Novel phosphodiesterases inhibitors from the group of purine-2,6-dione derivatives as potent modulators of airway smooth muscle cell remodelling

Article abstract of DOI:10.1016/j.ejphar.2019.172779

Airway remodelling (AR) is an important pathological feature of chronic asthma and chronic obstructive pulmonary disease. The etiology of AR is complex and involves both lung structural and immune cells. One of the main contributors to airway remodelling is the airway smooth muscle (ASM), which is thickened by asthma, becomes more contractile and produces more extracellular matrix. As a second messenger, adenosine 3′,5′-cyclic monophosphate (cAMP) has been shown to contribute to ASM cell (ASMC) relaxation as well as to anti-remodelling effects in ASMC. Phosphodiesterase (PDE) inhibitors have drawn attention as an interesting new group of potential anti-inflammatory and anti-remodelling drugs. Recently, new hydrazide and amide purine-2,6-dione derivatives with anti-inflammatory properties have been synthesized by our team (compounds 1 and 2). We expanded our study of their PDE selectivity profile, ability to increase intracellular cAMP levels, metabolic stability and, above all, their capacity to modulate cell responses associated with ASMC remodelling. The results show that both compounds have subtype specificity for several PDE isoforms (including inhibition of PDE1, PDE3, PDE4 and PDE7). Interestingly, such combined PDE subtype inhibition exerts improved anti-remodelling efficacies against several ASMC-induced responses such as proliferation, contractility, extracellular matrix (ECM) protein expression and migration when compared to other non-selective and selective PDE inhibitors. Our findings open novel perspectives in the search for new chemical entities with dual anti-inflammatory and anti-remodelling profiles in the group of purine-2,6-dione derivatives as broad-spectrum PDE inhibitors.

Full text of DOI:10.1016/j.ejphar.2019.172779

EuropeanJournalofPharmacologyxxx(xxxx)xxxx
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Full length article
Novel phosphodiesterases inhibitors from the group of purine-2,6-dione
derivatives as potent modulators of airway smooth muscle cell remodelling
Katarzyna Wójcik-Pszczołaa,∗, Grażyna Chłoń-Rzepab, Agnieszka Jankowskab, Eugenie Ellenc,
Artur Świerczekd, Krzysztof Pociechad, Paulina Koczurkiewicza, Kamil Piskaa, Anna Gawędzkae,
Elżbieta Wyskad, Małgorzata Knapik-Czajkae, Elżbieta Pękalaa, Reinoud Gosensc
a Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688, Kraków, Poland
b Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688, Kraków, Poland
c Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, 9713, AV, Groningen, the Netherlands
d Department of Pharmacokinetics and Physical Pharmacy, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688, Kraków, Poland
e Department of Analytical Biochemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-688, Kraków, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
Phosphodiesterases inhibitors
cAMP
ASMC hypertrophy
ASMC hyperplasia
Airway remodelling
Airway remodelling (AR) is an important pathological feature of chronic asthma and chronic obstructive pul-
monary disease. The etiology of AR is complex and involves both lung structural and immune cells. One of the
main contributors to airway remodelling is the airway smooth muscle (ASM), which is thickened by asthma,
becomes more contractile and produces more extracellular matrix. As a second messenger, adenosine 3′,5′-cyclic
monophosphate (cAMP) has been shown to contribute to ASM cell (ASMC) relaxation as well as to anti-re-
modelling effects in ASMC. Phosphodiesterase (PDE) inhibitors have drawn attention as an interesting new
group of potential anti-inflammatory and anti-remodelling drugs. Recently, new hydrazide and amide purine-
2,6-dione derivatives with anti-inflammatory properties have been synthesized by our team (compounds 1 and
2). We expanded our study of their PDE selectivity profile, ability to increase intracellular cAMP levels, meta-
bolic stability and, above all, their capacity to modulate cell responses associated with ASMC remodelling. The
results show that both compounds have subtype specificity for several PDE isoforms (including inhibition of
PDE1, PDE3, PDE4 and PDE7). Interestingly, such combined PDE subtype inhibition exerts improved anti-re-
modelling efficacies against several ASMC-induced responses such as proliferation, contractility, extracellular
matrix (ECM) protein expression and migration when compared to other non-selective and selective PDE in-
hibitors. Our findings open novel perspectives in the search for new chemical entities with dual anti-in-
flammatory and anti-remodelling profiles in the group of purine-2,6-dione derivatives as broad-spectrum PDE
inhibitors.
1. Introduction
COPD patients are currently not the target of any available treatment
for these diseases, although a recent study has shown promising effects
Airway remodelling (AR) is a collective term for pathological
structural alterations in the airways of patients with obstructive lung
diseases such as asthma or chronic obstructive pulmonary disease
(COPD) (Bergeron et al., 2010; Fehrenbach et al., 2017; Hogg et al.,
2004; James and Wenzel, 2007). AR leads to thickening of the airway
wall and consequently leads to airflow obstruction, thereby causing
breathing disorders and reduced airway patency (Aoshiba and Nagai,
2004; Jones et al., 2016). Nearly all airway structural cells as well as a
number of infiltrating inflammatory cells play an active, concerted role
in AR. Structural changes which occur in the airways in asthma and
of the novel prostaglandin D2 receptor antagonist fevipiprant (Saunders
et al., 2019). According to the Global Initiative for COPD and Asthma,
the main therapeutic strategies are based on administration of inhaled
or systemic corticosteroids, β2-agonists, anti-leukotrienes or cromones
(Global Initiative for Asthma, 2018; Global Initiative for Chronic
Obstructive Lung Disease, 2019). However, although these drugs in-
hibit inflammation and promote bronchodilation, they do not sub-
stantially affect AR (Beckett and Howarth, 2003; Bergeron et al., 2010;
Durrani et al., 2011; Nayak et al., 2018).
