Placental transporter‐mediated drug interactions and offspring congenital anomalies

Aims P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are efflux transporters expressed in the placenta, limiting their substrates from reaching the foetus. Our aim was to investigate if concomitant prenatal exposure to several substrates or inhibitors of these transporters increases the risk of congenital anomalies. Methods The national Drugs and Pregnancy database, years 1996–2014, was utilized in this population‐based birth cohort study. In the database, the Medical Birth Register, the Register on Induced Abortions, the Malformation register and the Register on Reimbursed Drug Purchases have been linked. The University of Washington Metabolism and Transport Drug Interaction Database was used to identify substrates and inhibitors of P‐gp and BCRP. We included singleton pregnancies ending in birth or elective termination of pregnancy due to foetal anomaly. Known teratogens were excluded. We identified women exposed 1 month before pregnancy or during the first trimester to P‐gp/BCRP polytherapy (n = 21 186); P‐gp/breast cancer resistance protein monotherapy (n = 97 906); non‐P‐gp/BCRP polytherapy (n = 78 636); and unexposed (n = 728 870). We investigated the association between the exposure groups and major congenital anomalies using logistic regression adjusting for several confounders. Results The prevalence of congenital anomalies was higher in the P‐gp/BCRP polytherapy group (5.5%) compared to the P‐gp/BCRP monotherapy (4.7%, OR 1.13; 95% CI 1.05–1.21), the non‐P‐gp/BCRP polytherapy (4.9%, OR 1.14; 95% CI 1.06–1.22), and to the unexposed groups (4.2%, OR 1.23; 95% CI 1.15–1.31). Conclusion The results suggest a role of placental transporter‐mediated drug interactions in teratogenesis.


| INTRODUCTION
Drug use during pregnancy is common. In Finland, more than half of pregnant women use prescription drugs during the entire course of pregnancy, and 1 out of 4 use 2 or more prescription drugs. 1 A web-based survey in several European countries reported use of prescription or over-the-counter drugs in nearly 80% of pregnancies. 2 A recent study from the USA reported that nearly 8% of pregnant women used more than 4 drugs during the first trimester. 3 Drug transporters play an important role in the absorption, disposition and toxicity of drugs. P-glycoprotein (P-gp, encoded by ABCB1 gene) and breast cancer resistance protein (BCRP, encoded by ABCG2 gene) belong to the ATP-binding cassette transporters. 4 They are expressed in several human tissues and considered the 2 most important drug transporters in the human placenta. 5 The level of P-gp expression in the placenta is similar to that in the liver and small intestine, 6 and the expression of BCRP in the human placenta is higher than in any other human tissue. 7 These efflux transporters are already expressed on the maternal blood-facing surface of the syncytiotrophoblast of the placenta in the first trimester. Both P-gp and BCRP transport their substrates out of the syncytiotrophoblast back into the maternal circulation and prevent the substrate from reaching the foetus. Both transporters are also involved in the regulation of several endogenous compounds entering the foetal circulation. 7 Their presence in the placenta suggests an important barrier in preventing drugs from entering the foetal circulation and protecting the foetus from exogenous chemicals. P-gp substrates include several commonly used pharmaceuticals such as cetirizine, calcium channel inhibitors, macrolides, opioids, selective serotonin reuptake inhibitors and secondgeneration antipsychotic drugs. 5 BCRP has many overlapping substrates with P-gp, but the individual substrates have different affinities to these transporters. Both P-gp and BCRP are inhibited by numerous drugs, and in addition, their substrates may competitively inhibit transport function. This could affect the function of the placental barrier and alter the degree of foetal exposure to the specific substrate. 8 Indeed, placental perfusion studies have shown that pharmacological blockade of P-gp function can increase the transfer of P-gp substrates to the foetal side by several-fold. 9,10 Particularly in the case where a transporter substrate is a teratogen, its teratogenic potential may increase if it is used together with another substrate or an inhibitor of the same transporter. 9 As teratogenesis is a dose-dependent phenomenon, higher exposure to a harmful agent would be expected to result in an increased risk of foetal adverse effects including congenital anomalies. The potential role of drug interactions involving transporters in teratogenesis is not known and research in this field is only starting to emerge. 10

| OBJECTIVES
The objective of the study was to investigate if concomitant use of 2 or more P-gp or BCRP drug transporter substrates or inhibitors during 1 month before pregnancy or first trimester is associated with an increased risk of major congenital anomalies in offspring, compared to use of only 1 drug classified as P-gp or BCRP drug transporter substrate or inhibitor, to use of 2 or more drugs not classified as P-gp or BCRP drug transporter substrates or inhibitors, and to no use of drugs.

