In vitro metabolism of oprozomib, an oral proteasome inhibitor: Role of epoxide hydrolases and cytochrome P450ss

Article abstract of DOI:10.1124/dmd.117.075226

Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent bond with the active site N-terminal threonine of the

Full text of DOI:10.1124/dmd.117.075226

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In Vitro Metabolism of Oprozomib, an Oral Proteasome Inhibitor: Role of Epoxide Hydrolases and
Cytochrome P450s
Zhican Wang, Ying Fang, Juli Teague, Hansen Wong, Christophe Morisseau, Bruce D. Hammock, Dan
A. Rock, Zhengping Wang
Department of Pharmacokinetics and Drug Metabolism (Z.W., Y.F., D.R., and Z.W.), and Clinical
Pharmacology Modeling & Simulation (H.W.), Amgen Inc., South San Francisco, CA 94080
Drug Metabolism and Pharmacokinetics (J.T.), Onyx Pharmaceuticals, an Amgen Subsidiary, South San
Francisco, CA 94080
Department of Entomology and Nematology (C.M., and B.H.), and UC Davis Comprehensive Cancer
Center, University of California, Davis, One Shields Avenue, Davis, California 95616
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Running Title Page
Running title: Metabolism of oprozomib by microsomal epoxide hydrolase
Corresponding author:
Zhengping Wang, PhD, Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., South San
Francisco, CA 94080. Phone: 650-766-6695; Email: zwang687@gmail.com
Number of text pages: 25
Number of tables: 1
Number of figures: 7
Number of supplemental tables: 1
Number of supplemental figures: 3
Number of references: 48
Number of words in the Abstract: 249
Number of words in the Introduction: 733
Number of words in the Discussion: 1492
Abbreviations
1-ABT: 1-aminobenzotriazole; BSA: bovine serum albumin; CYP: cytochrome P450; DDI: drug-drug
interaction; DMSO: dimethyl sulfoxide; EH: epoxide hydrolase; FA: formic acid; GSH: glutathione;
HLM: human liver microsome; IS: internal standard; LC: liquid chromatography; mEH: microsomal
epoxide hydrolase; MM: multiple myeloma; MS: mass spectrometer; NSPA: 2-nonylsulfanyl-
propionamide; PDA: photodiode array detector; PK: pharmacokinetics; RRMM: relapsed or/and
refractory multiple myeloma; sEH: soluble epoxide hydrolase; cis-SO: cis-stilbene oxide; trans-SO:
trans-stilbene oxide; TPPU: 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea; UPLC-
MS/MS: ultra-performance liquid chromatography tandem mass spectrometry
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Abstract
Oprozomib is an oral proteasome inhibitor currently under investigation in patients with hematologic
malignancies or solid tumors. Oprozomib elicits potent pharmacological actions by forming a covalent
bond with the active site N-terminal threonine of the 20S proteasome. Oprozomib has a short half-life
across preclinical species and in patients due to systemic clearance via metabolism. Potential for drug-
drug interactions (DDIs) could alter the exposure of this potent therapeutic therefore a thorough
investigation of pathways responsible for metabolism is required. In the present study, the major drug-
metabolizing enzyme responsible for oprozomib metabolism was identified in vitro. A diol of oprozomib
was found to be the predominant metabolite in human hepatocytes, which formed via direct epoxide
hydrolysis. Using recombinant epoxide hydrolases (EHs) and selective EH inhibitors in liver
microsomes, microsomal EH (mEH) but not soluble EH (sEH), was found to be responsible for
oprozomib diol formation. Co-incubation with 2-nonylsulfanyl-propionamide, a selective mEH inhibitor,
resulted in a significant decrease in oprozomib disappearance (>80%) with concurrent complete blockage
of diol formation in human hepatocytes. On the contrary, a selective sEH inhibitor did not affect
oprozomib metabolism. Pre-treatment of hepatocytes with the pan-CYP inhibitor 1-aminobenzotriazole
resulted in a modest reduction (~20%) of oprozomib metabolism. These findings indicated that mEH
plays a predominant role in oprozomib metabolism. Further studies may be warranted to determine
whether drugs that are mEH inhibitors cause clinically significant DDIs with oprozomib. On the other
hand, pharmacokinetics of oprozomib is unlikely to be affected by co-administered CYP and sEH
inhibitors and/or inducers.
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Introduction
The proteasome is a multicatalytic enzyme complex and key to the ubiquitin dependent pathway
for intracellular protein degradation (Kisselev and Goldberg, 2001; Ciechanover, 2005). The highly
regulated ubiquitin proteasome system affects a wide variety of cellular processes and is critical for
cellular homeostasis. Inhibition of proteasome leads to accumulation of misfolded proteins and the
induction of cell cycle arrest and apoptosis. Transformed cells, particularly multiple myeloma (MM)
cells, possess elevated levels of proteasome activity. Consequently, these actively proliferating malignant
cells are more sensitive to proteasome inhibitors than normal cells (Kumatori et al., 1990; Loda et al.,
1997; Li and Dou, 2000; Almond and Cohen, 2002). Proteasome inhibition has emerged as an effective
therapeutic strategy for the treatment of MM and some lymphomas. Bortezomib (Velcade®), the first
generation proteasome inhibitor, was approved in 2003 (Bross et al., 2004; Chauhan et al., 2005;
O'Connor et al., 2005). Carfilzomib (Kyprolis®), a peptide epoxyketone (Figure 1), represents a new
generation of proteasome inhibitors. It selectively binds to the N-terminal threonine of the proteasome via
two covalent bonds to form a morpholino ring (Harshbarger et al., 2015), and has demonstrated favorable
clinical safety and efficacy profiles compared to bortezomib (Kuhn et al., 2007). Carfilzomib has been
approved for use as a single agent and in combination with lenalidomide and dexamethasone in patients
with relapsed or/and refractory multiple myeloma (RRMM) (Thompson, 2013). Notably, carfilzomib in
combination with dexamethasone has doubled progression-free survival in patients with RRMM
compared to bortezomib and dexamethasone in the Phase 3 ENDEAVOR clinical trial (Dimopoulos et al.,
2016). The introduction of proteasome inhibitors and immunomodulatory drugs has revolutionized MM
treatment with the current overall survival of MM patients increased by 2-3 fold (Anderson, 2013).
