A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy

Pregnancy causes a multitude of physiological and biochemical changes in the women’s body. The levels of estrogens, progestins, growth hormones, and other factors needed for fetal growth increase with the progress in gestational age. The profound increase in the hormonal levels and the exposure of various organs to these hormones could affect both the expression and activity of several proteins involved in drug disposition. We studied the effects of mixture of female hormones at both circulatory and projected liver levels on drug-metabolizing enzymes using human hepatocytes and a cocktail approach.

Primary cultures of human hepatocytes are valuable resources to evaluate the impact of chemicals and various physiological and pathological conditions on the activity and expression of drug-metabolizing enzymes and transporters [23]. All the currently published studies typically use one substrate at a time to study the activities of drug-metabolizing enzymes using primary cultures of human hepatocytes. Given the difficulties associated with obtaining primary cultures of human hepatocytes, and the high cost associated with procuring them for studies, our group has developed a sensitive and specific CYP450 cocktail assay using human hepatocytes for simultaneously assessing the activity of several major CYP450 enzymes involved in the metabolism of almost 95% of drugs in clinical use [20]. We have performed incubations with substrates using primary cultures of freshly isolated human hepatocytes in our previously published studies and observed that the reaction rates remained linear for more than 4 h for several drugs tested [24, 25]. In the current report, the incubations were performed for 60 min only, and we utilized a cocktail of substrates to investigate the modulation of drug-metabolizing enzymes due to hormonal changes observed during pregnancy using primary cultures of human hepatocytes.

Both in vitro and animal models have been employed to elucidate the potential role of changes in hormone levels in altering the metabolism of multiple CYP450 substrates, but most of the studies used either one or two hormones and may not predict the overall outcomes observed in pregnancy [15, 18, 19]. Pregnancy is a dynamic state with many altered physiologic and metabolic functions where maternal hepatocytes are exposed continuously to changing combinations of hormones and growth factors. These combinations may act in a synergistic or antagonistic manner. Our studies were designed to evaluate the net effect of multiple hormones resembling hormone levels observed in plasma and projected liver concentrations to study the effects in primary cultures of freshly isolated human hepatocytes.

The mean of clinically observed plasma levels of female hormones reported in literature [9] were used for treatment at plasma levels. Previous studies in hepatocytes have used similar concentrations of hormones and used individual substrates for assessing the activity of drug-metabolizing enzymes [14]. We hypothesized that the combined effects of the increased levels of female hormones may be responsible for the alterations in expression and activity of various CYP450 enzymes in the liver and would contribute to the changes in the pharmacokinetics of several drugs in pregnant women.

The metabolism in mother and fetus and the growth and development of fetus are regulated by human growth hormone (hGH) which belongs to human placental lactogen (hPL) gene family. During early gestation, maternal circulation of growth hormone originates from the anterior pituitary gland, but during the second trimester onwards, pituitary growth hormone levels decline, and placental growth hormone levels increase gradually and reach a maximum of 15–25 ng/mL in the third trimester [26]. Placental growth hormone (pGH) is traditionally not detected in fetal serum during pregnancy. Recent findings by Mittal et al. report that pGH in fetal circulation is around 1% of the maternal circulation [27]. hGH differs from placental growth hormone by 13 amino acids and is differentially glycosylated [28], and differences in proteins may be related to localizing the activity in placental tissue. Despite high circulatory levels of pGH in late stages of pregnancy, the physiological role of placental growth hormone is not understood completely. Both hGH and pGH are expected to have similar pharmacological effects on the maternal liver, but the uptake of pGH in maternal hepatocytes is unknown. Our studies were designed to study the effect of different hormones on hepatocytes; it was reasonable to use hGH as a part of the hormone mixture in our studies in the absence of data about the localization and effect of pGH on hepatocytes in the mother.

