Acetylshikonin is a novel non-selective cytochrome P450 inhibitor

Article abstract of DOI:10.1002/bdd.2101

Acetylshikonin is a biologically active compound with anti-cancer and anti-inflammatory activity, which is isolated from the roots of Lithospermum erythrorhizoma. An inhibitory effect of acetylshikonin against CYP2J2 activity was discovered recently. Based on this result, this study was expanded to evaluate the inhibitory effects of acetylshikonin against nine different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using substrate cocktails incubation assay. Acetylshikonin showed a strong inhibitory effect against all P450s tested with IC₅₀ values of 1.4–4.0 μ m. Pre-incubation of acetylshikonin with HLMs and NADPH did not alter the inhibition potency, indicating that acetylshikonin is not a mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no inhibitory activity against CYP1A2, 2A6, 2E1 and 2J2, while it showed an inhibitory effect against CYP2B6, CYP2C19 and 2D6 with IC₅₀ values of 2.5, 3.6 and 0.5 μ m, respectively. Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could replace SKF-525A.

Full text of DOI:10.1002/bdd.2101

Short Communication
Acetylshikonin is a Novel Non-selective Cytochrome P450 Inhibitor
a
b
a
a
a


Jong Cheol Shon , Nguyen Minh Phuc , Won Cheol Kim , Jae Kyung Heo , Zhexue Wu ,
Hyunyoung Leea, and Kwang-Hyeon Liua
aBK21 Plus KNU Multi-Omics based Creative Drug Research Team, College of Pharmacy
and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu
41566, South Korea
bSaokim Pharmaceutical Company, Hanoi, Vietnam
These authors contributed equally to this work.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/bdd.2101
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Running title: Acetylshikonin as non-selective P450 inhibitor
Corresponding author: Kwang-Hyeon Liu
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook
National University, 80 Daehakro, Bukgu, Daegu 41566, South Korea
Voice: +82 53 950 8567; FAX: +82 53 950 8557; Email: dstlkh@knu.ac.kr
ABBREVIATIONS:
P450, cytochrome P450; HLM, human liver microsome; LC-MS/MS, liquid chromatography-
tandem mass spectrometry; SKF-525A, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl; SRM,
selected reaction monitoring
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Abstract
Acetylshikonin is biologically active compound with anti-cancer and anti-inflammatory
activity, which is isolated from the root of Lithospermum erythrorhizoma. We have recently
discovered a inhibitory effect of acetylshikonin against CYP2J2 activity. Based on this result,
we expanded our study to evaluate the inhibitory effects of acetylshikonin against nine
different cytochrome P450 (P450) isoforms in human liver microsomes (HLMs) using
substrate cocktails incubation assay. Acetylshikonin showed strong inhibitory effect against
all P450s tested with IC50 values of 1.4–4.0 M. Pre-incubation of acetylshikonin with HLMs
and NADPH did not alter inhibition potency, indicating that acetylshikonin is not a
mechanism-based inhibitor. SKF-525A, a widely used non-specific P450 inhibitor, had no
inhibitory activity against CYP1A2, 2A6, 2E1, and 2J2 while it showed inhibitory effect
against CYP2B6, CYP2C19, and 2D6 with IC50 values of 2.5, 3.6, and 0.5 M, respectively.
Our findings indicate that acetylshikonin may be a novel general P450 inhibitor, which could
replace SKF-525A.
Key words: acetylshikonin; cytochrome P450; inhibitor; SKF-525A
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Introduction
Identification of the specific cytochrome P450 (P450) enzymes responsible for the
biotransformation of drugs is important in drug discovery [1]. This information is essential
for understanding the drug-drug interaction potential and the inter-individual variability in
drug metabolism and pharmacokinetics. Appropriate inhibitors of the various P450 enzymes
are essential for conducting these in vitro studies [2].