Airway smooth muscle cells (ASMC) play a key role in AR as a result
∗ Corresponding author. ul. Medyczna 9, 30-688, Kraków, Poland.
E-mail address: katarzynaanna.wojcik@uj.edu.pl (K. Wójcik-Pszczoła).
https://doi.org/10.1016/j.ejphar.2019.172779
Received 17 September 2019; Received in revised form 24 October 2019; Accepted 4 November 2019
0014-2999/©2019ElsevierB.V.Allrightsreserved.
Pleasecitethisarticleas:KatarzynaWójcik-Pszczoła,etal.,EuropeanJournalofPharmacology,
https://doi.org/10.1016/j.ejphar.2019.172779

K. Wójcik-Pszczoła, et al.
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of ASMC proliferation (hyperplasia), increased cell size (hypertrophy),
increased extracellular matrix deposition and augmented cell migration
(Bentley and Hershenson, 2008; Chung, 2005; Salter et al., 2017;
Zuyderduyn et al., 2008). It has been shown that increased ASM mass is
associated with the severity of asthma and COPD (Baraldo et al., 2012;
Doeing and Solway, 2013; Hogg et al., 2004; James et al., 2012; Yan
et al., 2018). Increased ASMC proliferation has also been described in
these diseases (Johnson et al., 2001; Michaeloudes et al., 2017; Perry
et al., 2011). Several different growth factors and cytokines may be
responsible for evoking an ASMC remodelling response (Gawaziuk
et al., 2007; Michaeloudes et al., 2017). Among others, a special role is
attributed to transforming growth factor type β (TGF-β) which activates
genes responsible for the expression of profibrotic proteins. A large
body of evidence suggests an increased intracellular level of adenosine
or guanosine 3′,5′-cyclic monophosphate (cAMP or cGMP) may lead to
an inhibition of immune and lung structural cells' pro-inflammatory
response (Oldenburger et al., 2012; Raker et al., 2016). Among other
factors, high levels of cAMP and cGMP have been shown to contribute
to ASMC relaxation as well as have suppressive effects on proliferation,
extracellular matrix production and cell migration (Billington et al.,
2013). As phosphodiesterases (PDEs) are one of the most important
players that bring about a decrease in cAMP and/or cGMP intracellular
levels, targeted, anti-PDEs therapy has been put forth as a promising
approach for treating patients with asthma and COPD (Global Initiative
for Asthma, 2018; Global Initiative for Chronic Obstructive Lung
Disease, 2019). This may be especially important for corticosteroids-
resistant patients, a group that is particularly in need of effective
pharmacotherapy for asthma.
Recently, our team synthesized new hydrazide and amide deriva-
tives of 1,3-dimethylpurine-2,6-dione (theophylline) (compounds 1 and
2, Fig. 1A and B). Pharmacological studies of their biological activity
have revealed their strong anti-inflammatory properties. Both com-
pounds decreased the tumor necrosis factor α (TNF-α) concentration in
rats (Chłoń-Rzepa et al., 2018a, 2018b). This effect was caused, at least
in part, by PDE4B and 7A inhibition (Chłoń-Rzepa et al., 2018b,
2018a). In the current study, we aimed to investigate compounds 1 and
2 in the context of their potential to limit ASMC remodelling responses.
First, the importance of studying the PDE selectivity profile of the
compounds was demonstrated. We also examined their metabolic sta-
bility and tested their ability to raise the intracellular cAMP. Finally, we
used FBS and TGF-β1-treated ASMC cultured in the presence of com-
pounds 1 or 2 to study their effects on ASMC remodelling and compare
their efficacy against several PDE inhibitors.
butanehydrazide) and compound 2 (4-(8-butoxy-1,3-dimethyl-2,6-
dioxo-2,3,6,7-tetrahydro-1H-purin-7-yl)-N-(5-tert-butyl-2-hydro-
xyphenyl)butanamide) were used in the study. They were synthesized
according to the multistep procedure as described previously (Chłoń-
Rzepa et al., 2018b, 2018a). Briefly, to obtain compound 1, 8-bromo-
1,3-dimethyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione was treated with
benzylamine in refluxing 2-methoxyethanol. Next, the obtained 8-
benzylamine derivative was alkylated at the 7-position by treatment
with ethyl 4-bromobutyrate in the presence of potassium carbonate
(K2CO3) and a catalytic amount of benzyltriethylammonium chloride
(TEBA) in refluxing acetone. The treatment of the obtained ester with
hydrazine hydrate in anhydrous ethanol gave corresponding hydrazide.
