Vancomycin Pharmacokinetics in a Pregnancy Rat Model

ABSTRACT

Vancomycin usage is often unavoidable in pregnant patients; however, literature suggests vancomycin can cross the placental barrier and reach the fetus. Understanding the mass transit of vancomycin to the fetus is important in pregnancy. We aimed to (i) identify a relevant population pharmacokinetic (PK) model for vancomycin in pregnancy and (ii) estimate PK parameters and describe the mass transit of vancomycin from mother to pup kidneys. Pregnant Sprague-Dawley rats (i.e., trimester 1 and trimester 3) received 250 mg/kg vancomycin once daily for three days through intravenous injection via an internal jugular vein catheter. Vancomycin concentrations in maternal plasma and pup kidneys were quantified via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Multiple compartment models were fitted and assessed using a nonparametric approach with Pmetrics. A total of 10 vancomycin-treated rats and 48 pups contributed PK data. A 3-compartment model adjusted for trimester fit the data well (maternal plasma Bayesian, observed versus predicted R² = 0.978; pup kidney Bayesian, observed versus predicted R² = 0.999). The mean rate constant for vancomycin mass transit to the pup kidney was 0.72 h⁻¹ for trimester 1 dams and 0.75 h⁻¹ for trimester 3 dams. Median vancomycin concentrations in pup kidneys from trimester 3 were significantly higher than those in trimester 1 (8.62 versus 0.36 μg/mL, P < 0.001). Vancomycin transited to the fetus from the mother and was; kidney accumulation differed by trimester. This model may be useful for a translational understanding of vancomycin distribution in pregnancy to ensure efficacious and safe doses to both mother and fetus.

INTRODUCTION

Pregnancy is well known to alter the pharmacokinetics (PK) of many commonly used medications. Alterations in absorption, excretion, and metabolism of drugs are common during pregnancy (1). Pregnant women undergo several transformations to their organ systems because of the need to support a developing fetus. Some of the changes include alterations in plasma protein concentrations and increases in total body water, fat deposition, renal clearance, and cardiac output (2, 3). For example, maternal glomerular filtration changes during different stages of pregnancy, with a relative increase of 23.7% (95% confidence interval [CI], 10.7 to 36.7%) at <14 weeks (~first trimester), 37.6% (26.9 to 48.2%) at 15 to 21 weeks (~second trimester), 26.9% (15.7 to 38%) at 22 to 28 weeks (~third trimester), and 15.1% (8.6 to 21.7%) at 29 to 35 weeks (third trimester) (4).

Colonization with methicillin-resistant Staphylococcus aureus (MRSA) in pregnant women admitted to labor and delivery occurs at a rate of 2.1% (5). Nasal colonization with S. aureus has been documented in 4 to 14.3% of screened pregnant women (5, 6), and cesarean section infections caused by S. aureus account for 25 to 50% of surgical site infections in pregnancy (5). Unfortunately, MRSA infections have increased throughout the years due to the selection of resistance driven by increased antibiotic usage (5). Notably, maternal colonization is also linked to infant S. aureus colonization (7). With the increasing rate of cesarean delivery (i.e., surgical intervention) and neonatal infection risk caused by maternal colonization, the implications of S. aureus colonization in pregnancy are significant for both the mother and the baby. Consequently, vancomycin is a common treatment option for MRSA infections during pregnancy (8).

