Drug Metabolism Studies with the Incubated Hen’s Egg

Abstract

Previous investigations have shown that an ex vivo model on the basis of the incubated hen’s egg represents a potential alternative to animal experimentation for xenobiotic metabolism studies. This model is characterized by administration of the xenobiotic into the yolk sac and identification of metabolites in the allantoic fluid. In the present study, gentisic acid (2,5-dihydroxybenzoic acid, 2,5-DHBA, CAS 490-79-9) was used as a test substance. The oxidative fate of this salicylate metabolite in human and animal species could not be completely elucidated until now. Inoculation of 2,5-DHBA into the incubated hen’s egg in concentrations up to 8 mg/egg did not affect embryo viability. For the metabolism studies a dose of 2 mg/egg was chosen and 2,5-DHBA was identified as the main metabolite in both the free and conjugated form (glucuronide/sulfate). Two minor metabolites were detected in addition. The first of these could be identified as 2,3,5-trihydroxybenzoic acid (2,3,5-THBA) and the second one as 2-hydroxy-5-methoxybenzoic acid (2-H-5-MBA). 2,3,5-THBA has been characterized as a metabolite of 2,5-DHBA in a vertebrate species for the first time. Since 2,3,5-THBA can be methylated to the guaiacol derivative, 2,5-dihydroxy-3-methoxybenzoic acid (2,5-DH-3-MBA) can not to be excluded as a further possible metabolite. The metabolites were identified by comparative TLC with the authentic reference substances as well as additionally by GC and GC-MS analysis of the trimethylsilyl derivatives. The structures of the synthesized reference substances were confirmed by MS, ¹H and ¹³C NMR spectral data.

Key words

CAS 490-79-9 - Gentisic acid, drug metabolism, ex vivo model - 2-Hydroxy-5-methoxybenzoic acid - Incubated hen’s egg, alternative to animal experimentation - Salicylates - 2,3,5-Trihydroxybenzoic acid