In the final step, the obtained hydrazide was condensed with 2,3,4-
trihydroxybenzaldehyde in methanol in the presence of a catalytic
amount of hydrochloric acid (HCl). To obtain compound 2, 8-bromo-
1,3-dimethyl-2,3,6,7-tetrahydro-1H-purine-2,6-dione was alkylated at
the 7-position with ethyl 4-bromobutanoate in the presence of K2CO3
and a catalytic amount of TEBA in refluxing acetone. Next, the obtained
ester was treated with sodium 1-butoxide in butan-1-ol, then hydro-
lysed using potassium hydroxide in water-acetone mixture and acidified
with concentrated HCl to obtain the corresponding acid. In the final
step, the obtained acid was condensed with 2-amino-4-(tert-butyl)
phenol in N,N-dimethylformamide in the presence of di (1H-imidazole-
1-yl)methanone. Both evaluated compounds were dissolved in di-
methylsulfoxide (DMSO, PAN-Biotech, Germany). Reference sub-
stances: theophylline (1,3-dimethyl-7H-purine-2,6-dione), 1-methyl-3-
(2-methylpropyl)-7H–purine-2,6-dione (IBMX), BRL-50481, KW-3920,
KW-6002, roflumilast, cilomilast and forskolin (Sigma Aldrich, St.
Louis, MO, USA) as well as vinpocetine, milrinone and rolipram
(Cayman Chemical, Ann Arbor, MI, USA), were dissolved in DMSO and
culture medium according to the manufacturer's protocol. The final
DMSO concentration in culture medium did not exceed 0.9%. Vehicle
control was performed in all experiments. No harmful effects of DMSO
were demonstrated at the applied concentrations and incubation time
for ASMCs. No changes in ASMCs' morphology, viability and pro-
liferation, even at longer culture time with DMSO were observed. In
addition, mixtures of selective PDE inhibitors, MIX 1 and MIX 2 were
prepared by combining vinpocetine, milrinone, roflumilast and BRL-
50481 in 1:1:1:1 and 1:0.2:2:6 ratios, respectively.
2.2. In vitro PDE inhibition assay
The PDE inhibitory activity of compounds 1 and 2 was evaluated
using the PDE-Glo™ Phosphodiesterase Assay (Promega Corporation,
Madison, WI, USA). Human recombinant PDEs were purchased from
SignalChem, Richmond, Canada. The optimal amount of PDE in the
reaction mixture and the optimal PDE reaction time was determined
individually for each enzyme subtype according to the manufacturer's
instructions. Aliquots of 1X PDE-Glo Reaction buffer (6.5 μl) containing
appropriate amounts of purified human recombinants PDE1B, PDE2A,
2. Materials and methods
2.1. Study compounds
Compound 1 (4-(8-(benzylamino)-1,3-dimethyl-2,6-dioxo-2,3,6,7-
tetrahydro-1H-purin-7-yl)-N’-(2,3,4-trihydroxybenzylidene)
Fig. 1. Compounds 1 and 2 as PDE4B and 7A
inhibitors. Chemical structures of 4-(8-(benzyla-
mino)-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-
1H-purin-7-yl)-N’-(2,3,4-trihydroxybenzylidene)
butanehydrazide - compound 1 (A) and 4-(8-bu-
toxy-1,3-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-
1H-purin-7-yl)-N-(5-tert-butyl-2-hydroxyphenyl)
butanamide – compound 2 (B) are presented.
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PDE3A, PDE4D, PDE5A, or PDA10A were added to a 384 well plate
(Thermo Scientific, USA). Both tested compounds were dissolved in
DMSO and a serial dilution using 1X PDE-Glo Reaction buffer was
performed. After the addition of 1 μl of diluted inhibitors to each well,
2.5 μl of cAMP solution was added to initiate the reaction. In the case of
PDE2A and PDE5A, 2.5 μl of cGMP solution was used instead of the
cAMP solution. After an appropriate incubation time at 30 °C, 2.5 μl of
PDE-Glo™ Termination Buffer and 2.5 μl of PDE-Glo™ Detection
Solution were added to each well. After 20 min of incubation, 10 μl of
Kinase-Glo® Reagent was pipetted into each well and 10 min later lu-
minescence was measured using a microplate luminometer (POLARstar
Omega, BMG LABTECH, Ortenberg, Germany). All data were expressed
as the mean of the percentage of the relative PDE activity from three
independent determinations ( S.D.). IC50 values were obtained by
non-linear regression using Phoenix WinNonlin v. 7.0 (Certara, USA).
assays were performed by incubation of the cells with alamarBlue®
reagent (Invitrogen, Life Technologies, USA) and dissolved in Hank's
Balanced Salt Solution (HBSS, 10% vol/vol; Gibco, Thermo Scientific,
USA) for 30 min at 37 °C. After the observed colour change, the super-
natant was transferred to 96-well plates and the absorbance was mea-
sured at 570 and 600 nm. The percent difference in reduction between
treated and control cells was calculated according to the manufacturer's
protocol.