In 2015, the FDA removed pregnancy categories (A, B, C, D, X) and replaced these categories with descriptive sections outlining various subsections related to pregnancy. The package labeling suggests limited data for vancomycin usage in pregnant women (9). In a small clinical toxicology study, 10 pregnant women with serious staphylococcal infections were administered vancomycin (10). Vancomycin was found in fetal cord blood in two infants, showing that vancomycin crosses the placenta and is present in cord blood. While the study was limited in the number of subjects, the authors concluded that vancomycin could be administered safely during pregnancy without risk of toxicity (i.e., nephrotoxicity, hearing loss) to the fetus. However, the identification of vancomycin in cord blood indicates that the fetus may achieve systemic exposures and be subject to toxicity risks similar to the mother. In pregnant rats and rabbits, vancomycin administration was found to cause slight to moderate cortical tubular nephrosis in mothers but did not appear to cause any fetal malformation when given during organogenesis at the humanized doses (rats, doses up to 200 mg/kg ~32 mg/kg/day humanized equivalent [70 kg patient, ~2,250 mg/day]; rabbits, doses up to 80 mg/kg ~25mg/kg/day humanized equivalent [70 kg patient, ~1,800 mg/day]) (11). An upper limit maximum in the rat (based on an upper limit maximum of 40 mg/kg/day in humans) is 250 mg/kg after allometric scaling. In our previous work, vancomycin administered to pregnant rats at different trimesters resulted in elevated concentrations of kidney-injury molecule 1 (KIM-1) in pup kidneys (12). Thus, for pregnant women, determining vancomycin fetal transfer rates and magnitude of accumulation in fetal kidneys is important. Here, we sought to identify a relevant PK model for pregnant rats and their pups and estimate vancomycin’s mass transit from the dam to pup.

RESULTS

Characteristics of the animal cohort.

A total of 10 animals received vancomycin and had plasma concentrations sampled. All 10 dams and 48 pups contributed to the PK data (Fig. 1). Each dam had a median of 7 plasma concentrations that contributed to the model build. Median baseline weight was significantly different between trimester 1 and trimester 3 dams (195 g versus 319.5 g, P = 0.0095; Table 1). Median observed kidney concentrations in pups were also significantly different between trimester 1 and trimester 3 protocols (0.36 μg/mL versus 8.62 μg/mL; P < 0.001; Table 1).

FIG 1.

Animal dosing flow chart for trimester 1 and 3 dams.

TABLE 1.

Summary of characteristics for dams and pups^a

Characteristic	Data for:					P value
	All dams contributing PK data	Trimester 1 dams	Trimester 3 dams	Trimester 1 pups	Trimester 3 pups
No. of animals	10	4	6	36	12
Median (IQR) baseline wt (g)	307.5 (195.5–326.3)	195 (193.3–196.8)	319.5 (307.8–335.8)			0.0095
Median (IQR) observed kidney concn (μg/mL)				0.36 (0.193–0.568)	8.62 (6.94–10.15)	<0.001

^aItalicized values refer to the groups compared in the statistical analysis (Wilcoxon rank sum test). IQR, interquartile range; PK, pharmacokinetic.

Vancomycin PK models.

A three-compartment model adjusted for trimester was selected over the other models explored given overall better performance and Bayesian and population maternal plasma/pup kidney observed versus the predicted R² of 0.978/0.999 and 0.814/0.897 and the Akaike information criterion (AIC) of 559.9, respectively (Table 2). A schematic of the final model can be found in Fig. S1 in the supplemental material. Trimester was the only covariate found to have a significant relationship with volume for both dams and pups in the base three-compartmental model. Further, the clinical significance of trimester was considered when choosing the model adjusted for trimester given the known physiological changes documented during different trimesters of pregnancy (1, 4). Total body weight (TBW) was not found to have a significant covariate relationship with volume of distribution (VD) for VD_{trim 1 dams} or VD_{trim 3 dams} (P values > 0.05, R² < 0.2) and did not significantly improve the model (AIC, 566.2; P > 0.05). Eight support points were identified from the final model. Specific PK parameters differed by trimester group. The final model’s population mean (coefficient of variation percentage) parameter values for elimination rate constant by trimester (Ke_{trim 1 dams} and Ke_{trim 3 dams}), dam volume by trimester group (VD_{trim 1 dams} and VD_{trim 3 dams}), rate to/from central/peripheral compartment (K12, K21), transfer rate to pup compartment by trimester (K13_{trim 1 dams} and K13_{trim 3 dams}), and pup compartment volume (VD_{trim 1 pups} and VD_{trim 3 pups}) were 5.24 h⁻¹ (56.61%) and 0.34 h⁻¹ (61.66%), 0.08 L (77.12%) and 0.20 L (31.5%), 7.58 h⁻¹ (29.89%) and 2.36 h⁻¹ (44.88%), 0.72 h⁻¹ (62.49%) and 0.75 h⁻¹ (14.83), and 27.69 L (47.32%) and 19.04 L (20%), respectively (Table 3). A lower volume of distribution (0.08 L [77.12%] and 0.20 L [31.5%]) and a higher elimination rate constant (5.24 h⁻¹ [56.61%] and 0.34 h⁻¹ [61.66%]) were observed in mothers of the trimester 1 group compared to mothers of the trimester 3 group. However, the drug transfer rate constants between mother and fetus were comparable between the two groups (0.72 h⁻¹ [62.49%] and 0.75 h⁻¹ [14.83]). Vancomycin was accumulated in pup kidneys of both groups with volumes of distribution observed at 27.69 L (47.32%) and 19.04 L (20%) in the pups of the first trimester group and the third trimester group, respectively. In the model predictive performance, observed versus Bayesian predicted concentrations, bias, imprecision, and the coefficient of determination (R²) were −0.0187 μg/mL, 1.25 (μg/mL)², 97.8% for plasma_dams and 0.0208 μg/mL, 0.0107 (μg/mL)², and 99.9% for pups’ kidneys, respectively (Fig. 2).

