Z-338 - Cerep

Involvement of CYP2C8 and UGT1A9 in the
metabolism of a novel gastroprokinetic agent, Z-338
S. Furuta 1 , E. Kamada 1 , T. Sugimoto 1 , Y. Kawabata 1 ,
X. C. Wu 2 , J. Skibbe 3 , E. Usuki 3 , A. Parkinson 3
and T. Kurimoto 1
1
Central Research Laboratories, ZERIA Pharmaceutical
Co., Ltd., Saitama, Japan
2
Cerep Inc., WA, USA
3
XENOTECH, L.L.C., KS, USA

Introduction
Z-338 is a novel gastroprokinetic agent, which is
synthesized by Zeria Pharmaceutical Co., Ltd (Fig. 1).
Z-338 is under development for the treatment of
functional dyspepsia, and is now in clinical trials.
The purpose of the study was to evaluate possible
involvement of cytochrome P450 (CYP) enzyme(s) and
UDP-glucuronosyltransferase (UGT) enzyme(s)
responsible for metabolizing Z-338.
Potential interactions of Z-338 with CYP enzymes were
also investigated, and we compared its inhibitory
potentials with cisapride and Z-338 analog, ID951551.

CYP2C8
(>>CYP3A4 or 1A1)
MeO
O
N
H
S
N
O
N
H
N
H
MeO
O
N
H
S
N
O
N
H
N
MeO
OH
Z-338 deisopropyl metabolite
MeO
MeO
MeO
O
OMe
OH
N
H
Z-338
S
N
N
H
ID951551
O
N
UGT1A9
(>>UGT1A8)
MeO
MeO
CYP2C8
(>CYP3A4 or 1A1)
MeO
MeO
O
OGlu
N
H
O
OMe
S
N
H
N
S
N
O
O
N
H
Z-338 glucuronide
ID951551 deisopropyl metabolite
N
H
N
N
H
OCH 3
H 3 CO
CYP3A4
OCH 3
H 3 CO
F O(CH 2 ) 3 N NHCO
NH 2
HN
NHCO
NH 2
Cisapride
Cl
N-dealkylcisapride
Cl
Fig.1 Metabolic Scheme of Z-338, ID951551 and Cisapride

Method: In vitro metabolism study
HPLC methods that were validated for the quantification of Z-338,
Z-338 deisopropyl metabolite and ID951551 were employed.
Z-338 and ID951551 were incubated with a panel of recombinant
human enzymes to investigate possible involvement of CYP and
UGT enzymes responsible for the metabolism of Z-338 and
ID951551. rCYP and rUGT enzymes were obtained from Gentest
Corporation (Woburn, MA) or Cypex Ltd, (Scotland, UK) . Once
initial rate conditions were established, Km and Vmax were
determined for the dealkylation of Z-338 by human liver
microsomes (HLM), recombinant CYP2C8 and recombinant
CYP3A4. HLM was prepared by XenoTech (Lenexa, KS).
An antibody inhibition experiment was performed to determine the
relative contribution of CYP2C8 and CYP3A4 to the metabolism of
Z-338 and ID951551 by HLM. Polyclonal antibodies and intact
rabbit serum were obtained from Nosan Corporation (Yokohama,
Japan).

Method: In vitro inhibition study
The following marker substrate activities were used to evaluate the
potential inhibitory effects on CYP isoforms: 7-ethoxyresorufin
deethylation activity (CYP1A1/2), coumarin hydroxylation activity
(CYP2A6), 7-benzyloxyresorufin debenzylation activity (CYP2B6),
dibenzylfluorescein debenzylation activity (CYP2C8), tolbutamide
hydroxylation activity (CYP2C9), (S)-mephenytoin hydroxylation
activity (CYP2C19), bufuralol hydroxylation activity (CYP2D6),
chlorzoxazone hydroxylation activity (CYP2E1), testosterone 6βhydroxylation
activity and terfenadine hydroxylation activity
(CYP3A4). HLM was obtained from the International Institute for
the Advancement of Medicine (Exton, P.A., USA) , and rCYP
enzymes were obtained from Gentest Corporation (Woburn, MA) or
Cypex Ltd, (Scotland, UK) .
The Ki values were calculated from Dixon plots based on a
competitive inhibition model.