| MATERIAL AND METHODS
This is a population-based cohort study using national register data Drugs and Pregnancy database contains data from births, terminations of pregnancy, and prescription drug purchases since 1 January 1996.
In the project, the beginning of pregnancy is calculated from the best clinical estimation of gestational age at birth. This estimation is primarily based on ultrasound but if this is not available, the estimation is based on the day of last menstrual period, both registered in the Medical Birth Register. If this information is not available, the beginning of pregnancy is calculated by subtracting 280 days from the date of birth. Pregnancy trimesters have been divided into first (until 84 days' gestation), second (days 85-182) and third (day 183 until birth).
The study material presented here is based on 1 080 655 singleton pregnancies identified from the Drugs and Pregnancy database between 1 January 1996 and 31 December 2014. Of these pregnancies, 1 075 349 ended in birth and 5306 ended in elective termination of pregnancy due to foetal anomaly. For the pregnancies in our cohort, we identified all reimbursed drug purchases from 1 month prior to pregnancy until the end of the first trimester of pregnancy. The International Anatomic Therapeutic Chemical classification codes of these purchases were compared to those identified in the University of Washington (UW) Metabolism and Transport Drug Interaction

What is already known about this subject
• P-glycoprotein and breast cancer resistance protein are efflux transporters expressed in the placenta.
• Polypharmacy may lead to inhibition of the transporters and increased foetal exposure to their substrates.
• The potential role of drug interactions involving transporters in teratogenesis is not known.

What this study adds
• The results suggest a role of placental drug transporter interactions in teratogenesis.
• This study should direct future research to assess the role of drug transporter -mediated drug interactions in teratogenesis.
Database (DIDB) to define exposure groups (see below). Pregnancies exposed to known teratogens were excluded from study material (Table S1; Figure 1). The utilization of sensitive health register data for scientific research and the data linkages in the Drugs and Pregnancy project have been approved by the register administrators and the national data protection authority.

| DESCRIPTION OF THE REGISTERS INCLUDED IN THE STUDY
The Medical Birth Register (MBR), maintained by the THL since 1987, is a nationwide register collecting data on maternal demographic characteristics, medical history including reproductive history, smoking, diagnoses during pregnancy and delivery, and neonatal outcome data up to 6 days' age. Data in the MBR include all live and stillbirths with gestational age of 22 weeks or more or birth weight of 500 g or more. The register data are confirmed and complemented from the maternity hospital records in cases of conflicting or missing information. The definitions and variables included in this registry are based on established international concepts and use the 10 th version of the WHO International Classification of Diseases (ICD) since 1996.
The PIN is required for a patient to access subsidized health care, which means that there are virtually no births in Finland that take place without being registered in the MBR. 1,11,12 The Register on Congenital Malformations (maintained by the THL requiring continuous drug treatment. We excluded pregnancies exposed to proven and suspected teratogens from the study material ( Figure 1, Table S1).

| DEFINITION OF EXPOSED AND UNEXPOSED GROUPS
• P-gp/BCRP polytherapy group. Women with purchases of 2 or more drugs that are substrates or inhibitors of either P-gp or BCRP (n = 21 186; Figure 1).
• P-gp/BCRP monotherapy group. Women with purchase(s) of 1 drug that is either a substrate or an inhibitor of either P-gp or BCRP (n = 97 906).
• Non-P-gp/BCRP polytherapy group. Women with purchases of 2 or more drugs that are not substrates or inhibitors of P-gp or BCRP (n = 78 636).
Exposure classified by drug transporter, but allowing overlap between transporter-specific substrates and inhibitors • P-gp polytherapy group. Women with purchases of 2 or more drugs that are substrates or inhibitors of P-gp (n = 17 790).
• P-gp monotherapy group. Women with purchase(s) of 1 drug that is either a substrate or an inhibitor of either P-gp (n = 93 993).
• Non-P-gp polytherapy group. Women with purchases of 2 or more drugs that are not substrates or inhibitors of P-gp (n = 83 122).
• BCRP polytherapy group. Women with purchases of 2 or more drugs that are substrates or inhibitors of BCRP (n = 2068).
• BCRP monotherapy group. Women with purchase(s) of 1 drug that is either a substrate or an inhibitor of BCRP (n = 34 409).
• Non-BCRP polytherapy group. Women with purchases of 2 or more drugs that are not substrates or inhibitors of BCRP (n = 134 353).
Exposure classified by drug transporter, but allowing no overlap between transporter-specific substrates and inhibitors • Pure P-gp polytherapy group. Women with purchases of 2 or more drugs that are substrates or inhibitors of P-gp (n = 11 467).
• Pure P-gp monotherapy group. Women with purchase(s) of 1 drug that is either a substrate or an inhibitor of P-gp (n = 75 529).
• Pure BCRP polytherapy group. Women with purchases of 2 or more drugs that are substrates or inhibitors of BCRP (n = 157).
• Pure BCRP monotherapy group. Women with purchase(s) of 1 drug that is either a substrate or an inhibitor of BCRP (n = 11 533).

