Enzyme Kinetics of GTI-2040, a Phosphorothioate Oligonucleotide Targeting Ribonucleotide Reductase

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 (∼total metabolism) because of sequential metabolism. In HLMs, a biphasic binding was observed for GTI-2040 with high- and low-affinity constants (K_ds) of 0.03 and 3.8 μM, 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 μM 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-20409s 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.