Download Expression and Characterization of Cynomolgus Monkey

Supplemental material to this article can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/12/13/dmd.113.055491.DC1
1521-009X/42/3/369–376$25.00
DRUG METABOLISM AND DISPOSITION
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.113.055491
Drug Metab Dispos 42:369–376, March 2014
Expression and Characterization of Cynomolgus Monkey
Cytochrome CYP3A4 in a Novel Human Embryonic Kidney
Cell–Based Mammalian System s
Sindhuja Selvakumar, Priyadeep Bhutani, Kaushik Ghosh, Prasad Krishnamurthy,
Sanjith Kallipatti, Sabariya Selvam, Manjunath Ramarao, Sandhya Mandlekar, Michael W. Sinz,
A. David Rodrigues, and Murali Subramanian
Pharmaceutical Candidate Optimization (P.B., M.S.) and Applied Biotechnology (Si.S., K.G., P.K., S.K., Sa.S.), Biocon Bristol-Myers
Squibb Research and Development Center (BBRC), Syngene International Limited, Plot No. 2 & 3, Bommasandra IV Phase,
Bangalore, India; Bristol-Myers Squibb, Wallingford, Connecticut (M.W.S.); Bristol-Myers Squibb, Pennington, New Jersey (A.D.R.);
and Bristol-Myers Squibb India Ltd. BBRC, Bangalore, India (M.R., S.M.)
Received October 18, 2013; accepted December 13, 2013
Cynomolgus monkeys are a commonly used species in preclinical
drug discovery, and have high genetic similarity to humans,
especially for the drug-metabolizing cytochrome P450s. However,
species differences are frequently observed in the metabolism of
drugs between cynomolgus monkeys and humans, and delineating
these differences requires expressed CYPs. Toward this end,
cynomolgus monkey CYP3A4 (c3A4) was cloned and expressed in
a novel human embryonic kidney 293-6E cell suspension system.
Following the preparation of microsomes, the kinetic profiles of five
known human CYP3A4 (h3A4) substrates (midazolam, testosterone,
terfenadine, nifedipine, and triazolam) were determined. All five
substrates were found to be good substrates of c3A4, although
some differences were observed in the Km values. Overall, the data
suggest a strong substrate similarity between c3A4 and h3A4.
Additionally, c3A4 exhibited no activity against non-h3A4 probe
substrates, except for a known human CYP2D6 substrate (bufuralol), which suggests potential metabolism of human cytochrome
CYP2D6-substrates by c3A4. Ketoconazole and troleandomycin
showed similar inhibitory potencies toward c3A4 and h3A4,
whereas non-h3A4 inhibitors did not inhibit c3A4 activity. The
availability of a c3A4 preparation, in conjunction with commercially
available monkey liver microsomes, will support further characterization of the cynomolgus monkey as a model to assess CYP3Adependent clearance and drug-drug interactions.
Introduction
prediction can be accomplished in a logical, mechanistic manner if
monkey-human species differences are proven for the implicated
P450. Given that greater than 90% homology exists for most of the
P450s between the two species, cynomolgus monkey CYP2C20,
2C43, 2C75, and 3A8 have been renamed CYP2C8, 2C9, 2C19, and
3A4, respectively (Iwasaki and Uno, 2009; Emoto et al., 2013). In
addition, it has been proposed that 2B17 and 2B30 be renamed to 2B6,
2C74 to 2C8, 2C83 to 2C9, 2F6 to 2F1, 3A64 to 3A4, 3A66 to 3A5,
and 4F45 to 4F2 (Uno et al., 2011).
While excellent homology between human and cynomolgus monkey P450s would indicate similar PK of their respective substrates,
a number of studies have found that cynomolgus monkeys are characterized by higher first-pass metabolism than humans (Sietsema,
1989; Chiou and Buehler, 2002; Ward and Smith, 2004; Nishimura
et al., 2007; Komura and Iwaki, 2008; Takahashi et al., 2009, 2010).
One study found that 75% of 16 tested compounds in cynomolgus
monkeys showed significantly lower bioavailability than humans,
even though clinically these are orally administered drugs (Takahashi
et al., 2009; Nishimuta et al., 2011). The authors showed that the
During drug discovery and development, heavy reliance is placed
on utilizing preclinical species to predict human pharmacokinetics
(PK), pharmacology, and toxicity. Cynomolgus monkeys (Macaca
fascicularis) are extensively used in this regard since they are one of
the phylogenetically closest species to humans, apart from nonhuman
primates. A comprehensive assessment has suggested that human PK
is most reliably predicted from monkeys more so than from rats or
dogs, which show a significantly different drug-metabolizing enzyme
homology from humans (Ward and Smith, 2004). Among drugmetabolizing enzymes, cytochrome P450s (P450s) play the most
significant role in determining human PK parameters, and hence
understanding monkey-human P450 species differences is critical to
predicting human exposures of novel chemical entities based on
monkey data. Alternatively, excluding monkey data from a human PK
dx.doi.org/10.1124/dmd.113.055491.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: c3A4, cynomolgus cytochrome CYP3A4; c3A5, cynomolgus cytochrome CYP3A5; CID, compound identifier; Clint, intrinsic
clearance (Vmax/Km); CPR, NADPH–cytochrome P450 reductase; Fg, the fraction escaping intestinal (gut) metabolism; h3A4, human cytochrome
CYP3A4; HEK, human embryonic kidney; HLM, human liver microsomes; IC50, the inhibitor concentration at which the metabolism of a given
substrate is reduced by 50%; Km, the substrate concentration at half of Vmax; KTZ, ketoconazole; MDZ, midazolam; MkLM, monkey (cynomolgus)
liver microsomes; P450, cytochrome P450; PCR, polymerase chain reaction; PK, pharmacokinetics; TAO, troleandomycin; TRZ, triazolam; TST,
testosterone; Vmax, the maximal rate of formation of a metabolite (enzyme capacity).
369
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ABSTRACT
370
Selvakumar et al.
