The in vitro hydrolysis of isoeugenyl acetate and eugenyl acetate I. Glenn Sipes Department of Pharmacology, University of Arizona, Tucson, AZ
Objective Determine the extent of hydrolysis for isoeugenyl acetate and eugenyl acetate in various biological matrices
Hydrolytic Reaction R R O O OH O O IA, R= EA, R= ester alcohol M.W 206.24 M.W 164.20
Specific Aims • Determine if skin, blood, and liver possess hydrolytic activity towards isoeugenyl acetate and eugenyl acetate • Establish stoichiometric relationship for the disappearance of the esters and the formation of the alcohols over time • Determine kinetic constants for the hydrolytic conversion to the corresponding alcohols in the respective tissues. • Assess the integrated metabolism of the esters in monolayers of SD rat hepatocytes, an intact cellular system
Isolation and Treatment • Reversed-phase alpha Male SD rat bond C18 column • Mobile phase: acidified water and acetonitrile • Monitoring wavelengths of 254 nm Liver Skin Blood and 265 nm Hepatocyte Subcellular Isolation HPLC Fractionation Incubate sample IA or EA 37 o C prep ~500 M
Hydrolysis of EA by rat hepatic microsomes - (HPLC Profile) DAD1 B, Sig=265,4 Ref=550,50 (CS010903\008-0901.D) mAU 350 300 250 A. 0.5 min 200 150 eugenol 100 50 0 10 12 14 16 18 20 22 24 26 28 min DAD1 B, Sig=265,4 Ref=550,50 (CS010903\013-1501.D) mAU 350 300 250 B. 5 min 200 150 100 50 0 10 12 14 16 18 20 22 24 26 28 min DAD1 B, Sig=265,4 Ref=550,50 (CS010903\015-1701.D) mAU 400 350 300 C. 20 min 250 eugenyl 200 acetate 150 100 50 0 10 12 14 16 18 20 22 24 26 28 min
Ester Hydrolysis in hepatic microsomal preparations 600 600 Concentration (nmol/ml) Concentration (nmol/ml) 500 500 400 400 300 300 200 200 100 100 0 0 0 10 20 30 0 10 20 30 Incubation Time (min) Incubation Time (min) A. Human Male B. SD Male Rat • Isoeugenyl-acetate concentration : 532 nmol/ml, (0.11 mg/ml) N=3 • Human female hepatic tissue also hydrolyzed IA to isoeugenol within 30 min • Hydrolytic activity towards eugenyl-acetate is comparable for the respective tissues
Ester Hydrolysis in human blood and plasma preparations 600 500 Concentration (nmol/ml) Concentration (nmol/ml) 500 400 400 300 300 200 200 100 100 0 0 0 10 20 30 40 0 10 20 30 40 50 60 70 Incubation Time (min) Incubation Time (min) A. Blood:PBS (1:3) B. Plasma:PBS (1:10) • Isoeugenyl-acetate concentration : 488 nmol/ml, (0.10 mg/ml) N=2 • Blood and plasma from male SD rats also hydrolyzed both IA and EA. • Studies indicate increased carboxylesterase activity in plasma preparations
Hydrolysis of IA by rat skin microsomes - (HPLC Profile) DAD1 B, Sig=265,4 Ref=550,50 (DC092503\012-1001.D) mAU 80 70 60 50 A. 1 min 40 30 isoeugenol 20 10 0 10 12 14 16 18 20 22 24 26 28 min DAD1 B, Sig=265,4 Ref=550,50 (DC092503\018-1601.D) mAU 120 100 80 B. 20 min 60 40 20 0 10 12 14 16 18 20 22 24 26 28 min DAD1 B, Sig=265,4 Ref=550,50 (DC092503\020-1801.D) mAU 120 100 80 isoeugenyl C. 60 min 60 acetate 40 20 0 10 12 14 16 18 20 22 24 26 28 min
Ester Hydrolysis by rat skin microsomes and cytosol 400 500 Concentration (nmol/ml) Concentration (nmol/ml) 400 300 300 200 200 100 100 0 0 0 10 20 30 0 10 20 30 Incubation Time (min) Incubation Time (min) A. Microsomes A. Cytosol • Isoeugenyl-acetate concentration : 488 nmol/ml, (0.10 mg/ml) N=4 • Microsomal Incubations consisted of 0.0375 mg/ml protein • Cytosol Incubation consisted of 0.1 mg/ml protein • Studies with eugenyl-acetate are comparable
