Hammid Anam, Fallon John K, Lassila Toni, Salluce Giulia, Smith Philip C, Tolonen Ari, Sauer Achim, Urtti Arto, Honkakoski Paavo
School of Pharmacy, University of Eastern Finland, Yliopistonranta 1 C, 70210 Kuopio, Finland.
Division of Pharmacoengineering and Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Campus Box 7355, Chapel Hill, North Carolina 27599-7355, United States.
Mol Pharm. 2021 Mar 1;18(3):1305-1316. doi: 10.1021/acs.molpharmaceut.0c01154. Epub 2021 Feb 17.
Hydrolytic reactions constitute an important pathway of drug metabolism and a significant route of prodrug activation. Many ophthalmic drugs and prodrugs contain ester groups that greatly enhance their permeation across several hydrophobic barriers in the eye before the drugs are either metabolized or released, respectively, hydrolysis. Thus, the development of ophthalmic drug therapy requires the thorough profiling of substrate specificities, activities, and expression levels of ocular esterases. However, such information is scant in the literature, especially for preclinical species often used in ophthalmology such as rabbits and pigs. Therefore, our aim was to generate systematic information on the activity and expression of carboxylesterases (CESs) and arylacetamide deacetylase (AADAC) in seven ocular tissue homogenates from these two species. The hydrolytic activities were measured using a generic esterase substrate (4-nitrophenyl acetate) and, in the absence of validated substrates for rabbit and pig enzymes, with selective substrates established for human CES1, CES2, and AADAC (d-luciferin methyl ester, fluorescein diacetate, procaine, and phenacetin). Kinetics and inhibition studies were conducted using these substrates and, again due to a lack of validated rabbit and pig CES inhibitors, with known inhibitors for the human enzymes. Protein expression levels were measured using quantitative targeted proteomics. Rabbit ocular tissues showed significant variability in the expression of CES1 (higher in cornea, lower in conjunctiva) and CES2 (higher in conjunctiva, lower in cornea) and a poor correlation of CES expression with hydrolytic activities. In contrast, pig tissues appear to express only CES1, and CES3 and AADAC seem to be either low or absent, respectively, in both species. The current study revealed remarkable species and tissue differences in ocular hydrolytic enzymes that can be taken into account in the design of esterase-dependent prodrugs and drug conjugates, the evaluation of ocular effects of systemic drugs, and in translational and toxicity studies.
水解反应是药物代谢的重要途径，也是前药活化的重要途径。许多眼科药物和前药含有酯基，在药物分别被代谢或释放（水解）之前，这些酯基极大地增强了它们穿过眼内几个疏水屏障的渗透能力。因此，眼科药物治疗的发展需要全面了解眼部酯酶的底物特异性、活性和表达水平。然而，文献中此类信息匮乏，尤其是对于眼科常用的临床前物种，如兔子和猪。因此，我们的目的是获取关于这两个物种七种眼组织匀浆中羧酸酯酶（CESs）和芳基乙酰胺脱乙酰酶（AADAC）的活性和表达的系统信息。使用通用酯酶底物（乙酸对硝基苯酯）测量水解活性，并且在没有针对兔和猪酶的经过验证的底物的情况下，使用为人类CES1、CES2和AADAC建立的选择性底物（d - 荧光素甲酯、荧光素二乙酸酯、普鲁卡因和非那西丁）。使用这些底物进行动力学和抑制研究，同样由于缺乏经过验证的兔和猪CES抑制剂，使用人类酶的已知抑制剂。使用定量靶向蛋白质组学测量蛋白质表达水平。兔眼组织在CES1（角膜中较高，结膜中较低）和CES2（结膜中较高，角膜中较低）的表达上表现出显著差异，并且CES表达与水解活性的相关性较差。相比之下，猪组织似乎只表达CES1，并且CES3和AADAC在这两个物种中似乎分别表达水平较低或不存在。当前研究揭示了眼部水解酶在物种和组织方面存在显著差异，这在设计依赖酯酶的前药和药物偶联物、评估全身药物的眼部效应以及在转化和毒性研究中都应予以考虑。
