Metabolic Activation of Pirfenidone Mediated by Cytochrome P450s and Sulfotransferases

Article abstract of DOI:10.1021/acs.jmedchem.9b02073

Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis. Idiosyncratic drug reactions, due to clinical application of pirfenidone, have been documented, even along with death cases resulting from acute liver failure. The present study aimed at the investigation of metabolic activation of pirfenidone possibly participating in the reported adverse reactions. Pirfenidone-derived glutathione/N-acetylcysteine (GSH/NAC) conjugates were detected in microsomal/primary hepatocyte incubations after exposure to pirfenidone. The GSH/NAC conjugates were also observed in bile and urine of rats given pirfenidone, respectively. The observation of the conjugates suggests the formation of a quinone methide intermediate derived from pirfenidone. The intermediate was possibly generated through two pathways. First, pirfenidone was directly metabolized to the quinone methide intermediate via dehydrogenation; second, pirfenidone was oxidized to 5-hydroxymethyl pirfenidone, followed by sulfation to a benzyl alcohol-sulfate derivative. The findings facilitate the understanding of the mechanisms of pirfenidone-induced idiosyncratic toxicity and assist medicinal chemists to minimize toxicities in the development of new pharmaceutical agents.

Full text of DOI:10.1021/acs.jmedchem.9b02073

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Article
Metabolic activation of pirfenidone mediated
by cytochrome P450s and sulfotransferases
Shenzhi Zhou, Wei Li, Min Tian, Na Zhang, Xiaojing Yang, Weiwei Li, Ying Peng, and Jiang Zheng
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02073 • Publication Date (Web): 09 Jul 2020
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Metabolic activation of pirfenidone mediated by cytochrome
P450sand sulfotransferases
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Shenzhi Zhou,† Wei Li,† Min Tian,† Na Zhang,† Xiaojing Yang,† Weiwei Li,*‡ Ying
Peng,*† and Jiang Zheng*†‡§
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†Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang,
Liaoning 110016, P. R. China
‡State Key Laboratory of Functions and Applications of Medicinal Plants, Guizhou
Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, Guiyang,
Guizhou 550025, P. R. China
§Key Laboratory of Environmental Pollution, Monitoring and Disease Control (J.Z.),
Ministry of Education, Guizhou Medical University, Guiyang, 550025, P. R. China
Corresponding Authors
Jiang Zheng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua
Road, 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, 9
Beijing Road, Guiyang, Guizhou, 550004, P. R. China
Email: zhengneu@yahoo.com
Tel: +86-24-23986361; Fax: +86-24-23986510
Ying Peng, PhD
Wuya College of Innovation, Shenyang Pharmaceutical University, No. 103, Wenhua
Road, Shenyang, Liaoning, 110016, P. R. China
Email: yingpeng1999@163.com
Tel: +86-24-23986361; Fax: +86-24-23986510
Weiwei Li, PhD
Guizhou Provincial Key Laboratory of Pharmaceutics, Guizhou Medical University, 9
Beijing Road, Guiyang, Guizhou, 550004, P. R. China
Email: weiweili0615@163.com
Tel: +86-24-23986361; Fax: +86-24-23986510
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ABSTRACT
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Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis.
Idiosyncratic drug reactions, due to clinical application of pirfenidone, have been
documented, even along with death cases resulting from acute liver failure. The
present study aimed at the investigation of metabolic activation of pirfenidone possibly
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participating in the reported adverse reactions.
Pirfenidone-derived GSH/NAC
conjugates were detected in microsomal/primary hepatocyte incubations after exposure
to pirfenidone. The GSH/NAC conjugates were also observed in bile and urine of rats
given pirfenidone, respectively. The observation of the conjugates suggests the
formation of a quinone methide intermediate derived from pirfenidone.
The
intermediate was possibly generated through two pathways. First, pirfenidone was
directly metabolized to the quinone methide intermediate via dehydrogenation; second,
pirfenidone was oxidized to 5-hydroxymethyl pirfenidone, followed by sulfation to a
benzyl alcohol-sulfate derivative. The findings facilitate the understanding of the
mechanisms of pirfenidone-induced idiosyncratic toxicity and assist medicinal
chemists to minimize toxicities in the development of new pharmaceutical agents.
