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Elucidating Time-Dependent Inhibition of
Cytochrome P450 Enzymes and Reactive
Metabolite-Induced Hepatotoxicity of Duloxetine
Chun Yip Chan, Lee Sun New, Han Kiat Ho, Eric Chun Yong Chan
Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science Drive 4, Singapore 117543
Results and Discussion
Introduction
Time-dependent inhibition studies
• The TDI assay was validated using furafylline, known to cause irreversible TDI of CYP1A2.
• Concentration and time-dependency was demonstrated.
ln (% CYP1A2 activity remaining)
(A)
Drug-induced hepatotoxicity
Apart from drug interactions,
irreversible TDI can also cause
hepatotoxicity
•
10
20
(A)
30
0.00
0
40
40
60
(B)
5.0
5.0
5.0
S
NH2
Figure 1. Structure of duloxetine showing known toxicophores and their potential bioactivation products.
4.5
500 M
M
5 M
500
M
100M
4.0
250 M
20 M
10 M
4.5
100M
M
50
75 M
150 M
200 M
4.0
750 M
1000 M
3.5
3.0
0 M
0.03
0.005
0.004
0.003
3.5
0 M
0.001
100
200 M
3.5
3.0
0
500
1000
1500
0
50
100
150
200
0.00
0
0.04
0.03
0.02
0.01
250
10
20
30
0
50
150
200
250
0.00
2.5
Concentration ( M)
40
Concentration ( M)
2.5
0
10
20
30
40
0
10
20
30
40
Preincubation time (min)
Preincubation time (min)
Inhibited HLM +
probe substrate (~5x Km)
0 M
4.5
20 M
75 M
100 M
150 M
200 M
10 M
4.0
M
50 M
3.5
2.5
0.0020
3.0
4.5
10 M
50 M
75 M
100 M
150 M
20 M
0
50
100
150
200
0.010
10 M
50 M
100 M
0
200 M
20 M
75 M
150 M
4.5
200 M
0.010
0.000
4.0
0.0025
0 M
0.015
kobs (min-1)
Time-dependent inhibition studies
(F)
5.0
5.0
kobs (min-1)
ln (% CYP2D6 activity remaining)
Methodology
(E)
5.0
ln (% CYP3A4/5 activity
remaining)
(D)
Preincubation time (min)
kobs (min -1)
• To investigate the nature of the TDI of duloxetine (i.e. whether it is reversible or irreversible).
• To profile the reactive metabolites of duloxetine, in particular those of the thiophene ring.
• To examine the role of irreversible TDI and reactive metabolites in duloxetine-induced
hepatotoxicity and drug-drug interactions.
250
0.008 0
Concentration (M)
0.0015
Transfer aliquot,
dilute 10x
Primary incubation
Secondary incubation
100
Concentration ( M)
0.000
3.0
Objectives
50 M
10 M
20 MM
150
75
MM
4.0
0.002
0.02
0.01
4.5
kobs (min-1)
ln (% CYP1A2 activity remaining)
O
Duloxetine + human liver
microsomes (HLM) + NADPH
20
Concentration (M)
(C)
Naphthyl ring, bioactivated
to generate quinones or
epoxides
Thiophene ring, bioactivated to
generate epoxides, ring opening
or S-oxidation products
0
Figure 5. (A) Determination of
residual activity of CYP1A2
(incubated with 0-50 μM
furafylline). The slope of each
line is known as k obs
. (B) k obs
graph showing a hyperbolic
increase of k obswith increasing
concentration of furafylline,
which is characteristic of TDI.
With duloxetine, no irreversible TDI was observed with CYP1A2, CYP2B6, CYP2C19, CYP2D6 and
CYP3A4/5, and time-dependency was not observed.
No reports on the reactivity of the
thiophene ring in literature
Drug interactions observed in vivo
with CYP2D6 but not CYP1A2
0.02
1
0
0.04
Preincubation time (min)
Naphthyl ring is metabolized to a
reactive epoxide which is trapped by
glutathione [3]
Irreversible TDI causes clinically
significant drug interactions
2
ln (% CYP3A4/5 activity remaning)
Not known if the TDI is reversible or
irreversible
3
ln (% CYP2C19 activity remaining)
Naphthyl ring and thiophene ring
could generate reactive metabolites
which can cause hepatotoxicity
0.06
0 M
10 M
25 M
50 M
kobs (min-1)
Duloxetine causes time-dependent
inhibition (TDI) of CYP1A2, CYP2B6,
CYP2C19 and CYP3A4/5 [2]
0.08
4
ln (% CYP2B6 activity remaining)
Drug-drug interactions
(B)
5
kobs (min-1)
Significance
-1)
•
Duloxetine is a selective serotonin-norepinephrine reuptake inhibitor (SNRI) approved to treat
major depressive disorder and diabetic peripheral neuropathic pain in Singapore in 2006.
Post-marketing reports have revealed that duloxetine causes hepatotoxicity [1].
kobs (min
•
50
250
0.006
100
150
200
0.005
Transfer aliquot
0.004
0.0010
2.0
0.002
0.0005
0
0.0000
LC/MS/MS analysis
50
100
150
200
250
0.000
Concentration ( M)
Concentration (
M)
Figure 2. Schematic showing methodology of TDI studies. Negative controls were prepared by excluding NADPH.
