Tofacitinib Is a Mechanism-Based Inactivator of Cytochrome P450 3A4

Article abstract of DOI:10.1021/acs.chemrestox.9b00141

Tofacitinib (TFT) is an oral JAK inhibitor which has been approved for the treatment of moderately and severely active rheumatoid arthritis. TFT was found to show concentration-, time-, and NADPH-dependent inhibition of CYP3A4, and irreversibility of the inactivation was also observed. Incubation (40 min, 37 °C) of recombinant CYP3A4 with TFT at 200 μM resulted in >70% loss of CYP3A4 activity. Estimated k_inact and K_I were 0.037 min^-1 and 93.2 μM, respectively. GSH and superoxide dismutase/catalase revealed minor or little protection against the CYP3A4 inactivation. Furthermore, ketoconazole attenuated TFT-mediated CYP3A4 inactivation. Epoxide and α-keto-aldehyde intermediates of TFT were trapped and characterized in microsomal incubations, respectively. The aldehyde intermediate is believed to be the key for the enzyme inactivation. Multiple P450 enzymes, including CYPs2C19, 3A4, 2D6, and 1A2, participated in the metabolism of TFT to the epoxide, while the formation of the aldehyde was mainly catalyzed by CYP3A4. In conclusion, TFT was proven to be a mechanism-based inactivator of CYP3A4.

Full text of DOI:10.1021/acs.chemrestox.9b00141

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Article
Tofacitinib is a mechanism-based inactivator of cytochrome P450 3A4
Xiucai Guo, Wei Li, Qingmei Li, Yan Chen, Guode Zhao, Ying Peng, and Jiang Zheng
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00141 • Publication Date (Web): 15 Aug 2019
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Tofacitinib is a mechanism-based inactivator of cytochrome P450 3A4
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Xiucai Guo†, Wei Li†, Qingmei Li†, Yan Chen†, Guode Zhao‡, Ying Peng*, † and Jiang
Zheng*, †, §
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‡
†Wuya College of Innovation, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, Shenyang, Liaoning, 110016, P.R. China;
§State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of
Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550004, P.
R. China
Correspondence authors:
Jiang Zheng, Ph.D.
1. Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, Liaoning, 110016,
P. R. China;
2. State Key Laboratory of Functions and Applications of Medicinal Plants, Key Laboratory of
Pharmaceutics of Guizhou Province, Guizhou Medical University, Guiyang, Guizhou, 550004, P.
R. China.
The two corresponding units contributed equally to this work.
Email: zhengneu@yahoo.com
Tel: +86-24-23986361 Fax: +86-24-23986510
Ying Peng, Ph.D.
Wuya College of Innovation, Shenyang Pharmaceutical University, PO Box 21,103 Wenhua
Road, Shenyang 110016, P. R. China
Email: yingpeng1999@163.com
Tel: +86-24-23986361 Fax: +86-24-23986510
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Abstract
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Tofacitinib (TFT) is an oral JAK inhibitor which has been approved for the
treatment of moderately and severely active rheumatoid arthritis. TFT was found to
show concentration-, time- and NADPH-dependent inhibition of CYP3A4, and
irreversibility of the inactivation was also observed. Incubation (40 min, 37 °C) of
recombinant CYP3A4 with TFT at 200 μM resulted in >70 % loss of CYP3A4 activity.
Estimated kinact and KI were 0.037 min-1 and 93.2 μM, respectively. GSH and
superoxide dismutase/catalase revealed minor or little protection against the CYP3A4
inactivation. Furthermore, ketoconazole attenuated TFT-mediated CYP3A4
inactivation. Epoxide and -keto-aldehyde intermediates of TFT were trapped and
characterized in microsomal incubations, respectively. The aldehyde intermediate is
believed to be the key for the enzyme inactivation. Multiple P450 enzymes, including
CYPs2C19, 3A4, 2D6, and 1A2, participated in the metabolism of TFT to the epoxide,
while the formation of the aldehyde was mainly catalyzed by CYP3A4. In conclusion,
TFT was proven to be a mechanism-based inactivator of CYP3A4.