Parameters for Kinetic Analysis • Formation of isoeugenol and eugenol was measured in skin and hepatic tissue preparations • Skin Protein Concentrations: 0.0375 (M) & 0.1 mg/ml (C) • Hepatic Protein Concentrations: 0.0125 (M) & 0.1 mg/ml (S-9) • Dosing Range: IA [ 5 to 495 M], EA [39 to 970 M] • Duration of incubation: skin (15 min) and hepatic (3 min) • Kinetic Data was normalized to nmol / min / mg of protein
Concentration Dependent Ester Hydrolysis 800 4000 Rate (nmol/min/mg of protein) Rate (nmol/min/mg of protein) 600 3000 400 2000 200 1000 0 0 0 250 500 750 1000 1250 0 100 200 300 400 500 600 M of Eugenyl-acetate M of Isoeugenyl-acetate • Formation of eugenol by male SD rat skin microsomes (left) N=2 • Formation of isoeugenol by human male hepatic microsomes (right) N=3
Kinetic Constants for Alcohol Formation K m V max CL int ( M) (nmol/min/mg protein) (ml/min) Subcellular Fraction IA EA IA EA IA EA Rat Skin Cytosol 97 114 137 216 0.7 0.5 Rat Skin Microsomes 505 638 190 223 2.7 2.9 Rat Hepatic S-9 52 60 110 173 0.5 0.4 Rat Hepatic Microsomes 3822 3829 91 97 42.0 39.6 Human Male Microsomes 3795 3656 72 89 52.5 41.3 Human Female Microsomes 3072 2748 51 52 60.1 52.9 • CL int = intrinsic metabolic clearance (Vmax / Km) • Note the similarities between human and rat hepatic microsomes with respect to the intrinsic metabolic clearance of EA and IA
Integrated Metabolism of the esters in an intact cellular system 600 600 Concentration (nmol/ml) Concentration (nmol/ml) 500 500 400 400 300 300 200 200 100 100 0 0 0 5 10 15 20 25 0 5 10 15 20 25 Incubation Time (min) Incubation Time (min) A. B. • Isoeugenyl-acetate (A) concentration: 538 nmol/ml (0.11 mg/ml) • Eugenyl-acetate (B) concentration: 483 nmol/ml (0.10 mg/ml) • Within approximately 20 minutes hydrolytic conversion was complete
Summary • IA and EA are rapidly hydrolyzed by tissue esterases to the corresponding alcohols, isoeugenol and eugenol. • Stoichiometric conversion to the alcohols was observed in hepatic, blood, and skin preparations when incubated with IA and EA. • When the localization of esterases was evaluated, the most extensive activity was observed in the hepatic microsomal fraction. • Human hepatic and blood preparations displayed similar rates of hydrolysis to that of rodents when incubated under the same conditions.
Conclusions • Absorption of IA and EA into the systemic circulation would be minimal due to hydrolytic activity in skin, blood as well as the liver • Toxicological data presently available for isoeugenol and eugenol can be used for the safety assessment of the esters • Animal data can be extrapolated to humans for rational safety evaluation for the use of esters in fragrance and food products • Slow rate of hydrolysis of esters in skin correlates with their decreased sensitization potential
NOELs based on HRIPT Compound NOEL 2 % Isoeugenyl-acetate isoeugenol 0.2-0.5 % eugenyl-acetate 8 % eugenol ~ 5 %
Potential reaction pathways for skin sensitization IA EA Hydrolysis OH OH OCH 3 OCH 3 Conjugation Conjugation Oxidation Demethylation / Oxidation O O OCH 3 O p-quinone methide o-quinone
Acknowledgements • Lab Members: • David Castro • Cathy Sweet • Bob Kuester • Research Associate: • Aniko Solyom
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