SKF-525A (2-diethylaminoethyl-2,2-diphenylvalerate-HCl) is one of the most widely used
non-selective P450 inhibitor, and has been used to determine whether a drug is metabolized
by P450 enzymes [3, 4]. SKF-525A has inhibitory effects against CYP2B6, CYP2C8,
CYP2C9, CYP2C19, CYP2D6, and CYP3A, but not against CYP1A2, CYP2A6, or CYP2E1
(the IC50 values for these three are  50 M) [4-6]. Another useful non-specific P450
inhibitor is 1-aminobenzotriazole (ABT), which is thought to inactivate P450 enzymes
following complex formation with metabolic intermediates (MI) [2]. ABT inhibits the
activities of most P450s; however, it does not inhibit CYP2C9-mediated diclofenac 4’-
hydroxylation [4, 6]. In addition, the inhibitory activities of SKF-525A and ABT against
CYP2J2, which is responsible for the metabolism of drugs including albendazole, astemizole,
ebastine, and terfenadine [7], have not been reported. These results collectively demonstrate
that using ABT and SKF-525A as non-specific P450 inhibitors is not optimal.
Acetylshikonin (CAS 54984-93-9) is a bioactive compound derived from the roots of
Lithospermum erythorhizon and has been shown to have anti-cancer and anti-inflammatory
activities [8, 9]. It also showed strong noncompetitive inhibitory effects against CYP2J2-
mediated astemizole O-demethylation [10]. In the present study, we investigated the
inhibitory effects of acetylshikonin against ten major P450 isoforms (CYP1A2, 2A6, 2B6,
2C8, 2C9, 2C19, 2D6, 2E1, 2J2, and 3A) in order to evaluate the possibility of using it as a
nonselective general P450 inhibitor in phenotyping studies. In addition, we compared the
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P450 inhibitory potential of acetylshikonin with that of the currently used non-specific P450
inhibitors SKF-525A and thelephoric acid.
Materials and Methods
Chemicals and Reagents. Acetylshikonin ( 98.0%) and thelephoric acid (95.8%) were gifts
from the National Development Institute of Korean Medicine (Daegu, Korea). Astemizole,
dextrorphan, midazolam, omeprazole, tolbutamide, O-desmethylastemizole, 1’-
hydroxymidazolam, hydroxyomeprazole, and hydroxytolbutamide were purchased from
Toronto Research Chemicals (North York, Canada). Glucose-6-phosphate (G6P), glucose-6-
phosphate dehydrogenase (G6PDH), nicotinamide adenine dinucleotide phosphate (NADP+),
SKF-525A, shikonin (CAS 517-89-5), acetaminophen, amodiaquine, bupropion,
chlorzoxazone, coumarin, dextromethorphan, phenacetin, N-desethylamodiaquine,
hydroxybupropion,
hydroxychlorzoxazone,
hydroxycoumarin,
uridine
5’-
diphosphoglucuronic acid (UDPGA), mebendazole (MBZ, internal standard (IS)), and
terfenadine (TFD, IS) were obtained from Sigma-Aldrich (St. Louis, MO). Solvents were of
high-performance liquid chromatography (HPLC) grade, and all other reagents and chemicals
were of analytical grade (≥ 98%, Fisher Scientific Co., Pittsburgh, PA). Pooled human liver
microsomes (HLMs, H0630) were purchased from XenoTech (Lenexa, KS).
Microsomal Incubation
Reversible inhibition study: All incubations were performed in triplicate, and the data are
presented as average values. The inhibitory effect of acetylshikonin, shikonin, and SKF-
525A against CYP2J2-mediated astemizole O-demethylase activity was evaluated using
pooled HLMs. In brief, the incubation reaction mixtures contained 0.25 mg/ml HLMs,
astemizole (1 M) and inhibitor (0.5 – 50 M) in 0.1 mM phosphate buffer (pH 7.4), and
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were pre-incubated for 5 min at 37C. The reaction was initiated by the addition of NADPH-
generating system (containing 1.3 mM NADP+, 3.3 mM G6P, 3.3 mM MgCl2, and 500
unit/ml G6PDH). The final volume of the incubation mixture was 100 μl. After a 15 min
incubation period, the reactions were stopped by adding ice-cold acetonitrile containing 15
ng/ml terfenadine as the internal standard (IS). After centrifugation, aliquots (1 l) were
injected into a liquid chromatography-tandem mass spectrometry system (LC-MS/MS) as
described previously [11].