The success of bortezomib and carfilzomib has inspired efforts to develop oral proteasome
inhibitors with improved flexibility in dose administration, specifically oral administration, for improved
patient compliance and convenience. Ixazomib (Ninlaro®) and oprozomib, representing peptide boronate
and peptide epoxyketone, respectively, are two orally bioavailable proteasome inhibitors under clinical
investigation (Rajan and Kumar, 2016). Ixazomib in combination with lenalidomide and dexamethasone
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administrated once-weekly demonstrated efficacy in a Phase 3 study (Moreau et al., 2016). Oprozomib
(Figure 1) is being evaluated in Phase 1/2 clinical trials in patients with hematological malignancy or
solid tumors (Infante et al., 2016). Similar to carfilzomib, oprozomib inhibits primarily the
chymotrypsin-like activity of the constitutive and immunoproteasomes via covalent modification of the
N-terminal threonine (Zhou et al., 2009). It has shown promising clinical activity in patients with RRMM
either as a single agent or in combination with pomalidomide and dexamethasone (Hari et al., 2015; Shah
et al., 2015; Vij et al., 2015).
Oprozomib is a tripeptide analogue (Figure 1) of carfilzomib that was developed to improve
absorption. It was believed that smaller peptides were likely to be more effectively delivered across the
small intestine (Hamman et al., 2005; Zhou et al., 2009). When dosed via oral gavage to Balb/C mice at
30 mg/kg, more than 80% proteasome inhibition was observed in blood, liver and adrenal gland one hour
post administration (Zhou et al., 2009). Furthermore, oprozomib demonstrated improved metabolic
stability in vitro across multiple species compared to carfilzomib. Nonetheless, oprozomib still displayed
high systemic clearance and short half-life (Fang et al., 2015). This is not surprising because both the
peptide backbone and the epoxyketone warhead could be susceptible to metabolism (Yang et al., 2011;
Wang et al., 2013). The rapid clearance of oprozomib was primarily mediated by metabolism, supported
by the observation that parent compound was a minor component in urine and bile samples from rats, as
well as urine samples from human (in-house unpublished data). In addition, oprozomib has shown a
relatively large inter-patient pharmacokinetic (PK) variability (Infante et al., 2016).
The objective of the present study was to characterize the major metabolic pathways and key
enzymes responsible for oprozomib metabolism in vitro using cryopreserved human hepatocytes,
recombinant enzymes, and liver cellular fractions in the presence and absence of selective enzyme
inhibitors. Deciphering the role of various enzymes responsible for oprozomib elimination is important in
drug-drug interaction (DDI) risk assessment and mitigation of exaggerated pharmacology of this potent
therapeutic. Particularly, it is important for CYP-mediated DDIs since CYP isoforms are responsible for
the primary metabolism of the majority of small molecule drugs on the market (Wienkers and Heath,
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2005). Defining the primary metabolic routes could also help shed light on the observed oprozomib PK
variabilities in patients.
Materials and Methods
Materials. All chemicals and reagents were obtained from Sigma (St. Louis, MO) and liquid
chromatography (LC) solvents were obtained from Fisher Scientific (Pittsburgh, PA). Oprozomib and
authentic metabolite standards were synthesized and characterized at Onyx Pharmaceuticals, an Amgen
subsidiary (South San Francisco, CA). Deuterated oprozomib (d5-oprozomib, five deuterium on the
benzyl ring) was obtained from PepTech Corp (Bedford, MA). A selective mEH inhibitor, 2-
nonylsulfanyl-propionamide (NSPA) was previously synthesized (Morisseau et al., 2008), and a selective
sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was previously
characterized (Rose et al., 2010). The structures of these two inhibitors are shown in Supplemental
Figure 1. Human recombinant microsomal epoxide hydrolase (mEH, # Cri 08.17, purity 80%) and
soluble epoxide hydrolase (sEH, # Cri 71.16, purity 95%) were prepared and characterized as described
previously (Morisseau et al., 2000; Morisseau et al., 2011). Supersomes containing cDNA-expressed
human CYPs, CYP reductase and cytochrome b5 were purchased from BD Bioscience (San Jose, CA).
Pooled human cryopreserved hepatocytes (Lot# FDX, 20 donors, mixed gender) and media were
purchased from BioreclamationIVT (Baltimore, MD). Liver subcellular fractions including male or
female liver microsomes, and cytosols (10 donors per gender, age: 21 – 58) were purchased from Sekisui
Xenotech (Kansas City, KS). Pooled, gender mixed liver microsomes were prepared by mixing an equal
volume of male (10 donors) and female (10 donors) liver microsomes.
Metabolic profiling of oprozomib in human hepatocytes. Pooled cryopreserved human hepatocytes
were thawed at 37 °C, centrifuged at 65 g in 45 ml thawing medium, and re-suspended in maintenance
medium. Cell viability and density were measured by a trypan blue exclusion assay. For metabolic
profiling, suspensions (1.0 ml) containing hepatocytes (0.5 million cells/ml) were incubated with 10 µM
of oprozomib for 1 hour. Reactions were conducted in 37 °C cell culture incubator with 100% relative
humidity, 95% air, 5% CO2. Final incubation contained 0.2% of dimethyl sulfoxide (DMSO). Reactions
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were terminated by addition of an equal volume of acetonitrile with 0.1% formic acid (FA). Control
incubation was prepared as follow: a mixture containing hepatocytes only was incubated for 1 hour,
quenched with acetonitrile with 0.1% FA, and then spiked with 10 µM of oprozomib. Supernatants (600
µl) were concentrated under a nitrogen stream and then reconstituted in 100 µl of 40% acetonitrile/water
(0.1% FA). Metabolite identification was conducted using a Thermo Oribtrap Q Exactive high-resolution
mass spectrometer (MS) equipped with a Thermo Accela LC system. Metabolite structures were assigned
based on the accurate mass obtained from a full scan mode at a resolution of 70,000 and MS2
fragmentation pattern from data-dependent acquisition at a resolution of 17,500. For the quantification of
oprozomib diol and PR-025 [(S)-2-amino-1-((R)-2-methyloxiran-2-yl)-3-phenylpropan-1-one] diol
formation in human hepatocytes, cells (0.25 ml, 0.5 million cells/ml) were incubated with 2 µM of
oprozomib in 12-well, collagen-coated plates (Thermo Scientific, CA). At each time point (0, 10, 20, 30,
and 60 min), reactions were terminated by the addition of an equal volume of acetonitrile containing 100
ng/ml internal standard (IS) d5-oprozomib. After centrifugation, supernatants were collected and
analyzed for oprozomib and the major metabolites by LC-MS/MS as described below.
