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In vitro and in vivo metabolism of
repaglinide: Modeling clinicallyrelevant drug-drug interactions
Joanna Barbara, Ph.D.
Director of Analytical Services, XenoTech LLC.
Pacific Northwest Biosciences Winter Seminar
March 3, 2014
XenoTech’s integrated service capabilities
XT Consulting Department
Expert data review and study consultation
Drug Metabolism
Enzyme Induction
Metabolic stability and species comparison
In vitro studies in cultured hepatocytes
Metabolite characterization/ID
(human and animal)
Reaction phenotyping (CYP & UGT)
Ex vivo studies in animals
Bioanalytical
Customized services
Toxicity & mechanistic studies
Non-GLP Bioanalysis
Enzyme Inhibition
GLP and non-GLP in vitro
study support
Transporters
XenoTech and Sekisui
Evaluate potential for direct, and metabolismdependent inhibition(MDI or TDI)
In vitro studies in mono-layer cell lines for uptake
Mechanistic studies (direct or MDI)
Bi-directional assay for efflux transporters
Non-CYP enzymes (e.g., UGT, MAO, AO)
Membrane-based vesicles and ATPase assays
Sekisui Medical (Conducted in Japan)
RI synthesis (radiolabeling), preclinical in vivo PK studies, QWBA, plasma protein binding,
humanized chimeric mice (PXB), biomarker analysis, pharmacological receptor assays
Products
2
Overview
• Enzymatic biotransformation and drug-drug
interactions
• Introduction to repaglinide and project background
– Repaglinide as a probe substrate
• Investigating mechanism of drug-drug interactions
– In vitro metabolism
• Evaluating rat as a preclinical model
– In vivo metabolism
• Conclusions
3
Enzymatic biotransformation of drugs
• Cytochrome P450 (CYP) enzymes are responsible for
biotransformation of ~70% hepatically-cleared drugs
Esterase
CY P
UG T
UGT
e ste ra se
FM O
CYP
NA T
M AO
Hepatic clearance route by enzyme type
Data adapted from: Cassarett and Doull’s Toxicology (2001) C. Klaassen (Ed),
New York, NY: McGraw-Hill
4
Drug-drug interactions (DDI)
• Altered enzymatic biotransformation can lead to
clinically-relevant drug-drug interactions between coadministered drugs, a key safety consideration
• In preclinical drug development, DDI risk is assessed by
evaluating
– Major clearance routes (e.g., mass balance, CYP phenotyping)
– Enzyme inhibition potential
– Enzyme induction potential
– Transporter involvement and inhibition potential
5
Cytochrome P450 inhibition
• CYP inhibition has potential to result in
– Black box label warnings
– Withdrawal from market
O
O
O
N
F
N
N
H
N
OH
Terfenadine: withdrawn 1997
(victim drug)
Mibefradil: withdrawn 1998
(perpetrator drug)
Co-administration with CYP3A4
inhibitors (e.g., ketoconazole)
reduced clearance of the drug
and resulted in cardiotoxicity
caused by terfenadine
accumulation
Mibefradil inhibits CYP3A4 and
can cause elevated levels of
coadministered drugs cleared by
these enzymes. Life-threatening
interactions can occur with bblockers and other
antihypertensives
6
Repaglinide uses
• Repaglinide is an insulin secretagogue used to normalize
postprandial hyperglycemia in patients with type 2
diabetes
OH
OH
CH3
O
H 3C
O
NH
CH3
CH3
O
H3C
O
O
NH
O
CH3
O
N
NH
Repaglinide
OH
Dicarboxylic acid (M2)
• Major human metabolite in vivo is the dicarboxylic acid
(van Heiningen et. al., 1999)
• Other oxidative metabolites and glucuronide conjugate
van Heiningen et al. Eur, J. Clin. Pharmacol. Exp. Ther. 1999; 55(7): 521-525.