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Introduction
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Cytochrome P450 enzymes are a family of heme proteins involved in the
metabolism of exogenous and endogenous compounds.1-2 Pharmaceutical agents are
one of the major categories of substrates. Among various drug-metabolizing P450
enzymes, CYP3A4 plays a critical role in the metabolism of marketed drugs up to 50%,
and the enzyme shows high-levels of protein expression in small intestine and liver.1-2
Mechanism-based inactivation (MBI) of P450s remains an important issue in drug
discovery and development. MBI provides the potential to amplify the effects of drug-
drug interactions by altering drug clearance after other drugs are co-administered and
finally results in safety issues during drug use.3-5
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Tofacitinib (TFT), an oral JAK inhibitor, was approved by the US Food and Drug
Administration for the treatment of moderately to severely active rheumatoid arthritis
in adult populations.6 TFT is an inhibitor of JAK1, JAK2, and JAK3, and slightly
decreases the activity of tyrosine kinase 2. In a cellular environment, TFT inhibits
signaling preferentially by heterodimeric receptors associated with JAK1 and/or
JAK3.6 By inhibition of JAK1 and/or JAK3, TFT is able to block signaling through the
receptors containing a common gamma chain for some cytokines, such as interleukin-
2, -4, -7, -9, -15, and -21, which are essential for lymphocyte proliferation, activation,
and function.6 Therefore, modulation of multiple aspects of the immune response may
be caused by the inhibition of cytokines. In the clinic, TFT is widely used to treat many
diseases such as psoriasis7, rheumatoid arthritis8-9, and inflammatory bowel disease10.
Meanwhile, many clinical side effects have been reported, such as headache,
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nasopharyngitis, nausea, elevated transaminases, leukopenia, neutropenia, and
lymphopenia.9, 11-13 However, the mechanistic basis of the side effects induced by TFT,
especially liver injury, are unclear.
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The major metabolic pathways of TFT include glucuronidation, oxidation of the
side-chain of the piperidine ring, N-demethylation, oxidation of the pyrrolopyrimidine
and piperidine rings.14 Although phase I metabolism of TFT has been well studied, it is
far from clear for phase II metabolism of TFT, and the correlation between metabolism
and the adverse effects of TFT remains unknown. In the present work, we investigated
the metabolic activation of TFT by trapping reactive metabolites generated in
microsomal incubations. On the basis of enzyme kinetics, we established that
mechanism-based inactivation of CYP3A4 is induced by TFT. These findings provide
new understanding of mechanisms of TFT toxic action needed to inform clinical
applications.
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Materials and methods
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Materials and reagents. Tofacitinib (TFT, >98%) was purchased from Dalian Meilun
Biotechnology Co., Ltd. (Dalian, China). Human recombinant P450 enzymes were
acquired from BD Gentest (Woburn, MA). Nicotinamide adenine dinucleotide
phosphate (NADPH), ketoconazole (KTZ), glutathione (GSH), S-hexylglutathione, N-
acetyl-L-cysteine (NAC), testosterone, ammonium acetate, Nα-acetyl-L-lysine (NAL),
superoxide dismutase (SOD) and catalase from bovine liver were obtained from Sigma-
Aldrich Chemical (St. Louis, MO). Mixed male human liver microsomes (HLMs) of
Mongolia people were purchased from Research Institute for Liver Diseases (Shanghai)
Co., Ltd. (Shanghai, China). Dialysis membrane (cut off: 3,500 Da) was acquired
from Beyotime Biotechnology (Shanghai, China). 1-Hydroxybenzotriazole
monohydrate (HOBt), di-tert-butyl dicarbonate (Boc2O), propranolol, β-alanin,
cyanoacetic acid, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride
(EDCI), and Raney nickel (< 150μM) were obtained from Aladdin Technology
(Shanghai, China). N-Methyl-N-((3R,4R)-4-methylpiperidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine hydrochloride (1 in Scheme 1) was purchased from J & K
Scientific Ltd. (Beijing, China). Distilled water was purchased from Wahaha Co. Ltd.
(Hangzhou, China). All the other reagents and chemicals were either analytical or high-
performance LC grade obtained from various sources.