2.6. RNA isolation and quantitative PCR
Real-time polymerase chain reaction (qPCR) was used to detect and
quantify the expression of specific human genes: ACTA2, CNN1,
COL1A1, FN1, and VCAN. ASMC were seeded in 6-well plates
(2 × 105 cells/well) in standard culture medium. After 24 h of culture
they were serum-starved for 3 days to induce cell cycle arrest. The cells
were incubated with the study compounds and TGF-β1 for 24 h and
total RNA was extracted after 24 h incubation using the TRI Reagent®
Solution (Applied Biosystems, Foster City, California, USA) according to
the manufacturer's protocol and RNA concentration was determined
2.3. Metabolic stability in human liver microsomes
Glucose-6-phosphate,
NADP+,
glucose-6-phosphate
dehy-
drogenase, buffers and human liver microsomes (HLM) were supplied
by Sigma Aldrich (St. Louis, MO, USA). Pentoxifylline (PTX) was ob-
tained from Polpharma (Kraków). Reaction mixtures, consisting of
tested drugs (20 μM), HLM (0.4 mg protein/ml) in phosphate buffer
(pH = 7.40; 0.1 M) were preincubated for 10 min, at 37 °C on an
Eppendorf ThermoMixer®. Next, NADPH-regenerating system (NADP
+, glucose-6-phosphate, glucose-6-phosphate dehydrogenase in phos-
phate buffer) was added to initiate the reaction. Tests without NADPH-
regenerating system were conducted in parallel and incubations were
conducted in duplicates. Total volume of the reaction mixture was
250 μl. At different time points an internal standard (PTX, 20 μM) was
added, and the reaction was quenched with ice-cold methanol. Tubes
were transferred for 1 h to −20 °C. Samples were then centrifuged for
10 min at 15,000 g and the obtained supernatants subjected to ultra
performance liquid chromatography coupled with mass spectrometer
analysis. In vitro half-life (t1/2) was assessed from the slope of linear
regression of ln% parent compound remaining versus the incubation
time. Then intrinsic clearance (CLint) was calculated as described pre-
viously (Singh and Solanki, 2012).
with
a NanoDrop spectrophotometer (ThermoFisher, Breda, the
Netherlands). Equal amounts of total RNA were reversely transcribed
using the Reverse Transcription System (Promega Corporation,
Madison, WI, USA). Real-time PCR assays were performed using the
PIXO™ Real-Time PCR System (Helixis, Carlsbad, California, USA) and
SYBR® Green Mastermix (Roche Diagnostics, Almere, the Netherlands).
PCR cycling was performed with denaturation at 94 °C for 30 s, an-
nealing at 59 °C for 30 s and an extension at 72 °C for 30 s. Specific
primers used to quantify the transcripts are listed in Table 1. The re-
lative abundance of specific mRNA transcripts was estimated based on
cycle threshold (CT) value and recalculated against the endogenous
reference 18S ribosomal RNA gene using the ΔCT method. All samples
were run in duplicates.
2.7. Immunoblotting
Western Blot analysis was performed according to standard pro-
tocol. Briefly, cells were seeded in 6-well plates (2 × 105 cells/well) in
standard culture medium. After 24 h of culture they were serum-starved
for 3 days to induce cell cycle arrest. The cells were incubated with the
study compounds and TGF-β1 for 48 h and then lysed in RIPA buffer
(RIPA Lysis and Extraction Buffer, Thermo Scientific, USA) and sup-
plemented with protease inhibitors (Pierce Protease Inhibitor Tablets,
Thermo Scientific, USA). Protein concentrations were measured using a
Bradford Reagent. Equal amounts of protein (15 μg of total protein)
were separated using 10% SDS-polyacrylamide gels and transferred
onto nitrocellulose membranes (Amersham Hybond™, GE™ Healthcare,
USA), at a constant voltage (35 V) in a transfer buffer at 4 °C. Blots were
2.4. ASMC culture
Human ASMC (n = 3 donors) immortalized by stable expression of
human telomerase reverse transcriptase (hTERT) and isolated from two
donors, was used in the study. The entire procedure of obtaining ASMC
has been described in detail by Gosens et al. (2006). Cells were main-
tained between passages 21–28 on Petri dishes in Dulbecco's Modified
Eagle Medium (DMEM; Gibco, Thermo Scientific, USA) supplemented
with 10% (vol/vol) fetal bovine serum (FBS; Gibco, Thermo Scientific,
USA) and an antibiotics mixture (Penicillin, Streptomycin, Amphoter-
icin B; Gibco, Thermo Scientific, USA). Cells were cultured in standard
conditions (5% CO2, 37 °C, 95% humidity). ASMC were serum-deprived
for all experiments in DMEM supplemented with ITS-A (Insulin-Trans-
ferrin-Selenium–Sodium Pyruvate Solution, Gibco, Thermo Scientific,
USA) and the antibiotics mixture. Compounds 1 and 2 were added at
final concentrations from 1 μM to 50 μM, 1 h before FBS or TGF-β1
(2 ng/ml, BD Biosciences, USA) treatment.
Table 1
List of primers used for detection of specific human genes.
Gene
Primers
5’→3′ forward
5’→3′ reverse
α-SMA
CCG GGA GAA AAT GAC TCA
AA
GAA GGA ATA GCC ACG CTC
AG
ACTA2
calponin-1
CNN1
ACA TTT TTG AGG CCA ACG
AC
CTC CCA CGT TCA CCT TGT TT
2.5. ASMC proliferation
collagen Iα1
COL1A1
fibronectin I
FN1
versican
VCAN
AGC CAG CAG ATC GAG AAC
AT
TCG AGG AGG AAA TTC CAA
TG
GGG AAC CTG GTG AAG AAA
CA
CGC CGC TAG AGG TGA AAT
TC
TCT TGT CCT TGG GGT TCT TG
The effect of compounds 1 and 2 on ASMC proliferation rate was
evaluated by using an alamarBlue® Assay. Cells were seeded in 24-well
plates (3 × 104 cells/well) in standard culture medium. After 24 h of
culture they were serum-starved for 3 days to induce cell cycle arrest.
