Drug Metabolism by the Mitochondrial Amidoxime Reducing Component (mARC): Rapid Assay and Identification of New Substrates

Abstract

For the development of new drugs, the investigation of their metabolism is of central importance. In the past, the focus was mostly on the consideration of established enzymes leading to oxidations such as cytochrome P450. However, reductive metabolism by the mARC enzyme system can play an important role in particular for nitrogen containing functional groups. A rapid test was established to give developers of new drugs in the preclinical stage the opportunity to test the metabolism by mARC. To demonstrate the relevance and validity of the new test system, known and potential substrates were applied to this new assay. All known substrates could be detected by the system. Furthermore, several new substrates were found including long-established drugs such as hydroxyurea and new compounds in development such as epacdadostat.

A Novel Prodrug of a nNOS Inhibitor with Improved Pharmacokinetic Potential

“…The turnover rate of the amidoxime 2 was tested by an enzymatic NADH assay [38] with the mARC enzyme system in comparison to the model substrate benzamidoxime (BAO). The turnover rate for the amidoxime 2 and BAO are clearly identical for mARC1 and very similar for mARC2.…”

“…BAO is an excellent substrate for mARC, which is completely converted to the corresponding benzamidine in vivo . Due to this known in vivo studies and to other known prodrugs which are activated by mARC, for example, upamostat and ximelagatran, [38] it can be expected that the amidoxime prodrug 2 will also be converted extensively in vivo , and is thus a very suitable prodrug for the amidine 1 and making it orally available.…”

“…The assay was performed as previously described. [38] The proteins were dissolved in 20 mM of 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0) in disposable micro UV cuvettes. Each batch contained 15 μg human mARC, 7 μg B5 and 0.16 μg B5R.…”

“…In order to optimize the pharmacokinetic properties of the nNOS inhibitor 1 , in this study we present the synthesis of amidoxime prodrug 2 (Figure 1), which was in vitro evaluated to ascertain the potential NOS inhibitory activity, as well as its bioconversion into the acetamidine 1 by mARC [38] . Compound 2 was also profiled for in vitro physicochemical properties, by determining lipophilicity, passive permeation through the human GI tract and blood–brain barrier (BBB) by a parallel artificial membrane permeability assay (PAMPA), and chemical, enzymatic, and plasma stability.…”

Drug metabolism in animal models and humans: Translational aspects and chances for individual therapy

“…It must also be pointed out that the metabolism neither in humans nor in animals is fully elucidated. Several enzymes that are involved in human xenobiotic metabolism are often not given sufficient attention, because of the strong focus on cytochrome P450 enzymes 29,30 . Furthermore, in the case of many substances, the occurrence of metabolites has been described without knowing the exact correlation in the biotransformation.…”