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INTRODUCTION
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Idiopathic pulmonary fibrosis (IPF) is a severe form of fibrosing interstitial
pneumonia, which may cause progressive loss of pulmonary functions and eventually
respiratory failure.1 The pathogenic factors of IPF are perplexing, including but not
limited to cigarette smoking, breathing dust, viral infections and exposures to the
contaminated environment.2 Briefly, the micro-injuries mentioned above disturb lung
repeatedly, resulting in death of epithelial cells. Then an inordinate wound healing
leads to imbalance between fibrotic and anti-fibrotic factors, which is involved in
excessive proliferation of myofibroblasts, and then the progressive remodeling results
in IPF ultimately.1,3
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Pirfenidone (5-methyl-1-phenyl-1H-pyridine-2-one, 1), a pyridone derivative, has
been authorized for the treatment of IPF in Japan, Europe, the United States, China, and
other countries.4 Pirfenidone is considered to exert an anti-fibrotic effect via the
reduction of transforming growth factor-β1, a pro-inflammatory and profibrotic
cytokine. Inhibition of other pro-inflammatory cytokines such as tumor necrosis
factor-α also contributes to its anti-inflammatory property.5,6 As an anti-oxidant,
pirfenidone can scavenge hydroxyl radical and superoxide anion.7,8
The
recommended oral dosage of pirfenidone is 801 mg a day in the first week, then the
dose is elevated to 1,602 mg/day in the second week, and final therapy is invariable to
2,403 mg/day (801 mg taken 3 times) from 15th day onwards.9,10 The most common
side effects of pirfenidone include gastrointestinal reactions, photosensitization, and
elevation of serum ALT/AST and bilirubin.9-11 It was reported that 2.9% of patients
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showed increased serum ALT/AST activities 3-fold greater than that of normal upper
limits.11 In 2017, a 77-year-old patient taking pirfenidone 600 mg/day was reported
to die, due to suffering acute liver failure.12 Another death case resulting from
pirfenidone-related acute liver failure was documented in 2019.13
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Previous studies indicated that pirfenidone is mainly metabolized by cytochrome
P450 enzymes, especially CYP1A2.9
Pirfenidone can be oxidized to 5-
hydroxymethyl pirfenidone (5-hydroxymethyl-1-phenyl-1H-pyridine-2-one, 2) by
hydroxylation of the methyl group, followed by further oxidation to a more polar
metabolite, 5-carboxy pirfenidone.9 Pirfenidone and 5-carboxy pirfenidone can also
undergo glucuronidation.9,14
Sulfation, catalyzed by sulfotransferases (SULTs), is one of phase II conjugation
pathways responsible for metabolism of various endogenous and exogenous
molecules.15,16
SULTs catalyze the transfer of a sulfonyl group from 3′-
phosphoadenosine-5′-phosphosulfate (PAPS) to an amino, hydroxyl or sulfhydryl
group of substrates.17 Like many drug metabolizing enzymes, SULTs tend to increase
hydrophilicity of substances and thus facilitate their excretion in urine. However,
SULTs are reportedly involved in bioactivating certain compounds in vivo, especially
for allylic alcohols, benzyl alcohols, and aromatic hydroxylamines.15,17 Sulfate is a
good leaving group, and the departure of the sulfonyl group may produce reactive
electrophilic intermediates, which may result in potential toxicities.18,19 For example,
aloe-emodin was reported to be metabolized to the corresponding sulfate by SULTs,
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which possibly induces cytotoxicity to HepG2 cells.20 SULT-mediated metabolism
of lucidin was found to generate an electrophilic metabolite that reacts with DNA.21
Idiosyncratic drug reactions (IDRs) vary from individual to individual, and it is
difficult to reproduce IDRs in preclinical research for the lack of generally applicable
animal models. IDRs are almost the most unpredictable risk in the drug development
phase.22 Because of their puzzling uniqueness and low occurrence, IDRs are usually
not observed until drugs are extensively consumed in patient population, which means
both a great loss of resources for the pharmaceutical industry and a fatal threat for
patients.22-24 Although most side effects of pirfenidone are tolerable and reversible by
drug discontinuation, the common anaphylactic reactions, such as photosensitization,
drug eruption, and drug induced lupus erythematosus, particularly along with 2 death
cases resulting from pirfenidone-related acute liver failure, have been catching our
attention. The recommended daily doses9,10 of pirfenidone are quite high, not only for
the maximum (2,403 mg/day) but also for the minimum (801 mg/day), which increases
the risk of idiosyncratic toxicity.25,26 Although the incidence of idiosyncratic effect of
pirfenidone is rare, its consequence is fatal and the mechanisms of its toxic action
remain unknown. Idiosyncratic toxicity is considered to be associated with metabolic
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activation and immune-mediation.22,27-29
Thus we initiated the investigation to
determine whether pirfenidone is bioactivated and to define the metabolic pathways
involved in the metabolic activation of pirfenidone.
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RESULTS
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Metabolic activation of pirfenidone by P450 enzymes.
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As an initial step, we incubated 1 in rat liver microsomes (RLMs) fortified with
cofactor NADPH and trapping agent glutathione (GSH) or N-acetylcysteine (NAC).
At least triplet experiments were conducted in all in vitro studies to exclude contingency.
A chromatographic peak with a retention time at 7.25 min was observed by monitoring
precursor/product (Q1/Q3) ion pair m/z 202/77 tentatively responsible for compound 2,
a primary oxidative metabolite of pirfenidone (Figure 1).
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Figure 1. Characterization of metabolite 2. Extracted ion (m/z 202/77) chromatograms
obtained from LC-MS/MS analysis of RLM incubations of 1 in the absence (A) or
presence (B) of NADPH. Extracted ion (m/z 202/77) chromatogram obtained from
analysis of bile collected from vehicle-treated (D) or 1-treated (E) rats and of synthetic
2 (C).