Compounds 1 and 2 (1–50 μM) and reference substances, roflumilast
and cilomilast (1–10 μM) were added 1 h prior to stimulation of ASMC
growth with FBS (10%, vol/vol) for an additional 48 h. Proliferation
ACA CAC GTC CAC CTC ATC
AT
CTT CCA CAG TGG GTG GTC
TT
TTG GCA AAT GCT TTC GCT C
18S rRNA
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washed with TBST buffer (150 mM NaCl, 50 mM Tris, 0.05% Tween
20), blocked with 5% skim milk in TBST and incubated overnight at
4 °C with the appropriate primary antibody (mouse monoclonal anti-α-
smooth muscle actin antibody, clone 1A4, dilution 1:1000, #A2547;
mouse monoclonal anti-collagen type I antibody, dilution 1:1000,
#SAB4200678; mouse monoclonal anti-GAPDH antibody, dilution
1:5000, #G8795, all from Sigma Aldrich, St. Louis, MO, USA). After
incubation with primary antibodies, membranes were washed 3 times
with TBST buffer and incubated with secondary, rabbit anti-mouse IgG
(whole molecule)-peroxidase antibodies (#A9044, dilution 1:3000,
Sigma Aldrich, St. Louis, MO, USA). The protein bands were visualized
using an enhanced chemiluminescence method (SuperSignal™ West
Pico PLUS Chemiluminescent Substrate, Thermo Scientific, USA) with a
Chemi Doc Camera (BioRad, Hercules, CA, USA). Relative Optical
Density (ROD) was measured according to NIH guidelines with ImageJ
Software.
we determined their ability to inhibit other key PDE isoforms. Com-
pounds 1 and 2 could both inhibit the activity of PDE1B, 2A, 3A, 4D,
and 10A isoenzymes (Fig. 2A and B, Table 2). Their potency, expressed
as the IC50 value, was comparable to commercially available inhibitors
of individual PDE isoforms and greater than those that of other pan-PDE
inhibitors – theophylline and IBMX (Table 2). Both compounds showed
particularly strong activity against the 1B and 3A isoforms (Fig. 2A and
B). The IC50 for compounds 1 and 2 against PDE1B inhibition was 0.07
and 2.69 μM, respectively, and in the case of PDE3A, 0.25 and
0.184 μM, respectively (Table 2). Both compounds showed virtually no
activity against PDE5A. For compound 1, the PDE inhibitory potency
increased according to the following sequence: 1B > 3A > 4B, 7A, 2A,
4D, 10A > 5A. In turn, for compound 2, this was as follows: 3A > 1B,
4B, 7A > 2A, 10A, 4D > 5A. As the PDE4D isoform is considered re-
sponsible for the inhibitors’ side effects (Hatzelmann et al., 2010;
Kawamatawong, 2017), it is also worth noticing the reduced potential
of both compounds for inducing nausea (compounds 1 and 2 have
higher IC50 for PDE4D in comparison to roflumilast and cilomilast
which exhibit PDE4D activity at a nano-molar level).
2.8. Migration assay
Migration assay was performed using 6.5-mm Transwell culture
plates with an 8.0-μm pore polycarbonate membrane (Corning
Incorporated, USA). ASMC were serum-starved and following har-
vesting were seeded (1.5 × 103 cells/well) in the upper chamber in
serum-free culture medium supplemented with or without compounds
1 and 2 or reference drugs. The lower compartment was filled with the
same culture medium both with and without studied compounds and
after 1 h TGF-β1 was added to the wells. After 24 h of incubation the
non-migrated cells were removed from the upper face of the mem-
branes using a cotton swab. The remaining ASMC were fixed with 4%
paraformaldehyde solution (Sigma Aldrich, St. Louis, MO, USA) and
stained with 0.5% crystal violet solution (Sigma Aldrich, St. Louis, MO,
USA) for 10 min. The number of migrated cells on the lower face of the
filter was counted under an inverted microscope (Nikon Eclipse TS 100)
using 10 randomly selected fields of view.
3.2. Compounds 1 and 2 elevate the intracellular cAMP level
As the selected purine-2,6-dione derivatives are strong pan-PDE
inhibitors, for the next step we decided to verify their potential impact
on intracellular cAMP levels. Both compounds significantly increased
the levels of this second messenger in ASMC (Fig. 3). Their effect was
comparable to another pan-PDE inhibitor – IBMX, as well as to a known
adenylyl cyclase activator – forskolin. Interestingly, the applied selec-
tive PDE inhibitors: vinpocetine (PDE1 inhibitor), milrinone (PDE3
inhibitor) roflumilast and cilomilast (PDE4 inhibitors) and BRL-50481
(PDE7 inhibitor) also slightly increased cAMP levels but their effect was
less pronounced (Fig. 3). As we wanted to confirm the additive effect of
pan-PDE inhibitors we also used the mixtures of selective PDE1, PDE3,
PDE4, and PDE7 inhibitors. We tested two mixtures, the first contained
equal amounts of individual inhibitors and the second comprised
amounts of inhibitors in a ratio dependent on the experimental activity
of compounds 1 and 2 against individual PDE isoforms. Both blends
caused an effect similar to the effect of purine-2,6-derivatives on in-
tracellular cAMP (Fig. 3).
