Characterization of in Vitro Biotransformation of New, Orally Active, Direct Thrombin Inhibitor Ximelagatran, an Amidoxime and Ester Prodrug

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:N-Hydroxylated amidines (amidoximes) can be used as prodrugs of amidines. The prodrug principle was developed in our laboratory for pentamidine and had been applied to several other drug candidates. One of these compounds is melagatran, a novel, synthetic, low molecular weight, direct thrombin inhibitor. To increase the poor oral bioavailability due to its strong basic amidine functionality selected to fit the arginine side pocket of thrombin, the less basic N-hydroxylated amidine was used in addition to an ethyl ester-protecting residue. The objective of this investigation was to study the reduction and the hydrolytic metabolism of ximelagatran via two mono-prodrugs (N-hydroxy-melagatran and ethyl-melagatran) to melagatran by in vitro experiments. New high-performance liquid chromatography methods were developed to analyze all four compounds. The biotransformation of ximelagatran to melagatran involving the reduction of the amidoxime function and the ester cleavage could be demonstrated in vitro by microsomes and mitochondria from liver and kidney of pig and human, and the kinetic parameters were determined. So far, one enzyme system capable of reducing N-hydroxylated structures has been identified in pig liver microsomes, consisting of cytochrome b 5 , NADH-cytochrome b 5 reductase, and a P450 isoenzyme of the subfamily 2D. This enzyme system also reduces ximelagatran and N-hydroxy-melagatran. The participation of recombinant human CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 with cytochrome b 5 and b 5 reductase in the reduction can be excluded. In summary, ximelagatran and N-hydroxy-melagatran are easily reduced by several enzyme systems located in microsomes and mitochondria of different organs.Melagatran is the active form of the novel, oral thrombin inhibitor ximelagatran (Gustafsson et al., 2001). Melagatran has suboptimal bioavailability because of the presence of a carboxylic acid, a secondary amine, and an amidine residue resulting in a charged molecule at physiological pH values. The prodrug ximelagatran was developed with a view to improving absorption. Ximelagatran comprises an ethyl ester group in place of the carboxylic acid and an N-hydroxyamidine group in place of the amidine. After protonation of amidines at the double-bonded nitrogen, cations are formed that are highly stabilized by mesomerism. Amidines are very strong bases (Albert et al., 1948) and protonated under physiological conditions. By introduction of an oxygen atom in the amidine functional group, the basicity is lowered by 5 pK a value. Amidoximes have been used as prodrugs for certain amidine-containing drugs, including pentamidine derivatives (Clement, 1993) and sibrafiban (Weller et al., 1996). It has been shown that certain amidoximes are rapidly reduced in vitro and in vivo to the amidines (Clement et al., 1988(Clement et al., , 1992Hauptmann et al., 1988). More recently, it has been demonstrated that the model compound benzamidoxime is reduced by microsom...

Highly Potent and Selective Substrate Analogue Factor Xa Inhibitors Containing D‐Homophenylalanine Analogues as P3 Residue: Part 2

“…The conversion rates were comparable with those of ximelagatran to melagatran. [15] However, based on the detection of the active inhibitor 27 in plasma using a spectrophotometric activity assay, all prodrugs were insufficiently present after oral administration to rats. At a dose of 5 mg kg…”

Drug-Induced Liver Injury in Humans: The Case of Ximelagatran

“…After oral administration, ximelagatran is rapidly absorbed and bioconverted to the active form, melagatran, in a two-step process involving ester cleavage and reduction of the amidoxime group (Eriksson et al 2003c). None of the major human CYP isoenzymes appear to be involved in either of these steps (Bredberg et al, 2003;Clement and Lopian 2003). Ximelagatran, melagatran, and the intermediate metabolites, ethyl-melagatran and hydroxy (OH)-melagatran, have also been shown not to inhibit CYP isoenzymes in vitro (Bredberg et al 2003).…”

Role of Metabolism in Toxicity of Drugs in Humans

“…Likewise, there is no in vitro and/or in vivo evidence suggestive of reactive metabolite formation with ximelagatran, the first orally active thrombin inhibitor that has been withdrawn from commercial use because of several cases of hepatotoxicity [99,100]. As such, ximelagatran does not contain any structural alerts, and its in vivo biotransformation in animals and humans consists of innocuous pathways involving ester hydrolysis and reduction of the amidoxime motif to afford the active ingredient melagatran (Scheme 11.14) [101]. Finally, the possibility that toxicity can arise from mechanisms other than reactive metabolite formation (e.g., direct mitochondrial toxicity) also needs to be taken into consideration.…”