Mechanisms of Altered Hepatic Drug Disposition during Pregnancy: Small Molecules

Abstract

Introduction:

Pregnancy alters the systemic exposure and clearance of many hepatically cleared drugs that are commonly used by obstetric patients. Understanding the molecular mechanisms underlying the changes in factors that affect hepatic drug clearance (blood flow, protein binding, and intrinsic clearance) is essential to more precisely predict systemic drug exposure and dose requirements in obstetric patients.

Areas covered:

This review (1) summarizes the anatomic, physiologic, and biochemical changes in maternal hepatic, cardiovascular, endocrine, and renal systems relevant to hepatic drug clearance and (2) reviews the molecular mechanisms underlying the altered hepatic metabolism and intrinsic clearance of drugs during pregnancy via a comprehensive PubMed search. It also identifies knowledge gaps in the molecular mechanisms and factors that modulate hepatic drug clearance during pregnancy.

Expert opinion:

Pharmacokinetic studies have shown that pregnancy alters systemic exposure, protein binding, and clearance of many drugs during gestation in part due to pregnancy-associated decreases in plasma albumin, increases in organ blood flow, and changes in the activity of drug-metabolizing enzymes (DMEs) and transporters. The changes in the activity of certain DMEs and transporters during pregnancy are likely driven by hormonal-changes that inhibit their activity or alter the expression of these proteins through activation of transcription factors.

1. Introduction

Medication use during pregnancy is on the rise. In the United States, it is estimated that over 80% of pregnant individuals take at least one medication during gestation.[1,2] Clinical trials rarely include pregnant people due to safety and ethical issues. As a result, pharmacological management of medical conditions during gestation is challenged by the lack of sufficient evidence on drug dosing, efficacy, and safety. Consequently, medications are often used off-label and obstetric care providers often rely on ‘best-guess’ treatment.[3,4] Clinical studies have shown that pregnancy alters pharmacokinetics (PK) of drugs; consistently observed PK features during gestation are increased drug clearance and decreased systemic exposure. Changes in the maternal anatomical, physiological, and biochemical processes have been suggested to affect drug disposition: absorption, distribution, metabolism, and excretion (ADME). [5,6] However, pregnancy-evoked changes in a drug disposition may vary depending on the physicochemical properties of the drug and the presence and magnitude of pregnancy-related effects on the disposition mechanisms of the drug.

As the primary site of drug biotransformation, the liver plays a key role in drug clearance.[7] Hepatic clearance is influenced by hepatic blood flow, plasma protein binding, and intrinsic clearance (biotransformation and transport).[8] Pregnancy-associated changes in hepatic drug clearance are well recognized and appear to stem from anatomical and physiological changes in the liver, cardiovascular, renal, and endocrine systems.[9,10] Thus, predicting the overall impact of pregnancy on hepatic drug clearance remains challenging and requires a comprehensive understanding of molecular and biochemical mechanisms governing hepatic metabolism and transport, protein binding, and hepatic blood flow. In this review, we (1) summarize the anatomic, physiologic, and biochemical alterations in maternal hepatic, cardiovascular, and renal systems relevant to hepatic drug clearance; (2) review the molecular and biochemical mechanisms underlying the altered hepatic metabolism and intrinsic clearance of drugs during pregnancy; and (3) identifies knowledge gaps in the molecular mechanisms and factors that modulate hepatic drug clearance during pregnancy.

The databases utilized for the literature search included PubMed Central, PubMed, and various internet search engines such as Google. This literature search was conducted from August to December 2024. All available literature was reviewed, and there were no restrictions regarding publication dates. In some instances, the reference lists of selected articles were also examined to identify additional relevant studies.

2. Pregnancy-associated anatomical, physiological, and biochemical changes impacting hepatic drug clearance

Alterations in maternal anatomy and physiology are the hallmarks of pregnancy and are believed to be the primary sources of the changes in drug PK during pregnancy. Although pregnancy alters the anatomy and physiology of most maternal organs and tissues, changes in the cardiovascular, system, endocrine system, kidney, and liver are believed to be the major contributors to the altered maternal drug clearance and systemic exposure during gestation. These anatomical and physiological alterations across multiple organ systems influence hepatic drug clearance mechanisms through altered hepatic drug metabolism and transport, blood flow to the liver, and decreased plasma protein binding. [11,12] In addition to changes in maternal physiology and anatomy, placental and maternal proteins and metabolites can also impact maternal hepatic drug clearance, but discussion of these mechanisms is beyond the scope of this review.

2.1. Cardiovascular

Pregnancy associated changes in cardiovascular system are well established and are considered as adaptive mechanisms to meet the maternal and fetal metabolic needs. The prominent pregnancy associated changes in cardiovascular physiology are increased cardiac output, systemic vasodilation, reduced peripheral vascular resistance, and increased blood volume due to expansion of plasma and enlarged red blood cells.[11–13] Plasma volume progressively expands up to 50% near term while total protein decreases from 6.7–8.6 g/dL in nonpregnant adults to 5.6–6.7 g/dL in the third trimester.[14,15] The increase in blood volume is essential to meet the growing metabolic demands of both the mother and fetus, as well as to accommodate vasodilation of the maternal systemic vasculature during pregnancy and to help compensate for blood loss during childbirth. Pregnancy related increase in progesterone, estradiol, and relaxin are associated with vasodilation of the systemic vasculature, while plasma expansion is related to increased water and electrolyte reabsorption in the kidney due to activation of renin-angiotensin-aldosterone system.[13,15–17]

2.2. Endocrine

In addition to the expanded plasma volume and other cardiovascular changes, there are remarkable increases in placental biosynthesis and systemic concentrations of pregnancy related hormones (PRHs) in maternal circulation.[18] For instance, the median plasma concentration of estrone increases from 0.9 ng/mL in the first trimester to 11.5 ng/mL in the third trimester, while estradiol increases from 2.2 ng/mL in the first trimester to 20.4 ng/mL in the third trimester. Similarly, the mean progesterone levels in maternal circulation increase from 25.6 ng/mL in the first trimester to 130 ng/mL in the third trimester. Maternal cortisol also moderately increases from 140 ng/mL in the first trimester to 286 ng/mL in the third trimester.[19,20] Additionally, pregnancy leads to changes in peptide hormones such as growth hormones and prolactin, as well as introducing pregnancy-specific peptide hormones like placental growth hormone (pGH) and placental lactogen (hPL). Prolactin and hPL levels in maternal circulation significantly increase to 218 ng/mL and 8700 ng/mL on average, respectively, in the third trimester. Similarly, the pGH can be detected in early pregnancy and gradually increases to over 20 ng/mL in the third trimester, while the pituitary growth hormone does not change in early pregnancy but gradually decreases to below nonpregnant concentrations beyond mid-gestation.[20–24]

2.3. Renal

Pregnancy-associated changes in the anatomy and physiology of the kidney are remarkable. The kidney length increases by up to 1.5 cm due to extended fluid retention and collecting system dilation. Similarly, the kidney volume expands by up to 30% due to vascular and interstitial volume increase in the organ. In early pregnancy, the renal plasma flow and glomerular filtration (GFR) increase by up to 80% and 60%, respectively, relative to nonpregnant controls. The altered GFR can have a small but significant impact on the excretion of albumin, a key drug-binding protein in plasma, and other nutrients such as glucose. Additionally, pregnancy appears to increase the tubular reabsorption of water, resulting in a hypervolemic state. Collectively, increased GFR and tubular water retention contribute to the expanded plasma volume, increased organ blood flow, and decreased albumin concentrations during gestation,[25–27] which in turn can impact hepatic clearance mechanisms.