To verify the structure of the oxidative metabolite, compound 2 was chemically
synthesized by hydration of 5-bromomethyl-1-phenyl-1H-pyridine-2-one (P-Br, refer
to Experimental Section 2.1., Scheme 4). Compound 2 was successfully synthesized
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and characterized by mass spectrometry and NMR (refer to Experimental Section 2.1.).
As expected, synthetic 2 shared similar retention time and fragmentation pattern with
the one observed in microsomal incubations. This allowed us to ensure the metabolic
hydroxylation of the methyl group on pyridone ring of 1 to 2.
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A pirfenidone-derived GSH conjugate (10, Scheme 1) with a retention time at 6.6
min (Figure 2) was detected in GSH-fortified RLM incubations by acquiring Q1/Q3
ion pair m/z 491/184 in positive mode (Figure 2B). The tandem mass spectrometric
(MS/MS) spectrum of GSH conjugate 10 was obtained by acquiring enhanced product
ion (EPI) scanning with a range of m/z 50 to 550 (Figure 2D). The mass spectrum of
the GSH conjugate was quite simple with limited fragments. The most abundant
fragment generated was ion m/z 184 resulting from the cleavage of the S−C bond from
the GSH moiety. And fragment ion at m/z 218 came from the cleavage of the C−S bond
of the GSH moiety. The neutral loss of the γ-glutamyl moiety from m/z 491 led to the
production of fragment ion m/z 362. The characteristic fragment ion at m/z 156
resulted from the elimination of CO from the pirfenidone portion.
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Figure 2. Characterization of GSH conjugate 10 generated in microsomal incubations.
Extracted ion (m/z 491/184) chromatograms obtained from LC-MS/MS analysis of
RLM incubations of 1 fortified with GSH in the absence (A) or presence (B) of NADPH.
D: MS/MS spectrum of GSH conjugate 10 produced in RLM incubations. Extracted
ion (m/z 491/184) chromatogram (C) and MS/MS spectrum (E) of synthetic GSH
conjugate 10.
Pirfenidone-derived NAC conjugate 11 (Scheme 1) was detected at the retention
time of 7.26 min by acquiring Q1/Q3 ion pair m/z 347/184 (Figure 3B). EPI scanning
was employed to acquire the MS/MS spectrum of NAC conjugate 11 as well (Figure
3D). Again, ion m/z 184 was found to be the most abundant fragment for the NAC
conjugate, which was from the cleavage of S−C bond of 1. NAC conjugate 11 showed
similar fragmentation pattern as GSH conjugate 10, such as fragment ions m/z 218 and
156 derived from the cleavage of C−S bond attached with the NAC moiety and from
the pirfenidone moiety, respectively. No such GSH and NAC conjugates were
observed in NADPH-free incubations. This suggests that the formation of the two
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metabolites was NADPH-dependent, implicating the involvement of P450s in the
generation of the pirfenidone-derived reactive metabolites.
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Figure 3. Characterization of NAC conjugate 11 generated in microsomal incubations.
Extracted ion (m/z 347/184) chromatograms obtained from LC-MS/MS analysis of
RLM incubations of 1 fortified with NAC in the absence (A) or presence (B) of NADPH.
D: MS/MS spectrum of NAC conjugate 11 produced in RLM incubations. Extracted
ion (m/z 347/184) chromatogram (C) and MS/MS spectrum (E) of synthetic NAC
conjugate 11.
Chemical synthesis was executed to verify the structure of the GSH and NAC
conjugates above. The synthetic products were analyzed by LC-MS/MS and Q-TOF
MS. The retention times and MS/MS spectra of the synthetic conjugates were similar
as those of the GSH/NAC conjugates detected the in vitro and in vivo studies (Figures
2C/2E, 3C/3E, 6C/6E, 7C/7E, 8C/8E). The structure of GSH conjugate 10 was further
confirmed by NMR (refer to Experimental Section 2.2.).
Scheme 1. Proposed pathways of metabolic activation of pirfenidone.
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P450 enzymes responsible for metabolic activation of 1.
Human recombinant P450s 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5
were individually incubated with 1 supplemented with NADPH and GSH. The
catalytic capabilities of these enzymes were evaluated by monitoring the formation of
GSH conjugate 10. The results shown in Figure 4A demonstrated that CYP3A4
dominated the catalysis responsible for the formation of quinone methide 3 (by
monitoring GSH conjugate 10), closely followed by CYP3A5. Figure 4B describes
the graphic contribution of each P450 enzymes to the metabolic activation after
normalization, according to their natural abundance in human liver microsomes.31
CYP3A4 contributed the most to the formation of quinone methide 3, followed by
CYP2A6 and CYP1A2.
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Figure 4. Role of individual human recombinant P450 enzymes in metabolic activation
of pirfenidone. A: Formation of GSH conjugate 10 in incubations of 1 with nine
individual human recombinant P450 enzymes was measured by LC-MS/MS. The most
abundant GSH conjugate 10 detected was normalized to 100%; B: The contribution of
each P450 enzyme was calculated based on the formation of GSH conjugate 10 after
normalization, according to the natural abundance of the corresponding P450 enzymes
in human liver microsomes. Data represent the mean ± SD (n = 3).
Metabolic activation of 1 and 2 in primary hepatocytes.
Bioactivation studies were also conducted in cultured rat primary hepatocytes.