Our studies were conducted in accordance with the guidelines prescribed by Pharmaceutical Research and Manufacturers of America (PhRMA) directives in determining the extent of inducers in in vitro and in vivo DDI [29]. All the hepatocytes used in our studies showed response to positive control for induction (rifampin 10 µM) with a ~ seven-fold increase CYP3A4 activity where a ~ two-fold increase is considered acceptable to evaluate the quality of hepatocytes. Additionally, a 50% reduction in CYP3A4 activity was observed with ketoconazole treatment also (Fig. 1). Our previous studies in hepatocytes also show a 30–50% reduction which is similar to the inhibition observed in current studies [24, 25, 30]. The response to positive control for both induction and inhibition from all donors is in agreement with the reported observations by Papageorgiou et al. [16]. The positive controls rifampin (induction) and ketoconazole (inhibition) were used in the current investigation to examine the response of the hepatocytes modulation of CYP3A4. Additionally, it was not practical to use a positive control for each and every enzyme tested. Positive control for CYP3A represents the overall response of the hepatocytes to treatments.

Observations from clinical studies show that clearance of multiple CYP3A4 substrates was increased during pregnancy. Changes in drug dosing are needed for pregnant women to maintain optimal therapeutic effects [2,3,4,5, 31]. The clearance of midazolam and digoxin, CYP3A4 substrates, was significantly higher during pregnancy compared to postpartum [3], and glyburide dose-normalized plasma concentrations were ~ 50% lower in pregnant women than in non-pregnant subjects [4]. The pregnancy-mediated changes in the pharmacokinetics of drugs metabolized by CYP450 enzymes could be attributed to changes in the expression and activity of the respective drug-metabolizing enzymes. Because of its unusually poor substrate selectivity, CYP3A4 is sensitive to reversible or irreversible inhibition by a range of medications. Mechanism-based CYP3A4 inhibition is defined by NADPH, time, and concentration-dependent enzyme inactivation. Additionally, other characteristics associated with medications and patients can also affect the clinical outcomes resulting in difficulty to predict drug-drug interactions involving CYP3A4 inactivation.

Results from our studies show a statistically significant increase in CYP3A4 mRNA and protein expression as well as activity at predicted concentrations in the liver. The four-fold increase in CYP3A4 mRNA observed in our studies is in concurrence with reported values by Choi et al., using similar concentrations of progesterone [14]. Additionally, the observations in our study are consistent with results from our clinical studies in pregnant women during the third trimester where dextromethorphan N-demethylation was used as a marker of CYP3A4 activity [2]. The extent of midazolam exposure (AUC) was reduced by 50% during pregnancy compared to postpartum [3], and our results show a 25% increase in CYP3A4-mediated metabolism in the simulated pregnancy state. A corresponding change in CYP3A4 protein and mRNA levels is observed. Our study model and design allowed incubation of human hepatocytes with hormone mixtures for 72 h; but during pregnancy, hepatocytes are exposed to increased levels of hormones. With maternal hepatocytes getting sustained exposure of higher levels of hormones, the observed difference in magnitude of effect between clinical studies and human hepatocyte studies is reasonable.

In our studies, hepatocytes were treated with hormones every 24 h, but physiological regulation of hormones during pregnancy is continuous and maintained throughout pregnancy. Studies reporting the use of high concentrations of these hormones such as estradiol, progesterone individually or in combination with placental growth hormone, growth hormone, and cortisol in human hepatocyte systems reported a significant increase in the expression of CYP3A4 activity, which is in agreement with our data [14, 16]. A positive correlation (R2 = 0.7532) was observed between CYP3A4 protein and mRNA expressions in hepatocytes treated with predicted liver concentrations of hormones. The increase in mRNA expression is consistent with higher CYP3A4 protein levels and a corresponding increase in CYP3A4 activity.

CYP3A4 and CYP3A5 are the major forms of CYP3A expressed in the human liver and gut. Though CYP3A4 is the most abundant CYP450 isoform accounting for about 30% of the total hepatic P450 enzyme in humans [32], it is responsible for about 60% of the P450-mediated metabolism of currently available drugs [33] and has a wider substrate range than CYP3A5 [34]. It is difficult to tease out the contributions of 3A4 and 3A5 individually due to the large overlap in their substrate specificities. Both proteins contribute to the metabolism of different classes of drugs. Furthermore, observations from our study are in accordance with clinical observations demonstrating that clearance of CYP3A4 substrates such as midazolam, methadone, and nifedipine was increased during pregnancy [3, 5, 35].