7
Repaglinide metabolism
• Major biotransformation routes described (Bidstrup et
al., 2003)
CYP2C8
metabolism
M0-OH
M4
CYP2C8
probe
CYP3A4
metabolism
M1
M2
M5
Bidstrup et al. Br. J. Clin. Pharmacol. 2003; 56: 305-314.
8
Repaglinide M4 formation and antibody inhibition
Roles for
CYP2C8
CYP3A4
Bidstrup et al. Br. J. Clin. Pharmacol. 2003; 56: 305-314.
9
Repaglinide metabolized by CYP3A4/2C8 and UGT1A1
• Repaglinide therefore has potential for DDIs
with other drugs cleared hepatically by
CYP3A4 and 2C8 and UGT1A1
• According to the University of Washington
Drug Interaction Database, repaglinide is
known for interactions with 10 drugs
– Flucloxacillin and rifampin cause increased CL
– Gemfibrozil, clarithromycin, cyclosporine,
deferasirox, telithromycin, itraconazole,
trimethoprim cause >40% increase in AUC
10
Gemfibrozil and repaglinide
• Type 2 diabetics have 2-4-fold increased risk of
macrovascular disease
• Gemfibrozil is used to reduce triglycerides (TG) in
patients with certain dyslipidemias
– Almost 30% TG reduction in diabetics compared to
placebo group
Vinik and Colwell Diabetes Care 1993; 16(1): 37-44.
• In patients concommitant administration has
resulted in up to 8-fold plasma increase in
repaglinide Backmann et al. Drug Metab. Dispos. 2009; 37(12): 2359-66.
– Reports of severe, prolonged hypoglycemia
11
Gemfibrozil dosing and pharmacokinetics
• Gemfibrozil usually dosed at 600 mg twice a day or
less commonly 900 mg once daily
• PK parameters after a single oral dose
Parameter
600 mg dose
900 mg dose
Cmax (µg mL-1)
28.8 ± 4.1
40.8 ± 12.6
tmax (h)
1.8 ± 0.8
1.8 ± 0.8
AUC0-8 (µg h mL-1)
80.3 ± 10.3
132.1 ± 35.3
CL (L-1)
7.1 ± 0.9
6.6 ± 1.6
Vd (L-1)
11.6 ± 2.1
12.5 ± 3.4
t1/2 (h)
1.1 ± 0.2
1.3 ± 0.1
Rouini et al. Int. J. Pharmacol. 2006; 2: 75-78.
12
Gemfibrozil metabolism
• Metabolized in liver to 4 major metabolites but the
glucuronide metabolite is a potent CYP2C8 inhibitor
Baer et al. Chem. Res. Toxicol. 2009; 22(7): 1298-1309.
13
In vitro experiments with repaglinide
• Initially worked to establish a simple CYP2C8 assay in
vitro to complement the in vivo application of
repaglinide
• Noted discrepancies using reference material
potential issues with some of the analytical work
described in the literature
• Subsequently needed to re-establish the specificity
of the CYP2C8/CYP3A4 metabolism
14
Repaglinide in human liver microsomes (HLM)
• High-resolution LC UV chromatogram (254 nm)
HLM-30 min
RD117007_MSE_05Apr12_008 Sb (3,40.00 ); Sm (SG, 40x1)
4: Diode Array
50 mM Repaglinide
254
Range: 1.102e-2
0.5 mg/mL HLM
30 minutes; 37°C; pH 7.4
NADPH-generating system
8.72
Repaglinide
9.0e-3
8.0e-3
7.0e-3
6.0e-3
AU
5.0e-3
4.0e-3
3.0e-3
2.0e-3
1.0e-3
Probe metabolite
Hydroxyrepaglinide
(M4)
Major in vivo metabolite