Epoxide hydrolysis by recombinant human mEH and sEH in vitro. Recombinant human EHs were
kept at -80 °C until use. Briefly, incubations (0.1 ml) were conducted in 0.1 M Tris-HCl buffer (pH 9.0
for mEH; pH 7.5 for sEH) containing 0.1 mg/ml fatty acid-free bovine serum albumin (BSA) (Morisseau
et al., 2000; Morisseau et al., 2011). Enzyme activities of mEH and sEH (5 µg/ml) towards epoxide
hydrolysis were confirmed using their corresponding probe substrates (50 µM) cis-stilbene oxide (cis-SO)
and trans-stilbene oxide (trans-SO), respectively. In order to determine whether oprozomib is a substrate
for recombinant mEH or/and sEH, enzymes were pre-warmed for 5 min at 37 °C before the addition of
oprozomib (2 µM) to initiate the reaction. The enzyme concentration used was from 1 – 10 µg/ml for
mEH, and up to 100 μg/ml for sEH. After 0, 10, 20 or 30 min as indicated, reactions were terminated by
addition of 2 volumes of acetonitrile containing 0.1% FA and supernatants were collected for LC-MS/MS
analysis.
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Kinetics of oprozomib epoxide hydrolysis in recombinant mEH and human liver microsomes
(HLMs). Reaction kinetic parameters (Vmax and Km) were determined using recombinant mEH (2 µg/ml,
calculated based on 80% purity), oprozomib (10 – 600 µM) in 0.1 M Tris-HCl buffer (pH 9.0) containing
0.1 mg/ml BSA. Kinetic studies in male and female HLMs, were conducted in 0.1 M potassium
phosphate buffer (pH 7.4) containing 0.1 mg/ml BSA. The final microsomal protein concentration was
optimized to be 0.5 mg/ml and oprozomib was 10 – 600 µM. The final DMSO concentration was 0.5%.
Oprozomib was added to initiate the reaction after pre-warming enzymes for 5 min. Incubations (0.2 ml)
were conducted at 37 °C for 30 min and terminated by addition of 2 volumes of acetonitrile containing
100 ng/ml of IS d5-oprozomib. For kinetic studies of cis-SO, incubations (0.4 ml) were optimized and
performed using 4 μg/ml recombinant mEH or 0.1 mg/ml microsomal proteins, and cis-SO (1 – 200 μM)
at 37 °C for 10 min. The reaction was terminated by addition of one volume acetonitrile containing 50
μM trans-SO as IS. Supernatants were collected by centrifugation and analyzed by either UPLC-MS/MS
or UPLC linked with a photodiode array detector (PDA) as described below. Each set of data was fit to a
simple Michaelis-Menten kinetics model using nonlinear regression data analysis (GraphPad Prism v.6).
Each experimental reaction condition was conducted in triplicate.
NSPA was used as the selective mEH inhibitor to inhibit mEH enzyme activities in HLMs
(Morisseau et al., 2008). Briefly, pre-warmed pooled HLMs (0.5 mg/ml) were incubated with oprozomib
(10 μM) in the absence or presence of NSPA (0.02 – 100 μM) in 0.1 M potassium phosphate buffer (pH
7.4, 0.1 mg/ml BSA) at 37 °C. After 15 min, reactions were quenched with 2 volumes of acetonitrile
containing 0.1% FA and supernatants were isolated for quantitative analysis. The inhibitory effects of
NSPA on cis-SO (50 μM) hydrolysis were also determined under the similar conditions except for a lower
microsomal protein concentration (0.1 mg/ml) and shorter incubation time (10 min). The IC50 values
were estimated using nonlinear regression data analysis (GraphPad Prism v.6).
Inhibition of oprozomib epoxide hydrolysis in human hepatocytes in the presence of EH inhibitors.
Pooled cryopreserved human hepatocytes were thawed out and re-suspended in hepatocyte maintenance
medium, and cell viability and density were measured. Incubation mixtures (0.1 ml) contained human
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hepatocytes (0.5 million cells/ml), oprozomib (2 μM) or cis-SO (50 μM), and inhibitors NSPA or TPPU
(10 μM). The final solvent DMSO concentration was 0.2%. At each time point (0, 2, 5, 10, 20, 40, and
60 min), reactions were terminated by the addition of 2 volumes of acetonitrile containing IS either d5-
oprozomib or trans-SO. After centrifugation, supernatants (150 μl) were collected and analyzed using
LC-MS/MS or LC-PDA as described below.
Inhibition of CYP-mediated oprozomib metabolism in human hepatocytes by 1-aminobenzotriazole
(1-ABT). The role of CYPs on oprozomib metabolism in human hepatocytes was investigated using a
pan-CYP inhibitor 1-ABT. Pooled cryopreserved human hepatocytes were thawed at 37 °C, centrifuged
and re-suspended in hepatocyte maintenance medium. Incubations (0.25 ml) containing hepatocytes (0.5
million cells/ml) were pre-incubated with 0.5 mM 1-ABT or solvent (0.1% DMSO) for 30 min.
Oprozomib was added at a final concentration of 2 µM to initiate the reactions. Reactions were
conducted in a 37 °C cell culture incubator with 100% relative humidity, 95% air, 5% CO2. At each time
point (0, 2, 5, 10, 20, 40, and 60 min), reactions were terminated by the addition of an equal volume of
acetonitrile containing IS d5-oprozomib. After centrifugation, supernatants were collected for the
quantification of oprozomib disappearance and PR-176 formation by LC-MS/MS.
Metabolic stability of oprozomib in HLMs and recombinant CYPs. Metabolic stability of oprozomib
in pooled HLMs (mixed gender) was conducted in the presence and absence of cofactor NADPH. The
value of intrinsic clearance was determined by the rate of parent compound disappearance (Obach, 1999).
