Drug Metabolism and Disposition, 2015, 43(12):1938-1945.
Dose of phenobarbital and age of treatment at early life are two key factors for the persistent induction of cytochrome P450 enzymes in adult mouse liver.
Yun-Chen Tien1, Ke Liu2, Chad Pope1, Pengcheng Wang2, Xiaochao Ma2, and Xiao-bo Zhong1
1Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, Connecticut.
2Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania.
Drug treatment of neonates and infants and its long-term consequences on drug responses have emerged in recent years as a major challenge for health care professionals. In the current study, we use phenobarbital as a model drug and mouse as an in vivo model to demonstrate that the dose of phenobarbital and age of treatment are two key factors for the persistent induction of gene expression and consequential increases of enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult livers. We show that phenobarbital treatment at early life of day 5 after birth with a low dose (<100 mg/kg) does not change expression and enzyme activities of Cyp2b, Cyp2c, and Cyp3a in adult mouse liver, whereas phenobarbital treatment with a high dose (>200 mg/kg) significantly increases expression and enzyme activities of these P450s in adult liver. We also demonstrate that phenobarbital treatment before day 10 after birth, but not at later ages, significantly increases mRNAs, proteins, and enzyme activities of the tested P450s. Such persistent induction of P450 gene expression and enzyme activities in adult livers by phenobarbital treatment only occurs within a sensitive age window early in life. The persistent induction in gene expression and enzyme activities is higher in female mice than in male mice for Cyp2b10 but not for Cyp2c29 and Cyp3a11. These results will stimulate studies to evaluate the long-term impacts of drug treatment with different doses at neonatal and infant ages on drug metabolism, therapeutic efficacy, and drug-induced toxicity throughout the rest of life.
Cytochromes P450s (P450s) belong to a superfamily of enzymes highly expressed in the liver and intestine that are responsible for metabolizing up to 75% of current prescription drugs. Therefore, drug responses are largely dependent on the expression and activities of these P450s. Higher expression levels of the enzymes increase the metabolism rates of substrate drugs and lead to lowered therapeutic efficacy if the drugs are cleared from the system too quickly or increased risk of adverse effects if metabolism produces toxic metabolites. However, significant variability exists in the basal expression of these P450s among patients beyond what genetics and polymorphisms can explain. Predicting how an individual will respond to drug therapy is a significant clinical challenge, which is reflected by the substantial number of patients experiencing serious adverse drug reactions each year. The goal of our research is to better understand why humans display such vast differences in P450-mediated drug metabolism and the environmental factors and mechanisms driving this phenomenon.
Several P450 families, which are important for drug metabolism, including CYP3A4 and CYP2C9, are capable of having their expression induced or temporarily upregulated by specific xenobiotics that act as inducers. Numerous commonly used drugs are known inducers and are often the causative agents of drug-drug interactions, affecting the efficacy and safety of other drugs metabolized by P450s that are taken concurrently. While P450 induction is a temporary effect in adults with enzyme levels returning to normal once the inducing drug is cleared, research over the past 30 years has indicated this might not be the case for neonatal and infant patient populations. Works done in rodent models have shown that treatment with an inducing drug, particularly phenobarbital, at the neonatal age leaves lasting effects on P450 expression and activity into adulthood. Mice and rats that received phenobarbital as young pups exhibit significantly elevated expression of several P450s as adults compared to their age-matched counterparts that only received vehicle treatment. Although the mechanisms behind this observation are just beginning to be elucidated, we hypothesized that the lasting effects on P450 expression are dependent upon the age at which the animals are treated and the dose of the inducing drugs. The hypothesis was tested in a published work (Tien et al., 2015).
Our study used the mouse as an animal model since human studies would take many years and introduce numerous confounding factors. Phenobarbital was chosen as a model drug since it is known to be a potent inducer of CYP3A, CYP2B, and CYP2C enzymes in both humans and mice and its effects are well documented. Phenobarbital is also a commonly used antiepileptic drug, especially in pediatric populations as it is given to newborns to control seizures, so it is clinically relevant. To investigate the effect that the dose of phenobarbital has on producing the permanent upregulation of P450 expression, we treated mice at Day 5 of age with a single dose of phenobarbital over a range of concentrations from 20 to 250 mg/kg bodyweight. The mice were sacrificed at age Day 60, the age, at which they are considered mature adults, and we collected their livers for further analysis of P450 expression and activities. We found significant upregulation of Cyp3a11, Cyp2c29, and Cyp2b10 enzymes in the animals that had to be treated with at least 100 mg/kg or a higher dose of phenobarbital when compared to age-matched vehicle treated mice. Doses below 100 mg/kg did not produce any statistically significant differences in enzyme expression between groups. This suggests that there is a dose-dependent factor to produce permanent changes to P450 expression following neonatal drug exposure and lower doses of an inducer may not be sufficient to cause lasting effects.