TABLE 2.

PK model building comparison^a

Model	–2LL	AIC	Bayesian bias (μ/mL) (central)	Bayesian imp (μg/mL2) (central)	R2 Bayesian (central)	Bayesian bias (μg/mL) (pup kidneys)	Bayesian imp (μg/mL2) (pup kidneys)	R2 Bayesian (pup kidneys)
Two-compartment base model	712.2	723.3	–0.278	1.04	0.626	0.135	0.134	0.906
Three-compartment base model	543.2	558.8	–0.576	1.39	0.965	0.791	1.2	0.97
Three-compartment model adjusted for trimesterb,d (as depicted in Fig. S1)	534.0	559.9	–0.019	1.25	0.978	0.021	0.011	0.999
Three-compartment model adjusted for TBW (VDdams × TBW/0.3075c)d	540.3	566.2	–0.157	1.16	0.952	−0.0724	0.0352	0.998

^aPK, pharmacokinetic; −2LL, −2 log-likelihood; AIC, Akaike information criterion; imp, imprecision; VD, volume of distribution; VD₁, volume term 1; VD₂, volume term 2; TBW, total body weight; dam, female mother; pup, offspring.

^bFinal model based on overall performance on regression of observed versus predicted concentrations, visual plots of parameter estimates, −2LL/AIC, and rule of parsimony. The clinical significance of the variable was also considered in evaluation.

^cMedian weight of the dams.

^dModels were not statistically different.

TABLE 3.

Population pharmacokinetic parameter value summary^a

Parameter	Mean	SD	CV (%)	Shrink (%)b
VDtrim 1 dams (L)	0.08	0.065	77.12	0.43
VDtrim 3 dams (L)	0.20	0.062	31.50	2.53
VDtrim 1 pups (L)	27.69	13.10	47.32	9.02
VDtrim 3 pups (L)	19.04	3.81	20.0	3.83
Ketrim 1 dams (h–1)	5.24	2.96	56.61	0.65
Ketrim 3 dams (h–1)	0.34	0.21	61.66	4.92
K13trim 1 dams (h–1)	0.72	0.45	62.49	1.57
K13trim 3 dams (h–1)	0.75	0.11	14.83	7.16
K12 (h–1)	7.58	2.27	29.89	5.99
K21 (h–1)	2.36	1.06	44.88	0.64

^aValues were rounded to the nearest hundreds decimal point for consistency. VD, volume of distribution; Ke, elimination rate constant; K13/K31, transfer rate to/from pup compartment; K12/K21, rate to/from central/peripheral compartment.

^bRatio of the mean posterior parameter value variance across all subjects to the total population parameter value variance.

FIG 2.

