Enantioselectivity of debrisoquine 4-hydroxylation in Brazilian Caucasian hypertensive patients phenotyped as extensive metabolizers.
Debrisoquine (D), an antihypertensive drug metabolized to 4-hydroxydebrisoquine (4-OHD) by CYP2D6, is commonly used as an in vivo probe of CYP2D6 activity and can be used to phenotype individuals as either extensive (EMs) or poor metabolizers (PMs) of such drugs as beta-adrenergic blockers, tricyclic antidepressants, and class 1C antiarrhythmics. This report describes reversed-phase HPLC systems by which D and 4-OHD or S-(+) and R-(-)-4-OHD in urine are more selectively quantified without the need for derivatization techniques. We also studied the urinary excretion of R-(-)- and S-(+)-4-hydroxydebrisoquine in EM hypertensive patients in order to determine weather 4-OHD formation exhibits enantioselectivity. Twelve patients with mild to severe essential hypertension were admitted to the study. They received a single tablet of Declinax containing 10 mg debrisoquine sulfate. All the urine excreted during the following 8 h was collected. The debrisoquine metabolic ratio (DMR) was calculated as % of dose excreted as D/% of dose excreted as 4-OHD and the debrisoquine recovery ratio (DRR) was calculated as % of dose excreted as 4-OHD/% of dose excreted as D+4-OHD. Debrisoquine and its metabolite were determined in urine by HPLC using a reversed-phase Select B LiChrospher column, a mobile phase of 0.25 N acetate buffer, pH 5-acetonitrile (9:1, v/v) and a fluorescence detector. The limit of quantitation was determined to be 25.0 ng/ml for D and 18.75 ng/ml for 4-OHD. Intra- and inter-day relative standard deviations (RSDs) were less than 10%. All hypertensive patients studied showed a DMR of less than 12.6 or a DRR higher than 0.12 and were classified as EMs. Direct enantioselective separation on chiral stationary phase involved resolution of S-(+)-4-OHD and R-(-)-4-OHD on a Chiralcel OD-R column with a mobile phase of 0.125 N sodium perchlorate, pH 5-acetonitrile-methanol (85:12:3, v/v/v). The quantitation limit of each enantiomer was 3.75 ng/ml of urine. Intra- and inter-day RSDs were less than 10% for each enantiomer. A high degree of enantioselectivity in the 4-hydroxylation of D favouring the S-(+) enantiomer was observed, resulting in R-(-)-4-OHD not detected in the urine of the EM hypertensive patients studied.
Cerqueira
P M
PM
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Brazil.
Mateus
F H
FH
Cesarino
E J
EJ
Bonato
P S
PS
Lanchote
V L
VL
D000328:Adult; D000368:Aged; D000959:Antihypertensive Agents / Q000378:metabolism / Q000627:therapeutic use / Q000652:urine*; D001938:Brazil; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003647:Debrisoquin / Q000031:analogs & derivatives / Q000378:metabolism / Q000627:therapeutic use / Q000652:urine*; D005260:Female; D006801:Humans; D006973:Hypertension / Q000188:drug therapy / Q000208:ethnology / Q000652:urine*; D008297:Male; D008875:Middle Aged; D008968:Molecular Conformation; D010641:Phenotype; D015203:Reproducibility of Results; D044465:White People
D000959:Antihypertensive Agents; C012359:4-hydroxydebrisoquin; D003647:Debrisoquin