Materials and Methods
Materials. Glycerol [PubMed compound identifier (CID) 753], potassium
phosphate buffer (PubMed CID 516951), EGTA (PubMed CID 6207), human
NADPH–cytochrome P450 reductase (CPR), potassium chloride (PubMed CID
4873), sucrose (PubMed CID 4988), MDZ (PubMed CID 4192), 19-OH MDZ
(PubMed CID 107917), nifedipine (PubMed CID 4485), triazolam (TRZ;
PubMed CID 5556), 4-OH TRZ (PubMed CID 128260), phenacetin (PubMed
CID 4754), acetaminophen (PubMed CID 1983), dextromethorphan (PubMed
CID 5360696), dextrorphan (PubMed CID 5360697), diclofenac (PubMed CID
3033), 49-OH-diclofenac (PubMed CID 116545), coumarin (PubMed CID
323), 7-hydroxycoumarin (PubMed CID 5281426), bupropion (PubMed
CID 444), hydroxyl bupropion (PubMed CID 9837966), paclitaxel (PubMed
CID 36314), chlorzoxazone (PubMed CID 2733), ketoconazole (KTZ;
PubMed CID 47576), troleandomycin (TAO; PubMed CID 202225), furafylline
(PubMed CID 3433), clopidogrel (PubMed CID 60606), sulphaphenazole (PubMed
CID 5335), benzylnirvanol (PubMed CID 40633600), quinidine (PubMed CID
441074), diethyldithiocarbamate (PubMed CID 28343), terfenadine alcohol
(PubMed CID 3348), oxidized nifedipine (PubMed CID 128753), montelukast
(PubMed CID 5281040), and tranylcypromine (PubMed CID 441233) were
purchased from Sigma (St. Louis, MO). 19-Hydroxy bufuralol (PubMed CID
162836), h3A4, testosterone (TST; PubMed CID 6013), 6b -hydoxy TST
(PubMed CID 65543), 6a-hydroxytaxol (PubMed CID 10056458), bufuralol
(PubMed CID 71733), and hydroxyl chlorzoxazone (PubMed CID 2734)
were purchased from BD Biosciences (San Jose, CA). Pooled male human
liver microsomes (HLM) and monkey liver microsomes (MkLM) were
purchased from Xenotech (Lenexa, KS). Liquid chromatography–grade
acetonitrile, dimethylsulfoxide, and ethanol were purchased from E Merck
Limited (Mumbai, India). MultiScreen Solvinert filter plates (0.45 mM, low
binding hydrophilic PTFE [Polytetrafluoroethylene]) were purchased from
Millipore (Billerica, MA). All aqueous solutions were prepared in Mili-Q
water (Millipore). F17 media, pluronic F68, and glutamine were obtained from
Invitrogen (Carlsbad, CA); tryptone N1 from Organotechnie (La Courneuve,
France); and PEI (Polyethylenimine) from Polysciences (Warrington, PA) for the
cell culture maintenance. The primary antibodies used for Western blot
experiments were anti-h3A4 (C-17) IgG (Santa Cruz, Dallas, TX) and antihuman
CPR antibody (Abcam, Cambridge, UK). The secondary antibodies used were
donkey antigoat IgG-AP (alkaline phosphatase) (Santa Cruz) and goat antirabbit
IgG-AP conjugate (Bio-Rad, Hercules, CA). All other chemicals were of
analytical grade.
Cloning of c3A4 and Cynomolgus NADPH-P450 Reductase. c3A4 and
cynomolgus CPR were cloned from cynomolgus monkey liver total RNA
obtained from Biochain (Newark, CA). cDNA was prepared using Protoscript
First Strand cDNA Synthesis Kit from New England BioLabs (Ipswich, MA).
Primers for amplifying c3A4 were designed against the common chimpanzee
(Pan troglodytes) CYP3A4 sequence, and primers for amplifying cynomolgus
CPR were designed against the rhesus (Macaca mulatta) CPR sequence. KOD
(Thermococcus kodakaraensis) enzyme (Novagen, Billerica, MA) was used for
polymerase chain reaction (PCR) amplification, and PCR products were gel
purified using Qiagen’s gel extraction kit (Venlo, The Netherlands). DNA
fragments were cloned into a pTZ57R/T vector using standard molecular
biology techniques, and clones were confirmed by DNA sequencing.
Mammalian Expression Constructs. Both c3A4 and cynomolgus CPR
were PCR amplified using their de novo clones as templates and cloned into
a pDONR221 vector from Invitrogen to create an entry clone. Subsequently,
these genes were moved into a pTT Gate vector which was a derivative of the
original pTT vector (Durocher et al., 2002) through LR reaction using LR
clonase from Invitrogen, as per the manufacturer’s protocol. Final expression
constructs were sequence confirmed and used for transient transfections.
Expression of c3A4 in HEK293-6E Cells. The HEK293-6E cells were
cultured in serum-free F17 medium supplemented with 4 mM glutamine and
0.1% pluronic F68. The cells were maintained at 37°C in a 5% CO2 atmosphere
with shaking at 130 rpm in the tissue culture incubator. On the day of
transfection, the cells were seeded at a cell density of 1.1 106 cells/ml and
cotransfected with 1 mg of c3A4 construct and 200 mg of P450 reductase
construct (5:1 ratio) per liter of culture using PEI as a transfection reagent
(Durocher et al., 2002). After 24 hours of transfection, cells were fed with 0.5%
of tryptone N1 to increase recombinant protein production (Pham et al., 2005)
and incubated for 48 hours.
Preparation of Microsomes from HEK293-6E Cells. The transfected
HEK293-6E cells were centrifuged at 3000 rpm for 15 minutes, and the pellet
was washed with 1 phosphate-buffered saline for 10 minutes. The cell pellet
was resuspended in hypotonic buffer (100 mM potassium phosphate buffer, pH
7.5, 1 mM EGTA, 25 mM KCl, 10% glycerol), incubated for 20 minutes at 4°C,
and spun at 1000 g for 20 minutes. The cell pellet was further lysed using a
dounce homogenizer in isotonic buffer containing 0.25 M sucrose and subjected to differential centrifugation at 1000 g, 12,000 g, and 100,000 g.
The final pellet was resuspended in 100 mM potassium phosphate, pH 7.5, and
10% glycerol and dialyzed against the same buffer overnight. All of the
previously described steps were carried out at 4°C. The protein content was
determined by Bradford’s method, and the sample was further analyzed by
Western blot analysis and activity analyses.