2.4. Liver

The liver plays a central role in the metabolism of energy, endogenous molecules, and xenobiotics.[7,28] Thus, the change in the hepatic anatomy and physiology to meet this increased demand for the metabolic function during pregnancy is inevitable. In humans, the liver volume increases by 15% in late pregnancy relative to early pregnancy.[29] Similar observations were also made during rodent pregnancy and has been, associated with hepatocyte proliferation during gestation.[30] In human pregnancy, the mechanisms of increased liver size remains largely unknown. Liver function tests showed that serum transaminases, bilirubin, lactate dehydrogenase, and gamma-glutamyl transferase are not impacted during normal pregnancy while the two-fold increase observed in the maternal serum level of alkaline phosphatase is linked to placental secretion of the enzyme.[31] The impact of pregnancy on the hepatic drug clearance is widely observed in part due to altered expression and function of hepatic DMEs and transporters.[11]

3. Mechanisms Underlying Hepatic Drug Clearance during Pregnancy

Hepatic drug clearance is a function of protein binding, blood flow, and intrinsic clearance.[32] In this section, we discuss the molecular mechanisms and consequences of pregnancy-related changes in each of the three factors in the hepatic clearance of drugs.

3.1. Protein Binding

Since only the unbound fraction of drugs are available for target action or organ clearance, plasma protein binding plays a fundamental role in drug PK and pharmacodynamics.[33] Albumin and α-1-acid glycoprotein (AGP) are major drug transporters in plasma, that largely mediate plasma drug binding. While acidic drugs preferably bind to albumin, basic drugs can associate with plasma proteins including α-1-acid glycoprotein, albumin, and lipoproteins. The liver synthesizes and immediately excretes albumin into the circulation at high concentration (10–15 g/day); however, its concentration can decrease due to physiological or disease states such as inflammation, liver impairment, malnutrition, kidney impairment, and pregnancy.[34–36] During pregnancy, albumin plasma concentrations decrease from 4.1–5.3 g/dL in nonpregnant to 2.3–4.3 g/dL in the third trimester.[14] Additionally, AGP appears to decrease during pregnancy.[37–39] The fall in plasma total protein concentration is considered a dilutional effect of the expanded plasma that decreases concentration of albumin, the most abundant protein in human plasma. However, the concentration of most proteins of hepatic origin in maternal circulation remain unchanged during gestation, suggesting the presence of other factors contributing to lower albumin concentration during pregnancy. Studies have shown that plasma albumin levels were lower in individuals on oral contraceptives composed of progestin and ethinylestradiol, synthetic analogs of progesterone and estrogens, respectively. Thus, in addition to the dilutional effect of the expanded plasma, progesterone and estradiol can inhibit albumin synthesis resulting in the fall of plasma albumin concentration.[39–42]

Pregnancy-related reduction in plasma albumin and AGP levels can significantly affect hepatic drug clearance during gestation, depending on individual medication extraction ratios. For the clearance of drugs with a high extraction ratio (i.e. high unbound clearance), the hepatic blood flow is the limiting factor. On the other hand, for the clearance of drugs with a low extraction ratio (low unbound clearance), protein binding and intrinsic enzyme activity are the limiting factors. For example, although serum levels of AGP progressively decrease and the unbound fraction of the drug is higher during gestation, pregnancy did not change the total clearance of propranolol, a high protein binding and high extraction ratio drug. However, pregnancy increased apparent clearance of phenytoin, a low extraction ratio and high protein binding drug, by over 50% in the third trimester owing to decreased protein binding and increased CYP enzyme activity.[38,43–49]

3.2. Hepatic Blood Flow

Pregnancy increases maternal blood volume and cardiac output. In healthy pregnancy, the cardiac output significantly and progressively increases from first trimester to third trimester and falls postpartum.[13,50] As a result, blood flow increases to organs such as the liver and kidney has been reported.[27,51] In the third trimester, pregnancy increases portal vein and total blood flow, but not arterial blood flow, by 150 and 160%, respectively.[50] Other studies, however, suggested that hepatic blood flow remains unchanged during gestation.[52,53] The discrepancies in the observations might stem from the techniques of hepatic blood flow measurement. Easterling et al. study used noninvasive doppler ultrasound and reported a significant increase in total hepatic flow due to increased portal venous flow[50] while others used invasive techniques such as hepatic vein catheterization and indocyanine green and observed no change in hepatic blood flow.[52,53]

The effect of hepatic blood flow on the hepatic drug clearance is well recognized. However, the impact of hepatic blood flow is limited by the extraction of ratio of the drug in the liver. For drugs with high extraction ratio, hepatic clearance increases proportionally with increases in hepatic blood flow and presentation of the drug to the liver is considered as a rate limiting step. Thus, perturbations in the intrinsic clearance mechanisms (e.g., DME expression and activity) of the drug do not significantly impact its hepatic clearance. For instance, buprenorphine, a widely used anti-opioid disorder drug, is a high protein binding and a high extraction ratio drug. In nonpregnant individuals, it has been suggested that the hepatic clearance of buprenorphine is flow dependent and unimpacted by alterations in hepatic metabolism.[54,55] However, rifampicin, a DME inducer, decreased buprenorphine plasma concentration by 70% in nonpregnant opioid-dependent individuals, [56] suggesting that induction of hepatic DMEs may also contribute to the decreased buprenorphine systemic exposure in vivo. This suggests that altered activities of hepatic DMEs might contribute to the observed higher clearance of buprenorphine during gestation.[57] Buprenorphine is extensively metabolized to buprenorphine glucuronide (via UGT1A1, 1A3, and 2B7), norbuprenorphine (via CYP3A4 and 2C8), and norbuprenorphine-glucuronide (via UGT1A1, 1A3).[57,58]. In contrast, propranolol is a high extraction ratio and a high protein binding drug that is extensively metabolized (via CYP2D6 and CYP1A2) in the liver. However, pregnancy does not alter the disposition of propranolol, suggesting that neither a pregnancy related increase in hepatic flow nor pregnancy associated change in CYP2D6 and CYP1A2 activity alter propranolol clearance and exposure during pregnancy. [46,59–61]

3.3. Intrinsic clearance (enzyme activity)

Phase I and II hepatic DMEs and transporters are central to hepatic clearance of drugs and their metabolites. [62–64] Hepatic phase I DMEs are microsomal membrane or cytosolic proteins that primarily catalyze oxidation, reduction, or hydrolysis reactions. Cytochrome P450s (CYPs) are the main phase I DMEs, but the list also includes flavin monooxygenases (FMOs), carboxylesterases (CESs), and aldehyde oxidases (AOX).[65] On the other hand, phase II DMEs catalyze conjugation reactions to facilitate the excretion of drugs or their metabolites. The main phase II DMEs include uridine 5'-diphospho-glucuronosyltransferas (UGT), glutathione S-transferases (GST), N-acetyltransferases (NAT), and sulfotransferases (SULT).[66] Hepatic transporters mediate drug uptake to the hepatocytes for metabolism and translocate drugs or their metabolites to the bloodstream or bile.[62] DMEs are widely expressed in the liver and demonstrate substantial interindividual differences in expression and function owing to changes in individual physiology due to diseases such as metabolic dysfunction and pregnancy, exposure to chemicals, and genetics.[67,68]

Studies have consistently shown that pregnancy alters the clearance and systemic exposure of many drugs in part due to altered hepatic drug metabolism and transport.[5] For instance, the plasma ratio of 4β-hydroxycholesterol to cholesterol (a CYP3A metabolism biomarker [69]) is higher during pregnancy relative to nonpregnant or postpartum, suggesting increased metabolic activity of CYP3A enzymes during pregnancy.[70,71] Furthermore, pregnancy also increases the clearance and systemic exposure to CYP3A probe substrate midazolam, and clinically relevant CYP3A metabolized drugs nifedipine and buprenorphine.[57,72,73] Similar to CYP3A, pregnancy has been reported to alter the clearance of various drug substrates of other major CYPs enzymes such as CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1.[5,74] In addition to CYPs, pregnancy alterations in the clearance of drug substrates of UGTs, including UGT1A1, UGT1A4, UGT2B7, and transporters such as P-glycoprotein (P-gp) have been reported.[57,72,75,76] The mechanisms of the altered activities of hepatic DMEs and transporters remain largely unknown. However, it has been hypothesized that pregnancy-mediated hormonal changes activate steroidal and xenobiotic nuclear receptors, thereby changing hepatic expression and function of DMEs and transporters. In vitro models of xenobiotic metabolism and transport in human hepatocytes suggest that pregnancy related hormone (PRH) mediated alterations in the transcriptional regulation of certain DMEs and transporters contributes to the altered function during pregnancy.[77–79] In this section, we will review the proposed potential mechanisms (Table 1) underlying pregnancy changes in major drug-metabolizing CYPs, UGTs, and transporters. Due to insufficient evidence in the literature on the pregnancy-related changes in sulfotransferases, glutathione S-transferases, and arylamine N-acetyltransferases are beyond the scope of this manuscript.