(A to D) Best fit plot for observed versus predicted plasma vancomycin concentrations for dam individual/Bayesian (A) and population (B) and pup individual/Bayesian (C) and population (D) utilizing the final 3-compartment trimester-adjusted model.

DISCUSSION

This pregnancy animal model provides evidence that vancomycin accumulates in developing pup kidneys when dams are dosed with short courses (~3 days) of vancomycin, regardless of trimester. Notably, these findings show detectable vancomycin concentrations in pup kidneys long after the last vancomycin dose dams received. The accumulation rates noted in this study, along with the finding from our previous work that trimester 1 injury exceeds trimester 3 injury, provides insight into the prolonged vancomycin residence in kidneys (12). Finally, our final PK model’s low shrinkage values (<10%) for all estimated PK parameters demonstrates that interindividual variability of the animals was captured. Rats from the trimester 2 group (n = 6) did not have pup vancomycin kidney concentrations quantified due to processing errors and were thus excluded from the PK model analysis. Consequently, our results and analysis are only specific to trimester 1 and trimester 3 rats (n = 10).

Drugs are used in more than half of all pregnancies for maintaining maternal health, but evidence-based guidelines for drug use and dosing during pregnancy are limited (13). Further, for medications approved before 2001 (i.e., vancomycin), manufacturers are now required to remove the previously documented pregnancy category (categories A through X). Consequently, clinicians must rely on available literature to guide the dosing and appropriateness of therapeutic agents. Human studies in pregnancy are not often performed, and most information is derived from animal studies, uncontrolled studies, or postmarketing surveillance (14). Guidelines recommend assessment of PK during pregnancy to ensure appropriate clinical use and that adequate drug exposure is achieved during pregnancy. However, drug doses and regimens’ safety and efficacy are established in phase 3 clinical trials, where pregnant women are often excluded. Frequently, data and recommendations for specific drug usage during pregnancy are unavailable or not listed in the package insert. A study by Andrade et al. found that there was not enough information about risk or safety for over 90% of medications approved by the FDA between 1980 and 2000 for usage in pregnancy (15). Consequently, treatment and dosing strategies in pregnancy are often extrapolated from standard nonpregnant adult doses. It is essential to conduct PK studies in pregnancy, given the physiological changes that can occur, potentially impacting the absorption, metabolism, and excretion of drugs and drug-related exposure toxicity to the developing fetus.

To our knowledge, this is the first rat model to evaluate and describe the mass transit of vancomycin from the dam plasma to the pup kidney during various trimesters. We did not identify other significant covariate effects (e.g., weight) on PK parameters; however, outbred animal studies are purposefully homogenous, and the parameters were already separated by trimester. This study found that the mean estimated rate constant for vancomycin’s transit to pups and median observed concentrations in pups were similar in trimesters 1 and 3. For trimester 1 dams, the rate constant for vancomycin mass transfer to pup kidney and median observed pup kidney concentration were 0.72 h⁻¹ and 0.36 μg/mL, respectively. These values increased to 0.75 h⁻¹ and 8.62 μg/mL in trimester 3 dams. During pregnancy, increases in cardiac output, stroke volume, maternal heart rate, blood flow to kidneys, and total body weight occur during trimester 1 and usually peak during trimester 2 (16, 17). As such, the slightly increased rate constant estimate for vancomycin transfer from the dam to pup kidney in trimester 3 dam could be influenced by some or all of these increased physiological processes. Although the increase of transfer constant was slight, we observed a 22.9-fold increase (8.62 μg and mL/0.36 μg/mL, respectively) of vancomycin concentration in the pup kidneys in the trimester 3 group compared to the trimester 1 group. It could be that transfer rates are similar but that the pup kidneys have more time to clear the vancomycin prior to birth in the trimester 1 versus trimester 3 group. More studies looking at this are warranted given the small total number of dams in our pilot study. A recent systematic review by Pariente and colleagues summarized studies of pregnancy-associated pharmacokinetic changes with various xenobiotics (18). However, data on vancomycin were absent. As we did not directly measure fetal drug clearance, our estimates of the amount of vancomycin that remains in the pup kidney after delivery are predicated. However, this study design is still useful for understanding the total accumulation of vancomycin with treatment courses during different trimesters. Even when trimester 3 dams had undetectable vancomycin plasma concentrations, there were clinically relevant pup kidney concentrations observed (median [interquartile range (IQR)], 8.62 μg/mL [6.94 to 10.15]).

