Title: Pediatric development of glucuronidation: the ontogeny of hepatic UGT1A4. DMD #15214 2 Running Title: Ontogeny of UGT 1A4

Abstract This paper reports on the development of Uridine diphosphate glucuronosyltransferase (UGT) enzyme activity in pediatric livers. The substrates 4-methylumbelliferone (4MU) and trifluoperazine (TFP) were used as probes for general glucuronidation and specific UGT1A4 activity respectively. The activity of hepatic β -glucuronidase enzymes was also determined so as to investigate the balance between glucuronide clearance and systemic re-circulation. UGT activity towards 4MU reached maximum levels by 20 months of age, while the activity of β -glucuronidase was highest in the neonatal liver and decreased to steady-state adult levels by 4 months. The average Vmax and Km for UGT1A4 in pediatric samples were 151.9 ± 63.5 pmol/min/mg protein and 14.4 ± 9.6 µM respectively. Average Vmax was understandably low due to developmental dynamics, but Km was similar to those reported elsewhere. When a constant rate of enzyme development is assumed, maximum activity of UGT1A4 occurs at 1.4 years of age. When the intrinsic hepatic clearance of TFP was scaled with an allometric model, hepatic clearance of TFP by UGT1A4 did not reach maximum levels until 18.9 years of age and scaled results underestimated reported in vivo clearances in adult males. No significant differences in UGT activities or hepatic clearances were observed with gender or ethnicity. The developmental dynamics of most drug metabolizing enzymes are unknown and this paper contains the first description of the development of a single UGT isoform in childhood. Ultimately work such as this is important for predicting drug responses and for developing and evaluating new medications in children. DMD #15214 4 Introduction

The Development of UDP-Glucuronosyltransferases 1A1 and 1A6 in the Pediatric Liver

UDP-glucuronosyltransferases (UGTs) are critical for the metabolism and clearance of drugs, chemicals, and hormones. The development of UGT1A1 and 1A6 was studied in 50 pediatric liver samples using bilirubin, serotonin activity assays, and Western blot as well as pharmacokinetic scaling. UGT activity developed age dependently in pediatric liver. Maximal activity of 0.7690 nmol · min · −1 mg protein−1 was observed for UGT1A1 at 3.8 months. For UGT1A6, activity matured at 14 months (4.737 nmol · min · −1mg protein−1). Protein expression was not age-dependent, and activities did not correlate to protein levels for either enzyme. The in vitro activities were used to calculate normalized hepatic clearances using both allometric scaling and a physiologically based pharmacokinetic model. For UGT1A1, allometry predicted normalized adult clearances of 0.0070 l · h−1 · kg−1 at 3.0 (well stirred) and 2.8 years (parallel tube), whereas the Simcyp model showed normalized clearances of 0.0079 l · h−1 · kg−1 at 2.6 (well stirred) and 2.5 years (parallel tube). For UGT1A6, only the Simcyp well stirred model converged at 0.3524 l · h−1 · kg−1 at 12.6 months. These data imply independent regulation of UGT1A1 and 1A6 where activity has matured after 6 months to 1 year. Total hepatic clearance of substances mediated by these enzymes may mature concurrently or take longer because of other physiological factors. Late development of UGT enzymes may contribute to chemical, drug, and environmental toxicity

Neonatal development of hepatic UGT1A9:implications of pediatric pharmacokinetics

This article reports on the development of UDP-glucuronosyltrans-ferase 1A9 (UGT1A9) in neonatal and pediatric liver. The substrate 4-methylumbelliferone (4MU) with specific inhibition by niflumic acid was used to define specific UGT1A9 activity. Subsequently, in silico pharmacokinetic (PK) and physiology-based pharmacoki-netic (PBPK) modeling was used to determine UGT1A9 maturation and hepatic clearance. Modeled maximal enzyme activity was 27.9 nmol min1 mg protein1 at 4 months of age, which had high concordance with the average Vmax in 45 individual adult (>20 years) livers of 29.0 nmol min1 mg protein1. The activity of UGT1A9 ranged 7.5-fold in the adult population (4.1–54.5 nmol min1 mg protein1). Expression of UGT1A9 correlated with age only in children younger than 1 year (Spearman r 0.70). Activity correlated with expression up to 18 years of age (Spearman r 0.76). Furthermore, scaling intrinsic hepatic clearance of 4MU with an allometric PK model yielded a high clearance at birth and then fell to adult levels (1.3 l h1 kg1 at 18.1 years for well stirred or 1.4 l h1 kg1 at 18.7 years for parallel tube). The Simcyp PBPK models did not converge but showed an increase in clearance at under 1 year of age and then decreased to adult levels at approx-imately 20 years of age. Allometric scaling may be more accurate in cases of high-extraction drugs. Enzyme activities or hepatic clearances did not differ with gender or ethnicity. The UGT1A9 isoform has higher normalized clearance for 4MU at young ages, which may explain how other UGT1A9 substrates, such as propo-fol, have higher clearances in children than in adults