Briefly, oprozomib (1 µM) was mixed with liver microsomes (0.25 mg/ml) with or without 1 mM
NADPH in 0.1 M potassium phosphate buffer (pH7.4) containing 3.3 mM of MgCl2. The incubation
mixtures were kept at 37 °C for 1, 5, 10, 20, 30, and 40 min. The disappearance of oprozomib was
monitored using a LC-MS/MS method described below. In addition, a preliminary in vitro CYP
phenotyping study was conducted by incubating oprozomib (1 µM) with six major recombinant CYP
isoforms (125 pmol/ml) 1A2, 2D6, 3A4, 2C8, 2C9 and 2C19 individually, in the presence of 1 mM
NADPH at 37 °C for 0, 15, 30, and 60 min. The percentage of oprozomib remaining after incubation was
calculated by comparing the peak response with that from the zero-minute incubation.
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Inhibition of CYP activities by oprozomib. The relative potential of oprozomib to inhibit 6 major CYP
isoforms was assessed in pooled HLMs (mixed gender) with and without a 30 min pre-incubation. The
probe substrates used were phenacetin (CYP1A2), amodiaquine (CYP2C8), diclofenac (CYP2C9), S-
mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam and testosterone (CYP3A4/5). The
incubation mixtures (0.2 ml) contained oprozomib with various concentrations (0 – 30 μM), a probe
substrate, microsomal proteins and 1.5 mM NADPH in 0.1 M potassium phosphate buffer (pH 7.4) with 3
mM MgCl2 for 15 min. To examine time-dependent inhibition, oprozomib was pre-incubated with liver
microsomes for 30 min in the presence of NADPH, followed by the addition of probe substrates. The
concentrations of CYP probe substrates were approximately equal to their reported Km values. The
microsomal protein concentration was 0.05 or 0.1 mg/ml. The low protein concentrations were chosen to
minimize possible binding of oprozomib to microsomal proteins or lipids, as well as reduce oprozomib
metabolism via epoxide hydrolysis.
Quantification of oprozomib and its metabolites using LC-MS/MS. Quantification of oprozomib and
its major metabolites was performed using either an AB Sciex QTrap 4500 or 5500 MS equipped with an
electrospray ionization source. Chromatographic separation was achieved using a Waters XBridge BEH
C18 column (3.5 μ, 2.1 mm × 50 mm) and water (A)-acetonitrile (B) containing 0.1% FA as mobile
phases at a flow rate of 0.5 ml/min. The mobile phase started at 10% B for 0.5 min, and linearly
increased to 65% B within 1 min, held for 0.3 min before increasing to 95% B within 0.2 min, hold at
95% B for 1.5 min and returned to 10% B within 0.2 min. Multiple reaction monitoring of the transitions
m/z 533.0 -> 126.0, 551.3 -> 325.2, 206.0 -> 120, 224.0 -> 150.0, and 538.2 -> 126.1 was used to detect
oprozomib, PR-176, PR-025, PR-025 diol, and IS d5-oprozomib, respectively. Calibration curves were
generated by plotting the peak area ratio for each compound and IS versus the corresponding
concentrations of synthetic metabolite standards, and fitting with a linear regression equation.
Quantification of cis-SO, trans-SO, R,R-hydroxybenzoin and meso-hydroxybenzoin using LC-PDA.
Formation of R,R-hydroxybenzoin and meso-hydroxybenzoin from cis-SO and trans-SO hydrolysis was
used for measuring mEH and sEH activities in vitro, respectively. Quantification of these two diol
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products was performed using a Shimadzu LC-20AD system coupled with a PDA detector.
Chromatographic separation was achieved using a Waters XBridge BEH C18 column (2.1 mm × 100 mm,
3.5 μm) and water (A)-acetonitrile (B) containing 0.1% FA as mobile phases at a flow rate of 0.3 ml/min.
The mobile phase started at 10% B for 0.5 min, and linearly increased to 95% B at 5 min, hold for 2 min
before returning to initial 10% B within 0.5 min. The total running time was 9 min. Quantification of the
diol metabolites was calculated by fitting their peak area ratio (metabolites/IS) to the calibration curves
which generated by using authentic metabolite standards R,R-hydroxybenzoin and meso-hydroxybenzoin,
respectively.
Statistical analysis. GraphPad Prism v. 6 (GraphPad Software Inc., La Jolla, CA) was used for the
statistical analysis. Data represent mean ± S.D.. The means of the measured values of each treatment
group were compared using Student’s t-test. Means were considered statistically different from one
another if p < 0.05.
Results
Oprozomib metabolism in human hepatocytes. Incubation in cryopreserved human hepatocytes with
oprozomib for 60 min led to the formation of several metabolites, which were detected by LC-MS/MS
(Figure 2). A diol of oprozomib (also known as PR-176) was formed from direct epoxide hydrolysis
(Figure 2A). This appeared as the predominant metabolite based upon both MS ion signals and
ultraviolet absorbance. A peptide bond cleavage product with m/z at 245.0587 (cleavage on bond 2) was
also observed, albeit at relatively low levels. Another low abundance metabolite with m/z at 224.1276
(diol of PR-025) resulted from peptide cleavage (cleavage on bond 1) and epoxide hydrolysis was
detected. In addition, multiple trace metabolites including from hydroxylation or/and de-methylation,
direct GSH conjugation, and the combination of oxidation and epoxide hydrolysis or GSH conjugation,
were also observed.
Quantification of oprozomib disappearance and its major metabolite formation in hepatocyte
incubation was performed using synthesized standards. As shown in Figure 2B, PR-176 was readily
formed after incubation with oprozomib (2 µM). After 60 min incubation, nearly 55% of total oprozomib
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was recovered as the diol, about 7% was recovered as the diol of PR-025, and 20% remained as
oprozomib. These quantitative results suggest that epoxide hydrolysis is the major metabolic pathway for
oprozomib in human hepatocytes. Other minor pathways, such as peptide cleavage, oxidation and GSH
conjugation, combined to contribute to the remaining 18% of oprozomib from the initial incubation. It is
worth mentioning that in rat hepatocyte incubations, two oprozomib conjugates via direct GSH
conjugation were identified as major metabolites, in addition to PR-176 (Supplemental Figure 2).
