In vitro inhibition and induction of human liver cytochrome P450 enzymes by NTBC and its metabolism in human liver microsomes

Abstract

2-(2-Nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC, also known as nitisinone and marketed as Orfadin) is an inhibitor of 4-hydroxyphenylpyruvate dioxgenase (HPPD) that is used to prevent the liver and kidney toxicity associated with tyrosinemia type 1, a metabolic disorder in the tyrosine catabolism caused by fumarylacetoacetate hydrolase (FAH) deficiency. Genetically modified mice deficient in FAH [Fah-/-/II2rg-/-Rag2-/- (FRG) mouse strain] can be repopulated with human hepatocytes to support, among other applications, studies of drug metabolism and disposition. To sustain mouse hepatocellular function prior to (and during) the repopulation with human hepatocytes, FRG mice are treated with NTBC to inhibit the formation of hepatotoxic levels of fumarylacetoacetate (FAA). In the present study, we investigated the metabolism of NTBC in human liver microsomes (HLM) and the potential for NTBC to inhibit or induce human cytochrome P450 (CYP) enzymes. NTBC (1, 10 and 100 µM) was incubated with multiple concentrations of NADPH-fortified pooled HLM (0.5, 1 and 2 mg /mL) for multiple incubation times (0, 30, 60, 120 and 240 min). Little-to-no loss of NTBC was observed, suggesting that NTBC undergoes little or no oxidative metabolism by human liver CYP enzymes and little-to-no ketone reduction by microsomal carbonyl reductase. These results are consistent with the long clinical plasma half-life (t1/2 ~ 52 –54h) reported for NTBC. In CYP inhibition experiments, performed with pooled HLM (0.1 mg/mL), NTBC caused direct inhibition of CYP2C9 (IC50 11 µM). NTBC caused no direct inhibition of CYP1A2, 2B6, CYP2C8, 2C19, 2D6 and 3A4/5. Furthermore, NTBC caused no metabolism‑dependent inhibition of any of the CYP enzymes evaluated. To evaluate CYP induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for three consecutive days with NTBC (1, 10, and 100 µM), after which microsomal CYP activities and mRNA expression were measured. NTBC (100 µM), had a negligible effects (< twofold) on CYP2B6 and 3A4/5 activity but caused a 7.60-fold increase in CYP1A2 activity. However, as a CYP1A2 inducer, NTBC (100 µM) was only 9% as effective as omeprazole. In summary, NTBC has no capacity to inhibit or induce human CYP enzymes which suggests that repopulated FRG mice undergoing NTBC treatment are suitable for studies of drug metabolism involving human CYP enzymes.

Full text

In vitro inhibition and induction of human liver cytochrome P450
enzymes by NTBC and its metabolism in human liver microsomes.
Jason N. Neat1, Andrea Wol1, Faraz Kazmi1, Pilar Prentiss1, David Buckley1, Elizabeth M. Wilson2,
John Bial2, Cedo M. Bagi3, Markus Grompe2 and Andrew Parkinson1 | 1XenoTech, LLC, Lenexa, KS,
2Yecuris Corporation, Portland, OR, 3Global Science & Technology, Pzer, Inc., Groton, CT
Presented at the 9th International ISSX Meeting, Istanbul, Turkey, September 2010
Poster 146, Drug Metabolism Reviews, Volume 42, Supplement 1, 2010
2-(2-Nitro-4-triuoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC, also known as nitisinone and marketed as Orfadin®)
is an inhibitor of 4-hydroxyphenylpyruvate dioxgenase (HPPD) that is used to prevent the liver and kidney toxicity
associated with tyrosinemia type 1, a metabolic disorder in the tyrosine catabolism caused by fumarylacetoacetate
hydrolase (FAH) deciency. Genetically modied mice decient in FAH [Fah-/-/II2rg-/-Rag2-/- (FRG) mouse strain] can
be repopulated with human hepatocytes to support, among other applications, studies of drug metabolism and
disposition. To sustain mouse hepatocellular function prior to (and during) the repopulation with human hepatocytes,
FRG mice are treated with NTBC to inhibit the formation of hepatotoxic levels of fumarylacetoacetate (FAA). In the
present study, we investigated the metabolism of NTBC in human liver microsomes (HLM) and the potential for NTBC
to inhibit or induce human cytochrome P450 (CYP) enzymes. NTBC (1, 10 and 100 µM) was incubated with multiple
concentrations of NADPH-fortied pooled HLM (0.5, 1 and 2 mg /mL) for multiple incubation times (0, 30, 60, 120 and
240 min). Little-to-no loss of NTBC was observed, suggesting that NTBC undergoes little or no oxidative metabolism
by human liver CYP enzymes and little-to-no ketone reduction by microsomal carbonyl reductase. These results are