Repaglinide
dicarboxylic acid
metabolite (M2)
Repaglinide
desaturation
metabolites
17.62
17.24
0.0
High abundance
Low abundance
-1.0e-3
Time
6.00
8.00
10.00
12.00
14.00
16.00
Unlabeled peaks are not related to repaglinide
15
Repaglinide human liver microsome metabolite profile
Component
tr (min) m/z
value
Mass
error
(ppm)
Mass
shift
Proposed biotransformation
M0-OH
5.48
469.2693
-1.9
+15.9940
Hydroxylation
C1
5.94
451.2590
-0.2
-2.0156
Dehydrogenation
C2
6.83
441.2369
4.7
-12.0384
O-deethylation + hydroxylation
M4
7.50
469.2700
-0.4
+15.9947
Hydroxylation
M1
7.95
385.2112
-3.9
-68.0641
N,N-didealkylation
M5
8.26
425.2436
-4.5
-28.0317
O-deethylation
Repaglinide
8.98
453.2738
-3.3
-0.0015
None
C3
10.09
471.2848
-0.9
+18.0095
Hydroxylation+ reduction
M2
10.30
485.2642
-5.1
+31.9889
C4
16.09
451.2583
-3.1
-2.0170
N-dealkylation + oxidation to the
carboxylic acid
Dehydrogenation
C5
17.23
451.2591
-1.3
-2.0162
Dehydrogenation
C6
17.52
451.2599
0.4
-2.0154
Dehydrogenation
16
Recombinant CYP panel for repaglinide substrate loss
• Incubating drug with individual enzymes can help narrow
down enzymes involved in metabolism
• Complicated by involvement of enzymes that would not be
involved in a more complete test system
Percentage of maximum
remaining(%)
100
80
60
40
CYP3A4?
Substrate loss
10 mM repaglinide
10 pmol/inc rCYP
20 minutes
35°C; pH 7.4
20
0
17
Inhibition experiments for CYP reaction phenotyping
• Simple test system to minimize variables
– HLM for cytochrome P450-mediated M0-OH, M1, M2, M4
and M5
• Use known chemicals (or antibodies) to inhibit
specific enzymes
– Mibefradil for CYP3A4 (metabolism-dependent)
– Gemfibrozil glucuronide for CYP2C8 (metabolismdependent)
• Assess the effect of the presence/absence of the
inhibitor on formation of the metabolite of interest
18
Selecting appropriate conditions for inhibition experiments
• Initial-rate conditions desirable
19
Metabolism-dependent CYP2C8 and 3A4 inhibition
2C8
3A4
Less clear
110
No inhibitor
100
Percentage of control (%)
90
Mibefradil
80
Gemfibrozil
glucuronide
70
60
50
40
30
20
10
0
C1
(M0-OH)
M0-OH
C4
(M4)
M4
C5M1
(M1)
C7M5
(M5)
Repaglinide metabolite
C11
(M2)
M2
20
Correlation data for major metabolites with HLM donor panel
21
Exploring the interaction further
• Nonclinical species have very limited use in modeling
human DDIs
• One major challenge is species differences in protein
expression and function (e.g., enzyme specificity)
• Rodent studies occur early on for most drugs
• Rat is not a good model for drugs cleared by CYP3A4
– Ortholog CYP3A1 has limited similarity and little overlap in
function
• The rat ortholog for CYP2C8 is CYP2C22 which has
demonstrated some very similar properties
• Could this DDI be modeled in the rat?