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CYP3A4 assay. The formation of 6β-hydroxyl testosterone from testosterone was
measured to monitor CYP3A4 activity by LC-MS/MS. Mass spectrometric analysis
was performed on an AB SCIEX TRIPLE QUADTM 5500 (Applied Biosystems, Foster
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City, CA) coupled with an LC system (Agilent 1260). A Symmetry C18 column (3.5
μm, 4.6×75 mm; Waters, Wexford, Ireland) was applied for chromatographic
separation at 25 °C. Mobile phases were composed of 0.1% (v/v) formic acid in
acetonitrile (solvent A) and in water (solvent B). The flow rate was set at 0.8 mL·min-
1. A gradient was employed as follows: 20-20% A for 0-1.0 min, 20%-55% A for 1.0-
6.5 min, 55-90% A for 6.5-7.0 min, 90-90% for 7.0-8.0 min, 90-20% A for 8.0-8.5 min,
and 20-20% for 8.5-10.5 min. Quantification was carried out with multiple reaction
monitoring (MRM) for the detection of both internal standard propranolol (m/z
260.1116.1, CE 25) and 6β-hydroxyl testosterone (m/z 305.2269.2, CE 21) in
positive ion modes.
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Determination of concentration-, time-, and NADPH-dependent inactivation of
CYP3A by TFT. The primary incubation mixtures consisted of MgCl2 (3.2 mM),
HLMs (0.5 mg protein·mL-1), and TFT at various concentrations (0, 25, 50, 75, 100,
and 200 μM) with total volume 0.2 mL in 0.1 M potassium phosphate buffer (PBS, pH
7.4). After pre-incubation for 3 min at 37 °C, the primary incubations were triggered
by adding NADPH (1.0 mM). In addition, similar incubations without NADPH were
carried out as a negative control to investigate the contribution of metabolism on TFT-
induced enzyme inhibition. Subsequently, at time points of 0, 10, 20, and 40 min of the
incubations, respectively, aliquots (40 μL) of the incubation mixtures were transferred
into the secondary incubation mixtures containing NADPH (1.0 mM), MgCl2 (3.2 mM),
and testosterone (200 μM) with a total reaction volume of 0.12 mL in 0.1 M PBS (pH
7.4). After that, further reactions (37 °C, 15 min) were performed to assess the
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remaining CYP3A activity, and then the reactions were terminated by mixing with ice-
cold acetonitrile (equal volume) containing propranolol (internal standard). The
resulting mixtures were subjected to centrifugation at 19,000 g for 10 min, and the
resulting supernatants were analyzed by LC-MS/MS for the assessment of 6β-hydroxyl
testosterone. To determine whether CYP3A4 or CYP3A5 was inactivated, TFT was
incubated with individual human recombinant CYP3A4 and CYP3A5 (50 nmol)
instead of HLMs, and similar time-course enzyme inhibition studies were performed as
above.
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Determination of competitive inhibitor protection. Ketoconazole was used to
explore its protective effect on TFT-produced inactivation of CYP3A. The primary
reaction mixtures contained HLMs (0.5 mg protein·mL-1), TFT (100 μM), NADPH (1.0
mM), and ketoconazole at various concentrations (0.005, 0.01, and 0.05 μM), and
aliquots (40 μL) of the mixtures were taken to determine the remaining testosterone 6β-
hydroxylase activities at 0, 10, 20, and 40 min post incubations. As control, in parallel,
incubations were operated without TFT or ketoconazole.
Determination of effects of GSH and catalase/SOD on TFT-induced enzyme
inactivation. With GSH added, the primary incubation mixtures containing TFT (100
μM), NADPH (1.0 mM) and HLMs (0.5 mg protein·mL-1) were incubated at 37 °C.
Aliquots (40 μL) were withdrawn for the remaining CYP3A activity determination at
various time points. In a separate study, incubations containing TFT, NADPH, and
HLMs were performed in the present or absence of catalase (800 unit/mL) and SOD
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(800 unit/mL). As control samples, equal volume of PBS (0.1 mM, pH 7.4) was
included, instead of GSH or catalase/SOD solution. After incubation at various time
point, the remaining testosterone 6β-hydroxylase activities were then assessed as
described above.
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Determination of irreversibility of inhibition. Incubations containing MgCl2 (3.2
mM), TFT (100 μM), and HLMs (0.5 mg protein·mL-1) were performed with NADPH
(1.0 mM) added to trigger the reactions at 37 °C. TFT was absent in control incubations.