This study expands from our previous work, examining the relationship of maternal vancomycin exposure and fetal kidney injury KIM-1 (12). Pups from dams dosed during trimester 1 had higher concentrations of KIM-1 than those dosed during trimesters 2 and 3 (1.229 ± 0.09 pg/μg versus 1.098 ± 0.06 pg/μg versus 0.849 ± 0.04 pg/μg, P < 0.02). The study results depicted an inverse relationship between the progression of trimesters and kidney damage caused by vancomycin. Pup kidneys from trimester 1 had the highest measured concentrations of KIM-1, while dam KIM-1 values were the lowest in trimester 1. It is unclear if prolonged vancomycin exposure to the pup kidney is the reason for additional injury or if it is due to vancomycin exposure at a critical point of nephrogenesis which may not entirely be drug concentration dependent. Other animal studies in rats (19–27), mice (28), pigs (29), and rabbits (30) have also shown vancomycin’s potential to cause kidney injury. Specific to pregnancy, Byrd and colleagues showed that in pregnant rats and rabbits, vancomycin administered at various doses up to the maximum humanized equivalent dose did not cause any fetal harm; however, sensitive assessments of kidney harm were not performed (11). Moreover, they found that vancomycin did cause slight to moderate cortical tubular nephrosis in mothers (11). More recently, experiments from rats have been linked to outcomes from human trials using more sensitive biomarkers (31). Further studies evaluating the risk for kidney injury in pregnancy are warranted to truly quantify the risk of toxicity to both mother and developing child.

There are several limitations to this study. First, pup kidneys were homogenized, and the vancomycin concentrations quantified reflect total concentrations in the pup kidney rather than specific nephron segment concentrations. While we were successful in estimating the terminal mass transit of vancomycin to pup kidney in our animal model, it is impossible to know the exact transfer rate of vancomycin when dosing is temporally distant (e.g., dose during trimester 1, harvest kidneys at birth). Such information is very difficult to obtain, as tissue sampling requires animal sacrifice.

In summary, this analysis showcases an explanatory PK model for the mass transfer and accumulation of vancomycin across the placental border to the kidney of the fetus. Our data show that this transfer is predictable in our pregnancy rat model. This model can help understand how vancomycin is distributed and accumulates in offspring kidney during pregnancy.

MATERIALS AND METHODS

This pharmacokinetic study analyzed samples from pregnant Sprague-Dawley rats that received vancomycin for a toxicology study that has been previously reported (12). All study methods were approved by the Institutional Animal Care and Use Committee (IACUC; protocol no. 2846) and conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, 8th edition (32).

Animals and dosing.

The full protocol has been previously described (12). In brief, 9-week-old pregnant Sprague-Dawley rats that received at least one dose of vancomycin were studied (n = 10). The rats were divided into 2 groups, group I (trimester 1, n = 4) and group II (trimester 3, n = 6) (Fig. 1). Rats were administered intravenous (i.v.) injections of vancomycin (250 mg/kg/day once daily for 3 days) in normal saline (NS) over 5 min via internal jugular vein catheter. The dose chosen was an allometrically scaled clinical dose (i.e., the 250 mg/kg daily dose in rats is equivalent to a 40 mg/kg daily dose in humans) (33, 34). For group I, the dosing occurred on gestational days (GD) 5, 6, and 7, and for group II, dosing occurred on GD 18, 19, and 20. The duration of three dosing days was selected in the primary study to assess toxicity at each trimester (12). All pups were delivered vaginally on GD 21; kidney collection/processing were performed as previously described (12).

Blood sampling.