Table 1.

Proposed potential molecular mechanisms of pregnancy related alterations in the activities major drug metabolizing cytochrome P450s (CYPs) and uridine diphosphate glucuronosyltransferases (UGTs) in human liver.

Enzyme	Activity during Gestation In Vivo	Potential Molecular Mechanisms of Altered Activity
CYP1A2	Decreased [82]	Inhibition CYP1A2 activity by estrogens [84,85]. Estradiol and progesterone did not impact expression in vitro.[79]
CYP2A6	Increased [91,92]	Induction of CYP2A6 by estrogen and progesterone mediated activation of ERα, CAR, and PXR [79,96,99,100]
CYP2B6	Increased [103,104,107]	Induction of CYP2B6 expression by estrogen and progesterone mediated activation of ERα, CAR, and PXR [79,110,112,113]
CYP2C8	Unknown (likely increased) [117,118]	Induction of CYP2C8 expression by estrogen, progesterone, and cortisol mediated activation of ERα, CAR, PXR and GR [79,119–121]
CYP2C9	Increased [47,124,125]	Induction of CYP2C9 expression by cortisol via activation of GR, PXR, and CAR [130,135]. Conflicting data has been reported regarding the effects of estrogens on the expression and activity of CYP2C9.
CYP2C19	Decreased [136,137]	Inhibition of CYP2C19 activity by estrogens and progesterone or repression of its expression by estrogen via ERα [134,137,139]
CYP2D6	Increased [140,141]	Unknown. PRHs do not alter the expression of CYP2D6 in human hepatocytes, and oral contraceptives do not alter the activity of the enzyme.[79,86]
CYP2E1	Unknown (likely increased)	Induction of CYP2E1 expression by placental lactogen mediated activation of phosphoinositide 3-kinase pathway in human hepatocytes [156]
CYP3A4	Increased [70,72,73]	Induction of CYP3A4 expression by cortisol, estrogens, and placental growth hormone mediated activation of GR, PXR, CAR, and GHR [120,159,162]
UGT1A1	Increased [75,168]	Induction of UGT1A1 expression by cortisol, estrogen, and progesterone mediated activation of GR, PXR, and CAR [77,171–173]
UGT1A4	Increased [174,175]	Induction of CYP3A4 expression by estrogen and progesterone mediated activation of ERα, PXR, and CAR [77,179,180]
UGT2B7	No effect [183]	PRH cocktails composed of estrogens, progesterone, cortisol and placental growth hormone do not impact the expression of UGT2B7 in human hepatocytes but decreased its activity toward labetalol metabolism. [77,171,172]
OAT1B1/B3	Decreased [199,200]	Suppression of OATP1B1/B3 expression or inhibition of activity by PRHs and their metabolites.[77,201,202]
P-GP	Increased [206,208]	Induction of P-gp expression by PRHs.[77]

3.3.1. Hepatic Drug Metabolizing Enzymes

CYP1A2.

CYP1A2 represents approximately 13% of total CYP proteins in the liver.[80] The enzyme catalyzes biotransformation of clinically important drugs (e.g. acetaminophen, clozapine), toxins (e.g. aflatoxin), and endogenous molecules (e.g. melatonin). In general, CYP1A2 mediates metabolism of 10–15% of drugs that undertake hepatic biotransformation. Common pregnancy relevant CYP1A2 substrate drugs include caffeine and acetaminophen. In humans, CYP1A2 is responsible for over 95% of caffeine primary metabolism and converts caffeine to 1,7-dimethylxanthine theobromine (3,7-dimethylxanthine), and theophylline (3,7-dimethylxanthine).[81] In pregnant individuals, caffeine oral clearance is significantly reduced and half-life increased to 10.5 h from 3 h in nonpregnant controls resulting in systemic accumulation.[82] Since caffeine is a low extraction ratio drug with low protein binding,[83] the observed reductions in caffeine hepatic clearance can be related to decreased CYP1A2 activity during pregnancy. However, the mechanisms of pregnancy-evoked decreases in CYP1A2 activity remain largely unknown.

CYP1A2 is constitutively highly expressed in the liver, and it can be induced by proton pump inhibitors such as omeprazole and inhibited by oral contraceptives. In individuals taking contraceptive combinations of ethinylestradiol and gestodene, CYP1A2 activity is significantly lower relative to nonusers, and the authors suggested that the observed decrease in CYP1A2 activity might be related to inhibition of the enzyme by the contraceptive.[84,85] In vitro studies in human liver microsomes showed that ethinylestradiol, not gestodene, inhibits CYP1A2 [86,87], suggesting that pregnancy associated decrease in CYP1A2 activity might be related to inhibition of the enzyme by estradiol (or its metabolites). Additionally, CYP1A2 is transcriptionally regulated by aryl hydrocarbon receptor, (AHR).[88] However, 1–10 μM estradiol or progesterone did not impact CYP1A2 mRNA expression in human primary hepatocytes.[79] It is likely that the pregnancy associated decrease in CYP1A2 activity stems from hormonal mediated inhibition of its activity. However, the precise mechanism of CYP1A2 regulation during gestation warrants further investigation.

CYP2A6.

CYP2A6 represents approximately 4% of total CYP proteins in the liver, and the gene is also expressed in multiple extrahepatic tissues, such as the lungs and breast.[80] This enzyme is responsible for the metabolism of multiple xenobiotics, including approximately 3% of approved drugs (including pilocarpine, valproic acid, and tamoxifen).[89] However, nicotine is the most significant clinically relevant CYP2A6 substrate drug. In the U.S., around 5.4% of pregnant individuals use nicotine during gestation,[90] and it is difficult for them to quit smoking due to an increased clearance of the substance during this period.[91] In humans, CYP2A6 contributes approximately 40–70% of nicotine metabolism, and pregnancy significantly increases the rate of this metabolism.[91,92] This suggests an increase in CYP2A6 activity during gestation.[89,91,92] It is important to note that since nicotine is a drug with a high hepatic extraction ratio, its clearance is primarily influenced by blood flow.[50,93,94] Thus, increased hepatic clearance of the drug may not accurately reflect CYP2A6 activity during pregnancy. In contrast, cotinine, the primary metabolite of nicotine and substrate for CYP2A6, is a low extraction ratio metabolite with low protein binding. Consequently, it is considered a better indicator of changes in CYP2A6 activity than nicotine itself.[95] The elevated ratio of 3'-hydroxycotinine to cotinine during pregnancy supports the notion of increased CYP2A6 activity during gestation.[92]

In vitro studies have suggested that increased CYP2A6 during gestation may be related to pregnancy-associated hormonal changes. In human primary hepatocytes exposed to estradiol or progesterone, CYP2A6 mRNA expression and metabolism of coumarin significantly increased in a hepatocyte donor-dependent manner, suggesting the increased CYP2A6 activity during gestation is related to increased expression of the enzyme.[79] Studies have also shown that estrogen induces CYP2A6 expression via estrogen receptor alpha (ERα).[96] Furthermore, nuclear receptor CAR activators 6-(4-Chlorophenyl) imidazo[2,1-b] [1,3]thiazole-5-carbaldehyde O-3,4-dichlorobenzyl) oxime (CITCO) and phenobarbital and PXR activator rifampicin also induce CYP2A6 in human hepatocytes.[79,97,98] Because estradiol can bind and activate CAR and PXR,[99,100] increased CYP2A6 activity during pregnancy may be driven by hormonal activation of nuclear receptors ERα, CAR, and PXR.