1.5
4.0
3.5
0
10
20
30
40
0
10
20
30
40
0
10
20
30
40
•
•
Distinguishing between reversible and irreversible TDI:
• A 10 times dilution step between the primary and secondary incubation ensures that any
reversible inhibitors formed in the primary incubation would be sufficiently diluted in the
secondary incubation and their inhibitory effect suppressed.
• Probe substrate concentrations of around 5 times the Km value ensures that substrates will
outcompete any reversible metabolites in the secondary incubation.
• Thus any inhibition observed in the secondary incubation will be irreversible.
CYP1A2, CYP2B6, CYP2C19, CYP2D6 and CYP3A4/5 in HLM were investigated using probe
substrates specific to these enzymes.
•
Preincubation time (min)
Preincubation time (min)
Preincubation time (min)
The TDI previously reported is actually reversible, thus irreversible TDI is therefore not responsible for
Figure 6. Determination of residual activity of (A) CYP1A2, (B) CYP2B6, (C) CYP2C19, (D) CYP2D6), (E) CYP3A4/5 using
CYP2D6
in vivo
drugsubstrate
interactions
and is also
the cause
of the
hepatotoxicity
of duloxetine.
testosterone
as probe
and (F) CYP3A4/5
usingnot
midazolam
as probe
substrate.
Each respective
inset shows the kobs
graph lacking a definite hyperbolic trend and hence no irreversible TDI.
Reactive metabolite trapping studies
• Duloxetine epoxide adducts were found with GSH and GSH-EE. No duloxetine adducts with the
thiophene ring were found with either GSH, GSH-EE or SCBZ, suggesting that the thiophene ring in
duloxetine is metabolically inert.
(A)
(B)
Reactive metabolite trapping studies
Duloxetine
+ HLM + NADPH +
Trapping agent
Sample clean-up
Transfer aliquot
Centrifuge, obtain
supernatant
Solid phase extraction
Incubation
LC/MS/MS and QTOF analysis
Figure 3. Schematic showing methodology of reactive metabolite trapping studies. Reactive metabolites generated
during incubation are adducted by the trapping agent. Negative controls were prepared by excluding NADPH.
• Duloxetine forms both soft and hard electrophiles hence soft nucleophiles (glutathione (GSH)
and glutathione ethyl ester (GSH-EE)) and a hard nucleophile (semicarbazide (SCBZ)) were
used as trapping agents to trap potential reactive metabolites.
• Analytical approach for reactive metabolite profiling:
(C)
De novo screening
Neutral loss scan at m/z
129 (ESI +ve), scan range
m/z 500-900
Precursor ion scan at m/z 272 (for
GSH) and m/z 300 (for GSH-EE)
(ESI -ve), scan range m/z 500-900
Compare to negative control to
eliminate false positives
Proceed if peaks
present in both
scans
Rational structure screening
Cl
Cl
(D)
N
SCBZ
N
N
N
S
Predict m/z of potential adducts from structure
of known duloxetine metabolites, trapping agent
and potential adducting sites
Ticlopidine
Ticlopidine-SCBZ adduct
Figure 7. Illustrated are the structures, fragmentation patterns
and corresponding accurate mass spectra of each adduct. (A)
Duloxetine-GSH adduct at m/z 621, (B) Duloxetine-GSH-EE
adduct at m/z 649. This adduct was previously reported by
other authors, and no new GSH/GSH-EE adducts were found.
(C) Ticlopidine-SCBZ adduct at m/z 260. (D) Conversion of
thiophene ring in ticlopidine to a pyridazine ring in the adduct.
Ticlopidine which contains a thiophene ring was used as a
positive control to evaluate the ability of SCBZ to trap the
thiophene ring. The formation of the adduct demonstrates the
ability of SCBZ to trap thiophene ring containing compounds.
No adduct to the thiophene ring in duloxetine was observed.
Full scan (ESI +ve)
Extract ion chromatograms at predicted
m/z of potential adducts
Compare to negative control
to eliminate false positives
Product ion scan (MS/MS) at predicted m/z of potential adducts (ESI +ve)
Confirmation of structure through accurate mass measurement
Figure 4. For GSH and GSH-EE, both arms were used as the analytical approach for reactive metabolite profiling. For
SCBZ, the rational structure screening approach was used.
References:
1. Hanje AJ, Pell LJ, Votolato NA, Frankel WL, Kirkpatrick RB. Clin Gastroenterol Hepatol 2006;4(7):912-7.
2. Paris BL, Ogilvie BW, Scheinkoenig JA, Ndikum-Moffor F, Gibson R, Parkinson A. Drug Metab Dispos 2009;37(10):2045-54.
3. Wu G, Vashishtha SC, Erve JC. Chem Res Toxicol 2010;23(8):1393-404.
Conclusion
•
•
•
Duloxetine exhibits reversible TDI with CYP1A2, CYP2B6, CYP2C19 and CYP3A4/5, and no TDI is
observed with CYP2D6.
The thiophene moiety in duloxetine is inert to bioactivation.
Hepatotoxicity of duloxetine is therefore not due to irreversible TDI or a reactive thiophene moiety.
This work was supported by NUS grant (R-148-000-100-112 to E. C. Y. Chan) and Department of Pharmacy Final Year Project grant (R-148-000-003-001)