Microsomal Catalyzed N-Hydroxylation of Guanabenz and Reduction of the N-Hydroxylated Metabolite:  Characterization of the Two Reactions and Genotoxic Potential of Guanoxabenz1

Clement, Bernd; Demesmaeker, Mark; Linne, Sabine

doi:10.1021/tx9502047

Cited by 43 publications

(39 citation statements)

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“…The reduction of other N-oxygenated basic nitrogen-containing functional groups by these liver preparations has also been described for N-hydroxylated guanidine, N-hydroxydebrisoquine (12), and amidinohydrazone guanoxabenz (30). Previous studies (4, 7) have also shown that liver microsomal preparations can also catalyze NADPH-dependent N-hydroxylation of these compounds, but the rates of N-hydroxylation were about 2 orders of magnitude lower than rates of reduction, which suggests that the latter reaction may prevent accumulation of toxic N-hydroxylated products.…”

Section: Discussionmentioning

confidence: 92%

“…Thus an amidoxime group can serve as a prodrug functionality for an amidino group. The same concept can be applied to guanidines and amidinohydrazones and their N-oxygenated derivatives (12,30). Therefore, strong basic nitrogen-containing functional groups, which are protonated under physiological conditions and are not absorbed as cations, can be made orally available by introducing a hydroxy group, which lowers the pK a values significantly.…”

Section: Met Ile Leu His Pro Asp Val Gln Arg Arg Val Gln Gln Glu Ile Aspmentioning

confidence: 99%

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Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

Clement

Lomb

Möller

1997

Journal of Biological Chemistry

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Drugs containing strong basic nitrogen functional groups can be N-oxygenated to genotoxic products. While the reduction of such products is of considerable toxicological significance, most in vitro studies have focused on oxygen-sensitive reductase systems. However, an oxygen-insensitive microsomal hydroxylamine reductase consisting of NADH, cytochrome b 5 , its reductase, and a third unidentified protein component has been known for some time (Kadlubar, F. F., and Ziegler, D. M. (1974) Arch. Biochem. Biophys. 162, 83-92). This report describes the isolation and identification of all of the components required for the reconstitution of an oxygen-insensitive liver microsomal system capable of catalyzing the efficient reduction of primary N-hydroxylated structures such as amidines, guanidines, amidinohydrazones, and similar functional groups. In addition to cytochrome b 5 and its reductase, the reconstituted system requires phosphatidylcholine and a P450 isoenzyme that has been purified to homogeneity from pig liver. The participation of cytochrome b 5 and NADH cytochrome b 5 reductase in cytochrome P450-dependent biotransformations has previously only been described for oxidative processes. The data presented suggest that this system may be an important catalyst in the reduction of genotoxic N-hydroxylated nitrogen components in liver. Their facile reduction by cellular NADH may be the reason why N-hydroxylated products can be missed by studies in vivo. Furthermore, the enzyme system is involved in the reduction of amidoximes and similar functional groups, which can be used as prodrug functionalities for amidines and related groups.The metabolism of nitrogen-containing functional groups has become a topic of considerable interest since the early discovery that N-hydroxylated intermediates are often responsible for the toxic and/or carcinogenic properties of aromatic amines, hydrazines, and amides (1). On the other hand, the more facile N-oxygenation of secondary and tertiary alkylamines to hydroxylamines and N-oxides was considered as a route for detoxication (2, 3), and it was generally assumed that the strongly basic nitrogen compounds were metabolically stable. However, we have demonstrated that even the protonated hydrophilic amidines (4 -6), as well as diamidines such as pentamidine and diminazene and also guanidines and amidinohydrazones, are capable of undergoing metabolic N-oxygenation by liver microsomal cytochrome P450 monooxygenases (7,8). The N-oxygenation of these functional groups produces more reactive metabolites, and the genotoxic properties of benzamidoxime are well known (9). During investigations of the metabolic fate of strongly basic N-hydroxylated xenobiotics, we observed that they were readily reduced both in vivo and in vitro by a microsomal system present in all mammalian species (rats, rabbits, pigs, and humans) tested to date (10 -13).Preliminary experiments indicated that this system (10) had many of the characteristics of the microsomal O 2 -insensitive hydroxylamine reductase descri...

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Section: Discussionmentioning

confidence: 92%

Section: Met Ile Leu His Pro Asp Val Gln Arg Arg Val Gln Gln Glu Ile Aspmentioning

confidence: 99%

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

Clement

Lomb

Möller

1997

Journal of Biological Chemistry

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“…Prior consent of the local medical ethics committee and from the donors was obtained for these studies. Human and pig microsomes from liver were obtained by ultracentrifugation as described previously (Clement et al, 1996). The kidney microsomes were prepared analogously.…”

Section: Methodsmentioning

confidence: 99%

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

Clement¹,

Lopian²

2003

Drug Metab Dispos

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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...

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“…Our study shows that XO mediates the N-reduction of guanoxabenz with a K m value in the micromolar range (65.2 Ϯ 4.5 µM with NADH and 39.0 Ϯ7.1 µM with xanthine as electron donor). Interestingly, Clement et al [31] have reported that rabbit microsomes can also catalyze N-reduction of guanoxabenz in the presence of NADH, the K m value for the reaction being about 34 µM. Moreover, these authors reported recently that the microsomal N-reduction is catalyzed by an enzyme system composed of cytochrome b 5 , NADH cytochrome b 5 -reductase and benzamidoxime reductase [32].…”

Section: Discussionmentioning

confidence: 99%

Identification of an N‐hydroxyguanidine reducing activity of xanthine oxidase

Dambrova

Uhlén

Welch

et al. 1998

European Journal of Biochemistry

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A guanoxabenz [1-(2,6-dichlorobenzylideneamino)-3-hydroxyguanidine; an N-hydroxyguanidine] reducing enzymatic activity of rat spleen cytosol was investigated. By means of protein purification and N-terminal amino acid sequencing, the reducing activity was shown to reside in xanthine oxidase. The action of the enzyme on guanoxabenz resulted in the formation of guanabenz [1-(2,6-dichlorobenzylideneamino)-3-guanidine]; the product formation could be monitored by HPLC and its identity was confirmed by NMR analysis. The reduction of guanoxabenz required xanthine or NADH as reducing substrates, while the process could be blocked by allopurinol, a selective inhibitor of xanthine oxidase. By using bovine milk xanthine oxidase, the guanoxabenz reducing activity of the enzyme was also verified. We conclude that guanoxabenz is a novel electron acceptor structure for xanthine oxidase.

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Microsomal Catalyzed N-Hydroxylation of Guanabenz and Reduction of the N-Hydroxylated Metabolite: Characterization of the Two Reactions and Genotoxic Potential of Guanoxabenz¹

Cited by 43 publications

References 24 publications

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

Isolation and Characterization of the Protein Components of the Liver Microsomal O2-insensitive NADH-Benzamidoxime Reductase

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

Identification of an N‐hydroxyguanidine reducing activity of xanthine oxidase

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