Immunoblot Analysis. Different concentrations of the microsome samples
were electrophoresed by SDS-PAGE and transferred onto the nitrocellulose
membrane. The blot was blocked with 5% skimmed milk powder for 2 hours at
room temperature and then incubated with goat anti-h3A4 IgG (1:500)
overnight. The blot was then incubated with donkey antigoat IgG-AP (1:5000)
for 45 minutes at room temperature, and the bands were visualized using the
nitro-blue tetrazolium and 5-bromo-4-chloro-39-indolyphosphate substrate. For
detection of CPR, incubation with rabbit anti-CPR antibody (1:500) for 4 hours
was followed by goat antirabbit IgG-AP (1:3000) for 1 hour.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
fraction of oral dose absorbed was similar between species, whereas
the fraction escaping intestinal (gut) metabolism (Fg) in cynomolgus
monkeys was close to zero, which was corroborated by higher intrinsic
clearance in cynomolgus monkey intestinal microsomes as compared
with human intestinal microsomes. Additionally, a much higher intestinal extraction was observed in monkeys, as compared with humans, for midazolam (MDZ), nifedipine, amitriptyline, propranolol,
and timolol, drugs metabolized by human CYP3A4 (h3A4), 2C19,
2D6, and 1A2, respectively, suggesting that the lower Fg in cynomolgus monkeys is not exclusive to h3A4 substrates (Akabane et al.,
2010; Yoda et al., 2012). Because of the high levels of CYP3A5
expression in the jejunum, it has been speculated that CYP3A5 could
play an important role in the low cynomolgus monkey Fg (Nishimuta
et al., 2011). Additionally, total P450 content (based on the carbon
monoxide difference spectrum) is ;700 pmol/mg in cynomolgus monkeys, but the immunoquantitated amount of CYP3A4 therein is lower
(;100 pmol/mg). In contrast, the ratio of spectral to immune-quantified
levels of CYP3A4 in human liver microsomes (HLM) is less than 2
(Uehara et al., 2010; Emoto et al., 2013). This raises the possibility
that novel cynomolgus monkey CYPs such as CYP4A and CYP4F
may be abundantly expressed in cynomolgus monkeys, which may
contribute further to species differences (Uehara et al., 2010; Emoto
et al., 2013).
Approximately 75% of the drugs on the market are cleared by
P450s, and CYP3A subfamily members are responsible for the
metabolism of more than 50% of such drugs. The homology between
cynomolgus CYP3A4 (c3A4) and h3A4 is 93% (amino acid sequence), while for cynomolgus CYP3A5 (c3A5) and human CYP3A5,
the homology is 91% (amino acid) (Emoto et al., 2013). The excellent
amino acid homology between human and cynomolgus monkey 3A
enzymes suggests similar enzyme behavior. However, no detailed
kinetic and inhibition analyses of c3A4 have been performed thus
far, although publications have detailed the expression and purification of c3A4 (Uno et al., 2007; Iwasaki et al., 2010; Ohtsuka et al.,
2010; Emoto et al., 2011). In this manuscript, we present a novel
human embryonic kidney (HEK) 293-6E system for the expression
of c3A4 together with a detailed in vitro characterization of the
enzyme. Species similarities and differences between h3A4 and c3A4
are also discussed in terms of substrate enzyme kinetics and inhibitory
potencies.
371
Expression and Characterization of Cynomolgus CYP3A4
200 mM substrate in c3A4, MkLM, and h3A4 with a protein concentration of
50 nM for c3A4 and h3A4 and 1 mg/ml for MkLM. Other incubation variables
remained identical to previously described conditions.
Inhibition Studies. Inhibition of c3A4, h3A4, and MkLM was assessed
using MDZ as a substrate at a concentration of 5mM, which was the determined
substrate concentration at half of Vmax (Km) for MDZ. The protein
concentration was 10 nM for c3A4/h3A4 and 0.5 mg/ml for MkLM with an
incubation time of 10 minutes, conditions under which the reaction was
determined to be in the linear range. The concentrations of KTZ ranged from
0.0025 to 0.16 mM with an extra solvent control not containing KTZ. The
inhibition potency of TAO, a mechanism-based inhibitor, was determined with
and without a primary incubation to determine the reversible and irreversible
IC50, and the shifted IC50 value. The reversible IC50 experiment, performed in
the absence of a primary incubation, was conducted with a protein concentration of 10 nM, MDZ concentration of 5 mM, and TAO concentrations
ranging from 0.1 to 40 mM, with a 10-minute incubation. The irreversible IC50
was determined after a 20-minute primary incubation wherein 0.1–40 mM TAO
was incubated with 10 nM c3A4/h3A4 in the presence of 2 mM NADPH. The
secondary incubation was performed for 10 minutes with a further supplementation of NADPH and the addition of 5 mM MDZ (final concentration).
The formation of 19-OH-MDZ was a surrogate for activity remaining.
The inhibition of non-h3A4 inhibitors on MDZ activity was also assessed in
c3A4 and MkLM. The list of inhibitors along with their concentration is
summarized in Supplemental Table 3. Furafylline (20 mM), tranylcypromine
(10 mM), clopidogrel (10 mM), montelukast (10 mM), sulphaphenazole (10 mM),
benzylnirvanol (10 mM), quinidine (10 mM), and diethyldithiocarbamate
(20 mM) were used to inhibit CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and
2E1, respectively. The incubation contained 5 mM MDZ, 10 nM c3A4 or 1 mg/ml
MkLM, and 2 mM NADPH and was performed for 30 minutes at 37°C in
100 mM KPO4 at pH 7.4. A 20-minute preincubation, in the presence of
NADPH, was performed for furafylline since it is a known mechanism-based
inhibitor of CYP1A2. The inhibitor concentrations were chosen to be many folds
over their IC50 for h3A4.