After incubation for 0 and 40 min, 40 μL of the incubation mixtures were withdrawn in
triplicate for enzymatic activity determination. At the same time, equal volume of the
incubation mixtures with/without TFT were respectively transferred into dialysis bags
and dialyzed against 1 L PBS (0.1 mM) for three times (2 h for each) at 4 °C. The
samples obtained from dialysis were allowed to stand to room temperature, followed
by determining the residual enzyme activities as described above.
Chemical Synthesis of TFT Analogs. The pyrroline and amino analogs of TFT (2 and
3 in Scheme 1) were synthesized, according to published literature with modification.15-
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To synthesize analog 2, starting material 1 (150 mg, 0.61 mmol) was dissolved in
methanol (10 mL), and Raney nickel (20 mg) was then added to catalyze the reaction.
The resultant mixture was placed into a sealed and hydrogen-purged reactor and stirred
with pressure 5 MPa at 45 °C for 3 days. Sample obtained was filtered and condensed
under vacuum, dissolved in CH2Cl2 (20 mL), and mixed with triethylamine (2 mL),
EDCI·HCl (1.22 mmoL), and HOBt (1.22 mmol). The resulting mixture was stirred at
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room temperature for 30 min, followed by addition of cyanoacetic acid (1.22 mmol)
and stirring for another 10 h at room temperature. After reaction completion monitored
by TLC (ethyl acetate: methanol = 9:1), the mixture was washed with water (10 mL)
for three times. The organic layer was collected, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. Purification of the product was achieved by silica
gel chromatography and semi-preparative HPLC. Product 2 (purity >98%) was
characterized by MS/MS (Figure S1), Q-TOF MS (Figure S4), and 1H NMR/13C NMR
(Figure S2) as shown in supporting information. The synthetic route of analog 2 is
shown in Scheme S1.
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To synthesize analog 3, a stirred solution of β-alanine (2.0 g, 22.5 mmol) in 10%
NaOH (30 mL) over ice-water bath was slowly mixed with 1.2 equiv. of Boc2O (6 mL,
26.0 mmol) in 20 min. The resulting mixture was kept at room temperature and stirred
for 14 h. The resulting mixture was washed with CH2Cl2. The aqueous layer was
acidified to pH 2~3 with 1.0 M H2SO4 and extracted with CH2Cl2. The extracts were
pooled, dried over anhydrous NaSO4, and concentrated under reduced pressure. The
resulting oil was dissolved in a minimum volume of CH2Cl2 and kept stirring with
petroleum ether added slowly over ice-water bath. The resulting white solid (3.39 g, 4)
was harvested by filtration. After that, compound 1 (100 mg, 0.41 mmol) was dissolved
in CH2Cl2 (20 mL), followed by addition of triethylamine (0.8 mL), EDCI·HCl (0.82
mmol), and HOBt (0.82 mmol) and stirring at room temperature for 30 min. The
resulting mixture was mixed with compound 4 (154 mg, 0.82 mmol) and stirred for 10
h at room temperature. Upon completion of the reaction as indicated by TLC (petroleum
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ether: ethyl acetate = 5:1), the reaction mixture was washed with water (10 mL) for
three times. The organic layer was collected, dried over anhydrous Na2SO4, and
concentrated under reduced pressure. Silica gel chromatography eluted with ethyl
acetate was employed to purify the product (5). An ethyl acetate solution saturated with
HCl was mixed with a solution of 5 in 2 mL ethyl acetate. After stirred for 2 h, the
solvent was removed by filtration to obtain faint yellow powder as chloride salt of
analog 3. The final product (3, >98%) was characterized by MS/MS (Figure S1), Q-
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1
TOF MS (Figure S4), and H NMR/13C NMR (Figure S3) as shown in supporting
information. The synthetic route of analog 3 is shown in Scheme S1.
