1. The document identifies the primary metabolite of clonidine as hydroxyclonidine and develops an LC-MS method to quantify both compounds using a deuterated internal standard. 2. It characterizes the kinetic parameters of clonidine hydroxylation in human liver microsomes and screens 17 cytochrome P450 isoforms, finding five that hydroxylate clonidine with varying potency. 3. Selective inhibition experiments show that CYP2D6, CYP1A2, and CYP3A4/5 contribute most to clonidine hydroxylation in human liver microsomes.

1. Identified primary metabolite hydroxyclonidine ( pos m/z 246) with hydroxyl group on dichlorophenyl ring Developed LS-MS method to quantify clonidine and hydroxyclonidine, using d 4 -hydroxyclonidine as internal standard ( pos m/z 250, monitored at 252) Developed microsomal / supersomal incubation assay to measure reaction rate CL -> OHCL Characterized kinetic parameters of clonidine hydroxylation in human liver microsomes: Vmax ~ 42.4 pmol/nmol P450/min Km ~ 2.0 µM clonidine

2. Screened 17 CYP supersomes for clonidine hydroxylation activity: 3A4, 3A5, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 1A1, 1A2, 1B1, 4F2, 4F3A, 4F3B Five isoforms hydroxylated the dichlorophenyl ring of clonidine: CYP2D6, 1A2, 3A4, 1A1, and 3A5 in decreasing potency Performed selective inhibition experiments in HLM to measure contribution of each isoform: 100 nM quinidine to inhibit CYP2D6 results in 66% decrease in product formation 20 µM furafylline to inhibit CYP1A2 results in 17% decrease 5 µM ketoconazole to inhibit CYP3A4/5 results in 22% decrease 20 µM troleandomycin to inactivate CYP3A4/5 results in 0% time-dependent inactivation

4. Kinetic parameters of clonidine hydroxylation in pooled HLM 26 Analyzed Number of points Km > 0.0 Km Constraints 2.527 Sy.x 153.3 Absolute Sum of Squares 0.9735 R² 24 Degrees of Freedom Goodness of Fit 1.577 to 2.508 Km 40.03 to 44.77 Vmax 95% Confidence Intervals 0.2255 Km 1.147 Vmax Std. Error 2.043 Km 42.40 Vmax Best-fit values

5. Screen of 17 common cytochrome P450 isoforms (activity expressed in pmol/nmol P450/min )

6. Screen of 17 common cytochrome P450 isoforms (activity expressed in pmol/nmol P450/min )

8. Kinetic parameters of clonidine hydroxylation in rCYP2D6 26 Analyzed Number of points Km > 0.0 Km Constraints 0.9244 Sy.x 20.51 Absolute Sum of Squares 0.9881 R² 24 Degrees of Freedom Goodness of Fit 8.835 to 11.85 Km 25.00 to 27.15 Vmax 95% Confidence Intervals 0.7314 Km 0.5191 Vmax Std. Error 10.34 Km 26.07 Vmax Best-fit values

14. 100 nM quinidine inhibits clonidine hydroxylation by approximately 66% (at 1µM clonidine), suggesting an equivalent contribution by CYP2D6 to clonidine hydroxylation in the Xenotech human liver microsome pool 5 µM ketoconazole inhibits clonidine hydroxylation by approximately 22% (at 1 µM clonidine), suggesting an equivalent contribution by CYP3A4/5 20 µM furafylline inhibits clonidine hydroxylation by approximately 17% (at 1µM clonidine), suggesting an equivalent contribution by CYP1A2 20 µM TAO exhibits more competitive inhibition of CYP3A4, than a time-dependent inactivation effect on clonidine hydroxylation At 1 µM clonidine, the sum of inhibition by quinidine / furafylline / ketoconazole is 105%