DUCURS poster 31 - eScholarShare
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Transcript DUCURS poster 31 - eScholarShare
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 is extensively metabolized to hydroxybupropion, its major
active metabolite, by cytochrome P450 2B6 (CYP2B6)1:
CH3
CH3
CH3
O
CYP2B6
Cl
H
N
CH3
OH
*
*
400
*
*
200
0
20
40
60
80
100
120
140
160
180
(b)
500
The limit of detection for our HPLC assay was 6.0 ng/ml for bu
and 10.0 ng/ml for hydroxybupropion.
450
400
350
A PK study in male CF-1 mice was conducted to study the DD
bupropion and a known CYP2B6 inhibitor ticlopidine.
300
*
250
*
200
150
20
200
SUMMARY:
550
40
60
80
100
120
140
160
180
200
Time (min)
Time (min)
Bupropion + Ticlopidine
Bupropion alone
Bupropion + Ticlopidine
Bupropion alone
CH3
Hydroxybupropion
Potent inhibitors of CYP2B6 have the ability to decrease the
metabolism of bupropion, leading to adverse drug toxicity.2
Analysis: Plasma and brain homogenate samples were spiked with
internal standard, extracted and analyzed by the previously described
HPLC assay.
Mice treated with ticlopidine and bupropion displayed a signific
greater AUC of brain hydroxybupropion and a trend for greater A
bupropion.
RESULTS:
12
Timolol (IS)
10
10
8
8
6
6
Hydroxybupropion
(a)
5000
*
4000
3000
*
2000
1000
0
mAu
mAu
4500
6000
12
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.
Figure 3: (a) Plasma bupropion (ng/ml ± SEM); and (b) hydroxybupropion 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).
7000
(b)
4000
CONCLUSIONS:
3500
20
4
40
60
80
Bupropion
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.
2
0
0
-2
4
6
8
10
12
14
16
18
20
22
140
160
180
200
2500
2000
*
24
26
Minutes
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.
20.0
20
*
40
60
80
100
*
120
140
160
180
Bupropion + Ticlopidine
Bupropion alone
Figure 4: (a) Brain bupropion (ng/g ± SEM); and (b) hydroxybupropion 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).
AUC0-∞
Treatment Group
(ng/l/h) ± s.d.
λz (hr-1) ±
s.d.
20.0
Plasma Bupropion PK
17.5
Bupropion alone
58700 ± 20000
0.78 ± 0.18
17.5
Bupropion
15.0
Bupropion + ticlopidine 91000 ± 18000**
15.0
0.45 ± 0.042*
Timolol (IS)
Analysis3:
HPLC
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.
Plasma Hydroxybupropion PK Bupropion alone
12.5
Hydroxybupropion
7.5
7.5
5.0
5.0
2.5
2.5
0.0
0.0
Bupropion + ticlopidine 56000 ± 4600*
Brain Bupropion PK
2
4
6
8
10
12
14
16
18
20
22
24
Bupropion alone
192000 ± 61000
Bupropion + ticlopidine 254000 ± 40000
Brain Hydroxybupropion PK
0
67500 ± 7100
0.078 (n=2)
1. 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).
2. 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).
3. K.K. Loboz, A.S. Gross, J. Ray, and A.J. McLachlan. HPLC a
bupropion and its major metabolites in human plasma. J Chr
B Analyt Technol Biomed Life Sci. 823:115-121 (2005).
4. M. Turpeinen, R. Nieminen, T. Juntunen, P. Taavitsainen, H.
and O. Pelkonen. Selective inhibition of CYP2B6-catalyzed b
hydroxylation in human liver microsomes in vitro. Drug Metab
32:626-631 (2004).
10.0
mAu
10.0
mAu
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.
12.5
200
REFERENCES:
Time (min)
Bupropion + Ticlopidine
Bupropion alone
-2
2
120
Time (min)
2
0
100
We were able to demonstrate a significant PK DDI interaction
bupropion and ticlopidine, indicating that our methodology can b
to future PK DDI studies between bupropion and other potent C
inhibitors, such as sertraline.2
3000
1500
4
The HPLC assay was applied in the PK study to measure plas
brain concentrations of bupropion and hydroxybupropion.
Mice treated with ticlopidine and bupropion displayed a signific
greater area under the curve (AUC) of plasma and bupropion an
hydroxybupropion.
CH3
Cl
Bupropion
600
Brain Hydroxybupropion (ng/g)
H
N
CH3
800
Brain Bupropion (ng/g)
O
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.
(a)
*
1000
Plasma hydroxybupropion (ng/ml)
Bupropion’s likely mode of action is inhibition of norepinephrine and
dopamine reuptake.1
Plasma Bupropion (ng/ml)
Bupropion (Wellbutrin) is an atypical antidepressant used to treat
depression and is also a non-nicotine aid to smoking cessation.1
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 6th day, mice were administered bupropion
50 mg/kg i.p. and sacrificed by CO2 asphyxiation at 30, 60, 90, 120, and
180 minutes post dose.
1200
Bupropion alone
560000 ± 62000
0.276 ± 0.042
1.032 ± 0.318
1.092 ± .0276
0.24 ± 0.066
26
Minutes
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.
ACKNOWLEDGEMENTS:
Bupropion + ticlopidine 322000 ± 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)
Research funding was graciously provided by several Drake Un
Research grants and the College of Pharmacy and Health Scien