Reactive metabolite trapping by NAL and NAC. Incubation mixtures with a total
volume of 0.2 mL contained TFT or 2 or 3 (100 μM), NAL (1.0 mM) or NAC (1.0
mM), and HLMs (0.5 mg protein·mL-1) or individual human recombinant P450
enzymes (including CYPs3A4 2A6, 2C9, 3A5, 2C19, 1A2, 2B6, 2D6 or 2E1; 100 nM
for each). The reactions were initiated by addition of NADPH (1.0 mM), and the
incubations were executed at 37 °C for 30 min. In negative control incubations,
NADPH was excluded. Termination of microsomal reactions was executed by addition
of ice-cold acetonitrile (equal volume) containing S-hexylglutathione (internal
standard), followed by vortexing and centrifugation at 19,000 g for 10 min. The
resultant supernatants were submitted to LC-MS/MS to seek designed NAC/NAL
conjugates. The same column was employed for chromatographic separation as
described above. Mobile phases consisted of acetonitrile (solvent A) and water
containing 5 mM ammonium acetate (solvent B). The flow rate was set at 0.8 mL·min-1,
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and gradient elution was applied as follows: 10-10% A for 0-2 min,10-40 % A for 2-
5.5 min, 40-90% A for 5.5-7.5 min, 90-90% A for 7.5-8.0 min, 90-10% A for 8.0-8.5
min, and 10-10% A for 8.5-11.0 min. MRM scan mode was used to detect metabolites
with m/z 474345 for NAC conjugates, m/z 472272, 488272, 474274, 490274
for NAL conjugates and m/z 392.2263.2 for S-hexylglutathione. The collision energy
(CE) was set at 50 for NAL conjugates, 35 for NAC conjugates and 20 for S-
hexylglutathione. MS2 scan mode was employed at a scan range for product ions from
m/z 50 to 500 in positive mode. The data were analyzed by analyst software of Applied
Biosystems/SCIEX.
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Determination of effects of TFT analogs on enzyme inactivation. The primary
incubation mixtures consisted of MgCl2 (3.2 mM), CYP3A4 (50 nM), and TFT or 2 or
3 (200 μM) in 0.1 M PBS (pH 7.4) with total volume 0.2 mL. The proportion of organic
solvent was kept under 1%. Control incubation was also conducted in the absence of
TFT or 2 or 3. After pre-incubation for 3 min at 37 °C, the primary incubations were
triggered by adding NADPH (1.0 mM). And at time points of 0, 10, 20, and 40 min
after incubation, the remaining CYP3A4 activities were assessed as above.
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Results
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Concentration-, Time-, and NADPH-dependent inhibition of CYP3A4 by TFT.
Residual CYP3A activity of HLMs exposed to TFT was determined by monitoring the
catalytic ability to oxidize testosterone to its 6β-hydroxyl metabolite. A natural
logarithm plot of percent residual activity against incubation time was employed.17 The
remaining enzyme activity of HLMs after exposure to TFT at 0 min was defined as 100%
for comparing the inactivation effects at various incubation time points and
concentrations. As displayed in Figure 1A, the remaining enzymatic activities
decreased following the increase of the incubation time and concentrations of TFT
applied. After HLMs were incubated with 200 μM TFT for 40 min at 37 °C, about 66%
of CYP3A activity was lost. Neither TFT-free nor NADPH-free microsomal
incubations showed such enzyme activity loss (Figure 1C). The rates (kobs) of the
observed enzyme inactivation were acquired from linear regression analyses of the
time- and concentration-dependent inhibition data. To estimate the concentration
required for maximal rate constant of inactivation (kinact) and half-maximal inactivation
(KI), a double-reciprocal plot of kobs with TFT concentrations was employed according
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to a published method , and the values of kinact and KI were found to be 0.037 min-1
and 93.2 μM, respectively (Figure 1B). Moreover, recombinant enzyme incubation
studies demonstrated that 76.7% of CYP3A4 activity was suppressed after 40 min
incubation with TFT (200 μM) at 37 °C (Figure 8), but no such enzyme inactivation
was observed with CYP3A5 (data not shown) under the same incubation condition.
Competitive inhibitor protection. Ketoconazole is a competitive inhibitor of CYP3A
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enzymes and would suppress the metabolism of TFT by CYP3A in HLM incubations.
As a result, ketoconazole revealed protective effect against TFT-induced time-
dependent inhibition (Figure 2). With the increase of ketoconazole concentrations (0,
5, 10, and 50 nM) applied in HLM incubations, decrease in TFT-induced (100 μM)
testosterone 6β-hydroxylase inactivation was observed (47.3±1.2%, 55.8±0.9%,
66.7±6.6%, and 87.3±2.9% in the remaining enzyme activity, respectively).
