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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: RESULTS: SUMMARY: ØDrug-drug interactions (DDIs) account for over 100, 000 deaths annually. 1 ØWe developed an HPLC with fluoremetic detection method to analyze mouse brain catecholamines. ØBupropion (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 ØSertraline (Zoloft ) is a widely used selective serotonin re-uptake inhibitor (SSRI) used in the treatment of depression and other mood anxiety disorders. 3 ØThe limit of detection for norepinephrine, dopamine and serotonin was 780 pg/ml, 3000 pg/ml and 400 pg/ml, respectively. Norepinephrine * Serotonin * Dopamine ØBupropion is metabolized by cytochrome P 450 2 B 6 (CYP 2 B 6), whereas sertraline, interestingly, is a potent in vitro inhibitor of CYP 2 B 6. 4 ØThe HPLC assay was successfully employed in a pharmacodynamic (PD) study. ** ** Isoproterenol ØSurprisingly, mice dosed with bupropion or bupropion + sertraline (compared to control) displayed significantly lower levels of DA, NE and 5 HT levels. ØCurrent results are not predicative of a neurochemical PD DDI between bupropion and sertraline. ØBupropion 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. ØFuture 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. 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). CONCLUSIONS: 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 Norepinephrine Control Bupropion + Sertraline Norepinephrine 25 ± 2. 8 15 ± 2. 5* 13 ± 2. 2* Dopamine 57 ± 11 43 ± 5* 32 ± 6. 3* Serotonin 25 ± 3. 5 17 ± 0. 6* 16 ± 2. 8* Dopamine 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. ØWe 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: Isoproterenol Serotonin 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 m. M 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 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 m. M potassium dihydrogen phosphate buffer containing 1% heptanesulfonic acid sodium salt (p. H 3. 32) and 11% acetonitrile. A Hypersil BDS (Phenomenex) C 18 analytical column (150 X 4. 6 mm i. d. ) with 5 µm particle size protected by C 18 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. 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 analyzed for catecholamine levels using the HPLC fluorometric assay. *p<0. 05 vs. control group 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 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. CYP 2 B 6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metab Dispos. 28: 1176 -1183 (2000). 3. C. L. De. Vane, 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 P 450 2 B 6. 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.