Oprozomib hydrolysis by recombinant human mEH but not sEH. Since epoxide hydrolysis was
identified to be the major metabolic pathway, recombinant human mEH and sEH were used to investigate
the capacity of the different EH isoforms to hydrolyze oprozomib epoxide. Formation of PR-176
increased linearly with both incubation time (up to 30 min, data not shown) and mEH concentration (0.5 –
10 μg/ml) as shown in Figure 3A. In contrast, no significant formation of PR-176 was observed when
oprozomib was incubated with recombinant sEH for 30 min, at enzyme concentration up to 100 μg/ml. In
a parallel experiment, both mEH and sEH can readily convert their corresponding probe substrates, cis-
SO or trans-SO, respectively, in a time-dependent manner (Figure 3B).
The kinetics of oprozomib epoxide hydrolysis were subsequently evaluated using recombinant
mEH. The highest oprozomib concentration (up to 600 µM) was chosen based on its aqueous solubility
in the phosphate buffer system. An optimized protein concentration (2 μg/ml) and incubation time (30
min) were applied in the study to ensure the linearity over the course of the reaction with sufficient
amounts of PR-176 formation for accurate quantification. As shown in Figure 3C, formation of PR-176
increased with oprozomib concentration up to 600 μM without reaching a plateau. Fitting the data into
the Michaelis-Menten equation estimated the values of Km and Vmax of oprozomib at 974 ± 112 μM and
109 ± 8.6 nmol/min/mg proteins, respectively. While the values of Km and Vmax could not be more
accurately determined under the current assay conditions, the estimation suggests a relatively high Km for
oprozomib epoxide hydrolysis with recombinant mEH. As a control, the values of Km and Vmax of cis-SO
were determined to be 56.9 ± 3.8 μM and 648 ± 17 nmol/min/mg proteins, respectively, which was
comparable to previously reported literature values (Morisseau et al., 2011).
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Inhibition of oprozomib epoxide hydrolysis by a selective mEH inhibitor in HLMs. NSPA is a
selective mEH inhibitor that has been previously characterized with recombinant rat and human mEH for
its potency (Morisseau et al., 2008; Morisseau et al., 2011). Here, we evaluated its potency in inhibiting
hydrolysis of cis-SO and oprozomib in HLMs. As seen in Figure 4A, NSPA significantly inhibited cis-
SO hydrolysis with a mean IC50 value of 0.35 μM, and oprozomib diol formation with a mean IC50 value
of 0.51 μM in HLMs. Kinetics of oprozomib diol formation in both male and female HLMs were also
determined (Figure 4B). Similar to what was observed in the kinetic study with recombinant mEH
(Figure 3), formation of PR-176 essentially increased linearly with oprozomib concentrations without
reaching a plateau. As a result, the values of Km and Vmax could not be accurately estimated using
nonlinear regression analysis. Considering the Km values were likely over 5-fold higher than 50 µM, the
apparent ratios of Vmax/Km were roughly calculated from the slopes of PR-176 formation rate over low
oprozomib concentration range (10 – 50 μM). As a result, the mean Vmax/Km values were estimated as
8.43 and 3.20 μl/min/mg protein in male and female HLMs, respectively. In addition, the mean values of
Vmax/Km towards cis-SO and oprozomib epoxide hydrolysis in liver microsomes from rats and dogs were
also estimated (Supplemental Table 1). As a control, the estimated mean Vmax/Km value for cis-SO were
comparable to what reported in literature (Kitteringham et al., 1996).
Effect of the mEH inhibitor NSPA on oprozomib metabolism in human hepatocytes. Both NSPA
and TPPU were used to further determine the importance of mEH and sEH, respectively, on oprozomib
metabolism in human hepatocytes (Figure 5). The metabolic stability of two inhibitors TPPU and NSPA
in human hepatocytes were first determined. TPPU was stable in human hepatocytes over a one-hour
incubation, whereas NSPA was labile with a half time (t1/2) ~15 min. Thus, a concentration of NSPA (10
μM) 20-fold higher than the measured in vitro IC50 values on mEH activity was used in the human
hepatocyte study. The inhibitory effect of NSPA in human hepatocytes was first confirmed using cis-SO
as the probe substrate; over 80% inhibition of cis-SO hydration was observed within 10-min incubation,
however, inhibition was reduced to 60% with a longer incubation time (up to 20 min) (data not shown).
The rate of oprozomib metabolism in human hepatocytes was significantly inhibited (~80%) by NSPA
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co-treatment (9.7 ± 0.9 μl/min/106 cells) compared to vehicle DMSO control (40.7 ± 1.4 μl/min/106 cells).
Consistently, formation of PR-176 was also significantly decreased (>90% inhibition) by NSPA treatment
up to 60-min incubation (Figure 5B). In contrast, the sEH inhibitor TPPU did not significantly inhibit
oprozomib metabolism (35.0 ± 1.5 μl/min/106 cells) and PR-176 formation (Figure 5).
Metabolic stability of oprozomib in HLMs in the presence and absence of NADPH. Metabolic
identification and quantitation results (Figure 2 & 5) in human hepatocytes have suggested a minor role of
CYPs on oprozomib metabolism. HLM contains high hepatic mEH activity, and is also a common in
vitro system for assessment of CYPs-mediated metabolism. Therefore, microsomes were expected to be a
relevant in vitro system to study the metabolism of oprozomib. Interestingly, in pooled HLMs, the
presence of NADPH significantly increased the intrinsic clearance of oprozomib by 3.5-fold, as compared
to that in the absence of NADPH (70.7 ± 2.9 vs. 20.2 ± 1.8 µl/min/mg protein) (Figure 6A). These results
indicated a significant role of oxidation pathways over epoxide hydrolysis for oprozomib metabolism in
HLMs, which is not consistent with what was observed in human hepatocytes (Figure 2).
Oprozomib showed time-dependent inhibition of CYP3A4 in HLMs. In vitro inhibition potential of
oprozomib on major CYP isoforms (CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4/5) was also evaluated using
pooled HLMs (Table 1). Oprozomib did not inhibit the activities of CYP1A2, 2C8, 2C9, 2C19 and 2D6
at concentrations up to 30 µM with or without a 30 min pre-incubation. Oprozomib (30 µM) reduced the
activity of CYP3A4/5 by 45% and 33% as measured by testosterone 6β-hydroxylation and midazolam 1´-
hydroxylation assays, respectively, without pre-incubation. The inhibitory effect was further enhanced
with 30 min pre-incubation, where the IC50 values decreased to 12 and 5.5 µM with midazolam and
testosterone as the substrate, respectively (Table 1). This time-dependent inhibition was
NADPH-dependent and resistant to dilution (data not shown). The ratio of the maximum inactivation rate
constant (kinact) to the concentration of the inactivator that gives half the maximum rate of inactivation
(KI) was about 2.0 and 1.4 ml/min/µmol, respectively, with midazolam and testosterone as marker
substrates. In addition, a preliminary CYP phenotyping study using recombinant CYP isoforms showed
oprozomib was a substrate of CYP3A4 (Figure 6B).