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Effects of GSH and catalase/SOD on the inactivation of CYP3A4. GSH is a
nucleophilic agent which can react with electrophilic intermediate(s) released from the
active site of host enzymes. After 40 min microsomal incubation at 37 °C, TFT
exposure (100 μM) resulted in significant CYP3A activity loss (38.9±7.5% activity
remained), and the presence of GSH demonstrated limited protection (49.9±6.5%
activity remained) against TFT-induced CYP3A inactivation. Besides, scavengers of
reactive oxygen species catalase and SOD showed no protection on the observed
inactivation of CYP3A.
Irreversibility of inactivation. Protein dialysis experiments were carried out to
determine the reversibility of TFT-induced CYP3A inhibition. The residual enzyme
activities were adjusted, according to that of control at time 0 min. As a result, compared
with those samples without dialysis disposition, there were no marked recoveries of
enzyme activities in samples with dialysis (Table 1).
Identification of reactive metabolites. NAC and NAL were used as nucleophilic
agents to trap potential reactive intermediates of TFT in microsomal incubations
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containing TFT and NADPH. By MRM transition of m/z 474345, three major NAC
conjugates were detected at retention time of 5.50 min for M1, 5.75 min for M2, and
6.25 min for M3 (Figure 3B). In comparison, no chromatographic peaks responsible for
M1-M3 were observed in NADPH-free microsomal incubations (Figure 3A), which
suggests that metabolism was involved in the formation of the three NAC conjugates.
The MS result showed that all three compounds displayed [M+H]+ ions at m/z 474.
Additionally, M1 showed fragment ions at m/z 474, 432, 369, 345, 310, 278, 244, 181,
165, and 98 (Figure 3C), M2 revealed ions at m/z 474, 432, 369, 345, 310, 278, 181,
165, and 98 (Figure 3D), and M3 displayed fragment ions of m/z 474, 345, 310, 272,
246, 181, and 165 (Figure 3E). The product ion at m/z 345 as shown in the spectra arises
from the indicative and characteristic neutral loss of 129 Da of NAC conjugates, and
ions m/z 165 and 310 were postulated to be derived from the fragment by the cleavage
of the tertiary C-N bond, which indicates that the three compounds were formed by
NAC conjugation with moiety 7H-pyrrolo[2,3-d]pyrimidine. Furthermore, no such
NAC conjugates were detected in analog 2-fortified incubations in the presence of NAC
under the same condition by monitoring the transition of m/z 476347 (data not shown).
In addition, by MRM of the transitions of m/z 472272 and 488272, two NAL
conjugates were detected at retention time of 6.09 min for M4 and 6.46 min for M5
(Figure 4B and 4E). No such chromatographic peaks were observed in microsomal
incubations without NADPH (Figure 4A and 4D). Likewise, the generation of the NAL
conjugates also required P450-mediated metabolism. The MS/MS spectrum of M4
showed fragment ions of m/z 488, 444, 400, 383, 272, 246, 235, 173, and 149 (Figure
15
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4C), and the spectrum of M5 displayed product ions of m/z 472, 430, 384, 272, 246,
173, and 149 (Figure 4F). The product ion at m/z 272 corresponded to the fragment
from cleavage of the C-carbonyl bond, and ion m/z 149 detected was postulated to be
derived from the fragment by the cleavage of the tertiary carbon-N bond of TFT, which
indicates that the carbonyl group on the right side of TFT was intact. Moreover, by
scanning the m/z 402149 transition, an aldehyde intermediate derived from TFT was
detected at retention time of 6.69 min, which was found to require P450-mediated
metabolism (Figure 7A and 7B). In addition, in NAL-fortified microsomal incubations
with analog 3 under the same condition, no such NAL conjugates were detected by
acquiring ion pairs m/z 472272 and 488272 (data not shown). Instead, two NAL
conjugations, as expected, were detected in incubations of analog 2 with NAL by
monitoring ion pairs m/z 474274 and 490274, and the saturation of pyrrolic moiety
did not block the formation of the electrophilic center which reacted with NAL (Figure
5).