Data Analyses. The velocity versus substrate concentration data from the
kinetics experiments were observed visually and from an Eadie-Hofstee plot,
and subsequently fit to the appropriate equation. For typical hyperbolic
kinetics, the Michaelis-Menten equation was used (eq. 1):
v¼
Vmax *½S
Km þ S
ð1Þ
For biphasic kinetics, wherein a low Km and Vmax site and a high Km and
Vmax site metabolize substrates simultaneously, eq. 2 was used (Tracy, 2003):
v¼
ðVmax1 *½SÞ þ ðClint2 *S2 Þ
Km1 þ S
ð2Þ
For substrate inhibition kinetics, wherein a low Vmax and high Km site
predominates metabolism at high concentrations, eq. 3 was used (Tracy, 2003):
v¼
Vmax
1 þ K½Sm þ ½S
Ki
ð3Þ
For homotrophic positive cooperativity (sigmoidal or allosteric metabolism),
eq. 4 was used (Tracy, 2003):
v¼
Vmax •½Sn
K9 þ ½Sn
ð4Þ
For a combination of allosteric and substrate inhibition, eq. 5 was derived
and used:
v¼
Vmax
n
½S
Km
1 þ ½S
n þ
Ki
ð5Þ
In all these equations, v represents velocity of formation of a metabolite
(amount of metabolite per unit time and unit protein concentration), Vmax the
maximal rate of formation of a metabolite (enzyme capacity), S the substrate
concentration, Km the substrate concentration at half of Vmax, Clint2 the slope of
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
Activity Determination. Initial activity of the preparation was assessed by
the formation of 19-OH-MDZ upon incubation of the pellet with 20 mM MDZ
in the presence of 2 mM NADPH in potassium phosphate buffer (pH 7.4) at 37°C
for 30 minutes. Only pellets that showed robust activity and a discernible CO
difference spectrum were considered for further scale-up. The CO difference
spectrum was determined by performing a 20 to 100 dilution of the pellet in
potassium phosphate buffer (100 mM, pH 7.4) and measuring the absorbance in
a single-beam Tecan (Maennedorf, Switzerland) UV absorbance detector by
scanning from 400 to 500 nm in 2-nm step sizes (Omura and Sato, 1964). An
extinction coefficient of 11.99 was used for the determination of the 3A4
activity.
CPR Activity. The amount of CPR present was determined as described
previously (Venkatakrishnan et al., 2000). Briefly, 90 ml of 0.45 mg/ml
cytochrome c, as a substrate, was mixed with 5 ml of differing concentrations of
purified human CPR (standards) or 5 ml of c3A4 (unknown). Five microliters
of NADPH (0.85 mg/ml) was added to catalyze this reaction, and the
absorbance was measured at 550 nm continuously at 25°C. Absorbances of
samples were subtracted from blanks not containing NADPH. The concentration of CPR in the samples was determined against a calibration curve of the
purified human CPR (Sigma).
Analytical Methods. A Waters Acquity UPLC (Ultra-performance Liquid
Chromatography) system (Milford, MA) coupled to an API 4000 liquid
chromatography–tandem mass spectrometry (AB-SCIEX, Toronto, Canada)
was used for all bioanalyses. The mass spectrometry conditions are summarized
in Supplemental Table 1. All analyses were conducted in positive mode ESI
(Electrospray ionization). Standard curves for the metabolites were made by
serial dilution in buffer. Separation was achieved on an Acquity BEH C18 (1.7
mM, 2.1*50 mm) UPLC column with mobile phase A consisting of 0.1%
formic acid in water and mobile phase B consisting of 0.1% formic acid in
acetonitrile. A flow rate of 0.6 ml/min was maintained starting at 85% A, and
reduced to 50% A over 1 minute. From 1 to 1.5 minutes, the %A was reduced
from 50% to 0%, and subsequently increased to 10% A from 1.5 to 1.7 minutes
in a linear fashion. Mobile phase A (10%) was maintained for 0.1 minute and
then increased to 85% A over 0.1 minute and kept at 85% A for another 0.1
minute for a total run time of 2 minutes. Alprazolam was used as the internal
standard for all analyses.
Enzyme Kinetics. Enzyme kinetics for MDZ, TST, terfenadine, nifedipine,
and TRZ were performed by initially determining linear conditions of time and
protein concentration in c3A4, h3A4, and MkLM for each substrate. The
consumption of substrate was ensured to be less than 20%. MDZ and
terfenadine kinetics were assessed with a concentration range of 0.015 to 100
mM in c3A4/h3A4 and up to 125 mM in MkLM for MDZ. The concentration
range for TST, nifedipine, and TRZ was 0.78–200 mM in all systems. Standard
incubation protocols were adopted wherein the appropriate amounts of protein
and phosphate buffer (100 mM, pH 7.4) were mixed in a Waters 96-well deep
plate, followed by the addition of substrate. This mix was preincubated at 37°C
for 5 minutes before the addition of an appropriate volume of NADPH to give
a final NADPH concentration of 2 mM. The protein concentration and
incubation times are summarized with the results of the kinetics experiments in
Table 1. The organic concentration in all incubations was less than 0.5%, and
all experiments were performed in triplicates.
The ability of c3A4 to metabolize non-h3A4 substrates was also assessed by
performing a single-concentration incubation at 25 mM substrate, 50 nM c3A4,
and 2 mM NADPH at 37°C for 30 minutes using the incubation protocol
described earlier. The substrates and metabolites were monitored, and the
results are summarized in Supplemental Table 2. 49-OH-Diclofenac, dextrorphan, 19-OH-bufuralol, acetaminophen, 7-hydroxycoumarin, hydroxybupropion, 6a -hydroxytaxol, and 6-OH-chlorzoxazone were the metabolites
monitored when diclofenac, dextromethorphan, bufuralol, phenacetin, coumarin, bupropion, paclitaxel, and chlorzoxazone, respectively, were incubated
with c3A4 to monitor the activity of 2C9, 2D6, 2D6, 1A2, 2A6, 2B6, 2C8, and
2E1, respectively. Expanded kinetics was performed for bufuralol, which was
the only non-h3A4 substrate to show metabolism. This experiment was
performed in both c3A4 and h3A4 with a 100 nM protein concentration and
a 15-minute incubation time. The formation of 19-OH-bufuralol was monitored
as a surrogate for activity. The concentration range of bufuralol was 1–100 mM.
To reconfirm the lack of metabolism of dextromethorphan, chlorzoxazone, and
coumarin, the incubation was repeated at three concentrations of 50, 100, and
372
Selvakumar et al.