Freshly isolated hepatocytes were incubated with parent drug 1 or primary metabolite
2. As expected, GSH conjugate 10 was detected in the cell culture system after
exposure to 1 (Figure 5B). Interestingly, GSH conjugate 10 was also found in 2-
treated hepatocytes (Figure 5C).
In a separate study, cells were respectively preincubated with ketoconazole (KTC,
a selective CYP3A inhibitor), 2,6-dichloro-4-nitrophenol (DCNP, an inhibitor of
SULTs), or vehicle (0.1 % DMSO), followed by treatment with 1 and assessment of
GSH conjugate 10 by LC-MS/MS. GSH conjugate 10 detected in vehicle-pretreated
group was normalized as 100%. As depicted in Figure 6B, KTC pretreatment resulted
in 74% inhibition of the formation of GSH conjugate 10, and DCNP caused 32%
inhibition of the generation of the GSH conjugate. Similar inhibition study was
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performed to define the metabolic pathways of 2. The presence of DCNP was found
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to attenuate the formation of GSH conjugate 10 by 51% in 2-treated hepatocytes (Figure
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6A).
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Figure 5. Characterization of GSH conjugate 10 generated in primary hepatocytes.
Extracted ion (m/z 491/184) chromatograms obtained from LC-MS/MS analysis of rat
primary hepatocytes treated with vehicle (A), 2 (B) or 1 (C).
Figure 6. Inhibitory effects of KTC and DCNP on the generation of GSH conjugate 10
in primary hepatocytes. The formation of GSH conjugate 10 in primary hepatocytes
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pretreated with vehicle was normalized to 100%. A: Formation of GSH conjugate 10
in 2-treated primary hepatocytes which were pretreated with vehicle or DCNP; B:
Formation of GSH conjugate 10 in 1-treated primary hepatocytes which were
respectively pretreated with vehicle, KTC, or DCNP. Data represent the mean ± SD
(n=3). ***p < 0.005 (mean ± SD).
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Formation of reactive metabolite of 1 via sulfation.
To further verify the speculated sulfation route participating in the bioactivation of
2, we incubated synthetic 2 with rat liver cytosol (RLC) fortified with GSH or NAC in
the presence of SULT cofactor PAPS. As expected, the corresponding GSH and NAC
conjugates were both detected in PAPS-spiked RLC incubations (Figure 7E and Figure
S1E). No such conjugates were observed in vehicle-spiked incubations. The
observed PAPS-dependent formation of the GSH/NAC conjugates from 2 provided
strong evidence for the participation of sulfation in the metabolic activation of 2.
An in vitro two-step incubation was carried out to further define the metabolic
pathway of pirfenidone bioactivation.
Compound 1 was incubated with RLMs
supplemented with NADPH, and the resultant incubation mixture which contains 2
(confirmed by the above experiment) was mixed with the secondary incubation mixture
containing RLC and GSH, followed by incubation at 37 °C. GSH conjugate 10 was
detected in the resulting incubation mixture (Figure 7). Noteworthy, the incubation
spiked with PAPS offered approximately 2.5-fold increase in the formation of GSH
conjugate 10, relative to that of the incubation spiked with vehicle (Figures 7B and 7C).
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Figure 7. Characterization of GSH conjugate 10 generated in RLC and RLM-RLC
incubation systems. Extracted ion (m/z 491/184) chromatograms obtained from LC-
MS/MS analysis of GSH-fortified RLM-RLC incubations of 1 without NADPH or
PAPS (A), with NADPH but without PAPS (B), and with NADPH and PAPS (C).
Extracted ion (m/z 491/184) chromatograms obtained from LC-MS/MS analysis of
RLC incubations containing 2 and GSH in the absence (D) or presence (E) of PAPS.
F: MS/MS spectrum of GSH conjugate 10 produced in 2-fortified RLC incubation.
Biliary and urinary metabolites derived from 1
In order to determine the metabolic activation of pirfenidone in vivo, biliary and
urinary GSH-related metabolites were determined by LC-MS/MS. GSH and NAC
conjugates 10 and 11 were detected in bile of rats treated with 1 (Figures 8B/8D, S2B).
Abundant urinary NAC conjugate 11 was observed in rats given 1 (Figures 9B/9D).
The two conjugates showed consistent mass spectral fragment patterns and retention
times with the synthetic conjugates as well as those of the corresponding metabolites
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detected in in vitro incubations. No such conjugates were observed in vehicle-treated
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Figure 8. Characterization of biliary GSH conjugate 10. Extracted ion (m/z 491/184)
chromatograms obtained from LC-MS/MS analysis of bile collected from vehicle-
treated (A) and 1-treated (B) rats. D: MS/MS spectrum of GSH conjugate 10 obtained
from bile of rats treated with 1. Extracted ion (m/z 491/184) chromatogram (C) and
MS/MS spectrum (E) of synthetic GSH conjugate 10.
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Figure 9. Characterization of urinary NAC conjugate 11. Chromatograms of extracted
ion (m/z 347/184) obtained from LC-MS/MS analysis of urine of vehicle-treated (A)
and 1-treated (B) rats. D: MS/MS spectrum of NAC conjugate 11 detected in urine of
rats treated with 1. Extracted ion (m/z 347/184) chromatogram (C) and MS/MS
spectrum (E) of synthetic NAC conjugate 11.