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P450 enzymes responsible for TFT bioactivation. To determine the catalytic
efficiency of various P450 enzymes for the metabolic activation of TFT, nine individual
human recombinant P450 enzymes (including CYPs3A4 2A6, 2C9, 3A5, 2C19, 1A2,
2B6, 2D6 and 2E1; 100 nM for each) were incubated with TFT in the presence of NAC
or NAL, follow by LC-MS/MS analysis. As shown in the Figure 6, the three TFT-NAC
conjugates were detected in incubation mixtures containing CYPs2C19, 3A4, 1A2, or
2D6, but the other P450 enzymes examined only played limited roles in the formation
of the reactive metabolite trapped by NAC. Differently, it appears that CYP3A4
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dominantly catalyzed the generation of the reactive metabolite trapped by NAL, and
the other P450 enzymes tested showed limited capabilities of the TFT bioactivation. As
expected, the aldehyde intermediate was formed by the catalysis of CYP3A4, and the
resulting aldehyde was captured by NAL to form M4 and M5 (Figure 7C).
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Effects of TFT analogs on CYP3A4 inactivation. To define the mechanisms of TFT-
induced CYP3A4 inactivation, analogs 2 and 3 were synthesized to probe the roles of
pyrrole and nitrile moieties in the inactivation induced by TFT. Analog 2 resulted in
significant inactivation after 40 min at 37 °C, although the inactivation was not as potent
as that of TFT (43.9±4.8% vs. 23.3±4.1% in the remaining enzyme activity), while
analog 3 barely inactivated CYP3A4 (Figure 8).
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Discussion
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Our present studies clearly demonstrated that TFT inhibits CYP3A in both
concentration- and time-dependent manners (Figure 1), with a kinact of 0.037 min-1 and
a KI of 93.2 μM. NADPH was indispensable for TFT-induced CYP3A inactivation,
which indicates that the observed enzyme inactivation was mediated by P450 enzymes.
Ketoconazole, a competitive CYP3A inhibitor, attenuated TFT-induced CYP3A
inactivation in a concentration-dependent manner (Figure 2), indicating that
ketoconazole and TFT competed with each other to enter the active site of CYP3A,
leading to the observed protective effect. Catalase and SOD, scavengers of reactive
oxygen species, failed to protect against enzyme inactivation, which allows us to
exclude the involvement of reactive oxygen species potentially formed in the enzymatic
reaction.18
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Mechanism-based enzyme inactivation is considered to result from covalent
modification of host enzymes. Exhaustive dialysis did not restore TFT-induced CYP3A
inhibition (Table 1), implying that covalent modification was involved in the enzyme
inactivation. In addition, the nucleophilic agent GSH exhibited limited ability to
attenuate TFT-induced CYP3A inactivation, indicating that reactive metabolites
released from the active site of the host enzyme did not induce the enzyme inactivation,
even though they reacted with nucleophilic amino acid residues (non-active site).
Interestingly, CYP3A5, unlike CYP3A4, was not susceptible to time-dependent
inhibition mediated by TFT, since CYP3A5 almost failed to catalyze the oxidation of
TFT to the reactive metabolites. Taken together, the evidence indicates TFT is a
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mechanism-based inactivator of CYP3A4.
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Characterization of reactive metabolites is an important part of mechanism-based
enzyme inactivation study. Structurally, TFT contains two potential sites for oxidative
metabolism, including pyrrole and cyanomethyl groups, which could be metabolized to
pyrrole-epoxide and aldehyde intermediates, respectively. Both of the two
intermediates are chemically reactive to nucleophiles, and may cause enzyme inhibition
and reported liver injury.19-20 Pyrrole-epoxide 6 and aldehyde 7 (Scheme 2) were
postulated to be the reactive intermediates responsible for covalent modification. NAC
and NAL were used as nucleophiles to trap the two intermediates on account of their
chemical properties. Three NAC conjugates derived from TFT were detected in NAC-
fortified microsomal incubations. Thiols are known to be reactive to epoxides. It is most
likely that NAC conjugate formation arose from reaction of NAC with the epoxide,
followed by dehydration. Analog 2 was synthesized to probe the role of metabolic
epoxidation in bioactivation of TFT. As expected, NAC-fortified microsomal
incubation of analog 2 failed to generate the corresponding NAC conjugates, which
implies that the double bond of the pyrrole moiety in TFT was required for the
formation of the NAC conjugates, and the loss of the unsatuation made the metabolic
epoxidation impossible. Interestingly, analog 2 was also found to inactivate CYP3A4
enzyme. In other words, the selective saturation of the double bond did not knock out
the inhibitory effect. This indicates that the epoxidation of TFT was not critical for the
observed CYP3A4 inactivation (Scheme 2).
