Metabolism of selegiline in human Identification, Excretion, and Stereochemistry of Urine Metabolism เมตาบอลิซึ่มของซิลิจีลีนในมนุษย์ นำเสนอโดย น. ส. อรทัย กฤษณานุวัฒน์ รหัสนักศึกษา

Introduction The chemical name is (R)-(-)-N,2- dimethyl-N-2-propynylphenethylamine hydrochloride It is a white to near white crystalline powder, soluble in water, chloroform and methanol Molecular weight of Boling point 80 °c

The structural formula of selegiline

Monoamine oxidase, L-dopamin Later, Heinonen et.al Methamphetamine, amphetamine, desmethyl – selegiline urine human p-hydroxyamphetamine, p-hydroxymetamphetamine rat

Material and Method d-, l-,R-,S-, or(+)- and (-)- systems of naming optical isomers l-Methamphetamine H CH 3 NH CH 3 l-Amphetamine H2NH2N CH 3 H d-Amphetamine NH 2 CH 3 H d-Methamphetamine H NHCH 3 CH 3 enantiomers of amphetamine and methamphetamine

Materials and Methods Chemical (R)-amphetamine sulfate (S)-amphetamine sulfate sigma (S)- methamphetamine (1R,2R)-norpseudoepedrine · HCl

(1R,2S)-norephedrine · HCl (1S,2R)-norephedrine · HCl Fluka (1S,2R)-ephedrine · HCl (1S,2R)-pseudoephedrine · HCl p- hydroxyamphetamine p- hydroxymethamphetamine Instd. p- hydroxynorephedrine p- chlorphetermine

Drug administration and sample collection - 10, 5, and 2.5 mg of selegiline.HCl - Urine sample collection at various times over 72 hr and stored at 4 °c

Isolation of Unconjugate Metabolism Urine 3 ml+ 100mg sodium bicarbonate: potassium carbonate (2:1) +150 ng instd. (p- chlorphentamine) 8 ml of diethylether-tert butanol (7:1) for metabolite extracted Organic layer was transfer into a 15ml glass centrifuge tube + 0.4ml of 0.06M HCl

Mixing, Centrifuge 5 min at 1200 g Organic layer was aspirate, dried

Isolation of Conjugate Metabolite Urine 3 ml adjusted ph 5.2 with 0.2M Sodium acetate buffer Incubate with 50 µ l of arylsulfatase / β - glucolonidase 52 °c Solution was neutralized with 5 M KOH and adjusted pH 9.6 with 200mg of sodium bicarbonate : potassium carbonate (2:1)

Quantification of Selegiline and Its Metabolite Dry residue was dissolved in 50 µ l of ACN:TFA (60:40) Titrated with MSTFA until color reaction changes red to yellow Sample was heated for 10 min at 60°c

Two drops of MSTFA added to the reaction mixture 2 µ l of the solution was injected into the GC/MS

GC/MS, GC-NPD HP 5890/5971A instrument, HP 9144 disk drive,HP Think Jet Printer HP fused-silica capillary column, 5% phenylmethylsilicon(SE-54) 17 min length, with 0.2 mm diameter, 0.33 µ l film thickness Temperature and column condition identical for GC/MS

Results and Discussion Identification of Metabolites by GC-NPD and GC/MS Comparison of the EI mass spectra and gas chromatographic retention times GC-NPD gave six peaks (A1-A6) N-trifluoroacetylation, O- trimethylsilylation showed 10 peaks(B1- B10)

Identification of the Stereoisomer of the Selegiline Metabolites Applicable to the quantification of trace optical isomer containing more than one reactive functional gr. The stereochemical identities of the metabolites confirmed by comparison of the GC RT of derivative of the extractd metabolites with of the authentic std.

Urinary excretion of metabolites Summarized in table 1 Trifluoroacetylated form, N- trifluoroacetylation-O-trimethlysilation derivative Detection limit of selegiline was ~ 0.3ng/ml other metabolites were were ~ 0.1ng/ml - Correlation(r) =0.999 between injection amounts and detection response

The major metabolite was (R)- methamphetamine for ~ 37% of dose β-hydroxylation and aromatic hydroxylation were minor metabolite Dealkylation, β-hydroxylation of selegiline lead to unconjugate Most of the p-hydroxylated of selegiline excreted conjugate ( %)

The sum of the amount of urinary metabolite in 2 days 44-58% dose-independent amount of selegiline % dose of dealkylated metabolites (Asia) % (Europeans) % dose of aromatic hydroxylate metabolites (Asia) % (Europeans)

Kinetic Studies Quantification of urine concentration of selegiline and the metabolite The excretion rate for selegiline and metabolite was calculated by division of the total amount via the urine collection interval Readily absorbed, rapidly distributed

ขอบคุณ อาจารย์ ธงชัย เตโชวิศาล ที่ปรึกษาสัมมนา Quesssion

Abundance 1.0E 2.0E 3.0E 4.0E A1 A2 A3 A4 A5 A6