title: Enzyme kinetics of GTI-2040, a phosphorothioate oligonucleotide targeting ribonucleotide reductase.
abstract: Enzyme kinetics of GTI-2040 (5'-GGC TAA ATC GCT CCA CCA AG-3'), a phosphorothioate ribonucleotide reductase antisense, were investigated for the first time in 3' exonuclease solution and human liver microsomes (HLMs), using the ion-pair high-performance liquid chromatogram method for quantification of the parent drug and two major 3'N-1 and 3'N-2 metabolites. Enzyme kinetics of GTI-2040 in 3'-exonuclease solution were found to be well characterized by the Michaelis-Menten model, using the sum of formation rates of 3'N-1 and 3'N-2 (approximately total metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high- and low-affinity constants (K(d)s) of 0.03 and 3.8 microM, respectively. Enzyme kinetics of GTI-2040 in HLMs were found to deviate from Michaelis-Menten kinetics when the total GTI-2040 substrate was used. However, after correction for the unbound fractions, the formation rate of total metabolites could be described by Michaelis-Menten kinetics. Using the free substrate fraction, the K(m) and V(max) of GTI-2040 were determined to be 6.33 +/- 3.2 microM and 16.5 +/- 8.4 nmol/mg/h, respectively. Using these values, in vitro hepatic intrinsic clearance (CL(int)) in HLM was estimated to be 2.61 +/- 0.56 ml/h. The CL(int) was then used to predict GTI-2040's in vivo intrinsic clearance in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 l/h, representing 24.1% of the observed in vivo mean scaled hepatic intrinsic clearance of 758.7 l/h in patients with acute myeloid leukemia. We concluded that the saturable nonspecific binding of GTI-2040 in HLMs complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLMs only partially predicted the in vivo intrinsic clearance.
authors.affiliation: Division of Pharmaceutics, College of Pharmacy, Ohio State University, Columbus, OH 43210, USA.
authors.forename: Xiaohui
authors.initials: X
authors.lastname: Wei
authors.forename: Guowei
authors.initials: G
authors.lastname: Dai
authors.forename: Zhongfa
authors.initials: Z
authors.lastname: Liu
authors.forename: Hao
authors.initials: H
authors.lastname: Cheng
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Xie
authors.forename: Rebecca
authors.initials: R
authors.lastname: Klisovic
authors.forename: Guido
authors.initials: G
authors.lastname: Marcucci
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Chan
mesh_terms: D000818:Animals; D017839:Crotalus; D016503:Drug Delivery Systems / Q000379:methods*; D005260:Female; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000097:blood / Q000188:drug therapy / Q000201:enzymology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000008:administration & dosage / Q000378:metabolism* / Q000494:pharmacology; D054735:Phosphorothioate Oligonucleotides / Q000008:administration & dosage / Q000378:metabolism* / Q000494:pharmacology; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D054735:Phosphorothioate Oligonucleotides; C464617:GTI2040; D012264:Ribonucleotide Reductases