Saturation of hepatic GSH conjugate 10 formation
We measured hepatic GSH conjugate 10 in mice treated with pirfenidone at various
dosages. As depicted in Figure 10, the formation of hepatic GSH conjugate 10
significantly increased with the increase of doses administered in mice at doses from
10 to 100 mg/kg. After that, the levels of GSH conjugate 10 reached a plateau with
the elevation of doses, i.e. 200, 300, and 400 mg/kg. Clearly, there is certain capacity
for the formation of the reactive metabolites resulting from pirfenidone metabolism as
well as for the generation of GSH conjugation.
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0
100
200
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Dose (mg/kg)
Figure 10. Dose-dependent formation of hepatic GSH conjugate 10. Levels of GSH
conjugate 10 was measured in liver samples harvested from mice given 1 at various
doses after 0.5 h treatment. Data represent the mean ± SD (n=3).
Protein covalent modification by reactive metabolites of 1
Electrophilic metabolites may cause protein covalent binding, which is regarded to
trigger the progression of cytotoxicity. To determine the protein modification, we
decided to completely digest protein samples and analyze the resulting cysteine adduct.
We succeeded in the detection of a cysteine adduct in proteolytic protein samples
obtained from liver tissues of mice treated with 1. The cysteine adduct eluted at
retention time of 6.24 min, and the Q1/Q3 ion pair monitored was m/z 305/184 (Figures
10B/10D).
possibly resulting from the elimination of the cysteine moiety. No such adduct was
observed in the digestion mixture obtained from vehicle-treated mice. Similar
The MS/MS spectrum demonstrated base fragment ion at m/z 184,
procedure for the synthesis of GSH conjugate 10 was employed to synthesize cysteine
adduct 12. The synthetic cysteine adduct revealed similar retention time and MS/MS
spectrum with those of the cysteine adduct observed in animals given compound 1
(Figures 10C/10E).
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Figure 11. Characterization of cysteine adduct 12. Extracted ion (m/z 305/184)
chromatograms obtained from LC-MS/MS analysis of proteolytic liver protein samples
harvested from vehicle-treated (A) and 1-treated (B) mice. D: MS/MS spectrum of
cysteine adduct 12 obtained from proteolytic liver samples harvested from 1-treated
mice. Extracted ion (m/z 305/184) chromatogram (C) and MS/MS spectrum (E) of
synthetic cysteine adduct 12.
We examined the time course and dose dependence of the hepatic protein
adduction in mice treated with 1. As depicted in Figure 12A, the hepatic protein
adduction reached a peak at 1 h after administration and then diminished in an hour.
As expected, the protein adduction increased with the increase of doses administered in
mice (Figure 12B).
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Figure 12. Time course and dose-dependent formation of pirfenidone-derived hepatic
protein adduction. A: Levels of cysteine adduct 12 detected in proteolytic liver samples
harvested from mice treated with 1 at various time points after administration; B: Levels
of cysteine adduct 12 detected in proteolytic liver samples harvested from mice treated
with 1 at various doses. Data represent the mean ± SD (n=3). **p < 0.01 (mean ± SD).
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Furthermore, inhibitors KTC and DCNP were employed to define the roles CYP3A
and SULTs in pirfenidone-induced protein modification. As shown in Figure 13, 52%
decrease in protein adduction derived from 1 was observed in KTC-pretreated mice.
Pretreatment of DCNP also attenuated the protein adduction (by 17%) resulting from
the metabolic activation of 1. This further provides the evidence for the proposed
pathways of pirfenidone bioactivation (Scheme 1).
Figure 13. Inhibitory effects of KTC and DCNP on 1-derived hepatic protein adduction.
Levels of cysteine adduct 12 detected in proteolytic liver samples harvested from 1-
treated mice which had respectively been pretreated with vehicle, KTC, or DCNP. Data
represent the mean ± SD (n=3). ***p < 0.005 (mean ± SD)
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DISCUSSION AND CONCLUSION
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Apparently, pirfenidone does not show severe prototype toxicity in clinic
medication but has reportedly revealed adverse effects similar to idiosyncratic reactions
(IDRs). Though the mechanisms of IDRs remain uncertain, it has been accepted that
metabolic activation is involved in IDRs. Thus, we initiated the study to determine
whether metabolic activation of pirfenidone takes place in vitro and in vivo.
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The present study started with microsomal incubations to determine the role of
P450s in the metabolic activation of pirfenidone. Pirfenidone 1 was incubated with
RLMs fortified with GSH or NAC. Both GSH and NAC are common nucleophilic
reagents, with a soft sulfhydryl group, used for trapping electrophilic metabolites
generated. And NAC is a supplementary trapping agent to eliminate the interference
of glutathione S-transferases.
primary oxidative metabolite in microsomal incubations.
conjugate 10 and NAC conjugate 11 were detected in the corresponding incubation
systems (Figures 1/2). No such conjugates were observed in NADPH-free
Hydroxylated pirfenidone 2 was found to be the
Additionally, GSH
microsomal incubations. This indicates that P450s participated in both oxidative
metabolism and metabolic activation of pirfenidone.