TABLE 1
Kinetic characterization of multiple substrates after incubation with c3A4 and MkLM
System
c3A4
Substrate
MDZ
Protein and Time Incubation
Conditions for Linearity
Km (mean 6 SE)
Vmax (mean 6 SE)
mM
pmol/min/pmol-3A4 or
pmol/min/mg-protein
6.3 6 2.2
123 6 12.6
10 nM, 10 min
MDZ
MDZ
MDZ
TST
TST
TST
TST
Terfenadine
Terfenadine
Terfenadine
Terfenadine
Nifedipine
Nifedipine
1.5
1.6
4.2
49.2
67.2
78
46.4
0.52
7.2
3.4
12.9
2.78
20.3
6
6
6
6
6
6
6
6
6
6
6
6
6
0.17
1.3
0.1
5.1
22.7
34
1.9
0.1
2.1
0.4
3.7
0.7
5.2
61.6
13.7
1091
446.3
3481
86
5260
31.9
116.6
24
643
3.5
384.2
6
6
6
6
6
6
6
6
6
6
6
6
6
13.9
2.1
31
17.8
471.6
3
80
1.0
4.9
2
62.5
0.2
29.9
0.5 mg/ml, 10 min
10 nM, 10 min
Unknown
10 nM, 15 min
0.25 mg/ml, 10 min
10 nM, 15 min
Unknown
15 min, 20 nM
15 min, 0.25 mg/ml
15 min, 20 nM
h3A4
HLM
c3A4
MkLM
h3A4
HLM
c3A4
MkLM
h3A4
HLM
Nifedipine
Nifedipine
Triazolam (1-OH)
Triazolam (1-OH)
Triazolam (1-OH)
Triazolam (1-OH)
Triazolam (4-OH)
Triazolam (4-OH)
Triazolam (4-OH)
Triazolam (4-OH)
18.8
25.3
38.5
1.1
0.5
107
30.3
13.6
3.2
238
6
6
6
6
6
6
6
6
6
6
3.1
9
8.7
0.9
0.1
17
8.8
5.4
0.6
21
1.5
2040
11.0
95.4
0.4
1300
6.1
307.9
2.3
3300
6
6
6
6
6
6
6
6
6
6
0.1
830
1.2
26.7
0.0
300
0.7
34.6
0.1
130
15 min, 10 nM
15 min, 10 nM
15 min, 0.25 mg/ml
15 min, 10 nM
15 min, 0.25 mg/ml
15 min, 10 nM
15 min, 10 nM
15 min, 0.25 mg/ml
15 min, 10 nM
the linear portion of the biphasic graph (Clint2 = Vmax2/Km2 since saturation is
not achievable), Ki the inhibition constant for substrate inhibition, and n Hill’s
coefficient for sigmoidal kinetics. Subscripts of 1 or 2 indicate the first or
second binding site, respectively. All fitting and analyses were performed using
GraphPad Prism (version 5.02; GraphPad, La Jolla, CA).
IC50 values of inhibitors were determined by fitting eq. 6, a three-parameter
inhibition model, in GraphPad Prism. The formation of 19-OH-MDZ was
monitored as a measure of activity remaining. An inhibitor-free solvent control
was also run to ensure that greater than 80% inhibition was observed.
EnzymeActivity ¼ MinimumActivity
þ
ðMaximumActivity 2 MinimumActivityÞ
1 þ ð10∧ ðInhibitorConc 2 LogðIC50ÞÞÞ
ð6Þ
Results
Expression of Active c3A4. Initially, c3A4 expression was
attempted in Sf9 and Hi5 baculoviral cells, with multiple P450:CPR
ratios, hemin concentrations, and addition times, but batch to batch
variability in activities was observed. Use of a dual vector, with both
c3A4 and cynomolgus CPR on the same construct, was also
attempted, but the variability persisted. c3A4 was, subsequently,
coexpressed with cynomolgus monkey CPR in HEK293-6E cells
following the suggested protocol (Durocher et al., 2002), and this cell
line afforded robust and reproducible activities. The HEK293 cell line,
stably expressing Epstein-Barr virus nuclear antigen-1, increased the
yield of both recombinant intracellular and secreted proteins. The use
of cationic polymer PEI as transfection reagent helped in lowering the
overall cost of the large-scale transient expression in HEK cells,
whereas the addition of tryptone N1 (Organotechnie) increased the
transient expression efficiency (Pham et al., 2005). After 48 hours of
transfection, cells were harvested and microsomes were prepared
Sigmoidal and substrate
inhibition, n = 2.4
Biphasic
Substrate inhibition
Hyperbolic
Hyperbolic
Sigmoidal, n = 2.8
Hyperbolic
Substrate inhibition
Hyperbolic
Sigmoidal, n = 1.5
Hyperbolic
Hyperbolic
Substrate inhibition
Sigmoidal and substrate
inhibition, n = 1.1
Hyperbolic
Hyperbolic
Hyperbolic
Biphasic
Hyperbolic
Hyperbolic
Hyperbolic
Hyperbolic
Hyperbolic
Hyperbolic
Reference
(Walsky and Obach, 2004)
(Rodrigues et al., 1995)
(Patki et al., 2003)
(Patki et al., 2003)
(Patki et al., 2003)
using standard procedure involving hypotonic shock treatment. Upon
expression and purification of c3A4, Western blots of the protein
showed a band for c3A4 at around 55 kD and a band for reductase at
around 72 kD, very similar to their respective molecular weights of 57
and 78 kD, respectively (Supplemental Fig. 1). The CO difference
spectra showed an active c3A4 concentration of 1.4 mM (Supplemental Fig. 2) with a protein concentration of 13.5 mg/ml. A significant 420 nm peak was also observed, indicative of inactive protein.
The concentration of reductase, as measured by the cytochrome
c assay, was 1.0 mM. Hence, the ratio of c3A4 to cynomolgus CPR
was approximate 3:2.
Enzyme Kinetics. Full kinetic profiles were obtained for five
substrates—MDZ, TST, terfenadine, nifedipine, and TRZ—after
incubation with c3A4, h3A4, and MkLM.