CYP2B6.

CYP2B6 is primarily expressed in the liver, but it is historically considered as a minor P450 isoform because it represents less than 1% total CYP content in the liver.[80] However, recent studies suggest that the enzyme contributes 1–10% of total CYP proteins in the liver.[101,102], and metabolizes 2–10% of clinically approved drugs including antineoplastics, antiretrovirals, and anesthetics.[102] Bupropion, methadone, and efavirenz are among known drug substrates of CYP2B6 with known clinical significance in obstetric patients. Studies have shown that pregnancy increases clearance of methadone and decreases its systemic exposure.[103,104] Since methadone is primarily metabolized by the CYP2B6 enzyme and is a low hepatic extraction ratio drug,[105,106] the increased clearance of the drug during pregnancy may suggest a gestation mediated increase in CYP2B6 activity. Similarly, the clearance of efavirenz appeared to increase during gestation and has been associated with pregnancy-evoked alterations CYP2B6 activity.[107] In contrast, pregnancy did not affect clearance of bupropion, a CYP2B6 probe substrate.[108,109]

In vitro studies in human primary hepatocytes cultures have shown that PRHs such as estradiol and progesterone induce the expression and activity of CYP2B6.[79,110] Mechanistic studies have shown that estradiol activated ERα binds its response element on the CYP2B6 promoter and induces the expression of the gene. Furthermore, CYP2B6 expression is upregulated in ERα positive breast cancer.[111] In addition to ERα, because CYP2B6 is a typical target gene for xenobiotic nuclear receptors CAR and PXR [112,113] and these receptors are activated by estradiol, it is likely that pregnancy can increase CYP2B6 expression and activity through ERα, CAR and PXR mediated transcriptional activation. However, it has not been studied whether crosstalk between the steroidal and the xenobiotic receptors contributes to CYP2B6 transcriptional regulation during gestation.

CYP2C8.

CYP2C8 is highly expressed in the liver and represents approximately 7% of the total CYP content in the liver.[114] The enzyme catalyzes the metabolism of endogenous substrates (e.g., arachidonic acids, retinoic acid) and therapeutic drugs relevant to pregnancy, such as paclitaxel, chloroquine, and amodiaquine. Clinical studies have shown that pregnancy increases clearance and decreases systemic exposure to paclitaxel, an anti-cancer drug and CYP2C8 probe substrate.[115,116] Similarly, the ratio of systemic exposure of chloroquine to desethylchloroquine, the major metabolite of chloroquine, was significantly lower in pregnancy, suggests an increased hepatic metabolism. However, in addition to CYP2C8, desethylchloroquine is also formed by CYP3A4, an enzyme with a known increase in activity during pregnancy.[117] In contrast, pregnancy did not impact the clearance of amodiaquine, another CYP2C8 probe drug.[118] Thus, it appears that the impact of pregnancy on the CYP2C8 activity remains unclear.

Transcriptionally, CYP2C8 is regulated by multiple nuclear receptors including PXR, CAR, glucocorticoid receptor (GR), and hepatic nuclear factor 4 (HNF4α).[119] Using human primary hepatocytes, Khatri et al. showed that a PRH cocktail composed of estrogens, progesterone, and cortisol significantly induced CYP2C8 mRNA and protein expression; at the protein level, these effects appeared to be largely driven by estradiol.[120] Other studies reported that progesterone and synthetic steroid dexamethasone induce CYP2C8 mRNA expression in human primary hepatocytes.[79,121]

CYP2C9.

CYP2C9 is the most abundant CYP2C isoform and contributes approximately 20% of total CYP proteins in the liver. The enzyme contributes to the oxidation of approximately 20% of therapeutic agents metabolized by CYP enzymes [122,123] including pregnancy relevant drugs phenytoin and glyburide. Studies have shown that pregnancy increased phenytoin clearance by two-fold resulting in a decreased systemic exposure by about 60% relative to nonpregnant individuals.[47,124] Similarly, pregnancy increased oral clearance of glyburide and formation clearance of its major metabolite 4-trans-hydroxyglyburide by over 2-fold subsequently decreasing glyburide systemic exposure by more than 50%.[125] Since phenytoin and glyburide are low hepatic extraction ratio and high protein binding drugs,[126,127] the increased total clearance of the drugs during gestation can be associated with pregnancy related decreases in plasma albumin and increases in hepatic biotransformation, suggesting that pregnancy increases hepatic CYP2C9 activity.

CYP2C9 is an inducible gene by multiple xenobiotics such as phenobarbital, rifampicin, and dexamethasone in hepatocytes and Huh7 cells. Transcriptionally, multiple nuclear receptors such as CAR, PXR, GR, ERα, and vitamin D receptors appear to regulate the CYP2C9 gene.[128–131] The effect of hormonal drugs such as oral contraceptives containing ethinylestradiol and levonorgestrel is inconsistent. Cherala et al. reported that ethinylestradiol-levonorgestrel containing oral contraceptives did not impact metabolism of tolbutamide in reproductive age women[132] while Sandberg et al. observed metabolism of losartan to its carboxylic acid metabolite was slower in women on oral contraceptives compared to those without.[133] Similarly, ethinylestradiol appeared a potent inhibitor in human microsomes and suppressor CYP2C9 in hepatocytes while estradiol did not have significant impact on the expression or activity of the enzyme[131,134]. On the other hand, Choi et al. reported estradiol increased CYP2C9 activity without impacting its expression.[79] CYP2C9 activity can be activated by positive heterotropic cooperativity;[135] however, such effects have not been studied with PRHs. Studies also showed that at lower concentration (0.1 μM), dexamethasone induced CYP2C9 mRNA in human hepatocytes[130], suggesting possible role of cortisol in the regulation of CYP2C9 during gestation. Based on the in vivo and in vitro data, pregnancy related increases in CYP2C9 may be related to activation of GR by cortisol which in turn potentiates PXR and CAR.

CYP2C19.

CYP2C19 is predominantly expressed in the liver and contributes to approximately 3% of total hepatic CYP content. The enzyme metabolizes about 8–10% of therapeutic agents including pregnancy-relevant drugs citalopram and proguanil. Heikkinen et al.[136] studied the disposition of citalopram in pregnant individuals and reported that the ratio of desmethylcitalopram (CYP2C19 and CYP3A4 metabolite) to citalopram was 23% lower in pregnant compared to postpartum individuals, suggesting decreased activity of CYP2C19. Desmethylcitalopram is further metabolized to didesmethylcitalopram by CYP2D6 and the ratio of plasma didesmethylcitalopram to desmethylcitalopram was 54% higher in pregnancy relative to postpartum, suggesting an increased CYP2D6 activity during gestation. Hence, it is inconclusive whether lower plasma desmethylcitalopram in pregnant individuals was due to decreased CYP2C19 and/or increased CYP2D6 activity.[136] Similar to citalopram, CYP2C19 also contributes to the hepatic biotransformation of proguanil to cycloguanil. In pregnant individuals, the ratio of proguanil to cycloguanil in plasma was significantly higher compared to postpartum in extensive CYP2C19 metabolizers, suggesting decreased CYP2C19 activity during gestation.[137]

Transcriptionally, CYP2C19 is regulated by multiple nuclear receptors such as HNF4α, PXR, and ERα.[138] Rifampicin induces CYP2C19 in human primary hepatocytes.[130] Furthermore, contraceptives appeared to impair the hepatic biotransformation of proguanil in CYP2C19 extensive metabolizers.[137] Similarly, estradiol and ethinylestradiol decreased the expression of CYP2C19 via ERα in in human hepatocytes and Huh7 cells and progestins were potent inhibitors in liver microsomes,[134,139] although another study showed that estradiol and progesterone did not impact the expression and activity of CYP2C19.[79] Overall, a pregnancy-associated decrease in CYP2C19 might be related to remarkable changes in maternal estrogen levels.

