Transcript DUCURS poster 26 - eScholarShare

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:
RESULTS:
SUMMARY:
Drug-drug interactions (DDIs) account for over 100,000 deaths
annually.1
We developed an HPLC with fluoremetic detection method to
mouse brain catecholamines.
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
The limit of detection for norepinephrine, dopamine and seroto
780 pg/ml, 3000 pg/ml and 400 pg/ml, respectively.
400
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
Norepinephrine
350
300
Serotonin
Dopamine
250
250
200
200
150
150
100
100
50
50
0
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Surprisingly, mice dosed with bupropion or bupropion + sertral
(compared to control) displayed significantly lower levels of DA,
5HT levels.
**
**
Current results are not predicative of a neurochemical PD DDI
bupropion and sertraline.
Future studies, such as microdialysis procedures, which explo
specific regions of the brain are required to accurately determine
signficant PD interaction exists between these two antidepressa
36
Minutes
OBJECTIVE:
CONCLUSIONS:
The overall aim of this study was to utilize a HPLC assay to evaluate
bupropion brain PD following repeated administration of sertraline in
mice.
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
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 7th day mice were administered a single dose of bupropion 50
mg/kg. At 60 minutes post-dose, mice were sacrificed by CO2
asphyxiation, then decapitated to remove brain tissues for
catecholamine analysis. (Animal studies received prior approval from
the Drake IACUC before initiation).
HPLC Assay6: 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.
The HPLC assay was successfully employed in a pharmacody
(PD) study.
*
300
Isoproterenol
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.
400
Isoproterenol
Serotonin
Norepinephrine
350
Dopamine
300
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
350
Control
Bupropion
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*
300
250
250
200
200
150
150
100
100
50
50
0
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
We successfully developed and employed an HPLC assay to
mouse brain catecholamines in a PD study. However, our curre
findings do not indicate a significant PD interaction between bup
and sertraline, and further studies are needed to fully investigate
potential drug-drug interaction.
REFERENCES:
400
mVolts
mVolts
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.
*
350
mVolts
mVolts
Bupropion is metabolized by cytochrome P450 2B6 (CYP2B6),
whereas sertraline, interestingly, is a potent in vitro inhibitor of
CYP2B6.4
400
36
Minutes
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.
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 adv
drug reactions in hospitalized patients: a meta-analysis of pr
studies. JAMA. 279:1200-1205 (1998).
2. L.M. Hesse, K. Venkatakrishnan, M.H. Court, L.L. von Moltke
Duan, R.I. Shader, and D.J. Greenblatt. CYP2B6 mediates th
hydroxylation of bupropion: potential drug interactions with o
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
(2002).
4. R.L. Walsky, A.V. Astuccio, and R.S. Obach. Evaluation of 22
for in vitro inhibition of cytochrome P450 2B6. J Clin Pharma
46:1426-1438 (2006).
5. S. Zisook, A. J. Rush, B. R. Haight, D. C. Clines, and C. B. R
Use of bupropion in combination with serotonin reuptake inh
Biol Psychiatry 59: 203-10 (2006).
6. M. A. Peat and J. W. Gibb. High-performance liquid chromato
determination of indoleamines, dopamine, and norepinephrin
brain with fluorometric detection. Anal Biochem 128: 275-80
ACKNOWLEDGEMENTS:
The authors would like to thank the Drake University College of
Pharmacy and Health Sciences and the Drake University Office
Provost for research funding.