1. HPLC-UV Analysis of Bupropion and Hydroxybupropion: Application to In Vivo Pharmacokinetic Drug-Drug Interaction Studies Between Bupropion and Potent Cytochrome P450 2B6 (CYP2B6) Inhibitors Jillissa C. Molnari, Bryant M. Moeller, and Alan L. Myers Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy and Health Sciences BACKGROUND:  Bupropion (Wellbutrin  ) is an atypical antidepressant used to treat depression and is also a non-nicotine aid to smoking cessation. 1  Bupropion’s likely mode of action is inhibition of norepinephrine and dopamine reuptake. 1  Bupropion is extensively metabolized to hydroxybupropion, its major active metabolite, by cytochrome P450 2B6 (CYP2B6) 1 :  Potent inhibitors of CYP2B6 have the ability to decrease the metabolism of bupropion, leading to adverse drug toxicity. 2 OBJECTIVE: The purpose of this study was to develop a high pressure liquid chromatography (HPLC) assay to quantify plasma and brain concentrations of bupropion and hydroxybupropion, and to evaluate in vivo pharmacokinetic (PK) drug-drug interactions (DDIs) between bupropion and potent CYP2B6 inhibitors. EXPERIMENTAL METHODS: Plasma Extraction: Plasma samples were extracted using a modification of a literature method. 3 Blank mice plasma samples (0.2 ml) were spiked with known concentrations of bupropion (6.5-850 ng/ml), hydroxybupropion (6.5-850 ng/ml), erythrohydrobupropion (6.5-850 ng/ml), threohydrobupropion (6.5-850 ng/ml), and internal standard (1000 ng/ml). Plasma samples were extracted with 0.5 M carbonate buffer (pH 10.0) and 1.5% 3-methyl butanol in n-heptane. The organic layer was separated and back-extracted into 0.1 M HCl and the bottom aqueous layer was evaporated under nitrogen at 45°C. The residue was reconstituted in mobile phase and an aliquot was injected onto the HPLC system. Brain Extraction: Brain tissue was homogenized in 0.01 M HCl and centrifuged at 4200 x g for 20 minutes. The supernatant was removed and extracted similar to plasma. HPLC Analysis 3 : Fifty microliters of reconstituted extract was injected onto an automated Shimadzu HPLC system. The mobile phase consisted of 25 mM potassium dihydrogen phosphate buffer, acetonitrile and triethylamine (75:25:0.1) adjusted to a final pH 6.4. The reversed phase analytical column was a Phenomenex Synergi Hydro C18 (Torrance, CA) protected by a Aqua C18 Security Guard (Phenomenex) cartridge. The column oven temperature was maintained at 30°C, and the flow was delivered at 1.0 ml/min. All analytes were detected by ultraviolet detection at 214nm. PK Study: Male CF-1 mice were administered ticlopidine 1 mg/kg, a known CYP2B6 inhibitor, 4 or sterile water 1 μl/g i.p. once daily for 5 days. On the morning of the 6 th day, mice were administered bupropion 50 mg/kg i.p. and sacrificed by CO 2 asphyxiation at 30, 60, 90, 120, and 180 minutes post dose. Sample Collection: Blood was collected via heart puncture into heparinized syringes and centrifuged at 1,800 x g for 15 minutes. Plasma was removed and stored at -80°C until analysis. Brain samples were surgically removed following decapitation and stored at -80°C until analysis. Analysis: Plasma and brain homogenate samples were spiked with internal standard, extracted and analyzed by the previously described HPLC assay. RESULTS: SUMMARY:  The limit of detection for our HPLC assay was 6.0 ng/ml for bupropion and 10.0 ng/ml for hydroxybupropion.  A PK study in male CF-1 mice was conducted to study the DDI between bupropion and a known CYP2B6 inhibitor ticlopidine.  The HPLC assay was applied in the PK study to measure plasma and brain concentrations of bupropion and hydroxybupropion.  Mice treated with ticlopidine and bupropion displayed a significantly greater area under the curve (AUC) of plasma and bupropion and hydroxybupropion.  Mice treated with ticlopidine and bupropion displayed a significantly greater AUC of brain hydroxybupropion and a trend for greater AUC of bupropion. CONCLUSIONS:  We were able to demonstrate a significant PK DDI interaction between bupropion and ticlopidine, indicating that our methodology can be applied to future PK DDI studies between bupropion and other potent CYP2B6 inhibitors, such as sertraline. 2 REFERENCES: 1.L.M. Hesse, K. Venkatakrishnan, M.H. Court, L.L. von Moltke, S.X. Duan, R.I. Shader, and D.J. Greenblatt. CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab Dispos. 28:1176-1183 (2000). 2.R.L. Walsky, A.V. Astuccio, and R.S. Obach. Evaluation of 227 drugs for in vitro inhibition of cytochrome P450 2B6. J Clin Pharmacol. 46:1426-1438 (2006). 3.K.K. Loboz, A.S. Gross, J. Ray, and A.J. McLachlan. HPLC assay for bupropion and its major metabolites in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 823:115-121 (2005). 4.M. Turpeinen, R. Nieminen, T. Juntunen, P. Taavitsainen, H. Raunio, and O. Pelkonen. Selective inhibition of CYP2B6-catalyzed bupropion hydroxylation in human liver microsomes in vitro. Drug Metab Dispos. 32:626-631 (2004). ACKNOWLEDGEMENTS: Research funding was graciously provided by several Drake University Research grants and the College of Pharmacy and Health Sciences. BupropionHydroxybupropion CYP2B6 Figure 1: HPLC-UV chromatogram of blank mouse plasma extract spiked with 25.0 ng/ml each of hydroxybupropion and bupropion, and 100 ng/ml IS. Bupropion Hydroxybupropion Timolol (IS) Figure 2: HPLC-UV chromatogram of a plasma samples collected 60 minutes post-dose from a mouse treated with ticlopidine1 mg/kg and bupropion 50 mg/kg. Figure 3: (a) Plasma bupropion (ng/ml ± SEM); and (b) hydroxy- bupropion levels (ng/ml ± ml) in mice treated with bupropion + ticlopidine (  ) or bupropion only (o). * p< 0.05 (ANOVA with Tukey’s post-hoc t-test). Figure 4: (a) Brain bupropion (ng/g ± SEM); and (b) hydroxy- bupropion levels (ng/g ± SEM) in mice treated with bupropion + ticlopidine (  ) or bupropion only (o). * p< 0.05 (ANOVA with Tukey’s post-hoc t-test). Hydroxybupropion Bupropion Timolol (IS) (a) * * (b) ** * * * (a) * * * * * Treatment Group AUC 0-∞ (ng/l/h) ± s.d. λ z (hr -1 ) ± s.d. Plasma Bupropion PKBupropion alone58700 ± 200000.78 ± 0.18 Bupropion + ticlopidine91000 ± 18000**0.45 ± 0.042* Plasma Hydroxybupropion PKBupropion alone67500 ± 71000.078 (n=2) Bupropion + ticlopidine56000 ± 4600*0.276 ± 0.042 Brain Bupropion PKBupropion alone192000 ± 610001.032 ± 0.318 Bupropion + ticlopidine254000 ± 400001.092 ±.0276 Brain Hydroxybupropion PKBupropion alone560000 ± 620000.24 ± 0.066 Bupropion + ticlopidine322000 ± 20000***0.72 ± 0.186** Table 1: Noncompartmental PK analysis of plasma and brain bupropion and hydroxybupropion concentration time curves using WinNonlin PK software. *p<0.05; **p<0.01; ***p<0.001 (Student’s t-test)