CYP2D6.

CYP2D6 is predominantly expressed in the liver and contributes approximately 4% of total hepatic CYP content.[80] The enzyme mediates the metabolism of endogenous molecules and numerous xenobiotics including about 25% of clinical drugs. Studies have shown that the hepatic metabolism of CYP2D6 substrates significantly increased during gestation. Relative to postpartum, the ratio of dextromethorphan to dextrorphan decreased by 53% in CYP2D6 extensive metabolizers during gestation.[140] Similarly, the oral clearance of metoprolol significantly increased by 1.8 and 2.8- fold in mid and late pregnancy, respectively, relative to postpartum,[141] suggesting a significant increase in CYP2D6 metabolic activity during gestation.

CYP2D6 is a unique cytochrome P450 enzyme in that it is not inducible, and the variability in its enzymatic activity between individuals largely arises from genetic differences.[142,143] Studies have found that at fertile age, females exhibit higher CYP2D6 activity compared to males, indicating a potential influence of sex hormones on the regulation of this enzyme.[144] However, prospective in vitro studies using human primary hepatocytes demonstrated that progesterone and estradiol did not significantly affect CYP2D6 expression.[79] Additionally, the use of oral contraceptives did not show a notable impact on CYP2D6 activity, with changes remaining below 11% when compared to non-users, as assessed by the dextromethorphan to dextrorphan ratio.[86] While it is known that nuclear receptors such as HNF4α, small heterodimer partner (SHP), and farnesoid X receptor (FXR) may transcriptionally regulate the CYP2D6 gene,[145] it remains largely unclear whether the increased activity of CYP2D6 during pregnancy is due to increased expression of the gene itself or if it results from other changes related to pregnancy, such as post-translational and epigenetic modifications.

CYP2E1.

CYP2E1 is primarily expressed in the liver and contributes to approximately 7% of total CYP protein in human liver.[80] The enzyme contributes to the metabolism of a limited number of drugs such as acetaminophen and isoniazid, and several low molecular weight procarcinogens. Acetaminophen is used by over 60% of pregnant individuals in the U.S.A while isoniazid is a first-line antituberculosis medication.[146–149]. Acetaminophen and isoniazid are known hepatotoxins following CYP2E1 mediated generation of free radicals.[150,151] Studies have shown that pregnancy did not impact acetaminophen and isoniazid PK.[152,153] Since the contribution of CYP2E1 in the metabolic elimination of these drugs is minor,[154,155] absence of pregnancy-evoked alterations on the PK of acetaminophen and isoniazid may not precisely demonstrate impact of pregnancy on CYP2E1 activity. Studies in human hepatocytes demonstrated that hPL, a placental hormone specific to pregnancy, induces CYP2E1 expression and activity via phosphoinositide 3-kinase pathway. [156]

CYP3A4.

CYP3A4 is primarily expressed in the liver and intestine. The enzyme accounts for approximately 30% of the total CYP content in the human liver and mediates the metabolism of over 30% of marketed drugs.[80,157] Moreover, CYP3A4 depicts broad substrate specificity and oxidizes a wide variety of endogenous molecules and xenobiotics including steroidal hormones, cholesterol, therapeutic drugs, and environmental chemicals.[157,158] CYP3A4 mediates the metabolism of multiple pregnancy-relevant drugs including nifedipine and buprenorphine. During pregnancy, the activity of the enzyme has been measured using an endogenous biomarker,[70] probe substrate midazolam,[72] and clinical drugs such as nifedipine and buprenorphine,[57,73] and confirmed that pregnancy significantly increases CYP3A4-mediated clearance. In a study of pregnant (n=100) and nonpregnant controls (n=59), Mlugu et al. reported that the plasma 4β-hydroxycholesterol to cholesterol ratio was significantly higher in pregnant groups compared to nonpregnant control.[70] Similarly, pregnancy increased apparent midazolam oral clearance by 108% and 1´-hydroxy midazolam formation clearance by 123%.[72] Consistent with the approximate 2-fold increase in 4β-hydroxycholesterol formation and midazolam clearance, the systemic exposure to norbuprenorphine, CYP3A4 mediated metabolite of buprenorphine, increased by 1.8-fold during gestation [57] and nifedipine half-life substantially decreased while clearance increased by approximately 4-fold.[73]

Collectively, the widely accepted consensus is that pregnancy increases CYP3A4 activity. Although the molecular mechanisms of altered CYP3A4 activity remain not fully understood, pregnancy-associated hormonal changes are thought to induce the transcriptional expression of the gene through activation of regulatory proteins such as PXR and GR.[159] This notion is based on multiple observations: (1) females express higher basal hepatic CYP3A4 than males in humans due the pattern and extent of hormones such as growth hormones (GH),[160] (2) sex steroids such as estradiol and cortisol bind and activate their natural receptors or xenobiotic nuclear receptors PXR and CAR,[159,161] and (3) PRHs increase the expression and activity of CYP3A4 in human primary hepatocytes in vitro.[78,79] During pregnancy, GH decreases beyond mid gestation, but placental GH (pGH) substantially increases in a gestational age dependent manner. In human primary hepatocytes, pGH induced CYP3A4 expression and activity.[162] Secretion of PRHs such as estradiol and cortisol remarkably increases during pregnancy.[18] Studies have shown that estradiol can bind and activate xenobiotic receptors CAR and PXR or its own receptor ERα.[100,161] However, oral contraceptives containing ethinyl estradiol, an ERα agonist, appears to decrease CYP3A4 activity.[163] In human hepatocytes, cortisol activated GR resulting in induction of CYP3A4 expression and activity either by inducing PXR or directly binding CYP3A4 promoter.[159] In general, clinical drug metabolism and PK studies have clearly shown that pregnancy increases CYP3A4 activity and in vitro mechanistic studies suggest that estrogens, cortisol, and pGH may be driving induction of CYP3A4 expression during gestation. However, the exact molecular mechanisms of CYP3A4 regulation during gestation in humans remain unclear.

UGT1A1.

UGT1A1 belongs to the large family of UGT superfamily. The enzyme is predominantly expressed in the liver, and accounts for approximately 14% of total hepatic UGTs.[164,165] UGT1A1 mediates metabolic clearance of multiple endogenous (e.g. bilirubin) and exogenous chemicals including therapeutic drugs such as irinotecan and buprenorphine.[57,166,167] Studies have shown that pregnancy increases clearance of UGT1A1 substrate drugs such as labetalol and raltegravir. In pregnant individuals, labetalol oral clearance increased from1.4-fold at 12 weeks to 1.6-fold at term relative to postpartum.[75] Similarly, a 50% reduction in the systemic exposure to raltegravir has been reported during pregnancy compared to postpartum.[168]

The mechanisms of altered UGT1A1 activity during pregnancy has not been established but can be associated with pregnancy triggered hormonal changes. Transcriptionally, UGT1A1 can be regulated by multiple nuclear receptors including GR, PXR, CAR, AHR, hepatocyte nuclear factor-1 alpha (HNF1α), peroxisome proliferator-activated receptor alpha, and liver X receptor.[169,170] Studies have shown that PRHs induce UGT1A1 expression and activity. In SCHH, Khatri et al observed PRHs increased the absolute protein concentration of UGT1A1 and the formation of the phenolic glucuronide of labetalol (UGT1A1 metabolite). Additionally, the absolute protein concentration of UGT1A1 and the metabolite concentration correlated both the media and cell lysate, and the effects were primarily through estradiol and progesterone.[171] Consistent with these observations, estradiol and progesterone appeared to mediate the increased UGT1A1 mRNA expression, absolute protein concentration, and activity in HepG2 and SCHH [77,172]. Although glucocorticoids activate GR to synergistically enhance PXR/CAR regulation of the UGT1A1 gene expression,[173] collectively, it appears that PRHs estrogens and progesterone induce UGT1A1 expression and activity.