To evaluate the inhibitory activity of acetylshikonin, shikonin, and SKF-525A against nine
other P450 isoforms, namely CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A, a
previously-developed substrate cocktail method was used [12, 13]. The substrate
concentration, the selected reaction monitoring (SRM) transitions, and the collision energies
determined for each metabolite are listed in Table 1. Following a 15-min incubation of HLMs
(0.25 mg/ml) in the presence or absence of the inhibitor, the reaction was terminated and the
mixtures were centrifuged. Aliquots of the supernatants were analyzed by LC-MS/MS as
described previously [12, 13], with some modifications. The inhibitory effect of
acetylshikonin was also evaluated for CYP3A-mediated testosterone and nifedipine
metabolism as previously described [14].
Time-dependent inhibition study: Time-dependent inhibition (TDI) study was measured
using an IC50 shift method. The HLMs (0.25 mg/ml) were pre-incubated with the inhibitor
(acetylshikonin or thelephoric acid) at five different concentrations (0.5–50 M) in the
presence of an NADPH-generating system for 30 min. Thelephoric acid, a known non-
specific and time-dependent P450 inhibitor [5], was used as a positive control. The reaction
was initiated by adding P450 substrate, samples were further incubated for 15 min, and then
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the reaction was terminated by the addition of 100 l ice-cold acetonitrile containing IS.
After centrifugation, aliquots of the supernatants were analyzed by LC-MS/MS.
Data Analysis. IC50 values were determined using the WinNonlin software (version 2.1,
Pharsight, Mountain View, CA).
Results and Discussion
In this study, we evaluated the inhibitory potential of acetylshikonin against ten P450
isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, and 3A) and compared them
with those of SKF-525A [3] and thelephoric acid [5], known non-specific P450 inhibitors.
SKF-525A, which is the most commonly used general P450 inhibitor in reaction phenotyping
studies, showed strong inhibitory effects against CYP2B6, CYP2C19, and CYP2D6, with
IC50 values of 2.5, 3.6, and 0.5 M, respectively, whereas it had no inhibitory effects against
CYP1A2, CYP2A6, CYP2E1, or CYP2J2 (IC50  50 M) (Table 1). In a previous study,
SKF-525A was also reported to weakly inhibit CYP1A2, CYP2A6, and CYP2E1 with an
IC50 in the range 50–1,100 M [5, 6]. Similar to the previous results [5], thelephoric acid,
which is isolated from Polyozellus multiplex, moderately inhibited CYP2C8-mediated
amodiaquine O-deethylation and CYP2C9-mediated tolbutamide hydroxylation, with IC50
values of 6.9 and 11.4 M, respectively. Thelephoric acid showed strong inhibitory activities
against CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP2E1, and CYP3A (IC50  5
M) (Table 2).
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However, acetylshikonin exhibited strong inhibitory activities against all P450 enzymes
tested, with IC50 values  5.0 M. These findings showed that the compound had similar
inhibitory potential against all ten P450 enzymes in the range 1.4–4.0 M (Table 2), and its
inhibitory effects were stronger than those of thelephoric acid, a well-documented non-
specific P450 inhibitor (2.0 M  IC50  11.4 M), and the commonly used SKF-525A.
Acetylshikonin also inhibited CYP3A-mediated testosterone and nifedipine metabolism with
IC50 values of 5.2 and 3.0 M, respectively, indicating that it inhibits CYP3A activity in a
substrate-independent manner. These experiments demonstrated that acetylshikonin is a more
potent non-specific inhibitor of these ten P450 isoforms than are SKF-525A and thelephoric
acid in HLMs. Similar to acetylshikonin, shikonin, a structural analogue of acetylshikonin,
showed strong inhibitory effects against ten P450 isoforms (IC50  5.2 M, Table 2).
We next investigated whether the presence of NADPH led to shifts in the IC50 (Table 2).
The inhibitory potential of acetylshikonin against the ten P450 isoforms in the presence of an
NADPH-generating system (0.7 M  IC50  1.8 M) was similar to that in untreated HLMs
(0.9 M  IC50  3.3 M), suggesting that acetylshikonin is not a time-dependent inhibitor.
Acetylshikonin (at 5 M) inhibited all P450 isoforms tested by more than 60% (Fig. 1),
whereas SKF-525A did not inhibit any of its target isoforms (CYP1A2, CYP2A6, CYP2C9,
and CYP2E1) by more than 10%. In a previous study, acetylshikonin inhibited CYP2J2-
mediated astemizole O-demethylase activity in a noncompetitive manner [10]. Therefore,
acetylshikonin is speculated to inhibit noncompetitively other nine P450 enzymes.