consistent with the long clinical plasma half-life (t1/2 ~ 52 –54 h) reported for NTBC. In CYP inhibition experiments,
performed with pooled HLM (0.1 mg/mL), NTBC caused direct inhibition of CYP2C9 (IC50 11 µM). NTBC caused no
direct inhibition of CYP1A2, 2B6, CYP2C8, 2C19, 2D6 and 3A4/5. Furthermore, NTBC caused no metabolism dependent
inhibition of any of the CYP enzymes evaluated. To evaluate CYP induction, freshly isolated human hepatocytes (n = 3)
were cultured and treated once daily for three consecutive days with NTBC (1, 10, and 100 µM), after which microsomal
CYP activities and mRNA expression were measured. NTBC (100 µM), had negligible eects (< twofold) on CYP2B6 and
3A4/5 activity but caused a 7.60-fold increase in CYP1A2 activity. However, as a CYP1A2 inducer, NTBC (100 µM) was
only 9% as eective as omeprazole. In summary, NTBC has no capacity to inhibit or induce human CYP enzymes which
suggests that repopulated FRG mice undergoing NTBC treatment are suitable for studies of drug metabolism involving
human CYP enzymes.
ABSTRACT

Table 1.
The results of studies conducted with new drug candidates in nonclinical
species like mouse, rat, dog and monkey are always of questionable relevance
to humans because of well documented species dierences in the function of
drug-metabolizing enzymes, such as cytochrome P450 (CYP) enzymes, and their
regulation, as reected, for example, in species dierences in the xenosensors
(like AhR, CAR and PXR) that mediate the induction of CYP enzymes. For this
reason, regulatory agencies advocate the use of in vitro studies with human-
derived test systems and their nonclinical counterparts (such as hepatocytes
or liver subcellular fractions) that permit, for example, an evaluation of species
dierences in the routes of metabolism of a drug candidate and the potential for
drug-drug interactions due to CYP inhibition or induction.
Transgenic mice in which one or more murine genes encoding a drug-
metabolizing enzyme or xenosensor have been replaced with their human
counterparts provide an in vivo test system with which to study selected aspects
of drug disposition in a more clinically relevant manner. For example, in mice
that have been "humanized" with respect to the xenosensor PXR, the expression
of CYP3A is regulated by enzyme-inducing drugs in a more clinically relevant
manner than occurs in wild-type mice (Gonzalez and Yu, 2006).
Genetically modied mice whose livers have been repopulated with human
hepatocytes potentially allow for the global expression of human hepatic
function. These so-called chimeric or humanized mice are immunocompromised
to prevent rejection of human hepatocytes, and they harbor other genetic
modications to promote the growth of inoculated human hepatocytes over
that of mouse hepatocytes. The PXB mouse, for example, is a urokinase-type
plasminogen activator (uPA)+/+/severe combined immunodecient mouse (uPA/
SCID mouse) that can be repopulated with human hepatocytes (in some cases
to more than 90%). Such mice have been used to support a number of ADME-
Tox applications, (Katoh and Yokoi, 2007; Katoh et al., 2007; Schultz et al., 2007).
However, uPA+/+ mice must be inoculated with hepatocytes at a young age
(within two weeks of birth) and are prone to bleeding disorders. They are also
prone to a relatively high rate of spontaneous reversion, which allows for the
expansion and repopulation of the liver with mouse hepatocytes.
The Yecuris FRG mouse (or Hepatomouse) can also be repopulated with human
hepatocytes to support ADME-Tox studies (Azuma et al., 2007; Strom et al.,
2010). The Hepatomouse is an immunocompromised mouse (due to genetic
deciencies in Rag2 and Il2rg) with a genetic deciency in fumarylacetoacetate
hydrolase (FAH) that leads to the accumulation of hepatotoxic levels of
fumarylacetoacetate (a catabolite of tyrosine) in mouse hepatocytes. The
build-up of this toxic intermediate can be controlled throughout the life of
the FRG mouse with either a low tyrosine diet or, more conveniently, with
NTBC (2-(2-nitro-4-triuoromethylbenzoyl)-1,3-cyclohexanedione, also known
as nitisinone). NTBC inhibits 4-hydroxyphenylpyruvate dioxgenase (HPPD),
another enzyme involved in tyrosine metabolism, and is marketed under the
brand name Orfadin® for the treatment of tyrosinemia type 1 (which is caused
by FAH deciency).