UGT1A4.

UGT1A4 is primarily expressed in the liver and other extrahepatic organs such as small intestine and kidney and represents approximately 13% of total hepatic UGTs.[165] The enzyme plays a critical role in the hepatic elimination of drug substrates such as lamotrigine.[174] During gestation, the clearance of drugs substrates of UGT1A4 appeared to significantly increase relative to nonpregnant levels, resulting in therapeutic failures. For instance, the clearance of total and free lamotrigine, a commonly prescribed antiepileptic drug, significantly increased in all trimesters peaking at the third trimester.[174,175] Lamotrigine is extensively metabolized in the liver and up to 80% of the dose can be recovered in the urine as lamotrigine N2-glucronide and lamotrigine N5-glucuronide.[176,177] Considering the low extraction ratio and moderate plasma protein binding properties of lamotrigine, it has been suggested that alterations in UGT1A4 activity plays a critical role in the interindividual differences in lamotrigine clearance in humans.[178] Thus, this suggests that the clinically observed increase in lamotrigine clearance during pregnancy is associated with increased activity of UGT1A4.

Mechanistic studies in human primary hepatocytes showed that PRHs increase the expression and activity of UGT1A4.[77,179] The expression of the enzyme is induced by typical PXR (e.g., rifampicin) and CAR (e.g., CITCO) inducers.[77] In SCHH exogenously exposed to cocktails of PRHs (estrone, estradiol, estriol, progesterone, cortisol, and pGH), we reported that the absolute protein concentration of UGT1A4 significantly increased with increasing concentration of the hormone cocktails, and the effects were primarily mediated by progesterone.[77] Similarly, Chen et al. reported that estradiol increased the UGT1A4 expression and its activity toward lamotrigine glucuronidation in HepG2 cells and the effects were mediated by specificity protein-1 (SP1) and ERα.[179] Furthermore, a 2.1-fold higher oral clearance of lamotrigine was reported in women when administered with combined contraceptives (ethinyl estradiol and levonorgestrel) than lamotrigine alone.[180] Thus, it appears that pregnancy hormone mediated induction of UGT1A4 expression during pregnancy appears to drive the increased hepatic activity of the enzyme during gestation.

UGT2B7.

Representing over 26% of total hepatic UGT proteins, UGT2B7 is the most abundant UGT in the human liver.[164,165]. It mediates the conjugation of multiple therapeutic drugs (e.g., labetalol, buprenorphine, zidovudine).[57,75,181] UGT2B7 mediates glucuronidation of 60–70% zidovudine and is considered as the primary elimination mechanism.[182] During gestation, the zidovudine peak concentration, elimination half-life, and clearance were comparable to the levels observed at postpartum, although areas under the concentration curve were slightly but significantly lower in the pregnant group.[183] Contrary to this observation, pregnancy appears to alter the oral clearance of UGT2B7 drug substrates such as labetalol and buprenorphine.[57,75,181] However, because other DMEs play a critical role in the hepatic clearance of these drugs, pregnancy related increases in labetalol and buprenorphine can be linked to alterations in the activities of other DMEs such as UGT1A1 in case of labetalol and UGT1A1 and CYP3A4 in the case of buprenorphine.[77,171,172,184] For instance, of the two major metabolites of labetalol, UGT2B7 mediates the formation of benzyl glucuronide (the major metabolite) in humans while UGT1A1 mediates the formation of labetalol phenolic glucuronide[185] Therefore, it has been suggested that the increased oral clearance of labetalol is related to the pregnancy-evoked increase in UGT1A1 expression and activity, and not changes in UGT2B7 activity.[75,171]. Thus, it appears unlikely that pregnancy significantly impacts UGT2B7 activity in humans.

Consistent with the clinical observations, in vitro models of drug metabolism in human liver cells did not show alterations in UGT2B7 expression and activity. In SCHH exogenously exposed to estrone, estradiol, estriol, progesterone, cortisol, and pGH in combination or individually, we did not observe changes in the absolute protein concentration of UGT2B7.[77] Similarly, estradiol, progesterone, and cortisol did not impact UGT2B7 expression in human liver cells.[171,172] However, PRHs decrease the formation of benzyl glucuronide via unclear mechanism.[171] Transcriptionally, nuclear factor E2-related factor 2 (NRF2) and FXR regulate UGT2B7 expression in liver cells.[186]

3.3.2. Hepatic transporters

In addition to DMEs, hepatic drug transporters are essential for drug disposition and the homeostasis of endogenous compounds (e.g., bile acids, hormones).[187] More than 50% of the 100 most prescribed drugs interact with transporters[188] and some of these drugs are commonly used during pregnancy.[189] Solute carrier (SLC) transporters and ATP-binding cassette (ATP) transporters are the two major families responsible for active drug transport. Hepatic drug basolateral uptake transporter, such as organic anion transporting polypeptide (OATP1B1/SLCO1B1, OATP1B3/SLCO1B3, and OATB2B1/SLCO2B1), sodium taurocholate cotransporting polypeptide (NTCP/SLC10A1), organic anion transporter (OAT2/SLC22A7), and organic cation transporter (OCT1/SLC22A1, OCT3/SLC22A3) have shown to be involved in drug-drug interactions (DDIs)[187,190] and can be one of the rate-limiting steps in hepatobiliary drug clearance.[191–193] For example, DDIs with OATP inhibitors have shown to increase the plasma concentration of OATP substrates, such as rosuvastatin, pravastatin and pitavastatin. In addition to DDIs, genetic variants in OATP have shown altered PK of OATP substrates.[194] The clinical impact of DDIs involving hepatic efflux transporters, such as breast cancer resistance protein (BCRP/ABCG2), multidrug resistance-associated proteins (MRPs/ABCC), p-glycoprotein (P-gp/ABCB1), bile salt export pump (BSEP/ABCB11), and bidirectional organic solute transporter (OST/SLC51), are challenging and data therefore are limited.[195] Studies have shown that pregnancy alters transporter substrate drug disposition.[5] In rodents, gestational age dependent changes in hepatic transporter gene expression and/or protein have been observed.[196,197] However, due to species differences in transporter function, human data on gene expression and protein levels is necessary, which is largely unknown.

OATP1B1/B3.

The uptake transporter OATP1B1 and OATP1B3 are mainly expressed in the liver, whereas OATP2B1 is ubiquitously expressed. They facilitate the transport of drugs or endogenous compounds into the hepatocytes.[198] Rosuvastatin, an OATPs/NTCP and BCRP substrate, is mainly cleared through the liver. Clinical PK data showing a 2-fold increased rosuvastatin systemic exposure during pregnancy suggest that OATPs are downregulated or inhibited during pregnancy.[199] In addition, rosuvastatin is also a BCRP substrate and excreted up to 70% through the liver, which may suggest downregulation or inhibition of BCRP as well, reducing the amount of rosuvastatin to be transported into the bile and more being available for basolateral efflux. Another study measuring bilirubin and bromosulfaphtalein, both substrates of OATP, showed impaired hepatic excretion in the third trimester of healthy pregnancy.[200] In vitro studies in SCHH treated with PRHs have shown a decrease in OATP1B1 and OAT1B3 mRNA expressions[201] whereas BCRP mRNA expression and protein was mostly unchanged in this model in response to exposure to PRHs.[77,201] In addition, treating HepG2 cells with 10 μM progesterone for 24 hours decreased OATP1B1 and FXR proteins.[202] Additionally, the plasma concentrations of estrone 3-sulfate (an OAT1B1 substrate and inhibitor) and estradiol-17β-D-glucuronide (OATP1B1/1B3 inhibitor) significantly increase during pregnancy[203–205], suggesting a possible inhibition of the transporter. Altogether, the decreased function of OATPs may be related to decreased expression or inhibition of their activity by pregnancy related hormones and their metabolites.