In conclusion, we evaluated the inhibitory potential of acetylshikonin against P450 isoforms.
Acetylshikonin strongly inhibited the activities of ten P450 isoforms in a NADPH-
independent manner. Our results suggest that acetylshikonin can be used instead of SKF-
525A or thelephoric acid as a novel non-specific P450 inhibitor in reaction phenotyping
studies using HLMs.
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Footnotes
This study was supported by the Bio-Synergy Research Project [Grants NRF-
2014M3A9C4066459] and the National Research Foundation of Korea, Ministry of
Education [NRF-2016R1D1A1A09916782], Republic of Korea.
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Fig. 1. Inhibitory effects of 5 M acetylshikonin (Black), SKF-525A (White), and
thelephoric acid (Gray) on the enzyme activities of ten P450 isoforms in pooled human liver
microsomes (0.25 mg/ml, Xenotech H0630). The corresponding control activities of
phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion 4-hydroxylation,
amodiaquine N-deethylation, tolbutamide 4-hydroxylation, omeprazole 5-hydroxylation,
dextrorphan O-demethylation, chlorzoxazone 6-hydroxylation, astemizole O-demethylation,
and midazolam 1’-hydroxylation were 188, 57.0, 4.4, 41.7, 120, 137.5, 53.2, 282.4, 26.5, and
298.5 pmol/min/mg protein, respectively. The data are shown as the average of triplicate
determinations (n=3).
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Table 1. SRM parameters for the major metabolites of ten cytochrome P450s’ probe substrates used in all assays
P450 enzyme
1A2
Substrate
Phenacetin
Coumarin
Bupropion
Concentration (M)
Metabolite
Transition (m/z)
152>110
Collision energy (eV)
100
5
Acetaminophen
25
35
20
2A6
7-Hydroxycoumarin
Hydroxybupropion
163>107
2B6
50
256>238
2C8
Amodiaquine
1
N-Desethylamodiaquine
328>283
17
2C9
2C19
2D6
2E1
2J2
Tolbutamide
Omeprazole
100
20
5
4-Hydroxytolbutamide
5-Hydroxyomeprazole
Dextrorphan
287>89
362>214
258>157
184>120*
445>204
342>203
305>269
60
10
35
25
35
25
30
Dextromethorphan
Chlorzoxazone
Astemizole
50
1
6-Hydroxychlorzoxazone
O-Desmethylastemizole
1’-Hydroxymidazolam
6-Hydroxytestosterone
3A
Midazolam
5
Testosterone
2
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Nifedipine
2
Dehydronifedipine
-
345>284
472>436
30
25
IS
Terfenadine
15 ng/mL
* negative ionization mode
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Table 2. Inhibitory potential of acetylshikonin, shikonin, SKF-525A, and thelephoric acid
against the activities of ten cytochrome P450 isoforms in human liver microsomes.
P450
Marker activity
IC50 (μM)a
enzyme
Thelephoric
acid
Acetylshikonin
Time-
Shikonin SKF-525A
Time-
Reversible
Reversible Reversible
inhibition inhibition
dependent
inhibition
dependent
inhibition
inhibition
1A2
2A6
2B6
2C8
2C9
2C19
Phenacetin O-deethylase
Coumarin 7-hydroxylase
Bupropion 4-hydroxylase
Amodiaquine N-deethylase
Tolbutamide 4-hydroxylase
Omeprazole 5-hydroxylase
Dextromethorphan O-
4.0
3.8
2.0
1.4
3.3
2.5
4.9
4.6
2.1
2.3
3.4
3.9
3.0
2.5
2.3
1.7
2.8
2.2
> 50
> 50
2.5
3.5
2.0
2.3
6.9
11.4
2.8
26.6
24.4
3.6
2D6
2.5
3.7
2.5
0.5
2.6
demethylase
2E1
2J2
3A
Chlorzoxazone 6-hydroxylase
Astemizole O-demethylase
Midazolam 1’-hydroxylase
2.7
3.3
2.3
4.6
5.1
3.6
4.5
5.2
4.1
> 50
> 50
23.4
4.0
3.4
4.5
aValues are presented as average of triplicate determination (n=3).
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