Chemical synthesis was executed to verify the structure of oxidative metabolite 2
and GSH/NAC conjugates 10/11. To take the advantage of the good leaving group of
bromide, P-Br (refer to Experimental Section 2.1., Scheme 4) was synthesized as an
electrophilic synthon to generate a reactive carbocation. Compound 2 was generated
by hydration of P-Br. GSH or NAC was submitted to react with the reactive
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carbocation to form conjugates 10/11. This work assisted us to characterize these
metabolites in vitro and in vivo.
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Identification of reactive metabolites is a very important step for mechanistic
understanding of the chemistry of metabolic activation. The observed compound 2
allowed us to anticipate that 2 could be dehydrated to quinone methide 3 which
sequentially reacts with GSH to generate GSH conjugate 10. To determine the
anticipated dehydration, we incubated 2 with GSH or NAC in buffer systems at pH 5,
7, or 9. We failed to detect any pirfenidone-derived GSH/NAC conjugates in the
systems, suggesting that spontaneous dehydration of 2 did not take place even under
weak acidic and basic conditions. Furthermore, we incubated 2 with RLMs fortified
with GSH or NAC to determine whether cytochrome P450 enzymes would catalyze the
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formation of quinone methide 3 from 2.
Again, no corresponding GSH/NAC
conjugates were detected in the microsomal systems. Clearly, the generation of
quinone methide 3 resulted neither from spontaneous dehydration of 2 nor from P450
enzyme-mediated reactions starting from 2 (Scheme 1).
Despite this, metabolic activation of parent compound 1 mediated by P450
enzymes, along with the formation of 2, is evident. As for the formation of 2, we
proposed that the oxidative reaction is initiated by hydrogen abstraction at the benzylic
carbon to form radical 5 (Scheme 2), followed by oxygen rebound. The generation of
quinone methide 3 may be achieved by electron transfer of the nitrogen of radical 5
(single electron transfer, SET) to FeO3+ of P450 enzymes.30 Alternatively, SET goes
first to produce intermediate 7, followed by hydrogen abstraction to form quinone
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methide 3 (Scheme 2). The reaction of quinone methide 3 with trapping agents
GSH/NAC by Michael addition results in the production of conjugates 10/11.
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Scheme 2. Proposed mechanisms of pirfenidone oxidation mediated by P450s.
H3C
P450s
-e-
H3C
H2C
P450s
-e-, -H+
H2C
N
O
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( )
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P450s
GSH
-e-
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H2C
GS
HO
P450s
OH
P450s
-e-, -H+
N
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N
O
N
O
2
( )
6
( )
5
( )
10
GSH conjugate ( )
To further define the role of 2 in the metabolic activation, we treated rat primary
hepatocytes with 2. Interestingly, GSH conjugate 10 was detected in the cell culture
system but not in NADPH fortified RLMs, suggesting that 2 was bioactivated by non-
P450 enzyme systems.
As a benzylic alcohol derivative, compound 2 has the
possibility to be metabolized by SULTs to sulfate 9 (Scheme 3), a benzyl alcohol-
sulfate derivative. Benzyl-sulfates are known to be electrophilic species, due to the
departure of sulfonyl group, an excellent leaving group. We speculated that sulfate 9
reacts with GSH to form GSH conjugate 10 (Scheme 3) by SN1 or/and SN2 reactions.
To probe the hypothesis, we co-treated cells with SULT inhibitor DCNP. The
presence of DCNP was found to attenuate the formation of GSH conjugate 10,
indicating the involvement of sulfation in the metabolic activation of 2 (Figure 6A).
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Rat liver cytosol incubations supplemented with 2 were carried out, and the formation
of GSH/NAC conjugates was found to be PAPS-dependent (Figures 7D/7E and
S1D/S1E). This supports the speculated role that the sulfation pathway involves in
the metabolic activation of pirfenidone.
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Scheme 3. Proposed pathways of metabolic activation of pirfenidone via sulfation.
RLM-RLC 2-step incubations were carried out to define the pathway of
pirfenidone bioactivation. Pirfenidone was pre-incubated with RLMs in the presence
or absence of NADPH, followed by incubation with RLCs spiked with PAPS or vehicle.
NADPH was found to be required for the formation of conjugate 10, and the presence
of PAPS elevated the conjugation reaction (Figures 7A/7B/7C). This indicates that
P450s initiated the metabolic pathway, and SULTs participated in the rest of pathway
(Scheme 3). The observed inhibitory effects of KTC and DCNP in the 1-treated
hepatocyte incubations support the proposed metabolic pathway, suggesting that both
oxidation mediated by P450s and sulfation catalyzed by SULTs participated in the
metabolic activation of pirfenidone (Figure 6B).
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In a recombinant P450 assay, we found that CYP3A4 made the largest contribution
to the metabolic activation of 1, followed by CYP2A6 and CYP1A2 (Figure 4B).