P-gp.

P-glycoprotein is ubiquitously expressed and is an ATP dependent efflux transporter. In the liver it transports substrates from within the hepatocytes into the bile. Renal P-gp has been shown to be increased during pregnancy, as shown with a 2-fold increase in renal secretion clearance of the P-gp substrate digoxin.[72] However, PK data supporting a change in hepatic P-gp alteration are challenging, because there is a lack of data on intracellular hepatic concentrations. A slight reduction in plasma concentrations of citalopram, a substrate of P-gp, suggests an increase in hepatic P-gp during pregnancy.[206] Another study comparing systemic exposure of fexofenadine, also a P-gp substrate (mostly effected by intestinal P-gp[207], in non-pregnant to pregnant individuals in the third trimester showed no alteration and suggest that intestinal P-gp activity is not altered during pregnancy, which would confirm the reduction of citalopram may be due in part to increased hepatic P-gp activity during pregnancy next to enzymatic activity alterations during pregnancy. In addition, systemic exposure of indinavir and ritonavir, both P-gp substrates were decreased in the second and third trimester compared to postpartum suggesting that hepatic P-gp is increased during pregnancy.[208] In vitro studies in SCHH treated with PRHs (estrone, estradiol, estriol, progesterone, cortisol, and pGH) showed an increase of P-gp protein and the effects were primarily mediated by progesterone,[77] suggesting that that the increased activity of P-gp is related to induction of the gene by PRHs.

4. Knowledge Gaps in Molecular Mechanisms of Altered Hepatic Clearance in Pregnancy and limitations

Clinical PK studies have consistently demonstrated that pregnancy alters the systemic exposure and clearance of many drugs in part due to altered hepatic clearance.[5] Clinical and preclinical studies have shown that the changes in hepatic clearance of drugs during gestation is linked to decreased protein binding, increased organ blood flow, and altered intrinsic clearance.[209] DMEs and transporters are integral to hepatic intrinsic clearance, and the expression and function of these hepatic proteins are tightly regulated. Despite research efforts to understand the mechanisms underlying pregnancy-evoked alterations in the expression and function of DMEs and transporters in the last decade, there exists substantial knowledge gaps in the (1) molecular mechanisms regulating hepatic DMEs, transporters, and transcription factors such as nuclear receptors during pregnancy, (2) impact and mechanisms underlying the interaction between pregnancy and diseases such as diabetes on hepatic clearance, and (3) presence and magnitude of interactions between the pharmacogenetics of DMEs and transporters and pregnancy evoked changes in hepatic clearance.

Studies have suggested that the altered activity of DMEs and transporters significantly impacts the hepatic clearance of drugs during pregnancy. Table 1 summarizes proposed potential molecular mechanisms underlying alterations in the activities of major CYPs, UGTs, and transporters during pregnancy. However, the available clinical and pre-clinical data are insufficient to draw direct connections and define precise mechanisms of pregnancy-evoked alterations in intrinsic activities of hepatic DMEs and transporters during gestation. First, the available clinical PK data is incomplete for many drugs in that only exposure and clearance of parent drug is reported and information on metabolite formation clearance is often lacking. Furthermore, sufficient data on gestational age dependent hepatic clearance of many drugs is unavailable. As a result, it is challenging to estimate the changes in activity of the DMEs in each trimester and other mechanisms of drug elimination. Second, experimental animal models such as humanized mouse models may play a role in understanding mechanisms of regulation and function of DMEs and transporters. The application of these models in studying the mechanisms of drug metabolism and disposition is limited due to species differences in reproductive biology. For instance, CYP3A4 activity is increased in human pregnancy whereas CYP3A4 expression and activity is decreased in pregnant humanized mouse liver.[210] Furthermore, although pregnancy hormones cortisol and pGH induce CYP3A4 expression in human hepatocytes, these hormones are not secreted during mouse pregnancy.[162,211] Finally, in vitro studies investigating mechanisms of altered hepatic clearance has primarily focused on the hypothesis that PRHs influence the activities of certain DMEs and transporters by affecting their expression or inhibiting their function and reported an isoform-specific impact of PRHs on the DMEs and transporters. [74,77,79,172] However, the association between PRHs and activities of DMEs and transporters has not been established yet in part due to the lack of liver specimens from pregnant individuals. Furthermore, the physiological relevance of the duration of and exposure levels of the exogenous PRHs in vitro models such as human hepatocytes remain limitation. Thus, it remains challenging to establish whether PRH-mediated alterations in DME and transporter expression in liver is driving the observed alterations in hepatic clearance during pregnancy in vivo.

In non-pregnant populations, nuclear receptors such as PXR, CAR, GR, AHR, and ERα play a crucial role in the transcriptional regulation of major DME transporters, including CYP3A4, UGT1A1, and P-gp.[77,159,172] Although it has been reported that cortisol can induce CYP3A4 expression through the activation of GR and the induction of PXR in HepaRG cells, [159] there is limited data on the regulation of hepatic nuclear receptors relevant to drug disposition in experimental models of pregnancy. Understanding the regulation of PXR, CAR, GR, AHR, and ERα mediated transcriptional activation of DMEs and transporters by pregnancy related factors (such as PRHs) and crosstalk between the nuclear receptors is essential for future studies aimed at predicting hepatic clearance and DDIs during pregnancy for both existing and new drug molecules.

Pregnancy-related complications such as diabetes, hypertension, and infection are common among pregnant populations. Diabetes (pregestational and gestational) affects up to 9% of pregnancies, and it is one of the leading pregnancy-related complications in the U.S.A. In nonpregnant populations, diabetes appears to decrease the expression and activities of many DMEs and transporters.[212–214] Mechanistic studies have suggested that diabetes-related increase in inflammatory factors such as cytokines activates interleukin-6 (IL-6) and the tumor necrosis factor-alpha (TNF-α) activate Nuclear factor-kappa B (NF-κB) that in turn decreases expression of PXR and RXR or their dimerization, resulting in decreased activities of DMEs such as CYP3A4.[214] Gestational diabetes also appeared to increase inflammatory cytokines.[215] For instance, pregnancy increases oral clearance of labetalol; however, in diabetic pregnant women, systemic exposure increased by approximately 100% compared to the nondiabetic pregnant group (SR)-labetalol.[216], suggesting the presence of a potential pregnancy-diabetes interaction impacting labetalol clearance. Similar to diabetes, preeclampsia can also modify pregnancy-evoked changes in hepatic clearance by altering hepatic blood flow, protein binding, and activities of DMEs and transporters.[217–219] Decreased plasma albumin and hepatic blood flow are reported in pregnant women diagnosed with preeclampsia. Furthermore, pre-eclampsia can also alter the plasma levels of circulating hormones such as estrogens and progesterone. Furthermore, the complication also severely injures the liver [218,219] However, the hepatic clearance of many drugs in women diagnosed with pregnancy complications such diabetes and pre-eclampsia and the magnitude and molecular mechanisms of the combined impact of pregnancy-associated hormonal changes and disease-related physiological and biochemical changes on the expression and function of hepatic DMEs and transporters have been largely unstudied and warrants further investigation.