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Effect of the pan-CYP inhibitor 1-ABT on oprozomib metabolism in human hepatocytes. In this
experiment, the rates of oprozomib metabolism in human hepatocytes in the absence and presence of a
pan-CYP inhibitor 1-ABT were compared. After pre-incubation with 1-ABT (0.5 mM) to inactivate CYP
enzymes (in control experiments, 1-ABT reduced testosterone oxidation by 90%), oprozomib
disappearance was modestly reduced by ~20% (clearance with 1-ABT: 31.6 ± 1.4 μl/min/106 cells;
without 1-ABT: 41.8 ± 1.2 μl/min/106 cells) (Figure 7A). As expected, 1-ABT did not affect the
formation of PR-176 (Figure 7B). In addition, pre-treatment of hepatocytes with 0.5 mM 1-ABT
completely blocked the formation of oprozomib metabolites from direct CYP-mediated oxidation, as well
as secondary products through oxidation and epoxide hydrolysis as shown in Figure 2.
Discussion
Peptide epoxyketone drugs represent a new class of proteasome inhibitors that are highly
efficacious for the treatment of patients with MM. As these inhibitors act via covalent modification, they
induce prolonged pharmacodynamic effects in spite of a short duration of systemic exposure (Yang et al.,
2011; Wang et al., 2013). Proteasome activity recovery primarily relies on the rate of de novo proteasome
synthesis, with a half-life of about 20-72 hours across a variety of tissues in preclinical species, except for
blood after dosing with oprozomib (Zhou et al., 2009; Dou and Zonder, 2014). Different from the first
generation epoxyketone proteasome inhibitor carfilzomib, which is administered intravenously due to the
lack of oral bioavailability, oprozomib demonstrated improved metabolic stability and was orally
bioavailable (Zhou et al., 2009). Yet, oprozomib still displayed high systemic clearance, a short half-life
and relatively high PK variability in preclinical species and patients (Fang et al., 2015; Infante et al.,
2016).
To understand the parameters governing the elimination of oprozomib, a number of in vitro
metabolism studies were conducted. As summarized in Scheme 1, the in vitro metabolism data showed
that hydrolysis of oprozomib epoxide to form the diol PR-176 by EH is the major metabolic pathway in
human hepatocytes, with peptide cleavage by peptidases, oxidation by CYPs, and direct GSH conjugation
representing minor alternative metabolic pathways. Consistent with the results in human hepatocytes,
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PR-176 was also found to be a major circulating metabolite in dogs, the non-rodent toxicity species, as
well as human plasma samples from a first-in-human clinical study (in-house unpublished data).
Interestingly, GSH conjugation, an alternative pathway to detoxify xenobiotic epoxides, was minor in
human hepatocytes. Although cryopreservation may reduce the in vitro GSH-conjugating capacity in
both human and rat hepatocytes (Sohlenius-Sternbeck and Schmidt, 2005), two GSH conjugates of
oprozomib were identified as major metabolites in addition to diol in cryopreserved rat hepatocytes. In
addition, multiple GSH conjugates were observed for clozapine, a positive control for GSH adduct
formation in both rat and human hepatocytes (Supplemental Figure 3). Consistent with the in vitro
observation, no detectable GSH conjugates were observed in human plasma and urine samples; on the
other hands, GSH conjugates were observed in vivo in rat plasma samples (in-house unpublished data).
These data indicate GSH conjugation plays a minor role in oprozomib metabolism in humans.
The predominant role of EHs on oprozomib metabolism could have implications on potential of
DDI and therefore may influence development strategy. Several EHs are found in mammals, among
them, two major isoforms, mEH and sEH, have been extensively characterized (Fretland and Omiecinski,
2000; Kodani and Hammock, 2015). Both isoforms are expressed ubiquitously throughout the
mammalian body with the highest expression generally in the liver (Gill and Hammock, 1980; Pacifici et
al., 1988; Coller et al., 2001). Collectively, data from recombinant EHs (Figure 3), liver microsomes
(Figure 4) and hepatocytes in the presence or absence of selective inhibitors (Figure 5) indicate that the
epoxide ring opening of oprozomib was catalyzed by mEH, but not sEH. Therefore, oprozomib PK is
unlikely to be affected by the co-administration with sEH inhibitors. On the other hand, oprozomib is
primarily metabolized by mEH, and amide-containing drugs such as valpromide or progabide have been
shown to inhibit in vivo mEH activity (Fretland and Omiecinski, 2000; Kodani and Hammock, 2015).
Thus, further studies may be warranted to determine whether drugs that are mEH inhibitors cause
clinically significant drug-drug interactions with oprozomib. Moreover, mEH is involved in the
metabolism of numerous xenobiotics, including 1,3-butadiene, naphthalene, carbamazepine and
phenytoin (Decker et al., 2009). It is possible that oprozomib acts as a mEH inhibitor that affects the
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clearance of co-administered drugs which are mainly metabolized by mEH. However, a preliminary in
vitro study in human liver microsomes showed that oprozomib does not significantly inhibit cis-SO
epoxide hydrolysis at a concentration up to 100 µM (data not shown).
The high variability of oprozomib PK observed in preclinical species and patients could be
attributed to variability of absorption and/or metabolism by mEH. Expression of mEH in ex vivo tissues
was shown to vary considerably among individuals (up to 8-fold) (Hassett et al., 1997), and that
expression is affected by age, sex, diseases, and environmental factors (Vaclavikova et al., 2015). In
addition, two human polymorphisms have been identified in the coding region of the mEH gene: exon 3
(a Tys113His mutation) resulting in a 50% decrease in enzyme activity in vitro and exon 4 (a His139Arg
mutation) resulting in a 25% increase in enzyme activity in vitro (Pinarbasi et al., 2010). The variant
alleles, however, appeared to exert only a modest impact on mEH enzymatic activities (within two fold)
in vivo (Kroetz et al., 1993; Hosagrahara et al., 2004). Based on this information and the high systemic
clearance of oprozomib, inter-individual difference in mEH activity may contribute to the high inter-
individual PK variability of oprozomib in patients (Infante et al., 2016), but it is unlikely to be the main
factor. Variable absorption, particularly associated with a narrow absorption window, may be another a
key factor to oprozomib PK variability (Teague et al., 2013).
