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High-Pressure Liquid Chromatography (HPLC) with Fluorometric Detection of Brain Catecholamines in a Pharmacodynamic Study Between Two Popular Antidepressants Bryant M. Moeller, Jillissa C. Molnari and Alan L. Myers Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy and Health Sciences BACKGROUND: Drug-drug interactions (DDIs) account for over 100,000 deaths annually. 1 Bupropion (Wellbutrin ) is a commonly prescribed antidepressant that is also used in the management of smoking cessation. 2 It works via inhibition of dopamine (DA) and norepinephrine (NE) re-uptake. 2 Sertraline (Zoloft ) is a widely used selective serotonin re-uptake inhibitor (SSRI) used in the treatment of depression and other mood and anxiety disorders. 3 Bupropion is metabolized by cytochrome P450 2B6 (CYP2B6), whereas sertraline, interestingly, is a potent in vitro inhibitor of CYP2B6. 4 Bupropion and sertraline are commonly co-prescribed in mentally ill patients, 5 yet the pharmacokinetic (PK) and pharmacodynamic (PD) consequences, such as a DDI, resulting from their co-use remains unknown. OBJECTIVE: The overall aim of this study was to utilize a HPLC assay to evaluate bupropion brain PD following repeated administration of sertraline in mice. EXPERIMENTAL METHODS: PD Study: Male CF-1 mice (30-35 g) were administered either sertraline i.p. 15 mg/kg daily or sterile water (5 µl/g) daily for 6 days. On the 7 th day mice were administered a single dose of bupropion 50 mg/kg. At 60 minutes post-dose, mice were sacrificed by CO 2 asphyxiation, then decapitated to remove brain tissues for catecholamine analysis. (Animal studies received prior approval from the Drake IACUC before initiation). Brain Extraction: Whole brain tissues were extracted using a modification of a literature method. 6 Tissues were homogenized in a solution of 0.1 M perchloric acid and 4 mM sodium bisulfate. Homogenate was centrifuged at 15,000 g for 15 min at 4 ° C. The supernatant was centrifuged at 7200 g for 10 min at 4 ° C. Fifty microliters of the new supernatant was analyzed by HPLC. HPLC Assay 6 : An automated Shimadzu HPLC system coupled to a fluorescence detector was used to measure mouse brain catecholamines. The mobile phase was a mixture of 89% 20 mM potassium dihydrogen phosphate buffer containing 1% heptanesulfonic acid sodium salt (pH 3.32) and 11% acetonitrile. A Hypersil BDS (Phenomenex) C18 analytical column (150 X 4.6 mm i.d.) with 5 µm particle size protected by C18 Security Guard (Phenomenex) cartridge was used to separate the compounds. The column oven temperature was set at 30 ° C, and the flow rate was maintained at 1.0 ml/min. The detector was set at excitation wavelength 290 nm and emission wavelength 330 nm. RESULTS: SUMMARY: We developed an HPLC with fluoremetic detection method to analyze mouse brain catecholamines. The limit of detection for norepinephrine, dopamine and serotonin was 780 pg/ml, 3000 pg/ml and 400 pg/ml, respectively. The HPLC assay was successfully employed in a pharmacodynamic (PD) study. Surprisingly, mice dosed with bupropion or bupropion + sertraline (compared to control) displayed significantly lower levels of DA, NE and 5HT levels. Current results are not predicative of a neurochemical PD DDI between bupropion and sertraline. Future studies, such as microdialysis procedures, which explore specific regions of the brain are required to accurately determine if a signficant PD interaction exists between these two antidepressants. CONCLUSIONS: We successfully developed and employed an HPLC assay to measure mouse brain catecholamines in a PD study. However, our current findings do not indicate a significant PD interaction between bupropion and sertraline, and further studies are needed to fully investigate this potential drug-drug interaction. REFERENCES: 1.J. Lazarou, B.H. Pomeranz, and P.N. Corey. Incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. JAMA. 279:1200-1205 (1998). 2.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). 3.C.L. DeVane, H.L. Liston, and J.S. Markowitz. Clinical pharmacokinetics of sertraline. Clin Pharmacokinet. 41:1247-1266 (2002). 4.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). 5.S. Zisook, A. J. Rush, B. R. Haight, D. C. Clines, and C. B. Rockett. Use of bupropion in combination with serotonin reuptake inhibitors. Biol Psychiatry 59: 203-10 (2006). 6.M. A. Peat and J. W. Gibb. High-performance liquid chromatographic determination of indoleamines, dopamine, and norepinephrine in rat brain with fluorometric detection. Anal Biochem 128: 275-80 (1983). ACKNOWLEDGEMENTS: The authors would like to thank the Drake University College of Pharmacy and Health Sciences and the Drake University Office of the Provost for research funding. Norepinephrine Dopamine Isoproterenol Serotonin Figure 1: Blank brain homogenate spiked with 10.0 ng/ml each of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and internal standard (isoproterenol; ISP). Approximate retention times for NE, DA 5-HT and ISP are 6, 13, 33 and 22 minutes, respectively Figure 3: Brain catecholamine concentrations in mice treated with either sterile water (control) or bupropion alone or bupropion + sertraline. Whole brain tissue was isolated 60 minutes post-dose, extracted and analyzed for catecholamine levels using the HPLC fluorometric assay. *p<0.05 vs. control group ControlBupropionBupropion + Sertraline Norepinephrine 25 ± 2.815 ± 2.5*13 ± 2.2* Dopamine 57 ± 1143 ± 5*32 ± 6.3* Serotonin 25 ± 3.517 ± 0.6*16 ± 2.8* Table 1: Tabulation of brain catecholamine concentrations depicted in Figure 3 above. Statistically significant differences between treatment groups were determined using ANOVA followed by Bonferroni’s t-test. *p< 0.05 vs. control group Figure 2: HPLC-fluorometric detection of brain catecholamines from a CF-1 mouse treated with a single dose of bupropion 50 mg/kg. The mean concentrations of NE, DA and 5-HT are 9.0 ng/ml, 20 ng/ml and 8.8 ng/ml, respectively. Norepinephrine Dopamine Isoproterenol Serotonin RESULTS: * * * * * *
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