Received for publication March 3, 2008.
Revised July 23, 2008.
Accepted for publication July 23, 2008.

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

Abstract

ABSTRACT: Enzyme kinetics of GTI-2040, a phosphorothioate ribonucleotide reductase antisense, was investigated for the first time in 3' exonuclease solution and human liver microsomes (HLM), using ion-pair HPLC 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 was found to be well characterized by Michaelis-Menten model, using the sum of formation rates of 3'N-1 and 3'N-2 (~total metabolism) because of sequential metabolism. In HLM, 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 HLM was 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 free substrate fraction, K_mand V_max of GTI-2040 were determined to be 6.33 ± 3.2 µM and 16.5 ± 8.4 nmol/mg/hr, respectively. Using these values, CL_int in HLM was estimated to be 2.61 ± 0.56 mL/hr. The CL_int was then used to predict GTI-2040’s in vivo intrinsic clearance (CL_int,h) in humans by a microsomal protein scaling factor, which gave a mean value of 182.7 L/hr, representing 24.1% of the observed in vivo mean CL_int,h1 of 758.7 L/hr in patients with acute myeloid leukemia. We concluded that the saturable non-specific binding of GTI-2040 in HLM complicated the interpretation of its enzyme kinetics, and scaled intrinsic clearance from HLM only partially predicted the in vivo intrinsic clearance.

Key words: enzyme kinetics, protein binding