Interestingly, CYP1A2 was found to be mainly responsible for the formation of 2
required for the sequential sulfation (Figure S3). Clearly, the expression of CYP1A2
is particularly important to sulfation-mediated metabolic activation of pirfenidone.
Biliary GSH conjugates are generally considered as biomarkers of exposure to
electrophilic species. Abundant biliary GSH conjugate 10 found in 1-treated rats (100
mg/kg, equivalent to its clinical single dosage) provides solid evidence for the
formation of electrophilic intermediate in vivo (Figure 8). NAC conjugate 11 detected
in bile and urine most likely results from further metabolism of GSH conjugate 10,
catalyzed by -glutamyltranspeptidase, dipeptidases, and N-acetyltransferase.
Protein covalent binding is an important biochemical mechanism for cytotoxicity.
Electrophilic species may react with cellular protein, especially when cellular GSH is
depleted. The resulting protein modification may cause permanent cellular damages.
As for the observed GSH conjugation in vitro and in vivo, we believed similar chemistry
would take place between the reactive intermediates and cysteine residues of protein.
The detection of cysteine adduct 12 (Figure 11) in proteolytic protein samples allowed
us to verify the protein adduction resulting from the metabolic activation of pirfenidone
in vivo.
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We successfully identified GSH conjugate 10 and NAC conjugate 11, along with
protein covalent binding, resulting from the metabolic activation of pirfenidone.
However, we failed to observe liver injury in mice (Figure S4) treated with pirfenidone
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at dose of 150 mg/kg (approximately equivalent to its clinical single dosage) and even
at 200 mg/kg (Figure S5). In addition, no cytotoxicity was found in cultured primary
hepatocytes after exposure to pirfenidone at concentration of 800 M (Figure S6). It
does not necessarily mean that pirfenidone is not hepatotoxic, since no animal models
are available for idiosyncratic toxicity investigation. The failure to observe hepatic
injury of pirfenidone in regular animals is understandable. The detected quinone
methide and sulfate metabolites of pirfenidone are electrophilic, and the two are
reactive to nucleophilic sites of cellular molecules. Protein covalent binding resulting
from the bioactivation of pirfenidone is evident. Further studies are needed to define
the correlation of the protein modification with pirfenidone idiosyncratic toxicities.
In conclusion, our study provided strong evidence for the formation of a quinone
methide reactive intermediate from pirfenidone in vitro and in vivo. The electrophilic
species may be generated by two metabolic pathways, including 1) direct oxidation of
pirfenidone mediated by P450 enzymes; and 2) hydroxylation of pirfenidone catalyzed
by P450 enzymes, followed by SULT-mediated sulfation. The reactive intermediates
reacted with sulfhydryl group of cysteine residues to form protein covalent binding.
Not only do the findings facilitate the understanding of the mechanisms of the metabolic
activation of pirfenidone but also provide medicinal chemists with the information
about bioactivation-alerting structure in rational drug design.
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EXPERIMENTAL SECTION
1. Chemicals and Materials. Pirfenidone (1, >98%) was supplied by Dalian Meilun
Biotech Co., Ltd. (Dalian, China). GSH, NAC, cysteine, NADPH, DCNP,
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dithiothreitol, and KTC were purchased from Aladdin Reagent Co., Ltd. (Shanghai,
China). Propranolol, PAPS, and collagenase IV were obtained from Sigma-Aldrich
(St. Louis, MO). Pronase E, chymotrypsin, and rat tail collagen I were acquired from
SalarBio (Beijing, China).
Distilled water was provided by Wahaha Co., Ltd.
(Hangzhou, China). All organic solvents were supplied by Fisher Scientific
(Springfield, NJ). All the reagents and solvents used were at least of analytical grade.
2.1. 5-Hydroxymethyl-1-phenyl-1H-pyridine-2-one (2). Synthesis of metabolite 2
started with bromination of 1 (Scheme 4), followed by hydration in alkaline aqueous
solution. Briefly, compound 1 (185.5 mg, 1.0 mmol) and NBS (178.6 mg, 1.0 mmol)
were mixed in 7.0 mL of CCl4, followed by addition of AIBN (20.0 mg, 0.12 mmol) to
trigger the reaction. The reaction mixture was magnetically stirred at 90 °C for 2 h
with refluxing, and the reaction was monitored by TLC. The resultant mixture was
filtered and the CCl4 solution was condensed to dryness. Product 5-bromomethyl-1-
phenyl-1H-pyridine-2-one (P-Br) was reconstituted in 8.0 mL 4 N NaOH and stirred at
40 °C for 4 h. The resulting mixture was submitted to a silica gel chromatography for
purification. The product obtained was characterized by Q-TOF MS and NMR.
HRMS: m/z 224.0692 [C12H11NO2Na+]. According to the prediction formula, the
error between the accurate mass and the corresponding theoretical mass was -4.5 ppm.
1H-NMR (DMSO, 600 MHz):  (ppm) 7.5 (m, 4H), 7.44 (tt, J = 7.4/1.8Hz, 1H), 7.39
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(m, 1H), 7.38 (m, 1H), 6.49 (dd, J = 2.4/8.1 Hz, 1H), 5.12 (t, J = 5.3 Hz, 1H,), 4.26 (d,
J = 4.7 Hz, 1H). HPLC purity: 98.1%.