| MATERNAL CHARACTERISTICS AND COVARIATES
Data on covariates were derived from the registers described above.
Covariates included maternal demographic, social and medical characteristics and use of other drugs as categorized in Table 1. Data on body mass index (BMI) were available only beginning from 2006.
Alcohol use is not routinely collected in the MBR and could therefore not be included in analyses.

| OUTCOME
A major congenital anomaly registered in the Congenital Malformation Register, using the International Classification of Diseases (ICD-9 Atlanta modification) coding system.

| STATISTICAL ANALYSES
All data in the Drugs and Pregnancy database have been pseudonymized. The prevalence of congenital anomalies was calculated in the different exposure groups. We assessed the association between the level of exposure (P-gp/BCRP polytherapy, P-gp/BCRP monotherapy, non-P-gp/BCRP polytherapy) and major congenital anomalies using logistic regression. First, association between level of exposure and maternal characteristics and other clinically relevant or plausible covariates was tested with χ 2 (Table 1). Multivariate models were then built based on all significant univariate covariates and subsequent removal of non-significant covariates from the model (P < 0.05) using stepwise regression (backward elimination). Model T A B L E 1 Maternal characteristics by exposure status to P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates or inhibitors

| RESULTS
The characteristics of the women in the 4 exposure groups are presented in Table 1 and pregnancy outcome (live birth, still birth, elective termination of pregnancy due to foetal anomaly) in each group is presented in Table S2. Women in the P-gp/BCRP polytherapy group were more than twice as likely as women in the P-gp/BCRP monotherapy group, 4 times as likely as women in the non-P-gp/BCRP polytherapy group, and >10 times as likely as women in the unexposed group to have a diagnosis of connective tissue disease. Similarly, severe mental disorders and asthma were several-fold more common in the Pgp/BCRP polytherapy group than in the other exposure groups (Table 1). The 30 most commonly used drugs in the P-gp/BCRP polytherapy group are presented in Table 2, and in more detail in the Pgp/BCRP polytherapy and monotherapy groups in Tables S3 and S4, respectively. The prevalence of major congenital anomalies was 5.5% in the P-gp/BCRP polytherapy group, 4.7% in the P-gp/BCRP monotherapy group, 4.9% in the non-P-gp/BCRP polytherapy group, and 4.2% in the unexposed group (Table 3). In crude and adjusted models, the risk was statistically significantly increased in the Pgp/BCRP polytherapy group compared to all other groups (Table 3).
Exposed to P-gp substrates or inhibitors. The prevalence of major congenital anomalies was 5.4% in the P-gp polytherapy group, 4.6% in the P-gp monotherapy group, and 5.0% in the non-P-gp polytherapy group ( Table 4). The risk estimates of major congenital anomalies associated with P-gp exposure were very similar to those of Pgp/BCRP exposure (Table 4).
Exposed to BCRP substrates or inhibitors. The prevalence of major congenital anomalies was 6.2% in the BCRP polytherapy group, 5.1% in the BCRP monotherapy group, and 4.9% in the non-BCRP polytherapy group (Table 5). In crude models, the risk of major anomalies was increased in the BCRP polytherapy group compared to all other exposure groups, but the risk estimates were attenuated after adjustment ( Table 5). The risk remained marginally increased when compared to the non-BCRP polytherapy group and remained statistically significant when comparing BCRP polytherapy to unexposed (