NTBC (2-(2-nitro-4-triuoromethylbenzoyl)-1,3-cyclohexanedione
Because NTBC is used to treat FRG mice at various times during the repop-
ulation of their livers with human hepatocytes and control the rate of cell
death in mouse hepatocytes, we conducted the present study to ascertain
whether NTBC has the potential to interact with human CYP enzymes either
as a substrate, inhibitor or inducer.
INTRODUCTION
MATERIALS & METHODS RESULTS
Figure 1 shows the eect of incubation time on the loss of NTBC (1, 10 and 100
μM) from incubations with human liver microsomes (1 mg/mL). Under the
conditions evaluated, little to no loss of NTBC was observed even when 1 μM
NTBC was incubated with 1 mg/mL HLM for 4 hours. These results suggest that
NTBC undergoes little or no metabolism by human liver CYP enzymes (or does
so at a very low rate) and that NTBC does not undergo ketone reduction by
microsomal carbonyl reductase. These results are consistent with the long clinical
plasma half-life (t1/2 ~ 52 – 54 h) reported for NTBC (Hall et al., 2001).
CF
3
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NO
2
OO
Chemicals and reagents
The sources of reagents used in this study have been described previously
(Robertson et al., 2000; Madan et al., 2003; Ogilvie et al., 2006; Paris et al., 2009).
NTBC was obtained by Yecuris Corporation through license under Swedish
Orphan AB (Stockholm, Sweden).
Test systems
Cultured human hepatocytes and pooled human liver microsomes (HLM) were
prepared at XenoTech, LLC (Lenexa, KS, USA).
Stability of NTBC in HLM
Analytical method set-up
To study the metabolism of NTBC based on substrate loss, an LC/MS/MS method
was developed and NTBC (analyte) quantitation was based on the following mass
transitions listed in Table 1:
Mass transitions
Compound Ion Transition (amu)
NTBC (substrate) 328 → 281
4-Hydroxydiclofenac-d4(internal standard) 314 → 270
Incubation of NTBC with human liver microsomes (HLM)
NTBC (1, 10 and 100 μM) was incubated with pooled human liver microsomes
(0.5, 1 and 2 mg protein/mL) at 37 ± 1°C in 0.2-mL incubation mixtures con-
taining potassium phosphate buer (50 mM, pH 7.4), MgCl2 (3 mM) and EDTA
(1 mM, pH 7.4) with and without cofactor (NADPH-generating system), at the nal
concentrations indicated. Reactions were started by addition of the cofactor, and
were stopped at designated times (0, 30, 60, 120 and 240 min) by the addition
of an equal volume of acetonitrile containing internal standard. Zero-time, zero-
cofactor (no NADPH), zero-substrate and zero-protein incubations served as
blanks. Samples were subjected to centrifugation (920 x g for 10 min at 10°C) to
remove precipitated protein. The supernatant fractions were analyzed by LC/MS/
MS to quantify the amount of unchanged NTBC.
Cytochrome P450 inhibition in human liver microsomes
NTBC was assessed for its ability to cause three types of CYP inhibition:
1. Direct inhibition, in which case NTBC and the CYP marker substrate were
added simultaneously and incubated for 5 min to determine CYP activity.
2. Time-dependent inhibition (TDI), in which case NTBC was incubated for 30
min with HLM in the absence of NADPH prior to the addition of CYP marker
substrate (followed by a 5-min incubation to determine CYP activity).
3. Metabolism-dependent inhibition (MDI) in which case NTBC was incubated
for 30 min with HLM in the presence of NADPH prior to the addition of CYP
marker substrate (followed by a 5-min incubation to determine CYP activity).
CYP inhibition experiments were conducted with conditions as previously
described (Ogilvie et al., 2006; Ogilvie et al., 2008, Paris et al., 2009).
Enzyme induction and toxicity in human hepatocytes
Cultured human hepatocytes from three donors were treated according to a
standard CYP induction protocol (Robertson et al., 2000; Paris et al., 2009). Briey,
after a two-to-three day adaptation period, hepatocytes were treated once daily
for three consecutive days with DMSO, (0.1% v/v; vehicle control) one of three
concentrations of NTBC (1, 10 and 100 μM) or one of three known prototypical
inducers, namely, omeprazole (100 μM), phenobarbital (750 μM) and rifampin
(10 μM). During treatment, medium was collected at 0, 24, 48 and 72 hours post
treatment and analyzed for lactate dehydrogenase release, an indicator of cell
toxicity (loss of membrane integrity) as described in the Roche Applied Science
Cytotoxicity Detection Kit (Catalog #1644793). Following treatment, hepatocytes
were either harvested for the preparation of microsomes as described by Paris
et al., 2009 for subsequent analysis of enzymatic activity or lysed with TRIzol
reagent to isolate RNA for analysis of mRNA expression by qRT-PCR as described
by Neat et al., 2009.