2.9. ELISA
Intracellular cAMP level was measured using a colorimetric cAMP
ELISA Kit according to the manufacturer's protocol (Cell Biolabs, INC.,
San Diego, CA, USA). ASMC were seeded in 6-well plates (2 × 105 cells/
well) in standard culture medium. After 24 h of culture they were
serum-starved for 2 h in HBSS. The study compounds and reference
substances were added to the cell culture and incubated for 30 min.
Whole cell lysates were used to quantify cAMP level. Total protein
content was measured by Bradford Assay and final cAMP concentra-
tions were normalized to total protein content in each sample. The
experiment was run twice in duplicates.
3.3. Compounds 1 and 2 are metabolically stable in human liver
microsomes
The metabolic stability of compounds 1 and 2 was quantified by
monitoring their disappearance over time in HLM supplemented with
an NADPH-regenerating system. Both compounds were determined to
be metabolically stable with CLint of 2.78 and 13.02 μL/min/mg for
compounds 1 and 2, respectively (Table 3). Two metabolites of com-
pound 2 were found: compound 2-M1 resulting from the hydroxylation
of the parent compound and compound 2-M2 – a product of hydro-
xylation and simultaneous oxidation of the methyl group at the tert-
butyl substituent to carboxylic acid. No metabolites of compound 1
were detected.
2.10. Data analysis
Unless indicated otherwise, statistical analysis was performed with
a GraphPad Prism (GraphPad Software, Inc., San Diego, California,
USA). The comparison of parameters between experimental conditions
was performed using a non-parametric Wilcoxon test for paired data.
Values presented in the graphs correspond to mean
standard error of
3.4. Compounds 1 and 2 inhibit ASMC proliferation
the mean (S.E.M.). Statistical significance was assumed at the level of
P < 0.05.
To investigate the potential effect of compounds 1 and 2 on ASMC
responses associated with remodelling, we first determined their impact
on cell proliferation. None of the selective reference PDE inhibitors
(PDE1 – vinpocetine, PDE3 – milrinone, PDE4 – roflumilast/cilomilast,
PDE7 – BRL50-481) reduced ASMC proliferation (Fig. 4A). At higher
concentrations, a slight decrease in cell proliferation was observed
when pan-PDE inhibitors, such as theophylline or IBMX were used
(Fig. 4B). However, a significant reduction in the proliferation rate was
observed for compounds 1 and 2. We demonstrated that in the range of
lower concentrations (1–10 μM), both compounds caused dose-
3. Results
3.1. Compounds 1 and 2 are non-selective but strong inhibitors of selected
PDE isoforms
Our previous research has shown that compounds 1 and 2 possess
strong anti-inflammatory properties and are inhibitors of PDE4B and 7A
isoenzymes (Fig. 1A and B, Fig. 2C and D, Table 2). To expand on this,
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Fig. 2. Compounds 1 and 2 PDE inhibitory activity. Plots represent curves obtained during determination of IC50 for compounds 1 and 2 against PDE1B (A),
PDE3A (B), PDE4B (C), and PDE7A (D). IC50 values obtained for compound 1 against PDE4B and 7A were published previously (Chłoń-Rzepa et al., 2018b, 2018a).
for the mixtures of individual PDE inhibitors (Fig. 4B). Both compounds
substantially limited ASMC proliferation, thus confirming the im-
portance of inhibition of PDEs 1, 3, 4, and 7 for the observed effect. To
rule out the involvement of adenosine receptors antagonism in the ef-
fects, we also examined the effect of adenosine receptor antagonists -
A1 (KW-3920) and A2A (KW-6002) on ASMC proliferation. Both com-
pounds had no effect on cell proliferation whatsoever (Fig. 4C).
Table 2
PDEs’ selectivity profile of compounds 1 and 2 in comparison to reference
compounds.
PDE isoform IC50 [μM]
1
2
Theophylline IBMX
Reference compounds
Vinpocetine 10.06
1B
0.07
9.16
0.25
10.70
2.69
10.89
0.18
> 1000
553.77
185.95
> 1000
> 1000
227.49
> 1000
35.70
9.22
0.71
–
45.09
1.16
2A
3A
4D
5A
10A
4B
EHNA
4.05
Milrinone
Rolipram
Zaprinast
Papaverine
0.23
4.27
6.98
0.04
3.5. Compounds 1 and 2 repress TGF-β1-induced ASMC contractile
phenotype marker expression
13.90
> 200 103.88
27.04
13.84
1.90a
5.43a
46.60b Rolipram
Roflumilast
1.10a
0.00049
2.10a
TGF-β1 stimulation in vitro of cultured ASMC promotes a contractile
phenotype which can be characterized by increased expression of
contractile genes such as ACTA2 (α-SMA) and CNN1 (calponin 1)
(Kumawat et al., 2016; Schuliga et al., 2013). We found that com-
pounds 1 and 2 both strongly reduced TGF-β1-induced ACTA2 and
CNN1 genes’ expression in ASMC (Fig. 5A and B). Both compounds
caused a significant and concentration-dependent decrease of α-SMA
and calponin-1 transcript levels. The ability of both compounds 1 and 2
to reduce TGF-β1-induced contractile gene expression was substantially
stronger than the effectiveness of reference substances, roflumilast and
cilomilast. At a concentration of 10 μM, compounds 1 and 2 were al-
most three times more efficacious in inhibiting α-SMA expression than
roflumilast and cilomilast. For calponin-1 expression, compounds 1 and
2 were respectively two and three times more efficacious than ro-
flumilast or cilomilast. We also performed immunoblotting, which
confirmed the inhibitory effects of compounds 1 and 2 on TGF-β1-
7A
8.10a
5.07a
> 1000
77.70b BRL-50481
Values were published previously -.