Blood samples were drawn from the right internal jugular vein catheter. Blood samples (0.25 mL) per animal were scheduled at 0, 15, 30, 60, 120, and 240 min following the 2nd dose with one additional sample obtained following the 3rd dose. Experiments were conducted over a 3-day sampling period, with samples collected at various time points important for estimation of the different PK phases of drug distribution/elimination. Each blood sample removed (0.25 mL aliquot) was replaced with an equivalent volume of NS to maintain euvolemia. Blood samples were immediately transferred to a disodium EDTA-treated (Sigma-Aldrich Chemical Company, Milwaukee WI) microcentrifuge tube and centrifuged at 3,000 × g for 10 min. Plasma supernatant was collected and stored at −80°C for batch sample analysis.

Chemicals and reagents.

All reagents used for vancomycin quantification have been previously described (12). Briefly, vancomycin hydrochloride (lot no. 591655DD) for injection was obtained commercially (Hospira, Lake Forrest, IL). All solvents were of liquid chromatography-tandem mass spectrometry (LC-MS/MS) grade. Polymyxin B (Sigma-Aldrich, St. Louis, MO) was used as the internal standard (12).

Determination of vancomycin concentrations in plasma and pup kidney.

Pup kidneys were homogenized by ultrasonic homogenization using a model 60 sonic dismembrator (Fisher Scientific, Hampton, NH) as previously detailed (12). Plasma and pup kidney concentrations of vancomycin were quantified using our previously validated LC-MS/MS method (12, 35). In brief, samples underwent protein precipitation with 0.1% formic acid in methanol and centrifugation prior to injection in an Agilent 1260 LC with a tandem 6420 triple quadrupole system (Agilent, Santa Clara, CA). The standard curves for rat plasma and pup kidney tissue homogenate were linear between concentrations of 0.5 to 100 μg/mL (r² = 0.993) and 0.5 to 100 μg/mL (r² = 0.9991), respectively. The lowest limit of quantitation (LLOQ) for vancomycin was 0.5 μg/mL. More than 92% of the analyte was recovered in all tested samples. Precision was <6.7% CV for all measurements, including intra- and interassay measurements. Vancomycin concentrations in pup kidneys by trimester group were averaged to represent one pup kidney value for each dam.

Vancomycin pharmacokinetic model.

We chose a 3-compartment base model as the most physiologically relevant structure for this animal model, considering vancomycin disposition in the dam plasma and pup kidneys and pregnancy status. Numerous PK models were fitted and assessed using the nonparametric adaptive grid (NPAG) algorithm within the Pmetrics package version 1.5.02 (Los Angeles, CA) for R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria) (36, 37). Each model parameter was described as a set of discrete support points with a maximum of one support point per animal (36). Model performance was also evaluated and compared utilizing a regression of observed versus predicted concentrations, visual plots of parameter estimates, Akaike information criterion (AIC), clinical significance, and the rule of parsimony. Untransformed relationships were explored, including total body weight (TBW) and rat trimester. Relationships between covariates and pharmacokinetic parameters were evaluated via stepwise linear regression via the PmStep function, where a P value of <0.05 was required for further exploration, and visually inspected and assessed with Spearman’s rho. The initial estimate of parameter weighting was accomplished using the inverse of the assay variance. The observation variance was proportional with a scalar (gamma) to assay variance (error = standard deviation [SD] × gamma, where SD = C₀ + C₁Y with inputs of C₀ = 0.5 and C₁ = 0.20 ng/mL [coefficients for assay variability polynomial]), where gamma = 2 (initial value). Using this assay error variance, all measurements (even below LLOQ) were fit as their actual values (38).

IACUC approval.

All study methods were approved by the Institutional Animal Care and Use Committee (IACUC; protocol no. 2846).

Statistical analysis.

Dam baseline TBW and observed concentrations in pup kidneys were compared across trimester groups. Differences were evaluated either using Student’s t test or the Wilcoxon rank sum test, as appropriate. All tests were two-tailed, with an a priori concentration of statistical significance set at an alpha of 0.05.