Osorio-Lozada Antonio, Surapaneni Sekhar, Skiles Gary L, Subramanian Raju
Pharmacokinetics and Drug Metabolism, Amgen Inc., Thousand Oaks, California 91320, USA.
Drug Metab Dispos. 2008 Feb;36(2):234-40. doi: 10.1124/dmd.107.019323. Epub 2007 Nov 15.
Fungal and bacterial microbes are known to mimic mammalian cytochrome P450 metabolism. Traditionally, microbial biotransformation screening and small scale-ups (<1 liter) are performed in shake-flask reactors. An alternative approach is the use of hollow fiber cartridge (HFC) reactors. The performance of HFC reactors is compared with shake-flask reactors using diclofenac as a model substrate. Actinoplanes sp. (American Type Culture Collection 53771) in a shake-flask reactor hydroxylated diclofenac (50 microM) with 100% turnover in less than 5 h. A scaled-up production resulted in the formation of 4'-hydroxy (169 mg, 54% yield), 5-hydroxy (42 mg, 13% yield), and 4',5-dihydroxy (25 mg, 7.7% yield) metabolites. HFC reactors with Teflon, polysulfone, and cellulose membranes were screened for nonspecific binding of diclofenac. Concentration-time profiles for turnover of 50 to 2000 microM diclofenac by Actinoplanes sp. were then determined at 22 and 30 degrees C in an HFC reactor. Cellulose-based HFC reactors exhibited the lowest nonspecific binding (87% of 50 microM diclofenac remaining after 5 h) and offered the best conditions for its biotransformation (100% conversion; < 5 h at 30 degrees C at 50 microM; 25 h at 500 microM). The time profile for substrate turnover was equivalent in both a cellulose membrane HFC reactor and shake-flask reactor. Two cellulose membrane HFC reactors were also tested to evaluate the reusability of the cartridges for diclofenac metabolism (50 microM, 22 degrees C, 15 h; 500 microM, 30 degrees C, 36 h). Up to seven reaction cycles with intermediate wash cycles were tested. At least 98% conversion was observed in each reaction cycle at both diclofenac concentrations.
已知真菌和细菌微生物可模拟哺乳动物细胞色素P450代谢。传统上，微生物生物转化筛选和小规模放大（<1升）在摇瓶反应器中进行。另一种方法是使用中空纤维柱（HFC）反应器。以双氯芬酸为模型底物，比较了HFC反应器与摇瓶反应器的性能。游动放线菌（美国典型培养物保藏中心53771）在摇瓶反应器中可使双氯芬酸（50微摩尔）羟基化，在不到5小时内转化率达100%。放大生产得到了4'-羟基（169毫克，产率54%）、5-羟基（42毫克，产率13%）和4',5-二羟基（25毫克，产率7.7%）代谢产物。对具有聚四氟乙烯、聚砜和纤维素膜的HFC反应器进行了双氯芬酸非特异性结合的筛选。然后在HFC反应器中于22和30℃测定游动放线菌对50至2000微摩尔双氯芬酸转化的浓度-时间曲线。基于纤维素的HFC反应器表现出最低的非特异性结合（5小时后50微摩尔双氯芬酸剩余87%），并为其生物转化提供了最佳条件（100%转化；在30℃、50微摩尔时<5小时；在500微摩尔时25小时）。在纤维素膜HFC反应器和摇瓶反应器中，底物转化的时间曲线是相同的。还测试了两个纤维素膜HFC反应器，以评估柱体对双氯芬酸代谢（50微摩尔，22℃，15小时；500微摩尔，30℃，36小时）的可重复使用性。测试了多达七个带有中间洗涤周期的反应循环。在两种双氯芬酸浓度下，每个反应循环中均观察到至少98%的转化率。