a
(Chłoń-Rzepa et al., 2018a, 2018b).
(Świerczek et al., 2017).
b
dependent inhibition of ASMC proliferation (Fig. 4B). At a concentra-
tion of 10 μM, the percent decrease in ASMC proliferation expressed as
the number of cells relative to the control, was 24% and 38% for
compounds 1 and 2, respectively. At higher applied concentrations,
compounds 1 and 2 caused further reduction of ASMC proliferation.
Compound 2 was particularly active in these ranges, as at a 50 μM
concentration, it caused a nearly complete reduction in ASMC pro-
liferation, in comparison to the control. A similar effect was observed
5

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Fig. 3. Compounds
1 and 2 elevate in-
tracellular cAMP levels in ASMC. ASMC
(n = 2 cell lines) were cultured in DMEM with
10% FBS for 24 h, then serum deprived in HBSS
for 2 h. All compounds were added for 30 min at
10 μM. Intracellular cAMP levels were measured
according to the manufacturer's protocol. Each bar
represents the mean cAMP level (fmol/ml/mg of
protein; from
4 independent repeats) S.E.M.
*P < 0.05 (in reference to control).
induced α-SMA protein level (Fig. 5C).
and 74% respective decrease in migrated cell number, Fig. 7A and B).
3.6. Compound 2 diminishes TGF-β1-induced extracellular matrix
components in ASMC
4. Discussion
Asthma and COPD are widespread diseases in both developed and
developing countries with increasing morbidity and mortality (Global
Initiative for Asthma, 2018; Global Initiative for Chronic Obstructive
Lung Disease, 2019). Current pharmacotherapy guidelines include nu-
merous drugs ranging from bronchodilators and corticosteroids to tar-
geted anti-inflammatory therapeutics such as monoclonal antibodies
(Durham et al., 2016; Global Initiative for Asthma, 2018; Global
Initiative for Chronic Obstructive Lung Disease, 2019). Unfortunately,
most of such drugs essentially do not affect airway remodelling or only
slightly inhibit individual characteristics of airway remodelling
(Beckett and Howarth, 2003; Durrani et al., 2011; Nayak et al., 2018).
Therefore, the search to find new chemical entities that could become
anti-remodelling drugs in the future is extremely urgent (Fromer, 2011;
Mullane, 2011). Here, we describe a novel class of PDE inhibitors with
improved anti-remodelling efficacies against several ASMC induced
responses such as proliferation, contractility, ECM protein expression
and migration.
Theophylline (1,3-dimethylpurine-2,6-dione), a non-selective PDE
family inhibitor has been available for the treatment of asthma or COPD
for almost 100 years. Although it has a good anti-inflammatory profile,
it is currently rarely used in clinical practice, mainly due to its narrow
therapeutic window and side-effects (Barnes, 2013a). To improve the
therapeutic use of PDE inhibition, numerous studies have aimed to
identify substances that might be used for the treatment of asthma or
COPD with more selective PDE inhibition profiles, especially with re-
spect to the PDE4 family (Kawamatawong, 2017; Mulhall et al., 2015;
Page, 2014). This isoform is expressed in inflammatory cells and lung
structural cells which are both involved in the pathogenesis of re-
spiratory diseases (Mulhall et al., 2015; Page, 2014). Pharmacological
As the studied compounds effectively reduced the level of con-
tractile proteins in ASMC, in the next experiments we investigated
whether they could also affect the level of ECM components synthesized
by these cells. To address this issue we estimated the level of COL1α1
(collagen Iα1), FN1 (fibronectin 1), and VCAN (versican) gene expres-
sion. Our experiments showed divergent effects of compounds 1 and 2
on the ECM components’ transcript levels in TGF-β1-induced ASMC
(Fig. 6). Whereas compound 2 was able to limit the expression of both
COL1α1 and FN1, compound 1 only inhibited the FN1 transcript level
(Fig. 6A and B). Roflumilast and cilomilast showed a limited impact on
the TGF-β1-induced ECM gene expression. Versican expression seemed
to be less affected under the action of both compounds as well as re-
ference substances (Fig. 6C). The decrease in this proteoglycan tran-
script level was only observed for compound 2 at the highest applied
concentration. To confirm these results, immunoblot analyses were also
performed. The experiments showed that both test compounds and
reference substances were able to diminish TGF-β1-induced collagen
levels in ASMC (Fig. 6D). However, in this case the action of especially
compound 2 was most profound.
