datasheet1_Hepatic Cytochrome P450 Abundance and Activity in the Developing and Adult Göttingen Minipig: Pivotal Data for PBPK Modeling.docx

The Göttingen Minipig is gaining ground as nonrodent species in safety testing of drugs for pediatric indications. Due to developmental changes in pharmacokinetics and pharmacodynamics, physiologically based pharmacokinetic (PBPK) models are built to better predict drug exposure in children and to aid species selection for nonclinical safety studies. These PBPK models require high quality physiological and ADME data such as protein abundance of drug metabolizing enzymes. These data are available for man and rat, but scarce for the Göttingen Minipig. The aim of this study was to assess hepatic cytochrome P450 (CYP) protein abundance in the developing Göttingen Minipig by using mass spectrometry. In addition, sex-related differences in CYP protein abundance and correlation of CYP enzyme activity with CYP protein abundance were assessed. The following age groups were included: gestational day (GD) 84–86 (n = 8), GD 108 (n = 6), postnatal day (PND) 1 (n = 8), PND 3 (n = 8), PND 7 (n = 8), PND 28 (n = 8) and adult (n = 8). Liver microsomes were extracted and protein abundance was compared to that in adult animals. Next, the CYP protein abundance was correlated to CYP enzyme activity in the same biological samples. In general, CYP protein abundance gradually increased during development. However, we observed a stable protein expression over time for CYP4A24 and CYP20A1 and for CYP51A1, a high protein expression during the fetal stages was followed by a decrease during the first month of life and an increase toward adulthood. Sex-related differences were observed for CYP4V2_2a and CYP20A1 at PND 1 with highest expression in females for both isoforms. In the adult samples, sex-related differences were detected for CYP1A1, CYP1A2, CYP2A19, CYP2E1_2, CYP3A22, CYP4V2_2a and CYP4V2_2b with higher values in female compared to male Göttingen Minipigs. The correlation analysis between CYP protein abundance and CYP enzyme activity showed that CYP3A22 protein abundance correlated clearly with the metabolism of midazolam at PND 7. These data are remarkably comparable to human data and provide a valuable step forward in the construction of a neonatal and juvenile Göttingen Minipig PBPK model.

哥廷根小型猪（Göttingen Minipig）作为非啮齿类动物，在儿科适应症药物的安全性评价中应用日益广泛。由于儿童在药代动力学与药效动力学方面存在发育变化，基于生理的药代动力学（PBPK）模型被用于更精准地预测儿童体内的药物暴露情况，并辅助非临床安全性研究的物种选择。这类PBPK模型需要高质量的生理数据以及吸收、分布、代谢、排泄（ADME）相关数据，例如药物代谢酶的蛋白丰度数据。此类数据在人类与大鼠中已有较多积累，但哥廷根小型猪相关数据仍较为匮乏。
本研究旨在通过质谱法，评估发育阶段哥廷根小型猪的肝脏细胞色素P450（CYP）蛋白丰度。此外，本研究还分析了CYP蛋白丰度的性别差异，以及CYP酶活性与CYP蛋白丰度的相关性。本研究纳入以下年龄组别：妊娠天数（GD）84~86（n=8）、GD 108（n=6）、出生后天数（PND）1（n=8）、PND 3（n=8）、PND 7（n=8）、PND 28（n=8）以及成年个体（n=8）。研究人员提取了肝微粒体，并将各年龄组的蛋白丰度与成年动物进行对比。随后，针对同一生物样本中的CYP蛋白丰度与CYP酶活性进行相关性分析。
总体而言，CYP蛋白丰度在发育过程中逐渐升高。但我们观察到，CYP4A24与CYP20A1的蛋白表达随时间保持稳定；而CYP51A1在胎儿阶段呈现高表达，随后在出生后首个月内出现下降，至成年阶段再次升高。在PND 1时，CYP4V2_2a与CYP20A1存在性别差异，两种同工型的表达均以雌性个体更高。在成年样本中，CYP1A1、CYP1A2、CYP2A19、CYP2E1_2、CYP3A22、CYP4V2_2a以及CYP4V2_2b均检测到性别差异，雌性哥廷根小型猪的对应蛋白表达水平均高于雄性。
CYP蛋白丰度与CYP酶活性的相关性分析显示，在PND 7时，CYP3A22的蛋白丰度与咪达唑仑的代谢水平显著相关。本研究所得数据与人类相关数据具有显著可比性，为构建新生与幼年哥廷根小型猪的PBPK模型提供了重要进展。