The ontogeny of drug metabolizing enzymes and transporters in the rat

doi:10.1016/j.reprotox.2008.09.010

Reproductive Toxicology

Volume 26, Issues 3–4, November–December 2008, Pages 220-230

https://doi.org/10.1016/j.reprotox.2008.09.010 Get rights and content

Abstract

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the findings during safety studies in juvenile animals.

The present study was designed to collect information on plasma concentrations of total protein and albumin, enzyme activity and mRNA expression of cytochrome P450 isoenzymes (CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1), carboxylesterase and thyroxin glucuronidation (T4-GT) activity in liver microsomes, and mRNA expression of transporters (Mdr1a/b, Mrp1–3 and 6, Bsep and Bcrp, Oct1–2, Oat1–3 and Oatp1a4) in liver, kidney and brain tissue during development in Sprague–Dawley rats. Enzyme activities were determined by measuring the metabolism of marker substrates; expression of mRNAs was assessed using RTq-PCR.

There were considerable differences in the ontogeny of the individual cytochrome P450 isoenzymes. In addition, ontogeny patterns of enzyme activity did not always parallel ontogeny patterns of mRNA expression.

Ontogeny of the transporters depended on the transporter and the organ studied. Changes in mRNA expression of the various transporters during development are likely to result in altered elimination and/or tissue distribution of substrates, with concomitant changes in hepatic metabolism, renal excretion and passage through the blood–brain barrier.

Consideration of the ontogeny of metabolizing enzymes and transporters may improve the design and interpretation of results of toxicity studies in juvenile animals.

Introduction

Many of the drugs currently used to treat children have not been appropriately studied in this specific group of patients. In clinical practice, children are generally treated as small adults, with the dose levels used in adults adjusted on the basis of body weight or body surface area. The pharmacokinetic behavior of many compounds in children is however clearly different from that in adults. At birth the various organ systems involved in absorption, distribution and hepatic and renal elimination are not yet fully developed, and undergo considerable changes with different rates and patterns of maturation to the adult situation [1], [2]. This may result in differences in systemic exposure and in differences in exposure in the individual organs.

In addition to these pharmacokinetic differences, therapeutic effects and safety and tolerability are also likely to be different.

Since December 2006 an EU regulation on the development of medicinal products for pediatric use has come into force, which makes adequate evaluation of efficacy and safety of new drugs in the pediatric population mandatory when the product is likely to be used in children [3].

To support the benefit/risk analysis of a medicinal product in children, studies in juvenile animals may be a valuable tool in obtaining information on parameters that cannot be adequately, ethically and safely assessed in pediatric clinical trials, such as severe or irreversible adverse effects and immediate or delayed adverse effects on growth and on developing organs.

Traditionally the rat is one of the species of first choice to be used in non-clinical safety studies as well as in juvenile toxicity studies. Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is however still limited and sometimes conflicting results are reported. The present study was performed to increase this knowledge. The primary focus was on the age shortly after birth until early adulthood of the animals (Day 42) to support the design of juvenile toxicity studies. The age differences from Day 42 onwards are commonly covered in general toxicity studies.

Many drugs are bound to plasma proteins, with the unbound fraction being assumed to be the active principle. The present study included investigation of the age-dependent changes in plasma concentrations of total protein and albumin.

The transport of molecules (including drugs) in and out of cells involves a large variety of transport proteins, each with their specific characteristics, tissue distribution and substrate specificity. Our study focused on representatives of the main classes, viz. the ATP-binding cassette (ABC) transporters and the Solute Carrier Family (Slc) transporters. Representatives of the ABC transporters included two multidrug resistance proteins (Mdr1a and Mdr1b), four multidrug resistance-associated proteins (Mrp1, 2, 3 and 6), the bile salt export pump (Bsep), and the breast cancer resistance protein (Bcrp). Representatives of the Slc family included two organic cation transporters (Oct1, Oct2), three organic anion transporters (Oat1, Oat2 and Oat3) and one organic anion transporting polypeptide (Oatp1a4). Levels of mRNA of the various transporters were studied in the liver, kidney and cerebellum.

Biotransformation is an important route of elimination for many drugs. Metabolism pathways are divided into Phase I and Phase II reactions. Phase I reactions are mainly catalyzed by cytochrome P450 (CYP) enzymes, a superfamily of heme-containing enzymes catalyzing a wide range of metabolic reactions, and by esterases. Most of the Phase I enzymes are found in the liver, but many are also present in a variety of other organs, including the kidney, brain, lung and intestines. Representatives of Phase I enzymes investigated in the present study include CYP1A1/2, CYP2B1/2, CYP2E1, CYP3A1/2, CYP4A1, and carboxylesterase. Phase II reactions catalyze conjugation reactions, including addition of glucuronic acid, sulphate or glutathione. In this study glucuronidation, which is the most important phase II reaction, was evaluated by measuring the thyroxin glucuronosyl transferase (T4-GT) activity. Enzyme activities and levels of mRNA of the metabolic enzymes were studied in the liver.

Section snippets

Animals

Sprague–Dawley rats were obtained from Charles River (Sulzfeld, Germany). The rats were housed in a restricted-access SPF rodent facility, with free access to water and feed (A04C fine ground diet, SAFE, Les Tremblats, Augy, France). The female rats were housed individually; the pups were kept with the mother animals until sacrifice. Rats up to 21 days of age were ordered as whole litters. The animals were anesthetized with ether and sacrificed by exanguination at gestation Day 21 (Day −1), the

Total protein and albumin concentrations in plasma

Plasma concentrations of total protein increased gradually from about 2.5 g/dl (males and females) at birth to about 5.2 g/dl (males) and 5.5 g/dl (females) at Day 42. Plasma albumin concentrations increased from about 1.8 g/dl at birth to about 3.8 g/dl (males) and 4.2 g/dl (females) at Day 42.

Liver weight and microsomal protein and CYP content

Liver weight increased exponentially from 0.27 g at birth to 5.49 g (males) and 4.63 g (females) at Day 42. Relative to body weight, the liver weight decreases from approximately 4% of body weight at birth to

Discussion

Knowledge of the ontogeny of the various systems involved in distribution and elimination of drugs is important for adequate interpretation of the results of safety studies in juvenile animals. Since the rat is one of the most frequently used species in non-clinical testing we used this species in our study on the age-dependent changes in cytochrome P450 isoenzymes and various transport proteins. The primary focus was on the age shortly after birth until early adulthood of the animals (Day 42)

Conflict of interest

None.

Acknowledgement

We thank Dr. H. Geys for the statistical analysis of the data.

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The ontogeny of drug metabolizing enzymes and transporters in the rat

Abstract

Introduction

Section snippets

Animals

Total protein and albumin concentrations in plasma

Liver weight and microsomal protein and CYP content

Discussion

Conflict of interest

Acknowledgement

J Biol Chem

Int J Biochem

Biochim Biophys Acta

Arch Biochem Biophys

J Hepatol

Ontogeny of hepatic and renal systemic clearance pathways in infants: Part I

Clin Pharmacokinet

Clinical pharmacology of the perinatal period and early infancy