• References

• 1 Graham GG, Roberts MS, Day RO, Rainsford KD. Pharmacokinetics and metabolism of the salicylates. In: Rainsford KD. ed Aspirin and related drugs. London: Taylor & Francis; 2004: 97-155
• 2 Astill BD, Fassett DW, Roudabush RL. The metabolism of phenolic antioxidants - 4. The metabolites of gentisic acid in the dog. Biochem J. 1964; 90 (1) 194-201
• 3 Chrastil J, Wilson JT. Quantitative estimation of salicylic acid and its metabolites by thin-layer densitometry. J Chromatogr. 1978; 152 (1) 183-189
• 4 Wilson JT, Howell RL, Holladay MW, Brilis GM, Chrastil J, Watson JT et al. Gentisuric acid: Metabolic formation in animals and identification as a metabolite of aspirin in man. Clin Pharmacol Ther. 1978; 23 (6) 635-643
• 5 DeBlassio JL, deLong MA, Glufke U, Kulathila R, Merkler KA, Vederas JC et al. Amidation of salicyluric acid and gentisuric acid: A possible role for peptidylglycine α-amidating monooxygenase in the metabolism of aspirin. Arch Bio-chem Biophys. 2000; 383 (l) 46-55
• 6 Sakamoto Y, Inamori K, Nasu H. Methylation of gentisic acid - Formation of 5-methoxysalicylic acid. J Biochem. 1959; 46 (12) 1667-1669
• 7 Dumazert C, El Ouachi M. Sur le métabolisme des salicylates. Ann Pharm Fr.. 1954; 12 (11) 723-730
• 8 Ionescu C, Caira MR eds Drug metabolism - Current concepts. Dordrecht: Springer; 2005
• 9 Amann R, Peskar BA. Anti-inflammatory effects of aspirin and sodium salicylate. Eur J Pharmacol. 2002; 447 (1) 1-9
• 10 Booth AN, Masri MS, Robbins DJ, Emerson OH, Jones FT, DeEds F. The metabolic fate of gallic acid and related compounds. J Biol Chem. 1959; 234 (11) 3014-3016
• 11 Masri MS, Booth AN, DeEds E. O-methylation in vitro of dihydroxy- and trihydroxy-phenolic compounds by liver slices. Biochim Biophys Acta. 1962; 65 (3) 495-500
• 12 Fötsch G, Gründemann E, Pfeifer S, Hiller K, Salzwedel D. Zur Struktur von Leiocarposid. Pharmazie. 1988; 43 (4) 278-280
• 13 Kiep L. Biotransformation von Salicylsäure im Kükenembryo (Gallus domesticus) [dissertation]. Halle-Wittenberg [DE); Martin-Luther-Universität; 1982
• 14 Kiep L, Bekemeier H. Biotransformation und Toxizität von Xenobiotica im Kükenembryo. Pharmazie. 1986; 41 (12) 868-872
• 15 Kiep L. Metabolism of xenobiotics in the incubated hen’s egg: Investigations with ethyl 4-hydroxybenzoate. ALTEX. 2005; 22 (3) 135-141
• 16 Grootveld M, Halliwell B. Aromatic hydroxylation as a potential measure of hydroxyl-radical formation in vivo. Biochem J. 1986; 237 (2) 499-504
• 17 Grootveld M, Halliwell B. 2,3-Dihydroxybenzoic acid is a product of human aspirin metabolism. Biochem Pharma col. 1988; 37 (2) 271-280
• 18 Schock Jr RU, Tabern DL. The persulfate oxidation of salicylic acid - 2,3,5-trihydroxybenzoic acid. J Org Chem. 1951; 16 (11) 1772-1775
• 19 Schock Jr RU, Tabern DL. Therapeutic compound and method of preparing the same. USP. 1953; 2: 641-609
• 20 Wiltshire H. Thin-layer chromatography. In: Venn RF. ed. Principles and practice of bioanalysis. London: Taylor & Francis; 2000: 149-159
• 21 Fötsch G, Pfeifer S, Bartoszek M, Franke P, Hiller K. Biotransformation der Phenolglycoside Leiocarposid und Salicin. Pharmazie. 1989; 44 (8) 555-558
• 22 Dupont I, Berthou F, Bodenez P, Bardou L, Guirriec C, Stephan N et al. Involvement of cytochromes P-450 2E1 and 3A4 in the 5-hydroxylation of salicylate in humans. Drug Meta b Dispos. 1999; 27 (3) 322-326
• 23 Ingelman-Sundberg M, Kaur H, Terelius Y, Persson JO, Halliwell B. Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Biochem J. 1991; 276 (3) 753-757
• 24 Strolin-Benedetti M, Brogin G, Bani M, Oesch F, Hengstler JG. Association of cytochrome P450 induction with oxidative stress in vivo as evidenced by 3-hydroxylation of salicylate. Xenobiotica. 1999; 29 (11) 1171-1180
• 25 Machala M, Nezveda K, Irizar A, Bu-Abbas A, Ioannides C. Expression and inducibility of cytochrome P450 proteins in the liver of chick embryo. Arch Toxicol. 1996; 71 (l–2) 57-63
• 26 Paolini M, Pozzetti L, Sapone A, Biagi GL, Cantelli-Forti G. Development of basal and induced testosterone hydroxylase activity in the chicken embryo in ovo. Br J Pharmacol. 1997; 122 (2) 344-350
• 27 Ourlin JC, Baader M, Fraser D, Halpert JR, Meyer UA. Cloning and functional expression of a first inducible avian cytochrome P450 of CYP3A subfamily (CYP3A37). Arch Biochem Biophys. 2000; 373 (2) 375-384
• 28 Axelrod J, Daly J. Phenol-O-methyltransferase. Biochim Biophys Acta. 1968; 159 (3) 472-478
• 29 Weinshilboum R. Methyltransferase pharmacogenetics. Pharmacol Ther. 1989; 43 (l) 77-90
• 30 Ignarro LJ, Shideman FE. Catechol-O-methyl transferase and monoamine oxidase activities in the heart and liver of the embryonic and developing chick. J Pharmacol Exp Ther. 1968; 159 (l) 29-37
• 31 Kiep L, Maderner S, Seifert K. Metabolism of metamizol in early stages of the incubated hen’s egg. Pharmazie. 2002; 57 (12) 829-833
• 32 Volz M, Fehlhaber HW, Siebenlist F. Biotransformation of metamizol (novalgin®/dipyrone®). Proceedings of the 5th European Meeting of the International Society for the Study of Xenobiotics; 1993 Sep 26-29; Tours, France, p. 70.
• 33 Wessel JC. Erstmaliger Nachweis von Oxalsäurederivaten bei der Biotransformation von Metamizol im bebrüteten Hühnerei und beim Menschen [dissertation]. Düsseldorf [DE); Heinrich-Heine-Universität 2004
• 34 Wessel JC, Matyja M, Neugebauer M, Kiefer H, Daldrup T, Tarbah FA et al. Characterization of oxalic acid derivatives as new metabolites of metamizol (dipyrone) in incubated hen’s egg and human. Eur] Pharm Sci. 2006; 28 (1–2) 15-25
• 35 Neugebauer M. Biotransformation von Arzneistoffen im Hühnerei - Eine Alternative zum Tierversuch [habilitation]. Bonn (DE); Rheinische Friedrich-Wilhelms-Universität; 1997
• 36 Steventon GB, Hutt AJ. The amino acid conjugations. In: Ioannides C. ed. Enzyme systems that metabolise drugs and other xenobiotics. Chichester: John Wiley & Sons; 2002: 501-520