mVolts
40
20
0
a
b
15.258
17.533
19.733
c
40
20
0
mVolts
Z-338
10 µM
60-min incubation
rCYP2C8 (40 pmol
P450/incubation)
a: Z-338 deisopropyl metabolite
b: I.S. (ID951551)
c: Z-338
0 5 10 15 20 25 30 35
Minutes
mVolts
40
20
a
13.750
b
c
ID951551
10 µM
5-min incubation
rCYP2C8 (40 pmol
P450/incubation)
0
18.000
0 5 10 15 20 25 30 35
Minutes
20.125
a: ID951551 deisopropyl metabolite
b: ID951551
c: I.S. (Z-338)
Fig. 2 HPLC Chromatograms of In Vitro Metabolism Study

CYP1A1
CYP1A2
CYP2A6
CYP2B6
CYP2C8
CYP2C9
CYP2C18
CYP2C19
CYP2D6
CYP2E1
CYP3A4
CYP3A5
CYP4A11
Z-338
100 µM
60-min incubation
40 pmol P450/incubation
(mean±SD, n=3)
0 2000 4000 6000 8000 10000
Metabolite formation (nM)
CYP1A1
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A4
ID951551
100 µM
5-min incubation
40 pmol P450/incubation
(mean, n=2)
0 500 1000 1500 2000 2500 3000
Metabolite formation (nM)
Fig. 3 Metabolite Formation from Z-338 and ID951551
incubation with a Panel of Recombinant CYP Enzymes

UGTControl
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A8
UGT1A9
UGT1A10
UGT2B7
UGT2B15
Percent reduction of Z-338 by
recombinant UGT isoforms
The values show the percentage reduction from initial value
(1000nM).
0.25 mg microsomal protain/incubation
UGT1A9: 30-incubation
Other UGTs:120-min incubation
Each value represents the mean±SD, (n=3).
0 20 40 60 80 100 120
Metabolism(nM)
Percent reduction from initial value
v (nmol/min/mg protein of expressed microsome)
8
7 UGT1A8
6
5
4
3
2
1
0
0 500 1000 1500
Concentration (M)
v (nmol/min/mg protein of expressed microsome)
16
14 UGT1A9
12
10
8
6
4
2
0
0 50 100 150
Concentration (M)
UGT1A8
Vmax :11.11 nmol/min/mg protain
Km = 1426.51 µM
Vmax/Km:0.008 mL/min/mg protein
UGT1A9
Vmax :15.24 nmol/min/mg protain
Km = 43.13 µM
Vmax/Km:0.353 mL/min/mg protein
Fig. 4 Metabolism of Z-338 by Human Recombinant UGT Isoforms

Rate
(pmol/min/mg protein)
160
140
120
100
80
60
40
20
0
0 0.4 0.8 1.2
Rate (pmol/min/mg protein)/
[Z-338] (µM)
Rate
(pmol/min/pmol P450)
24
20
16
12
8
4
0
0 0.035 0.07 0.105 0.14
Rate (pmol/min/pmol P450)/
[Z-338] (µM)
Rate
(pmol/min/pmol P450)
0.4
0.2
0
0 0.002 0.004 0.006 0.008
Rate (pmol/min/pmol P450)/
[Z-338] (µM)
Human Liver Microsomes rCYP2C8 rCYP3A4
Fig. 5 Eadie-Hofstee Plots of Z-338 Metabolism
Table 1 Kinetic Parameters of Z-338 by Human Liver Microsomes, Recombinant
CYP2C8 and CYP3A4 enzymes
Test System Km (µM)
Vmax
(pmol/ min/ mg protein
or pmol P450)
In vitro intrinsic clearance
(µL/ min /mg protein
or pmol P450)
HLM 318 ± 26 347 ± 26 1.09
rCYP2C8 152 ± 7 12.7 ± 0.3 0.0833
rCYP3A4 79.5 ± 1.9 0.534 ± 0.005 0.00676
Values are the mean ± standard error (n = 3).
In vitro intrinsic clearance = Vmax/Km