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Scheme 4. Synthetic route of P-Br.
H3C
Br
N
O
N
O
AIBN/NBS
CCl4
P-Br
1
( )
2.2. Synthesis of GSH/NAC/cysteine conjugates (10/11/12). Pirfenidone was
brominated as indicated above. The resulting P-Br (26.5 mg, 0.1 mmol) was
individually dissolved in 5.0 mL DMSO containing GSH (92.4 mg, 0.3 mmol), NAC
(49.0 mg, 0.3 mmol), or cysteine (36.3 mg, 0.3 mmol). The resultant mixtures were
stirred at room temperature for 3 h, and the reactions were monitored by LC-MS/MS.
The reaction mixture for synthesis of GSH conjugate 10 was submitted to a semi-
preparative HPLC system for purification, and the purified product was characterized
by HRMS and NMR. HRMS: m/z 491.1595. According to the prediction formula,
the error between the accurate mass observed in the HRMS system and the
corresponding theoretical mass was 1.2 ppm. 1H-NMR (D2O, 600 MHz):  (ppm)
7.8 (dd, J = 2.22/9.29 Hz, 1H), 7.6-7.5 (m, 3H), 7.6 (s, 1H), 7.4 (d, J = 7.33 Hz, 2H),
6.7 (d, J = 9.31 Hz, 1H,), 4.5 (dd, J = 5.34/8.45 Hz, 1H), 3.8 (s, 2H), 3.7 (t, J = 6.43
Hz, 1H), 3.6 (2H, s), 3.0 (dd, J = 5.16/14.2 Hz, 1H), 2.8 (dd, J = 8.64/14.16 Hz, 1H),
2.4 (t, J = 7.67 Hz, 2H), 2.1 (m, 2H). HPLC purity: 95.2%.
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3.1. Rat liver microsomal incubations. RLMs were prepared by following a published
method.32 Compound 1 or 2 was dissolved in a phosphate-buffered saline (PBS, 100
mM, pH 7.4) with MgCl2 (3.2 mM). The incubation systems consisted of substrate 1
or 2 at a final concentration of 50 M, RLMs (final concentration of 1.0 mg protein/mL,
assessed by BCA assay), and trapping agents GSH or NAC (1.0 mM). NADPH (2.0
mM) was added to initiate the reactions. NADPH was excluded in control group.
The volume of incubation mixtures was 250 L. After 45 min incubation at 37 °C,
the reactions were quenched by addition of 250 L ice-cold acetonitrile (1:1 v/v). The
resultant mixtures were vortex mixed for 2 min and then centrifuged at 19,000 g for 10
min. A 3.0 L aliquot of the resulting supernatants was individually injected into LC-
MS/MS for analysis. All incubation reactions were conducted in triplicate. In a
separate study, compound 2 was incubated in a buffer system fortified with GSH at pH
5, 7, or 9. The rest of the procedure was the same as that for microsomal incubations
described above.
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3.2. Recombinant P450 incubations. Incubations of 1 were conducted under similar
condition of microsomal incubations described above except that RLMs were replaced
by individual human recombinant P450 enzymes (0.1 nM), including CYPs1A2, 2A6,
2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. Reactions were terminated by addition of
an equal volume of ice-cold acetonitrile containing propranolol (5.0 ng/mL) as the
internal standard. The mixture solutions were vortex mixed and centrifuged as
described above, and then the supernatants (10 L) were analyzed by LC-MS/MS.
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3.3. Cytosol incubations of 1. RLCs were prepared from rat liver, according to a
published method.33 Liver microsomal incubations (final volume: 100 L) of 1 were
performed as described above, followed by mixing with the secondary incubation
mixtures (100 L) that consisted of GSH/NAC (1.0 mM) and RLCs at a final
concentration of 2.0 mg protein/mL. PAPS was added to initiate the sulfation reaction
at a final concentration of 250 M; control reactions were also conducted under the
same condition except for the addition of PAPS. The secondary incubations were
performed for 45 min at 37 °C. Incubations of 1 with rat liver S9 were also conducted
in the presence of NADPH and PAPS. Control groups were performed in the absence
of either NADPH or PAPS. Reactions in rat liver S9 were terminated by addition of
equal volume of ice-cold acetonitrile after 1 h incubation. Similar protocols were
followed for sample preparation as the above. The resulting supernatants (3.0 L)
were analyzed by LC-MS/MS.
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3.4. Cytosol incubations of 2. Incubation system contained 2 (50 M), MgCl2 (3.2
mM), PBS (100 mM, pH 7.4), RLCs (2.0 mg protein/mL), and GSH or NAC (1.0 mM).
PAPS (250 M) was subsequently added to the prepared incubation mixture to lunch
the reaction. Control reaction mixture lacked PAPS.
4.1. Animal experiments. All animal experiments were conducted in conformity to
regulations authorized by the Ethics Review Committee of Shenyang Pharmaceutical
University. Sprague-Dawley rats (220-240 g, male) and Kunming mice (18-22 g,
male) were both supplied by the Animal Center of Shenyang Pharmaceutical University.
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