Additionally, we also wanted to investigate whether there was a sensitive window of time, in which phenobarbital treatment must occur in order to produce permanent alterations to P450 expression. For this part of the study, we used a consistent single dose of 200 mg/kg phenobarbital, but treated mice at different ages between Day 5 and Day 25 of life. Once again, we waited until mice grew to Day 60 of age, at which point we sacrificed them and collected their livers for analysis of Cyp3a11, Cyp2c29, and Cyp2b10 enzyme expression and activities. Interestingly, we found that if phenobarbital treatment occurred past Day 10 of age, there were no significant differences in the expression or activity of any of the P450s at the adult age when compared to age-matched vehicle treated animals. Mice that received phenobarbital treatment on Day 10 did have significantly upregulated P450 expression when compared to control mice, however mice that received phenobarbital on Day 5 of age had even more dramatic increases in P450 expression. These findings suggest that in order to produce permanent alterations to P450 expression and activity, exposure to the inducing drug must occur before a certain age in early life.
We also considered whether multiple treatments of phenobarbital, rather than just a single injection, during the neonatal age would produce even greater overexpression of P450s in adulthood, indicative of an additive effect. However, there were no statistically significant differences in the expression of any of the enzymes tested among mice that received multiple doses of 200 mg/kg phenobarbital between age Day 5 and Day 15 and mice that received a single dose of 200 mg/kg phenobarbital at Day 5.
Since the completion of that study, our research has gone on to investigate how exposure to phenobarbital during the neonatal age alters the efficacy of other drugs administered in adulthood. Omeprazole, also known by the brand name Prilosec, is a commonly used gastric proton-pump inhibitor for the treatment of gastroesophageal reflex disease and peptic ulcers. Omeprazole is also known to be extensively metabolized by CYP3A4 and CYP2C19 into its inactivate metabolites in humans. Since phenobarbital induces these subfamilies of enzymes in both humans and mice, we hypothesized that neonatal treatment with phenobarbital affect the P450-mediated metabolism of omeprazole in adult life and therefore the drug’s therapeutic efficacy in mice. We have tested the hypothesis in a published work (Tien et al., 2017).
To test the efficacy of omeprazole in an animal model, the pH of the mouse’s stomach was measured following a course of treatment of the drug. Under normal conditions, omeprazole increases the pH of the mouse stomach as acid secretion is blocked. So, we compared the gastric pH following omeprazole treatment in adult mice that received phenobarbital at Day 5 of age versus mice that received a vehicle control. In accordance with our hypothesis, adult mice that received phenobarbital treatment as neonates did not have as significant of an increase in stomach pH following omeprazole treatment than mice that did not receive phenobarbital. Although pharmacokinetic studies are in progress to measure differences in omeprazole metabolite plasma concentrations, this observation may signify that the permanent alterations to P450 expression following neonatal inducer exposure can, in fact, affect clinically relevant drug responses later in life.
While the mechanisms explaining our findings are just beginning to be investigated, epigenetic factors are clearly the basis. Many chemical inducers, such as drugs like phenobarbital but also foods and environmental chemicals, are able to bind and activate nuclear receptors, which then change the chromatin structure of target genes to facilitate or repress their transcription. When the nuclear receptors are activated in early life while cells in the liver are still proliferating and differentiating, it is highly likely the epigenetic changes caused by phenobarbital exposure become permanent, causing the permanent upregulation of target gene transcription. We are currently studying whether other inducer drugs that are able to activate nuclear receptors also cause permanent alterations to P450 expression following neonatal exposure to prove the effect is not just unique to phenobarbital.
Our research is impactful since millions of pediatric patients are treated with a wide range of drugs for various conditions. Several of these drugs also have the ability to activate nuclear receptors and induce P450 expression. In the current clinical practice, physicians usually do not consider whether medications an adult patient received as an infant could have any effect on their response to their current drug regimen. The goal of our work is to prompt a reevaluation of the factors that should be considered when prescribing medicines, such as past drug exposure, to ensure safe and efficacious responses to therapy and a precision model of medicine. The concept is emphasized in a recent publication (Piekos et al., 2017).
Piekos SC, Pope C, Ferrara A, and Zhong XB. 2017. Impact of drug treatment at neonatal ages on variability of drug metabolism and drug-drug interactions in adult life. Curr Pharmacol Rep, 3(1):1-9.
Tien YC, Liu K, Pope C, Wang P, Ma X, Zhong XB. 2015. Dose of phenobarbital and age of treatment at early life are two key factors for the persistent induction of cytochrome P450 enzymes in adult mouse liver. Drug Metab Dispos. 43(12): 1938-1945.
Tien YC, Piekos SC, Pope C, Zhong XB. 2017. Phenobarbital treatment at a neonatal age results in decreased efficacy of omeprazole in adult mice. Drug Metab Dispos. 45(3): 330-335.
Acknowledgements: This study was supported by the National Institute of General Medical Sciences [Grant R01GM-087376 and R01GM118367] and the National Institute for Environmental Health Science [Grant R01ES-019487] (to X.B.Z); the National Institute for Diabetes and Digestive and Kidney Diseases [Grant R01DK090305] (to X.M).
Department of Pharmaceutical Sciences
School of Pharmacy
University of Connecticut
69 N Eagleville Road
Storrs, CT 06269
Xiao-bo Zhong, Ph.D.
Department of Pharmaceutical Sciences
School of Pharmacy
University of Connecticut
69 N Eagleville Road
Storrs, CT 06269