MDZ with c3A4 showed pronounced atypical kinetics, with both
homotrophic cooperativity (sigmoidal kinetics) and substrate inhibition being displayed (Fig. 1A). Data were fitted to the model
described by eq. 5. The Vmax value obtained was 128 pmol/min/pmolc3A4, the Km9 was 77 mM, and the Hill coefficient was 2.4 (Ki . 100
mM). Although a true Km could not be obtained since nonhyperbolic
kinetics was observed, it was still possible to fit the data to
a Michaelis-Menten equation (eq. 1) and obtain a composite Km of
6.3 mM. The Km and Vmax for MkLM were 1.5 mM and 61.6 pmol/
min/mg protein, while the corresponding Km values for h3A4 and
HLM are 1.6 and 4.2 mM. The Km values between all systems are
hence comparable, and the low Km values suggest that MDZ is a high
affinity substrate for both c3A4 and h3A4. The c3A4 Vmax was
;9-fold higher than h3A4, confirming that this recombinant expressed
enzyme is very active.
TST 6b-hydroxylation, another marker reaction for h3A4, was also
efficient in c3A4 and MkLM (Fig. 1B). Km values of 49.2 and 67.2
mM were obtained in c3A4 and MkLM, respectively, whereas the
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
MkLM
h3A4
HLM
c3A4
MkLM
h3A4
HLM
c3A4
MkLM
h3A4
HLM
c3A4
MkLM
Kinetic Mechanism
Expression and Characterization of Cynomolgus CYP3A4
corresponding Km values for h3A4 and HLM are 78 and 46.4 mM
(Walsky and Obach, 2004). While the reaction was hyperbolic in the
presence of c3A4, sigmoidal kinetics was observed with MkLM (eq.
4). The reaction appeared to be faster in c3A4 than h3A4, with a Vmax
of 446 compared with 86 pmol/min/pmol-P450. However, the relative
ratios of reductase and b5 can impact the velocity of the reaction. In
contrast, the reaction was faster in HLM as compared with MkLM
(5260 versus 3481 pmol/min/mg), although this is on a per milligram
basis. The Km values are similar across all four systems, making TST
6b-hydroxylation an attractive in vitro probe substrate to compare
activities between humans and cynomolgus monkeys.
Terfenadine alcohol formation showed hyperbolic kinetics (eq. 1) in
c3A4 and sigmoidal kinetics (eq. 4) in MkLM (Fig. 1C). Terfenadine
was an efficient c3A4 substrate, with a very low Km of 0.5 mM and
Vmax of 31 pmol/min/pmol-c3A4. The corresponding Km and Vmax
values in MkLM were 7.2 mM and 116 pmol/min/mg-MkLM,
respectively (Table 1). Similar kinetics have been reported previously
373
in the literature, with Km values ranging from 3 to 12 mM for h3A4
and HLM (Rodrigues et al., 1995). The Km value for c3A4 was
substantially lower than the three other comparative systems, while the
Vmax of c3A4 was similar to h3A4. The low Km in c3A4 as compared
with MkLM may suggest the involvement of a second lower affinity
enzyme in terfenadine oxidation, or altered nonspecific protein binding of the terfenadine to the milieu in MkLM studies.
c3A4 efficiently catalyzed the oxidation of nifedipine to oxidized
nifedipine with a low Km of 2.8 mM, which was around 10-fold lower
than the MkLM Km (Fig. 1D; Table 1), while MkLM, h3A4, and HLM
had similar Km values. Substrate inhibition was observed in c3A4 (eq.
4), whereas sigmoidal plus substrate inhibition was observed in
MkLM (eq. 5), similar to the mixed-atypical kinetics seen when MDZ
was incubated with c3A4. The Vmax values were comparable between
c3A4 and h3A4, but HLM had a 5-fold higher Vmax than MkLM (Patki
et al., 2003). The low Km in c3A4 as compared with MkLM may
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
Fig. 1. Enzyme kinetics of MDZ (A), TST (B), terfenadine (C), nifedipine (D), TRZ-19-OH (E), and TRZ-19-OH (F) when incubated with c3A4 and MkLM (inset figure).
19-OH-MDZ velocities are on the y-axis, whereas the x-axis represents MDZ concentration. S, substrate concentration.
374
Selvakumar et al.
values for their specific P450. This suggests that non-h3A4 inhibitors
are unlikely to inhibit c3A4.
Discussion
c3A4 has been previously expressed in Escherichia coli, and some
characterization has been completed with several substrates at one or
more concentrations (Uno et al., 2007; Iwasaki et al., 2010; Ohtsuka
et al., 2010; Emoto et al., 2011). However, extensive kinetic
characterization with multiple substrates, multiconcentration characterization with non-h3A4 substrates, and inhibition propensity with
non-h3A4 inhibitors has not been previously described. This paper
attempts to provide such data and, as a result, provide a broad data set
to understand c3A4 and its similarities and differences compared with
h3A4. In addition, this is the first report of HEK293 cells being used
to express c3A4, a robust system that may prove useful to researchers
attempting to express P450s of different species that are not amenable
to expression in baculoviral cells.
Several reports have provided c3A4 activity information for h3A4
and non-h3A4 substrates at one or two substrate concentrations
(Iwasaki et al., 2010; Emoto et al., 2011). For example, the alprazolam
4-hydroxylation kinetic profile was characterized for c3A4 and h3A4,
and was shown to display sigmoidal kinetics (Ohtsuka et al., 2010) in
both cases. c3A4 was characterized by an ;8-fold higher Vmax, a ;2fold lower Km, and a corresponding 13-fold higher intrinsic clearance
over h3A4.
Extensive characterization of rhesus monkey CYP3A64 (proposed
renaming to CYP3A4), expressed in a baculoviral system, was accomplished using TST, nifedipine, MDZ, and benzoxy-4trifluoromethylcoumarin as substrates, and it was found that Vmax,
Km, and Clint values were very similar between rhesus 3A64 and h3A4
for these four substrates (Carr et al., 2006). Sigmoidal kinetics were
also observed for some of these substrates, and KTZ was found to
inhibit 3A64 metabolism with an IC50 identical to h3A4 (TST as
substrate). Given the 100% homology between c3A4 and rhesus 3A64
enzymes, it is unsurprising that the Km values obtained were similar
for the common substrates between our study and the rhesus 3A64
study (Carr et al., 2006). Collectively, these data suggest that rhesus
3A64, h3A4, and c3A4 are characterized by similar kinetic properties.