Table 2 Inhibition of Z-338 and ID951551 on the enzyme activities of rCYP
isoforms
Isoforms Inhibitors IC 50 (mol/L) n H
rCYP2C8
(DBF substrate)
Z-338
ID951551
Quercetin
> 100
17
10
1.1
1.4
rCYP2C9
(7-MF substrate)
ID951551
Sulfaphenazole
> 100
0.20 1.1
rCYP3A4
(Testosterone substrate)
ID951551
Sulfaphenazole
21
0.86
1.2
2.3
Effect of Z-338 on the CYP2C8 enzyme activity
1/v
1.4
1.2
1
0.8
0.6
0.4
0.2
0
● DBF 0.25 µM
● DBF 0.5 µM
● DBF 1 µM
● DBF 2 µM
● DBF 2.5 µM
-0.2
-150 -100 -50 0 50 100 150 200 250
Z-338 (M)
Fig. 6 Inhibition of Z-338 on
CYP2C8 activities by
Dixon Plots
Z-338: Ki = 121µM
Quercetin : Ki = 5.8µM

Table 3 Inhibition constants (K i values, µM) on CYP isoforms of human liver microsomes by Z-338,
cisapride and specific CYP inhibitors on several enzyme metabolic activities
Z-338 Cisapride Inhibitor
CYP1A1&1A2
7-Ethoxyresorufin deethylation activity
225.5 148.1
0.021 (α-Naphthoflavone )
3.14 (Furafylline)
CYP2A6
Coumarin hydroxylation activity
CYP2B6
7-Benzyloxyresorufin debenzylation activity
CYP2C9
Tolbutamide hydroxylation activity
140.5 76.3 1.31 (8-Methoxypsoralen)
Not inhibited Not inhibited 2315.8 (Orphenadrine)
1030.8 15.7 1.16 (Sulfaphenazole)
CYP2C19
S-mephenytoin hydroxylation activity
>>10 4 164.0
8.72 (Omeprazole)
53.1 (Tranylcypromine)
CYP2D6
Bufuralol hydroxylation activity
135.1 101.9 0.042 (Quinidine)
CYP2E1
Chlorzoxazone hydroxylation activity
CYP3A4
2624.7 2254.3
33.8 (Anilline)
9.33 (Diethyldithiocarbamate)
Testosterone 6β-hydroxylation activity
>>10 4
0.95
0.30 (Ketoconazole)
Terfenadine hydroxylation activity
347.8
2.3
0.5 (Ketoconazole)
The K i values were calculated from Dixon plots based on a competitive inhibition model.

Anti-CYP2C8
Anti-CYP3A4
Z-338
Table 4 Summary of the relative contribution of CYP2C8 and CYP3A4 to the
metabolism of Z-338 by human liver microsomes
Clearance Specific Content Adjusted Clearance
Enzyme
Relative
(µL/min/pmol (pmol p-450/mg (µL/ min /mg
System
Contribution
P450)
protein)
protein)
rCYP2C8 0.0833 64 5.33 88%
Anti-CYP2C8
Anti-CYP3A4
ID951551
0 10 20 30 40 50 60 70 80 90
Inhibition (%)
rCYP3A4 0.00679 108 0.733 12%
r: recombinant
Clearance = (Vmax ÷ Km)
Specific content values are from Rodrigues, 1999.
Adjusted Clearance (µL/mg protein/min) = Clearance (µL/min/pmol P450) x Specific
Content (pmol/mg protein)
Relative Contribution (%) = (Adjusted Clearance (µL/ min /mg protein) ÷ Total
Adjusted Clearance (µL/min /mg protein)) x 100%
Fig. 7 Percent inhibition for Z-338 and ID951551when
incubated with human liver microsomes pretreated with
polyclonal antibodies against CYP2C8 and CYP3A4

Conclusion
Z-338 is predominantly metabolized to Z-338
glucuronide by UGT1A8 and UGT1A9
(probably UGT1A9>>UGT1A8).
Z-338 is also catalyzed to deisopropyl metabolite by
mainly CYP2C8.
Z-338 showed little inhibition on CYP2C8 activities,
which is significantly less than by its analog, ID951551.
This may be based on the fact that its major metabolic
pathway being glucuronidation rather than oxidation.
Z-338 is considered unlikely to cause significant drugdrug
interactions when coadministered with CYP
substrates at clinically effective doses.