| DISCUSSION
In this large, register-based study we found that concomitant use of multiple substrates or inhibitors of the drug transporters P-gp and BCRP during 1 month before pregnancy or first trimester was associated with an increased risk of major congenital anomalies in the offspring compared to their use in monotherapy, to use of 2 or more drugs other than P-gp or BCRP substrates or inhibitors, and to no drug use.
There are only 2 studies published to date that have tried to assess whether simultaneous use of transporter substrates increases malformation rate in humans. Both are register-based studies (malformation and pharmacy register) from the northern provinces of the Netherlands. In the first study, an increase in certain malformation types was seen when several substances affecting the P-gp system were used simultaneously during the first trimester of pregnancy. 13 In the second study, no effect on malformation rate was observed with concomitant use of substrates of other transporters. 14  in the literature. [16][17][18] In our study, antihistamines and drugs for asthma, together with macrolide antibiotics, proton pump inhibitors, oral glucocorticoids and selective serotonin reuptake inhibitors (citalopram, sertraline) were among the most commonly used drugs in the P-gp/BCRP polytherapy T A B L E 2 Number (n) of users of the most commonly used drugs classified as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) substrates (S) or inhibitors (I) in the P-pg/BCRP polytherapy group Two or more P-gp and/or BCRP substrates or inhibitors. 2 One P-gp or BCRP substrate or inhibitor. 3 Two or more drugs other than P-gp and/or BCRP substrates or inhibitors. 4 Adjusted by: birth year (child), maternal age, parity, cohabitation, smoking during pregnancy, socioeconomic status, body mass index, university hospital district, prepregnancy diabetes, connective tissue diseases, severe mental disorders, gestational diabetes.
T A B L E 4 Major congenital anomalies by exposure status to P-glycoprotein (P-gp) substrates or inhibitors Two or more P-gp substrates or inhibitors. 2 One P-gp substrate or inhibitor. 3 Two or more drugs other than P-gp substrates or inhibitors. 4 Adjusted by: birth year (child), maternal age, parity, cohabitation, smoking during pregnancy, socioeconomic status, body mass index, university hospital district, prepregnancy diabetes, connective tissue diseases, severe mental disorders, gestational diabetes.CI, confidence interval.
group (Table 2). While use of these drugs during pregnancy is generally considered safe, [19][20][21][22] we are not aware of studies investigating their teratogenicity when used concomitantly with other drugs not considered as teratogens.
The observed increased risk of congenital anomalies is not necessarily causally associated with drug transporter interactions but may have other possible explanations. First, pregnancy safety data for coxibes, meloxicam and itraconazole-all included in the commonly used drugs in Table 2-are limited or lacking and do not allow reliable risk assessment on their potential for teratogenicity. Further, conflicting results about teratogenicity have been reported for drugs commonly used in the P-gp/BCRP polytherapy group including erythromycin, 23 citalopram and paroxetine. 24,25 A study based on the Swedish Medical Birth Register reported an increased risk of cardiovascular anomalies associated with erythromycin use in early pregnancy. 23 Erythromycin has the potential to inhibit a cardiac potassium current channel and may induce cardiac arrythmias in the embryo predisposing to teratogenicity. 26 Further, serotonin (5-HT) has a role in regulating neural crest cell migration, and regulation of 5-HT by serotonin transporter has been demonstrated in cell culture to play a role in heart development. 27 However, most studies, including large numbers of exposed to erythromycin or citalopram and paroxetine, have been reassuring. 20,28 Further, the specific cardiac anomalies reported with paroxetine use are extremely rare 24 and consequently unlikely to offer an explanation to our results.
Second, some P-gp and BCRP inhibitors can also affect CYP enzymes. Most notably, several P-gp inhibitors also inhibit CYP3A4, whereas P-gp substrates are typically CYP3A4 substrates. 29 Third, polytherapy might be related more strongly to illness or illness severity than monotherapy or not using any drugs. Different pathological states, including diabetes, 30  Two or more BCRP substrates or inhibitors. 2 One BCRP substrate or inhibitor. 3 Two or more drugs other than BCRP substrates or inhibitors. 4 Adjusted by: birth year (child), maternal age, parity, cohabitation, smoking during pregnancy, socioeconomic status, body mass index, university hospital district, prepregnancy diabetes, connective tissue diseases, severe mental disorders, gestational diabetes.
When analysing P-gp and BCRP substrate/inhibitor use separately but allowing overlap between the groups, we found an increased risk of major congenital anomalies with P-gp polytherapy when compared to all the comparison groups. BCRP polytherapy was also associated with an increased risk in all comparisons but the risk turned insignificant after adjustment to confounders when compared to BCRP monotherapy group and remained marginally significant when compared to the non-BCRP polytherapy group.