Microsomes isolated from human hepatocytes at the end of the treatment
period were assayed for the following CYP activities: CYP1A2 (phenacetin
O-dealkylation), CYP2B6 (bupropion hydroxylation) and CYP3A4/5 (testosterone
6β-hydroxylation). Incubation conditions and the analytical methods to measure
metabolite formation by LC/MS/MS were those described by Paris et al., 2009.

Eect of incubation time on the loss of NTBC
(1, 10 and 100 µM) from incubations with human
liver microsomes (1 mg/mL)
Table 2.
Summary of in vitro evaluation of NTBC as an inhibitor of human CYP enzymes in HLM
Direct inhibition Time-dependent
inhibition
Metabolism-dependent
inhibition Potential
for time-
dependent or
metabolism-
dependent
inhibitionc
Zero-minute preincubation 30-minute preincubation
without NADPH
30-minute preincubation
with NADPH
Enzyme Enzyme reaction IC50 (μM)a
Maximum
inhibition at
100 μM (%)b
IC50 (μM)a
Maximum
inhibition at
100 μM (%)b
IC50 (μM)a
Maximum
inhibition at
100 μM (%)b
CYP1A2 Phenacetin O-dealkylation >100 NA >100 NA >100 2.3 Little or no
CYP2B6 Efavirenz 8-hydroxylation >100 2.9 >100 7.9 >100 8.3 Little or no
CYP2C8 Amodiaquine N-dealkylation >100 6.1 >100 5.4 >100 26 Little or no
CYP2C9 Diclofenac 4´-hydroxylation 11 90 10 90 9.1 91 Little or no
CYP2C19 S-Mephenytoin 4´-hydroxylation >100 9.7 >100 11 >100 12 Little or no
CYP2D6 Dextromethorphan O-demethylation >100 0.68 >100 2.1 >100 6.2 Little or no
CYP3A4/5 Testosterone 6β-hydroxylation >100 2.3 >100 NA >100 5.6 Little or no
CYP3A4/5 Midazolam 1´-hydroxylation >100 NA >100 NA >100 9.6 Little or no
a. Average data (i.e., percent of control activity) obtained from duplicate samples for each
test article concentration were used to calculate IC50 values. IC50 values were calculated
with XLFit.
b. Maximum inhibition (%) is calculated with the following formula and data for the
highest concentration of test article evaluated (results are rounded to two signicant
gures): Maximum inhibition (%) = 100% – Percent solvent control.
c. Potential for time-dependent or metabolism-dependent inhibition was assessed
by comparison of IC50 values with and without preincubation or NADPH and/or by
comparison of the maximum inhibition (%) with and without preincubation or NADPH
and by visual inspection of the IC50 plot.
NA. Not applicable. No value was obtained as the rates at the highest concentration of
NTBC evaluated (100 µM) were higher than the control rates.
120
100
80
60
40
20
00 50 100 150 200 250
100 µM
10 µM
1 µM
Incubation Time (min)
Substrate Remaining (%)
Eect of time on loss of NTBC
Figure 1.
Inhibition of CYP2C9 (diclofenac 4´-hydroxylation) by NTBC: IC50 determination
Figure 2.
Percent of Control Activity (%)
125
0
25
50
75
100
Zero-minute preincubation
30-minute preincubation minus NADPH
Time-Dependent Inhibition
[NTBC]
(µM)
0.1 10 1000
Percent of Control Activity (%)
125
0
25
50
75
100
Zero-minute preincubation
30-minute preincubation minus NADPH
Metabolism-Dependent Inhibition
[NTBC]
(µM)
0.1 10 1000
Table 2 shows a summary of the evaluation of NTBC as a direct, time-dependent
and metabolism-dependent inhibitor of human CYP enzymes in HLM.
NTBC caused direct inhibition of CYP2C9 with an IC50 value of 11 μM as shown
in Figure 2. There was little or no evidence that NTBC caused direct inhibition
of CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A4/5 (measured by
testosterone 6β-hydroxylation and midazolam 1´-hydroxylation), and the IC50
values were reported to be greater than the highest concentration of NTBC
studied (i.e., 100 μM). Furthermore, there was no compelling evidence that NTBC
caused time-dependent or metabolism-dependent inhibition of any of the CYP
enzymes evaluated.
