paclitaxel across meconium
samples, especially in the context of modifiers of paclitaxel metabolism and placental transport.
Since diagnosis of breast cancer during pregnancy is increasing due to demographic changes,
and these changes may alter transplacental exposures, recruitment of larger and
betterdescribed cohorts is reasonable. Second, the use of analytical screening before targeted
quantification may limit the ability to detect other chemotherapeutics in meconium. However, it
takes significant resources to develop and then validate methods in new biological matrices,
especially complex ones such as meconium. Future studies could take advantage of the
findings here to further this work with more complete data and biosample collection. The benefit
of meconium, in terms of ease of sample collection with a low burden on participants and
researchers, may make this feasible. However, one drawback of meconium is that collection is
more difficult to standardize since the meconium may pass at different times- corresponding
to different hospital branches, care-teams, or at home. It is however unlikely that difference in
handling between mothers may lead to differential levels of chemotherapeutics in meconium
as pre-labeled packaging was provided, paclitaxel would not be a likely exogenous
contaminant, and compounds detected in meconium by definition are stable enough to be detectable
in stored samples. Additional future work using cord tissue or more invasive samples
including amniotic fluid could provide additional information on concentrations of
chemotherapeutics in the fetal compartment.
The quantification of paclitaxel and its metabolites in meconium agrees with fetal
disposition quantified in previous pregnant baboon studies [
]. Similarly, ex vivo studies of human
placental transport of taxanes partially agrees with our results. Of particular relevance to
interpretation in this study, Berveiller, et al., performed transplacental studies of paclitaxel and
docetaxel in term placentas using a human perfused cotyledon placental model [
maternal concentrations mimicked an infusion of 90 mg/m2 of paclitaxel and 100 mg/m2 of
docetaxel used in clinical practice. Fetal transfer rate and placental uptake ratio were calculated
as transport parameters. After perfusion, the cotyledons underwent extraction to quantify
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docetaxel and paclitaxel using reverse-phase high-performance liquid chromatography.
Results showed that the transplacental transfer of paclitaxel and docetaxel were low and
similar. Large heterogeneity was also noted across placentas, similar to the meconium variability in
this current study. An explanation offered by the authors was the differential expression of
pglycoprotein (P-gp), a drug transporter which should decrease tissue accumulation in the
fetus. Authors found both variability in expression according to both gestational age and
between patients of similar gestational age. According to Hemauer et al., polymorphisms in
the MDR1 gene on the expression affect the activity of placental P-gp and therefore
P-gpmediated active transport of paclitaxel. When authors examined 200 human placentas there
was inter-individual variability and no correlation between P-gp protein expression and
transport activity of paclitaxel. Consequently, investigators hypothesized that genetic variation
could contribute to discrepancies between observed protein expression and activity of
placental P-gp there was a gene-dose effect, whereby heterozygous placentas (wild type/variant) had
intermediate level of P-gp uptake activity compared to placentas with the wild type (lowest
uptake) and homozygous variants (highest uptake).[
] In this current study we did not
perform genetic studies of mothers? or children?s MDR1 gene expression.
Heterogeneity in the amount of paclitaxel and its metabolites in different individuals may
indicate either differential maternal pharmacokinetics or different fetal pharmacokinetics.
Variations in pharmacokinetic parameters of paclitaxel have been reported from ABCB1
polymorphism and P-gp activity [
], and variations in toxicity have been reported by CYP3A4
]. When administered intravenously over 1 to 96 hours to adults, paclitaxel has
demonstrated the following pharmacokinetic characteristics: extensive tissue distribution;
approximately 90 to 95% plasma protein binding, average systemic clearance varying between
87 to 503 ml/min/m2 (5.2 to 30.2 L/h/m2); and <10% renal excretion of parent drug. In recent
trials in children and adults, paclitaxel elimination appeared to be saturable, the clinical
importance of which would be greatest when large dosages are administered and/or the drug is
infused over a shorter period. In these situations, achievable plasma concentrations are likely to
exceed the constant for elimination. Thus, small changes in dosage or infusion duration may
result in disproportionately large alterations in paclitaxel systemic exposure (10). Our findings
also agree with the descriptions of fecal excretion of paclitaxel metabolites in adults
acknowledging the major caveat that fecal elimination in adults is not equivalent to meconium deposition.
Regardless, this study should be taken into context within the clinical evidence base of the
effectiveness of taxol-based chemotherapy. No data in this study suggests or assumes toxic
levels of chemotherapeutics are achieved in the fetal compartment. In fact, the use of paclitaxel
and docetaxel have been reported in human pregnancies with reassuring neonatal outcomes
]. When looking at the developmental outcomes of infants exposed to chemotherapy,
prematurity had a stronger negative influence on development than chemotherapy . This
study does suggest that meconium analysis has utility as a measure of fetal exposure in
precision medicine for designing and testing dosing regimens to improve maternal delivery of
chemotherapeutics. Meconium analysis may also help understand human variability in maternal
pharmacokinetics and provide a more individualized approach to observational study of
developmental outcomes after cancer during pregnancy.
S1 Dataset. Minimal data set containing days of chemotherapy received until delivery and
pg/mg wet weight of meconium for paclitaxel (Pac), 3?-p-hydroxypaclitaxel (3-OH-Pac),
and 6?-hydroxypaclitaxel (6-OH-Pac) in 8 meconium samples.
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We acknowledge the contribution of research nurses in helping receive catalog and store the
samples, and the patient and patient families in transmission of samples.
Conceptualization: Elyce Cardonick, Nathaniel W. Snyder.
Formal analysis: Nathaniel W. Snyder.
Funding acquisition: Nathaniel W. Snyder.
Investigation: Robert Broadrup, Peining Xu, Mary T. Doan, Helen Jiang.
Resources: Elyce Cardonick, Nathaniel W. Snyder.
Supervision: Elyce Cardonick.
Validation: Robert Broadrup, Peining Xu, Mary T. Doan, Helen Jiang.
Visualization: Robert Broadrup, Peining Xu, Mary T. Doan, Helen Jiang.
Writing ? original draft: Elyce Cardonick, Nathaniel W. Snyder.
Writing ? review & editing: Elyce Cardonick, Nathaniel W. Snyder.
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