Additionally, our findings showed that projected hormone levels experienced by the liver during the late stage of pregnancy did not affect the expression or activity of CYP1A2, CYP2C9, CYP2C19, and CYP2D6. Though CYP2C9 activity showed an increasing trend with hormone treatment, the increase was not statistically significant. A longer duration of exposure and the continued presence of hormones throughout the entire culture period might have resulted in a significant increase in CYP2C9 activity. Estradiol and progesterone are rapidly eliminated from the body through hepatic metabolism [36, 37], and previous reports in hepatocyte studies show a rapid depletion of hormones within 4–8 h from hepatocyte maintenance medium [14, 38]. In our studies, hormones combination was replenished every 24 h, and the exposure of hepatocytes to hormones may not be similar to circadian modulation of physiological conditions. In pregnant women, liver cells are continuously exposed to hormones at higher levels [39] which may have a significant impact on the expression and activity of CYP450 enzymes. Overall, our results are consistent with the reports from Choi et al. where progesterone, the most abundant hormone during pregnancy, did not show significant effect on mRNA expression or enzyme activities of CYP1A2, CYP2C9, CYP2C19, and CYP2D6 [14].

Our study aimed to get the maximum information from each batch of freshly isolated primary human hepatocytes as they are a valuable resource and are sparsely available. We studied the impact of a mixture of pregnancy-related hormones in observed plasma and predicted liver concentrations during the third trimester of pregnancy on the expression and activity of major CYP450 enzymes and used a cocktail of CYP450 substrates to measure changes in enzyme activities. Our results show that female hormones in the third trimester predicted liver concentrations that increased the enzyme activity, mRNA, and protein expression of CYP3A4. The present study is in line with previous studies to utilize human hepatocyte model with cocktail of CYP450 probes to study the effect of hormones on drug metabolism. Our observations agree with data from previously reported in vitro and clinical studies [3, 16]. Though the results are consistent with clinical data for CYP3A4 metabolism, the discrepancies in the expression and activity of other CYP450 enzymes could be related to the rapid decline of the hormone levels during each 24 h of incubation, and the presence of dexamethasone in the medium could alter the activity of some P450 enzymes and an incubation period of 72 h that may not be long enough for inducing observable alterations in other enzymes using our current protocol. Ongoing studies in our lab are focused on evaluating the effect of more frequent media changes to replenish the hormones and better maintenance of hormones at constant levels using a flow through microphysiological system with human hepatocytes [40].

The outcomes of the research can be extrapolated to in vivo scenario as well as for the development of prediction models, in that the clearance of the medications metabolized by CYP3A4 will be higher during pregnancy. We characterized the effect of pregnancy on the changes in the expression and activity of drug metabolizing enzymes in the liver. Our data is critical in understanding the effects of medications such as nifedipine (antihypertensive), clindamycin (antibiotic), oxycodone (opioid analgesic), escitalopram (antidepressant), venlafaxine (anxiolytic), oseltamivir (antiviral), midazolam (anesthetic/sedative), indinavir (anti-HIV), ondansetron (prevent nausea), rosuvastatin (antihyperlipidemic), trazodone (antidepressant), and remdesivir (antiviral) that are used during and after pregnancy and are metabolized by CYP3A4. The pregnancy-mediated increase in hormonal concentration will significantly impact the concentration of these drugs, and plasma concentrations of female hormones should be considered as a covariate in computational models to explain variable impact of pregnancy on CYP3A activity. Further studies are recommended to evaluate the effect of pregnancy on the clearance of these medications in vivo. CYP3A4 is the enzyme that breaks down the most drugs in humans, so it is an important area to study when it comes to enzyme-based drug interactions during pregnancy. Some of these medications are sedatives such as diazepam, antidepressants such as amitriptyline, antiarrhythmics such as quinidine, antihistamines such as loratidine, and calcium channel antagonists such as nifedipine. Human hepatocyte studies were used to assess the change in clearance in the presence of drug-drug interactions, and the magnitude of change in clearance can be used to adjust the dose of affected drug. We have used this approach in dose optimization for anti-HIV drugs and anti-cancer drugs [24, 30].

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