Figure 3 shows the mean fold induction for both CYP activity and mRNA following
treatment of human hepatocytes with NTBC, daily, for three consecutive days.
Under the conditions of this study, where the positive controls caused anticipated
increases in CYP enzyme activities and mRNA levels, NTBC at 1 and 10 μM
concentrations caused no increase in either CYP1A2, CYP2B6 and CYP3A4/5
activity, or CYP1A2, CYP2B6 and CYP3A4 mRNA levels. In contrast, 100 μM NTBC
caused an increase in CYP1A2 activity and in CYP1A2 and CYP3A4 mRNA levels;
however, these increases were less than 40% of the positive controls, omeprazole
(CYP1A2) and rifampin (CYP3A4). At the highest concentration tested (100 μM)
and at the last time point (72 h) NTBC caused an increase in LDH release in two of
three cultures (data not shown). Additionally, the highest concentration of NTBC
(100 μM) caused a decrease in CYP2B6 and CYP3A4/5 activity (individual data not
shown) in two of three hepatocyte preparations, which was associated with cell
toxicity (based o LDH release).
• Little-to-no substrate loss of NTBC was observed when NTBC was incubated
for up to 4 hours with NADPH-fortied pooled HLM (1 mg/mL), suggesting
that NTBC undergoes little or no oxidative metabolism by human liver
CYP enzymes and little-to-no ketone reduction by microsomal carbonyl
reductase. These results are consistent with the long clinical plasma half-life
(t1/2 ~ 52 –54 h) reported for NTBC.
• In human liver microsomes, NTBC was a direct inhibitor of CYP2C9 (IC50 11 µM)
but, at concentrations up to 100 µM, NTBC caused no direct inhibition of CYP1A2,
2B6, CYP2C8, 2C19, 2D6 or 3A4/5. Furthermore, NTBC caused no metabolism-
dependent inhibition of any of the CYP enzymes evaluated.
• In cultured human hepatocytes NTBC had negligible eects (< twofold) on
CYP2B6 and 3A4/5 activity but, at the highest concentration tested (100 μM)
NTBC caused a 7.6-fold increase in CYP1A2 activity. However, as a CYP1A2
inducer, NTBC (100 μM) was only 9% as eective as omeprazole.
• In summary, with the exception of CYP2C9 inhibition, NTBC has little eect on
human CYP enzymes either as an inhibitor or inducer. Furthermore, NTBC is not
rapidly or extensively metabolized by microsomal CYP enzymes. The results
suggest that FRG mice repopulated with human hepatocytes and undergoing
(or having undergone) NTBC treatment are suitable for studies of drug
metabolism involving human CYP enzymes.
CONCLUSIONS

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REFERENCES
16825 W 116th Street | Lenexa, KS 66219 | 913.438.7450 | fax 913.227.7100 | xenotechllc.com
Figure 3.
The eects of treating cultured human hepatocytes with NTBC or prototypical inducers on microsomal
cytochrome P450 (CYP) enzyme activity and mRNA levels
Testosterone 6β-hydroxylation
Bupropion hydroxylation
Phenacetin O-dealkylation
0.1% Dimethyl
sulfoxide
1 µM NTBC
10 µM NTBC
100 µM NTBC
100 µM
Omeprazole
750 µM
Phenobarbital
10 µM Rifampin
0 20 40 60 80 100
5 10 150
0.1% Dimethyl
sulfoxide
1 µM NTBC
10 µM NTBC
100 µM NTBC
100 µM
Omeprazole
750 µM
Phenobarbital
10 µM Rifampin
5 10 150 20
0.1% Dimethyl
sulfoxide
1 µM NTBC
10 µM NTBC
100 µM NTBC
100 µM
Omeprazole
750 µM
Phenobarbital
10 µM Rifampin
10 20 300 40
Fold Increase
0 100 200 300 400
20 60 800 40
CYP1A2
0.1% Dimethyl
sulfoxide
1 µM NTBC
10 µM NTBC
100 µM NTBC
100 µM
Omeprazole
0.1% Dimethyl
sulfoxide
1 µM NTBC
10 µM NTBC
100 µM NTBC
750 µM
Phenobarbital
10 µM Rifampin
04 8 12
CYP2B6
10 20 300 40
Fold Increase
50
0.1% Dimethyl
sulfoxide
1 µM NTBC
10 µM NTBC
100 µM NTBC
10 µM Rifampin
Vehicle Control Prototypical InducerNTBC
CYP3A4
Cytochrome P450 activity Cytochrome P450 mRNA levels
Fold increase = treated / vehicle control