The expression of P-gp is known to vary with gestational age being highest during the first trimester, corresponding to the organogenetic period, and diminishing towards term. 34 unexposed. In the pure BCRP polytherapy group the odds ratios were increased (33-53% increased risk) but the results were not statistically significant. The wide confidence intervals in the pure BCRP polytherapy analyses might be related to the fact that the numbers included in the analyses were much lower (n = 157 in the pure BCRP polytherapy group) than in the primary analyses (n = 2,068). A possible explanation is that there is a different teratogenic potential between the P-gp and BCRP substrates/inhibitors and the pure P-gp and pure BCRP substrates/inhibitors.
It is also possible that within the group of individual drugs that are substrates or inhibitors to both P-gp and BCRP there may be drug transporter-mediated interactions that may predispose to teratogenicity. For example, several statins and bupropion are both Pgp and BCRP substrates/inhibitors and were not included in the pure P-gp and pure BCRP groups. While statin use has previously been contraindicated during pregnancy, research has been reassuring and statin use may in individual cases be continued until pregnancy is confirmed. 38 However, a recent study observed a 5-fold increased risk of congenital cardiac anomalies related to statin use. 39 Further, bupropion use in early pregnancy has been associated with an increased risk of cardiac anomalies in some but not all, studies. [40][41][42] While these conflicting results may be to some extent related to different study methodologies, the role of drug transporter interactions might also play a role in explaining the conflicting results.
Our study has several strengths. First, the data are derived from the Finnish national health registers covering 18 years and virtually all births and elective terminations of pregnancy due to foetal anomaly. The registers have extensive coverage, high quality and include data on several important potential confounders. 11,12 The PIN allows all women, their children and reimbursed drug purchases to be linked and followed in the registers in a reliable manner. We included all pregnancy terminations due to major foetal anomaly. This is important, as approximately 12% of all pregnancies with a major anomaly in Finland are terminated. 43 We excluded women who had purchased previously known teratogenic drugs during 1 month before the pregnancy or during the first trimester. Unlike previous research, we systematically utilized the UW DIDB to identify P-gp and BCRP substrates and inhibitors. This database is maintained by a team of research scientists with extensive knowledge in the field who assess published information continuously and it provides an unbiased tool to assess drug-drug interactions. It should be noted, however, that our definition of transporter substrate and inhibitor status was solely based on in vitro data. This does not necessarily translate to effect in vivo, but since placental transporter inhibition is still poorly characterized and our primary aim was to see if there is a signal indicating need for further studies, we chose this approach.
Our study also has some limitations. First, the registers do not have information on certain important confounders such as use of alcohol or illicit substances. Second, a registered drug purchase does not necessarily mean actual use and the results may be biased by compliance. In addition, we cannot confirm that the exposure in the polytherapy groups occurred simultaneously. However, several P-gp/BCRP substrates or inhibitors are drugs for chronic diseases such as psychiatric disorders and hypertension and simultaneous use is likely. Lastly, we had no information on over-the-counter drug use. The drug transporter substrate with the most significant over-the-counter use during the study period was cetirizine, a P-gp substrate. Based on the Finnish statistics on medicines, around 1.5-2% of the population used cetirizine between years 2002-2011. [44][45][46][47] However, regular use of cetirizine is likely to be covered by prescription. 44 In conclusion, polytherapy with P-gp and BCRP drug transporter substrates or inhibitors is associated with an increased risk of congenital anomalies. This study may direct future research to consider the role of drug transporter-mediated drug interactions in teratogenesis.
Specifically, studying certain substrate-inhibitor pairs would be an interesting direction for future studies.
takes the responsibility for the integrity of the work as a whole, from inception to the published article. All authors have given substantial contributions to conception and design of the work, or the acquisition, analysis, or interpretation of data. All authors have contributed to drafting of the work and revising it critically for important intellectual content, given final approval of the version to be submitted, and have given agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Study concept and design: All authors.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are not publicly available. According to the national data protection legislation, permission to obtain the research data must be applied from the Finnish Institution for Health and Welfare.