From our studies, for MDZ, low Km values (,10 mM) were
obtained in c3A4, MkLM, h3A4, and HLM, suggesting that this is
a high affinity substrate across species. A combination of sigmoidal
kinetics at the low substrate concentrations and substrate inhibition at
the high substrate concentrations was observed in c3A4, and hence the
profile was fitted to an equation derived from combining the sigmoidal
TABLE 2
Inhibition of c3A4- and MkLM-mediated MDZ 19-hydroxylation by KTZ and TAO
Incubation conditions were as follows: substrate: MDZ (5 mM), protein concentration: 10 nM
for c3A4 and 0.5 mg/ml for MkLM, incubation time: 10 minutes.
Fig. 2. Enzyme kinetics of bufuralol when incubated with c3A4 and h3A4. 19-OHbufuralol velocities are on the y-axis, whereas the x-axis represents bufuralol
concentration.
System
Inhibitor
c3A4
MkLM
h3A4
HLM
c3A4
MkLM
h3A4
HLM
KTZ
KTZ
KTZ
KTZ
TAO
TAO
TAO
TAO
na, not applicable.
IC50,
0 min
IC50,
20 min
mM
mM
0.03
0.016
0.037
0.025
1.1
20.5
.40
23
na
na
na
na
0.17
2.3
3.8
0.85
IC50
Shift
na
na
na
na
8x
9x
.10x
27x
Reference
(Parkinson et al., 2011)
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
suggest the involvement of a second enzyme/altered nonspecific protein binding, as in the case of terfenadine.
TRZ 1- and 4-hydroxylation kinetics were hyperbolic (eq. 1) in all
cases except in MkLM, where 1-hydroxylation showed biphasic
kinetics (eq. 2). TRZ 1-hydroxylation is shown in Fig. 1E, whereas
TRZ 4-hydroxylation is shown in Fig. 1F. TRZ 1-hydroxylation had
a lower Km and lower Vmax than 4-hydroxylation in HLM and h3A4
(Patki et al., 2003). The same trend was observed in MkLM as well,
but in c3A4, 1- and 4-hydroxylations had similar Vmax and Km values.
To determine whether c3A4 can metabolize non-h3A4 substrates,
a single concentration of substrate was incubated as described in the
Materials and Methods section. Of all the probe substrates for
CYP2C9, 2D6, 1A2, 2A6, 2B6, 2C8, and 2E1 tested, only bufuralol
(CYP2D6 substrate) showed any metabolism (Supplemental Table 2).
Hence, a follow-up multisubstrate concentration experiment was
performed with bufuralol concentrations ranging from 1 to 100 mM
with both c3A4 and h3A4 (Fig. 2). Saturation was not achieved even
at the highest bufuralol concentration in c3A4, suggesting a very high
Km reaction. The maximal velocity of 19-OH-bufuralol formation was
0.4 pmol/min/pmol-c3A4, suggestive of a reasonable amount of metabolism. h3A4 showed no metabolism of bufuralol across all concentration ranges.
Table 1 summarizes the time and protein conditions for each Vmax
and Km determination, the Vmax and Km values, and the corresponding
data in HLM and h3A4 from the literature.
Enzyme Inhibition. Inhibition of c3A4 by KTZ (reversible) and
TAO (mechanism-based) was also performed. The KTZ IC50 of
19-OH-MDZ formation was 0.03 mM in c3A4 and 0.016 mM in
MkLM (Table 2). The corresponding values for h3A4 and HLM are
0.04 and 0.02, respectively (Walsky and Obach, 2004). Hence, all the
IC50 values are similar to each other, confirming the suitability of KTZ
as a c3A4 inhibitor at low inhibitor concentrations.
TAO also behaved as a time-dependent inhibitor in c3A4, similar to
h3A4, MkLM, and HLM. The c3A4 IC50 at 0 minute was 1.2 mM,
suggesting competitive inhibition, and the IC50 at 30 minutes was 0.17
mM, an 8-fold IC50 shift suggesting time-dependent inhibition (Fig. 3;
Table 2). The corresponding IC50 shift in MkLM was 9-fold, although
the IC50 numbers at time zero and time 30 minutes were ;15-fold
higher (Fig. 3; Table 2). The effect of non-h3A4 inhibitors on c3A4
was also determined using the selective inhibitors for the various
human CYPs (Supplemental Table 3). As can be seen from the table,
none of the non-h3A4 inhibitors inhibited MDZ 19-hydroxylation in
c3A4 and MkLM even at concentrations several fold over their IC50
375
Expression and Characterization of Cynomolgus CYP3A4
and substrate inhibition equations. A similar profile was also noted for
nifedipine in MkLM. The existence of atypical kinetics suggests the
existence of multiple catalytic sites on c3A4 as with h3A4. With TST
as a substrate, the Km values for c3A4, h3A4, HLM, and MkLM were
similar to each other, whereas with nifedipine and terfenadine as
substrates, c3A4 had a Km value approximately 10-fold lower than
h3A4, HLM, and MkLM. For the latter two substrates, HLM, MkLM,
and h3A4 had similar Km values, suggesting that c3A4 has higher
affinity or lower nonspecific protein binding for these two substrates.
With TRZ as a substrate, c3A4, h3A4, and MkLM were equally
efficient in catalyzing both 19 and 49-hydroxylation pathways, whereas
in MkLM, 49-hydroxylation was about 3-fold more efficient that 19hydroxylation in terms of Vmax/Km values.
When the microsomal Vmax values are converted from a per
milligram to a per picomole basis using the relative abundance ratios
of P450s, direct comparisons can be made between expressed enzyme
Vmax values and microsomal Vmax values (Rodrigues, 1999; Uehara
et al., 2011). These values are summarized in Table 3. The ratio for
expressed enzyme Vmax to microsome Vmax was consistent between
humans and cynomolgus monkeys for three out of four substrates
(TST, terfenadine, and nifedipine). Viewed this way, c3A4 and h3A4
behaved very similarly for three substrates. Indeed, MDZ is
extensively used as an in vivo probe substrate for CYP3A4 activity
in both humans and cynomolgus monkeys and shows similar hepatic
extraction ratios in both species (Akabane et al., 2010; Yoda et al.,
2012).