In contrast to the major role of mEH, a minor role of CYP on oprozomib metabolism was
observed in human hepatocytes (Figure 7). Using the pan-CYP inhibitor 1-ABT, a modest decrease in
oprozomib disappearance (< 20%) was observed. This corroborates the result that oxidative products are
minor metabolites in human hepatocytes (Figure 2), as well as in plasma samples from patients (in-house
unpublished data). Thus, although oprozomib is a substrate of CYP3A4, co-administration of oprozomib
with other drugs that are CYP modulators would be unlikely to significantly alter the PK of oprozomib.
In addition, oprozomib did display time-dependent inhibition of CYP3A4/5 in HLMs (Figure 6),
however, the inhibitory effect was not manifested in vivo in a clinical DDI study where oprozomib was
administered orally and co-administered with midazolam (Tsimberidou et al., 2016).
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Choosing relevant in vitro systems is critical in understanding the metabolism and associated DDI
potentials for oprozomib and other peptide epoxyketone inhibitors. When studying oprozomib
metabolism in HLMs (Figure 6), a higher level of CYP-mediated metabolism was observed in contrast to
the results observed in human hepatocytes (Figure 7). Moreover, the high level of oxidative metabolites
in HLMs were also not consistent with the in vivo human metabolite profiling results (in-house
unpublished data). The distinct observation of oprozomib metabolism in HLMs vs. human hepatocytes
might be due to a significant loss of mEH activity during microsomal subcellular fractionation. In
hepatocytes, beside endoplasmic reticulum membrane, mEH is also present on the plasma membrane
facing the extracellular medium (Levy, 1996), thus a significant portion of the hepatocyte mEH activity
might be lost when preparing microsomes. On the other hand, CYP activities are concentrated in
endoplasmic reticulum membrane, which could explain why HLM incubation may bias metabolism
toward CYP oxidation.
Furthermore, it is worth discussing the limitations of using hepatocytes in evaluation of the
metabolism and extrapolation of the in vivo clearance of peptide epoxyketone analogues. Hepatocytes
provided consistent metabolic profiles for oprozomib with those from in vivo samples. However, the
rates of oprozomib disappearance and diol formation from in vitro hepatocyte incubation would under-
predict the in vivo clearance of oprozomib, which was measured higher than hepatic blood flow in
preclinical species (Fang et al., 2015). There are several possible reasons. First, the rate of diol formation
obtained from in vitro kinetic incubation may underestimate the efficiency of mEH. For EH-mediated
hydrolysis, the epoxide is rapidly trapped as a covalent hydroxyl alkyl enzyme intermediate. The rate of
diol formation is limited by the slow hydrolysis of the intermediate (Armstrong, 1999). Second, the
activity of epoxide hydrolases is generally not well characterized in commercial preparations. The effects
of reagents and in vitro preparation procedure on epoxide hydrolase activity have not been systematically
studied to the best of our knowledge. Third, extrahepatic metabolism also plays an important role in the
elimination of this series of compounds because EHs and peptidases are ubiquitously expressed
throughout the body. Therefore, the rate of metabolism in hepatocytes could be used simply as a tool to
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rank order analogues for prioritizing in vivo studies instead of extrapolating clearance during lead
optimization.
In summary, in vitro characterization of oprozomib metabolism revealed that epoxide hydrolysis
mediated by mEH, rather than sEH, is the major metabolic pathway for oprozomib metabolism, consistent
with findings from in vivo samples. Oxidative metabolism plays a minor role in oprozomib elimination.
These data suggest CYP and sEH activity modulators when co-administered with oprozomib would
unlikely significantly affect oprozomib PK. On the contrary, the results in the present study indicate that
mEH inhibitors administered concomitantly with oprozomib may affect oprozomib PK. Further studies
may be warranted to determine whether drugs that are mEH inhibitors cause clinically significant DDIs
with oprozomib. Hepatocytes serve as a good in vitro system to assess the metabolic profiles of peptide
epoxyketone inhibitors but generally under-estimate the rate of in vivo clearance of oprozomib.
Metabolic findings using liver microsomes should be interpreted with caution as the microsomal system
may be biased toward CYP-mediated metabolism and underestimates non-CYP metabolism for peptide
epoxyketones.
Acknowledgement
We thank to Drs. Larry Wienkers, Ji Ma, Brooke Rock, and Lixia Jin from the Department of
Pharmacokinetics and Drug Metabolism, Amgen Inc. for reviewing the manuscript and helpful
suggestions on study design and data analysis.
Authorship Contributions
Participated in research design: Wang, Fang, and Wang
Conducted experiments: Wang, Fang, Teague, and Wong
Contributed new reagents or analytic tools: Morisseau, Hammock
Performed data analysis: Wang, Fang, Teague, Wong, Morisseau, Rock, and Wang
Wrote or contributed to the writing of the manuscript: Wang, Fang, Morisseau, Hammock, Rock, and
Wang
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DMD # 75226
Footnotes
The work was in part supported by the National Institute of Environmental Health Sciences grant [R01
ES002710].
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DMD Fast Forward. Published on April 20, 2017 as DOI: 10.1124/dmd.117.075226
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 75226
Legends for Figures
Figure 1. Structures of carfilzomib and oprozomib. The numbers 1, 2, and 3 labeled on the structure
indicate three possible cleavage sites of oprozomib by peptidases. PR-025 is the cleavage product
containing an epoxyketone at site 1 of oprozomib.
Figure 2. In vitro metabolism of oprozomib in human hepatocytes. A) Representative ion
chromatograms of oprozomib metabolites formed in human hepatocytes. Dash line: control sample, cell
suspensions spiked with oprozomib; Solid line: cell suspensions after 1-hour incubation with oprozomib.