22
In vivo experiments in the rat (Xenometrics/XenoTech)
23
Repaglinide PK data in rat (n = 3 per group)
Repaglinide plasma concentration
(ng/mL)
• AUC increase in group 1 animals
Group 1:
Gemfibrozil + repaglinide
Group 2: Repaglinide only
• Gemfibrozil concentrations 16 – 125 µg mL-1
24
Repaglinide PK data in rat (n = 3 per group)
• Clear evidence of drug-drug interaction between
gemfibrozil and repaglinide in Group 1 animals
Parameter
Group 1
Group 2
Fold-change
Cmax (ng mL-1)
284.1 ± 85.2
66.2 ± 6.9
4.3-fold increase
tmax (h)
1.7 ± 0.6
1.2 ± 0.7
1.4-fold increase
AUC0-12 (ng h mL-1)
853.9 ± 344.7
242.8 ± 32.5
3.5-fold increase
AUC0-∞ (ng h mL-1)
837.5 ± 337.0
282.1 ± 43.5
3.0-fold increase
CLobs (L h-1 kg-1)
1299.3 ± 522.9
3622.0 ± 589.6
2.8-fold decrease
Vdobs (L kg-1)
5158.6 ± 3397.4
13154.8 ± 2483.3
2.6-fold decrease
t1/2 (h)
2.6 ± 0.8
2.6 ± 0.6
None
25
Repaglinide rat plasma (AUC pool) metabolite profile
Component
tr (min) m/z
Proposed biotransformation
Group
1
Group
2
RP1
3.10
441
O-Deethylation + hydroxylation
+
+
M0-OH
5.58
469
Hydroxylation
+
ND
RP2
5.78
469
Oxidation
+
ND
Repaglinide
glucuronide
7.17
629
Glucuronidation
+
+
M4
7.42
469
Hydroxylation
ND
ND
M1
7.81
385
N,N-Didealkylation
ND
ND
RP3
7.87
441
O-Deethylation + hydroxylation
+
ND
M5
8.30
425
O-deethylation
+
+
RP4
8.59
451
Dehydrogenation
+
ND
RP5
8.59
469
Repaglinide
8.89
453
Oxidation
None
+
+
ND
+
C3
9.74
471
Hydroxylation+ reduction
+
ND
M2
10.12
485
N-dealkylation + oxidation to the
carboxylic acid
+
ND
26
Relative abundance of major human metabolites
Percentage of observed maximum (%)
• Very low abundance metabolites in plasma
• Limited plasma sample volume
Plasma
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Group 1
Group 2
27
Bile metabolite profiles (0-12 h pools)
• Repaglinide predominantly excreted in bile in humans
– 90% excreted in feces; 8 % excreted in urine
van Heiningen et al. Eur, J. Clin. Pharmacol. Exp. Ther. 1999; 55(7): 521-525.
• Rat bile profiles contained 49 metabolites across the
two groups
– Oxidative metabolism
– Glucuronidation
– Sulfonation
• Initial focus has to be on metabolites of interest
28
Exploring the CYP inhibition in bile
• Relative abundance of CYP2C8 (in human) metabolites
decreased (~65%) with gemfibrozil dosing
Percentage of observed maximum (%)
100%
90%
80%
70%
60%
50%
Group 1
40%
Group 2
30%
20%
10%
0%
M0-OH
M4
29
Relative abundance of major human metabolites
Percentage of maximum observed (%)
• All of them decreased with gemfibrozil dosing
Bile
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Group 1
Group 2
• Not characteristic of a specific CYP inhibition interaction
30
Urine metabolite profiles (0-12 h pools)
• Huge differences between the treatment groups
– Without gemfibrozil treatment, only 7 metabolites
– With gemfibrozil treatment, 27 metabolites
Component
tr
(min)
m/z
Proposed
biotransformation
Group 1
Group 2
Hydroxylation
+
ND
Glucuronidation
+
ND
469
Hydroxylation
+
ND
7.83
385
N,N-Didealkylation
+
+
M5
8.30
425
O-Deethylation
+
ND
Repaglinide
8.89
453
None
+
+
M2
10.12
485
N-dealkylation + oxidation
to the carboxylic acid
+
+
M0-OH
5.58
469
Repaglinide
glucuronide
7.17
629
M4
7.42
M1
31
Metabolite abundance in the urine
Percentage of maximum observed (%)
• Even the metabolites detected in Group 2 urine are
present at relatively low abundance
Urine
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Group 1
Group 2
32
Biliary vs urinary excretion
• Gemfibrozil increases urine and decreases bile excretion
100
90
80
70
60
50
40
30
20
10
0
• Why?