While c3A4 shows an almost exclusive preference to metabolize
h3A4 substrates, earlier reports have indicated that c3A4 and c3A5
could metabolize dextromethorphan and bufuralol (2D6 substrates), in
addition to chlorzoxazone (2E1 substrate). Surprisingly, h3A4 was
also able to catalyze these reactions (Iwasaki et al., 2010; Emoto et al.,
2011). At 20 mM bufuralol concentration, h3A4 and c3A4 appeared to
be equally proficient at catalyzing 19-hydroxylation, whereas c3A5
was 3–4 times more efficient than h3A5 in catalyzing the reaction.
This trend was more pronounced at 200 mM bufuralol. MkLM showed
8-fold higher bufuralol 19-hydrxoylation activity than HLM at 20 and
200 mM bufuralol concentrations. Bufuralol 6-hydroxylation occurred
with equal velocities in liver microsomes at both concentrations in
both species (Iwasaki et al., 2010; Emoto et al., 2011). Hence,
bufuralol 6-hydroxylation does not show a species difference, and
bufuralol 19-hydroxylation species difference appears to be more due
to c3A5 than c3A4. Dextromethorphan O-dealkylation was catalyzed
only by c3A4 and c3A5 and not by h3A4/3A5, and MkLM also
showed higher velocities than HLM (Emoto et al., 2011). Dextromethorphan N-demethylation reaction velocity was similar in MkLM
and HLM, and both species 3A4s were able to catalyze this reaction
(Emoto et al., 2011). Hence, only the dextromethorphan O-dealkylation
TABLE 3
Comparison of Vmax values between liver microsomes and expressed enzymes by
converting liver microsomes from a per-milligram basis to a per-picomole basis by
utilizing enzyme levels in microsomes
MkLM 3A4 content was 29 pmol/mg (Uehara et al., 2011); HLM 3A4 content was 108 pmol/
mg (Rodrigues, 1999).
Vmax (pmol/min/pmol-3A4)
c3A4
MkLM
Vmax, c3A4/Vmax,MkLM
h3A4
HLM
Vmax, h3A4/Vmax,HLM
MDZ
TST
Terfenadine
Nifedipine
123.0
20.8
5.9
13.7
10.1
1.4
446.0
120.0
3.7
86.0
48.7
1.8
32.0
4.0
8.0
24.0
6.0
4.0
3.5
13.2
0.3
1.5
18.9
0.1
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
Fig. 3. Inhibition of c3A4 after a 0-minute (A) and 20-minute (B) preincubation with TAO, and inhibition of MkLM after a 0-minute (C) and 20-minute (D) preincubation
with TAO. The x-axis contains the log concentrations of TAO, whereas the y-axis contains 19-OH-MDZ concentrations, as a marker for c3A4 activity remaining.
376
Selvakumar et al.
Acknowledgments
The authors thank Punit Marathe and Ramaswamy Iyer for guidance,
support, and critical review.
Authorship Contributions
Participated in research design: Ghosh, Krishnamurthy, Subramanian.
Conducted experiments: Selvakumar, Bhutani.
Contributed new reagents or analytic tools: Selvakumar, Kallipatti, Selvam.
Performed data analysis: Bhutani, Subramanian.
Wrote or contributed to the writing of the manuscript: Ghosh, Krishnamurthy,
Ramarao, Mandlekar, Sinz, Rodrigues, Subramanian.
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Address correspondence to: Murali Subramanian, Biocon Bristol-Myers Squibb
Research and Development Center, Syngene International Limited, Biocon Park
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Downloaded from dmd.aspetjournals.org at ASPET Journals on June 12, 2017
was a clear species difference, although the higher activity (85- to 400fold) of the 2D enzymes over 3A enzymes may ameliorate the species
difference. Chlorzoxazone was only tested at 50 mM, and at this
concentration, c3A4/5 showed 4- to 5-fold higher velocities than
h3A4/5, although HLM and MkLM showed similar velocities
(Iwasaki et al., 2010; Emoto et al., 2011). It is unclear if this is
truly a species difference, since 50 mM is a very high concentration.
In our studies, 25 mM dextromethorphan did not show the
formation of dextrorphan, and 25 mM chlorzoxazone did not show
the formation of 1-OH-chlorzoxazone. These findings were reconfirmed in a repeat experiment conducted in triplicates at higher
substrate concentrations of 50–200 mM. In contrast, bufuralol showed
the formation of 19-OH-bufuralol when incubated with c3A4. While
bufuralol showed minor metabolism (,1%) when incubated at
25 mM, in a repeat multiconcentration study, a maximal velocity of
0.4 pmol/min/pmol-c3A4 was suggestive of low-level metabolism.
Saturation of metabolism was not observed, and h3A4 showed no
metabolism of bufuralol across all concentration ranges. Consistently
in microsomes, MkLM Vmax values for bufuralol 19-OH hydroxylation
was found to be 15-fold higher than HLM Vmax values, suggesting
higher intrinsic bufuralol clearance in MkLM as compared with HLM
(Mankowski et al., 1999). The c2D17 and c2D44 Vmax for bufuralol
hydroxylation ranges between 10 and 20 pmol/min/pmol-2D6 (Uno
et al., 2010). The maximal velocity is 0.4 pmol/min/pmol-c3A4 by
c3A4, but the abundance of c3A4 (39 pmol/mg) is more than 10-fold
higher than c2D (3.3 pmol/mg); hence, the amount adjusted to c3A4
(15.6 pmol/min/mg protein) rate is much closer to the cynomolgus
CYP2D6 bufuralol hydroxylation rate (33–66 pmol/min/mg protein),
and hence, c3A4 may play a significant role in bufuralol hydroxylation in MkLM.
In summary, we have extensively characterized a preparation of
c3A4 and compared it to both h3A4 and MkLM. Enzyme kinetic
parameters were obtained for five prototypical h3A4 substrates, and in
all cases, c3A4 showed robust activity with some differences in Km
values. In addition, the inhibition parameters for c3A4 with two
prototypical CYP3A inhibitors (KTZ and TAO) were comparable to
those obtained with h3A4. c3A4 was also able to metabolize bufuralol
to its 1-hydroxylated metabolite, although no metabolism of dextromethorphan or chlorzoxazone was observed.