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In addition, two NAL conjugates were detected in NAL-fortified microsomal
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incubations. The MS/MS spectra of the two metabolites displayed the core frame of
TFT but without the nitrile group. We speculated that the loss of nitrile resulted from
oxidative denitrilation (Scheme 2) and the resulting -keto-aldehyde reacted with the
trapping agent to form the two NAL conjugates. Metabolic activation of analog 3, a
product resulting from nitrile reduction of TFT, was investigated, and no such NAL
conjugates were detected in microsomal incubations. It is not surprising since analog 3
is unable to undergo oxidative denitrilation. Furthermore, analog 3 did not induce
CYP3A4 inactivation at all (Figure 8), which suggests that the oxidative denitrilation
was essential for TFT-induced CYP3A4 inactivation (Scheme 2). Additionally, two
analog 2-derived NAL conjugates were detected (Figure 5) in NAL-fortified
microsomal incubations after exposure to analog 2. This could explain why analog 2
remained the ability to inactivate CYP3A4 (Figure 8). This may also support the
proposed mechanism that oxidative denitrilation of TFT offered -keto-aldehyde 7 that
reacted with apoprotein or/and heme resulting in enzyme inactivation (Scheme 2).
Furthermore, it appears that the formation of the NAL-conjugates and -keto-aldehyde
was mediated by CYP3A4 rather than CYP3A5, which could explain why TFT failed
to inhibit CYP3A5 activity.
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Mechanism-based inactivation of drug-metabolizing enzymes could lead to severe
drug-drug interactions when other drugs are co-administered. Toxic and side effects
could be caused by resulting elevated plasma levels of the drug, increasing internal
exposure. Examples like this often occur: Paroxetine (a mechanism-based inactivator
of CYP2D6) co-administrated in patients with risperidone medication for schizophrenia
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or schizoaffective disorder caused 1.5-fold elevation of concentrations of risperidone;21-
22 Oral midazolam in healthy volunteers pretreated with verapamil (a mechanism-based
inactivator of CYP3A4) showed three times AUC elevation and the peak concentration
of midazolam was doubled compared with that pretreatment with placebo;23-24 Imatinib
(a mechanism-based inactivator of CYP3A4) significantly decreased the total-body
clearance of drug from plasma with a mean reduction of 70% for simvastatin.25-26 In
the present work, TFT was proven to be a mechanism-based inactivator of CYP3A4,
suggesting caution for the application of TFT, especially co-administrated with other
medicines, to avoid the likelihood of adverse drug-drug interactions.
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Metabolic activation of drugs is an important mechanism for drug-induced liver
injury, and reactive metabolites could react irreversibly with endogenous
macromolecules, which may result in the alternation of physiological function.27 The
discovery of the two reactive metabolites provides a potential biochemical basis for
liver damage induced by TFT, and suggests that further research is needed to determine
their contributions to TFT-induced liver injury.
In conclusion, strong evidence indicates that TFT acted as a mechanism-based
inactivator of CYP3A4 in a time- and concentration-dependent manner, and the
inhibition of CYP3A4 was irreversible. TFT was metabolized by HLMs and human
recombinant P450 enzymes to electrophilic pyrrole-epoxide and -keto-aldehyde
intermediates, and the latter may be responsible for the observed CYP3A4 inactivation.
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Supporting Information. [Additional figures and experimental procedures.]
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Abbreviations:
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TFT, tofacitinib; RSD, relative standard deviation; IS, internal standard; ESI,
electrospray ionization source; KTZ, ketoconazole; NAC, N-Acetyl-L-cysteine; EP,
entrance potential; MRM, multiple reaction monitoring; GSH, glutathione; DP,
declustering potential; NAL, Nα-Acetyl-L-lysine; CE, collision energy; NADPH,
nicotinamide adenine dinucleotide phosphate; CXP, collision cell exit potential; HOBt,
1-Hydroxybenzotriazole Monohydrate; SOD, superoxide dismutase; EDCI, N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride; HLMs, human liver
microsomes; Boc2O, Di-tert-butyl dicarbonate; PBS, potassium phosphate buffer; AUC,
area under the concentration-time curve.
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Funding sources
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This work was supported in part by the National Natural Science Foundation of China
(Grants 81430086, 81773813, 81830104).
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Notes
The authors declare no competing financial interest.
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