Bile
Urine
Percentage of total material (%)
Percentage of total material (%)
Group 1
Group 2
100
90
80
70
60
50
40
30
20
10
0
Bile
Urine
33
Systemic effects of gemfibrozil
• Metabolism-dependent CYP2C8 inhibitor
– Does not seem to account for all the metabolic profile
changes
– As yet, do not have evidence of CYP2C22 inhibition
• UGT1A1 inhibitor
Gan et al. Br. J. Pharmacol. 2010; 70(6): 870-80.
– Repaglinide glucuronidation occurs at least in part through
1A1 mediation
– Would not explain other effects
• OATP1B1 (SLCO1B1) hepatic uptake transporter
inhibitor Nakagomi-Hagihara et al. Xenobiotica 2007; 37(5): 474-486.
– Would severely reduce abundance of all metabolites in bile
– May also account for increased urinary excretion in Group 1
34
Human and rat OATPs
• Human OATP1B1 inhibition has been described as a
confounding factor in the repaglinide/gemfibrozil DDI
Kudo et al. Drug Metab. Dispos. 2012; 41(2): 362-371.
• OATP1B family comprises OATP1B1 and 1B3
• Only rodent ortholog for OATP1Bs is Oatp1b2
– Functions similarly to both
– Mice deficient in Oatp1b2 have shown some utility as
models for OATP1B studies
• Repaglinide PK has been shown to correlate with
OATP1B1 polymorphism
Niemi et al. Clin. Pharmacol. Ther. 2005; 77(6): 468-478.
Kallioski et al. Br. J. Clin. Pharmacol. 2008; 66(6): 818-825.
35
Back to the PK data
• The observed clearance, volume of distribution and
t1/2 data do support the transporter hypothesis
Parameter
Group 1
Group 2
Fold-change
Cmax (ng mL-1)
284.1 ± 85.2
66.2 ± 6.9
4.3-fold increase
tmax (h)
1.7 ± 0.6
1.2 ± 0.7
1.4-fold increase
AUC0-12 (ng h mL-1)
853.9 ± 344.7
242.8 ± 32.5
3.5-fold increase
AUC0-∞ (ng h mL-1)
837.5 ± 337.0
282.1 ± 43.5
3.0-fold increase
CLobs (L h-1 kg-1)
1299.3 ± 522.9
3622.0 ± 589.6
2.8-fold decrease
Vdobs (L kg-1)
5158.6 ± 3397.4
13154.8 ± 2483.3
2.6-fold decrease
t1/2 (h)
2.6 ± 0.8
2.6 ± 0.6
None
36
Next experiments
• Still have untapped potential in the liver samples
• They were flash frozen so cannot do
hepatocyte/transporter work
• Plan to make microsomes and measure CYP/UGT
activities to explore the inhibition independently
– CYP2C8/CYP3A4
– UGT1A1/1A3 (more complicated)
• Transporter work will need to be done in vitro
– Clear evidence of uptake interactions
– Efflux transporter issues may also be involved
37
Conclusions
• Individual CYP inhibition effects can be modeled well
in vitro; repaglinide does seem to have
CYP2C8/2C22-specific metabolites but not
necessarily as expected
• More complete systems have both advantages and
disadvantages
• In the case of gemfibrozil and repaglinide,
transporter inhibition appeared to be much more
involved in PK changes than CYP inhibition
– Still some work to be done
• Rodent utility in transporter studies needs further
study
38
Acknowledgements
• XenoTech
–
–
–
–
–
–
–
Phyllis Yerino
Forrest Stanley
Dr. Sylvie Kandel
Seema Muranjan
Chandra Kollu
Dr. David Buckley
Brian Ogilvie
• Xenometrics
– Dr. Kristin Russell
– Tom Haymaker
39
Thank you
Questions?
Joanna Barbara, Ph.D.
Division Director, Analytical Services
XenoTech, LLC
[email protected]
40

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