B) Quantitative oprozomib (OPZ) disappearance and metabolite formation in human hepatocytes. At
each time point, the levels of oprozomib, PR-176 and PR-025 diol in cell suspensions were quantified and
then normalized as the percentage of initial oprozomib concentration at zero-minute. Data represent
mean ± S.D. from three replicate incubations. PR-176 was the predominant metabolite formed via
epoxide hydrolysis within one-hour incubation.
Figure 3. Epoxide hydrolysis by recombinant microsomal and soluble EHs. A) Epoxide hydrolysis
of oprozomib by recombinant human mEH and sEH. Formation of PR-176 was quantified after
incubation of oprozomib (2 µM) with various concentrations of enzymes for 30 min. A pilot study has
shown a linear formation of PR-176 within 30 min under current conditions. B) Epoxide hydrolysis of
cis-SO and trans-SO (50 µM) by mEH and sEH (5 µg/ml), respectively. Significant diol product
formation was observed after incubation for 10 or 20 min, confirming the hydrolysis activities of EHs. C)
Kinetics of the formation of PR-176 using recombinant mEH. A range of oprozomib concentrations were
incubated with mEH (2 µg/ml) at 37 oC for 30 min. D) Kinetics of cis-SO hydrolysis using recombinant
mEH. A series of cis-SO concentrations were incubated with mEH (4 μg/ml) at 37 °C for 10 min. Data
represent mean ± S.D. from three replicate incubations. The Km and Vmax values were estimated by fitting
the curve into the Michaelis-Menten equation.
Figure 4. Inhibition and kinetics of PR-176 formation in HLMs. A) Inhibition of mEH activity by
NSPA in HLMs. Pooled HLMs (0.5 mg/ml) were incubated with oprozomib (10 μM) or cis-SO (50 µM)
in the presence of various concentrations of NSPA at 37 °C for 15 min as indicated. The IC50 values were
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DMD Fast Forward. Published on April 20, 2017 as DOI: 10.1124/dmd.117.075226
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 75226
estimated using nonlinear regression data analysis. B) Kinetics of PR-176 formation in male and female
HLMs was conducted in 0.1 M potassium phosphate buffer (pH 7.4, 0.1 mg/ml BSA). Incubations were
performed at 37 oC for 30 min. Data were fitted into the Michaelis-Menten equation for the calculation of
Km and Vmax values. Data represent mean ± S.D. from triplicate incubations.
Figure 5. Inhibition of epoxide hydrolysis of oprozomib in human hepatocytes by the mEH
inhibitor NSPA, but minimal inhibition by the sEH inhibitor TPPU. A) Percentage of oprozomib
remaining in human hepatocytes. B) Formation of diol PR-176 in human hepatocytes. Cryopreserved
human hepatocytes were incubated with oprozomib (2 μM) in the presence of NSPA, TPPU or vehicle
0.1% DMSO for various incubation times. Both oprozomib disappearance and PR-176 formation were
quantified using LC-MS/MS. Data represent mean ± S.D. from triplicate incubations. The percentage of
oprozomib was calculated using the remaining oprozomib concentration divided by zero-minute
oprozomib concentration.
Figure 6. Metabolism of oprozomib by HLMs and recombinant CYPs. A) Percentage of oprozomib
remaining in pooled HLMs in the presence or absence of cofactor NADPH. Initial oprozomib
concentration was 1 µM. B) CYP phenotyping on oprozomib oxidation using six major CYP isoforms.
Oprozomib was incubated with 6 recombinant CYP isoforms for various incubation times, individually.
The loss of oprozomib was quantified using LC-MS/MS assays. Data represent mean ± S.D. from
triplicate incubations.
Figure 7. Inhibition of CYP activities for oprozomib in human hepatocytes by 1-ABT. A)
Percentage of oprozomib remaining in human hepatocytes. B) Formation of diol PR-176 in human
hepatocytes. Cryopreserved human hepatocytes were incubated with oprozomib (2 μM) after pre-
incubation with 1-ABT (0.5 mM) or 0.1% DMSO as vehicle for 30 min. Both oprozomib disappearance
and PR-176 formation after various incubation times were quantified using LC-MS/MS. Data represent
mean ± S.D. from triplicate incubations. The percentage of oprozomib was calculated using the
remaining oprozomib concentration divided by zero-minute oprozomib concentration.
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DMD Fast Forward. Published on April 20, 2017 as DOI: 10.1124/dmd.117.075226
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 75226
Scheme 1. Proposed major metabolic pathways of oprozomib in human hepatocytes. Oprozomib is
predominantly metabolized by mEH in human hepatocytes, whereas CYPs, peptidases, and GST/GSH
play minor roles. Oxidative and cleavage products containing an epoxyketone could be further
metabolized via epoxide hydrolysis.
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DMD Fast Forward. Published on April 20, 2017 as DOI: 10.1124/dmd.117.075226
This article has not been copyedited and formatted. The final version may differ from this version.
DMD # 75226
Table 1. In vitro evaluation of oprozomib as an inhibitor of six human CYP enzymes. Results
representing the average data (i.e., percent of control activity) obtained from duplicate samples from each
test article concentration were used to calculate IC50 values. The IC50 values were calculated using
Graphpad Prism (v.5). The highest concentration of oprozomib tested in the assays was 30 µM.
Direct and time-dependent inhibition of human CYPs by oprozomib
Direct inhibition
0-minute pre-incubation
Inhibition at
IC50 (µM)
>30
Time-dependent inhibition
30-minute pre-incubation
Inhibition at
IC50 (µM)
>30
Enzyme
CYP1A2
CYP reaction
Phenacetin O-deethylation
Amodiaquine N-
dealkylation
Diclofenac 4´-
hydroxylation
S-Mephenytoin 4´-
hydroxylation
Dextromethorphan O-
demethylation
30 µM (%)a
21
30 µM (%)a
NA
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP3A4/5
CYP3A4/5
>30
>30
>30
>30
>30
>30
20
6.3
16
>30
>30
>30
>30
5.5
13
24
7.2
3.7
89
0.2
45
Testosterone 6β-
hydroxylation
Midazolam 1´-
33
12
68
hydroxylation
a: The inhibition (%) was calculated with the following formula and data for the highest concentration of
test article evaluated: Inhibition (%) = 100% – percent activity remaining of solvent control.
NA: Not applicable. No value was obtained as the rates at the highest concentration of oprozomib
evaluated (30 µM) were higher than the control rates.
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