In addition to disease, functional genetic polymorphisms in DMEs and transporters also introduce substantial interindividual differences in hepatic drug clearance.[220] For instance, CYP2D6 depicts substantial interindividual differences in its hepatic metabolism primarily due to its genetics, ranging across a spectrum from inactive or no enzyme activity to normal activity to ultrarapid activity.[221] During pregnancy, paroxetine (a CYP2D6 substrate) plasma concentrations decreased with gestational age in extensive or ultrarapid metabolizers while an opposite was observed in poor metabolizers.[222] Similarly, the oral clearance of a CYP3A substrate nifedipine was approximately 2.7-fold higher in CYP3A5 expressors than low expressors during pregnancy [223] while CYP3A5 expression level was not a significant predictor of plasma 4β-hydroxycholesterol to cholesterol ratios during pregnancy[70], suggesting a potential substrate specific impact of CYP3A5 pharmacogenetics on CYP3A metabolism during gestation. Since DMEs and transporters are prone to induction of expression or inhibition of their activity during gestation, a systemic examination of potential phenoconversion during pregnancy is lacking for majority of the proteins and warrants further investigation.

5. Conclusion

Clinical studies have shown that pregnancy alters the systemic clearance and exposure of multiple drugs that rely on hepatic clearance for elimination. Pregnancy impacts the hepatic clearance of many drugs due to decreased plasma protein binding, increased blood flow to the organ, and altered functions of hepatic DMEs and transporters. The change in plasma protein binding is attributed to lower plasma concentrations of drug-binding proteins such as albumin due to expanded plasma volume or decreased synthesis of albumin during pregnancy. Pregnancy appeared to increase hepatic blood flow; however, the observations are controversial and seem to be impacted by the methods used to measure the hepatic blood flow. Preclinical, clinical, and physiologically based PK (PBPK) modeling studies have consistently shown that pregnancy alters hepatic clearance of certain drugs by increasing or decreasing the activities of multiple DMEs and transporters in an isoform-specific manner. Understanding the mechanisms of the altered functions of hepatic DME and transporters has attracted significant research interest and has been thought to stem from the remarkable increase in the secretion of steroidal (estrogens, progesterone, and cortisol) and peptide (pGH and hPL) PRHs. In vitro studies in human liver cells have suggested that PRHs and their metabolites affect the functions of hepatic DMEs and transporters. However, it remains unknown whether the PRHs and their metabolites alter the expression or inhibit the activities of hepatic DMEs and transporters during pregnancy in vivo.

6. Expert Opinion

Over 80% of pregnant women take at least one medication during gestation. Despite the widespread use of drugs in this population, precise dosing, efficacy, and safety information are lacking for many medicines, and obstetric care often relies on provider experience and a ‘best guess’ treatment approach. PK studies are an essential part of drug development in establishing exposure and clearance parameters, and guiding dose optimization to maximize risk-benefit in patients. However, drug clinical trials rarely include pregnant individuals (often considered as drug orphans); consequently, lack of labeled indications or dosing recommendations for pregnancy remain the norm. Furthermore, due to remarkable pregnancy-associated changes in the maternal anatomy, physiology, and biochemical processes during gestation and its impact on the PK of many drugs, extrapolation of dosage and efficacy data from nonpregnant to pregnant population is considered ineffective and can be harmful to pregnant women.[209,224]

Notable physiological changes relevant to hepatic drug clearance include the altered function of the liver, kidney, endocrine, and cardiovascular systems, collectively impacting organ blood flow, plasma protein binding, and the activities of hepatic DMEs and transporters. We believe that understanding the magnitude and the molecular mechanisms of the impacts of pregnancy-related changes on the functions of key DMEs and transporters is essential for translating drug dosing and safety information from nonpregnant to the obstetric population and developing in silico tools such as PBPK models for improved quantitative prediction of drug exposure and clearance in pregnant groups.

The impacts of pregnancy on the systemic exposure and clearance of drug substrates of major hepatic DMEs and transporters are well documented. Although, in vitro studies in human liver cells suggest that PRHs are responsible for the altered activities of DMEs and transporters during gestation, the mechanisms of regulation of these proteins remain understudied and warrant further investigation. The mechanisms of hormone-evoked changes in the activities of these hepatic proteins can be due to (1) altered expression, (2) inhibition, and (3) heterotropic cooperativity. According to in vitro studies in human primary hepatocytes, induction of expression appears to be a plausible mechanism for many DMEs belonging to CYP and UGT superfamilies, and the effects are primarily driven by estrogens, progesterone, and cortisol; however, the precise molecular mechanism underlying the transcriptional activation remains unclear for most isoforms. On the other hand, for DMEs such as CYP1A2 andCYP2C19, inhibition and to a lesser extent suppression of their expression may explain pregnancy-related alteration in their activities. PRHs can alter the expression of the DMEs and transporters through activation of nuclear receptors or epigenetic mechanisms. Heterotropic cooperativity (stimulation) of metabolic activity of CYPs has been reported in vitro[225]. Although such effects are rarely observed in vivo and such mechanisms have not been explored with PRHs, estrogens and progesterone are substrates for CYPs and UGTs and can interfere with their metabolic activities.

Mechanistic studies to understand the alterations in the hepatic clearance of drugs during gestation are limited by the lack of clinical samples, such as liver tissue and limited utility of experimental animal models due interspecies differences in reproductive biology. Thus, human hepatocyte models such as SCHH have become the primary model in the study of mechanisms of altered hepatic clearance during gestation.[226] As research in the field progresses, advanced liver cell models such as 3D coculture organoids and liver-on-a chip systems could overcome some of the limitations, such as long-term exposure and role of other liver cells on DME and transporters. In addition, the assessment of changes in the protein concentration of DMEs and transporters in maternal plasma-derived liver-specific extracellular vesicles (EVs) will play a vital role in understanding the molecular mechanisms of alterations in hepatic drug clearance throughout pregnancy. Recent studies have indicated that maternal plasma-derived EVs exhibit alterations in the hepatic expression of DMEs such as CYP2D6 and CYP3A4.[227] Therefore, the quantification and systematic evaluation of DMEs and transporters in liver-specific EVs could serve as a valuable “liquid biopsy” tool for understanding the mechanisms behind the pregnancy-related changes in the activity of the enzymes and transporters.

Article Highlights.

• Medication use during pregnancy is widespread; however, clinical trials rarely include pregnant people due to safety and ethical issues, resulting in a substantial lack of sufficient evidence on drug dosing, efficacy, and safety during pregnancy. As a result, obstetric care providers often rely on best-guess treatment.

• Pregnancy alters the pharmacokinetics of many drugs, that are commonly prescribed for various complications in obstetric patients, due to the remarkable changes in the physiology of organs involved in drug absorption, distribution, metabolism, and excretion.

• The liver plays a paramount role in the clearance of many drugs and their metabolites. Hepatic clearance is a function of plasma protein binding, blood flow, and intrinsic clearance. Pregnancy decreases plasma protein binding, increases blood flow to the liver, and alters hepatic intrinsic clearance.

• Pregnancy-associated effects on hepatic intrinsic clearance are thought to arise from altered functions of drug-metabolizing enzymes (DMEs) and transporters due to pregnancy hormone-mediated changes in the expression (induction or suppression) and inhibition of the proteins.

• Human hepatocytes remain the primary model in the study of mechanisms of altered activities of DMEs and transporters during gestation. Advanced human liver cell models such as 3D coculture organoid systems, liver-on-a-chip, and liver specific extracellular vesicles in maternal plasma will play a vital role in understanding the molecular mechanisms of altered activities of DMEs and transporters.

• Future studies on the impact of pregnancy-disease or genetics interaction on hepatic drug clearance are essential for more precise prediction of systemic exposure of drugs during pregnancy.