3.7. Compounds 1 and 2 attenuate TGF-β1-induced ASMC migration
We further analyzed if our compounds may cause disturbances of
TGF-β1-induced ASMC migration. As demonstrated in Fig. 7A and B,
treatment of ASMC with compounds 1 and 2 in the presence of TGF-β1
decreased the migrated cell number by 42% and 47%, respectively.
Roflumilast and cilomilast also reduced ASMC migration, and in this
instance their effect was stronger than that of compounds 1 and 2 (75%
Table 3
Metabolic stability of compounds 1 and 2 in HLM.
Compound
Retention time [min]
m/z
CLint [μl/min/mg]
Metabolic reaction
1
2
4.86
7.82
5.52
5.97
522.3
486.4
531.3
502.3
2.78
13.02
–
–
–
2-M1
2-M2
hydroxylation; oxidation of methyl group at the tert-butyl substituent to carboxylic acid
hydroxylation
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Fig. 4. Compounds 1 and 2 reduce ASMC proliferation. ASMC (n = 3 cell lines) were cultured in DMEM with 10% fetal bovine serum (FBS) for 24 h, then serum
deprived in DMEM with ITS for 72 h. Compounds 1 and 2, reference inhibitors (PDE1 – vinpocetine, PDE3 – milrinone, PDE4 – roflumilast/cilomilast, PDE7 – BRL-
50481, theophylline, IBMX), adenosine receptor antagonists (KW-3920, KW-6002) and PDE inhibitors mixtures were added at increasing concentrations (1–50 μM)
1 h before FBS treatment. ASMC proliferation was measured after 48 h of incubation. Each bar represents mean percentile differences in alamar®Blue reduction
between treated and control cells ( S.E.M.). MIX 1 and MIX 2 - the final concentration of all inhibitors in the mixture is included in the X axis. Experiments were run
in duplicates for each ASMC culture.*P < 0.05 (in reference to control).
inhibition of PDE4 increases intracellular cAMP level and consequently
causes anti-inflammatory effects (Raker et al., 2016). Although the use
of inhibitors of a single PDE markedly improved the safety of using
compounds from this group, their effectiveness is suboptimal. Recent
findings indicate that dual inhibitors (PDE1-PDE4, PDE3-PDE4, or
PDE4-PDE7) may be more effective in preventing changes related to AR
(Maurice et al., 2014). This provides a rationale for discovering mole-
cules with a broader spectrum of PDE inhibition.
Our studies have shown that novel purine-2,6-dione derivatives are
non-selective, however strong PDE inhibitors. We have demonstrated
that they can significantly inhibit PDE4 as well as PDE1, PDE3 and
PDE7 isoenzymes, which are accompanied by an elevated intracellular
cAMP level. Although the role of PDE4 in asthma and COPD is well
established, the functional roles of PDE1, PDE3 or PDE7 sub-families in
airways disease are less known. All 3 of these isoforms are expressed in
bronchial epithelium, airway smooth muscle cells, pulmonary fibro-
blasts as well as inflammatory cells (Halpin, 2008; Oldenburger et al.,
2012). Several studies indicate that PDE3 and PDE7 inhibitors augment
the anti-inflammatory effects of PDE4 inhibitors (Maurice et al., 2014;
Page, 2014). It is believed that a broader spectrum of activity with si-
multaneous strong action not only improves the anti-inflammatory
properties of compounds but also provides a better safety profile. For
example, theophylline also exhibits activity towards different PDE
isoforms but inhibits them at much higher concentrations than com-
pounds 1 and 2 (Hatzelmann et al., 2010). Our aim was to obtain
compounds which due to their activity will be strong, second genera-
tion, pan-PDE inhibitors. Compounds 1 and 2 have relatively low IC50
values against individual PDE isoenzymes, including PDE1, PDE3, PDE4
and PDE7, which seems to be extremely important for the observed
pharmacological effects. These characteristics distinguish them from
the group of existing pan-PDE inhibitors, such as theophylline or IBMX.
It is worth emphasizing that in spite of adverse effects, theophylline is
still used in difficult cases to treat asthma. Therefore, the search for new
compounds with similar properties but with a preferential activity
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Fig. 5. Compounds 1 and 2 repress TGF-β1-induced contractile markers in ASMC. ASMC (n = 3) were cultured in DMEM with 10% FBS for 24 h, then serum
deprived in DMEM with ITS for 72 h. Compounds 1 and 2 and reference substances (roflumilast and cilomilast) were added at increasing concentrations (1–25 μM)
1 h before TGF-β1 (2 ng/ml) treatment. Expression of ACTA2 (A) and CNN1 (B) genes in comparison to 18S rRNA was quantified by qPCR, after 24 h of incubation.
Values represent means ( S.E.M.) of three ASMC cultures run in duplicates. (C) Representative immunoblot and densitometry analysis of α-SMA protein in ASMC
cultured in the presence of compounds 1, 2, roflumilast or cilomilast, and TGF-β1 was performed. ROD was measured and compared to GAPDH level. Each bar
represents the mean value ( S.E.M.) obtained from three ASMC cultures run in duplicates. *P < 0.05 - in reference to control, #P < 0.05 - in reference to TGF-β1-
treated ASMC.
profile is important. The strong activity of novel purine-2,6-dione de-
rivatives against different PDE isoforms makes them an interesting al-
ternative to theophylline, a broad spectrum PDE inhibitor and ro-
flumilast or cilomilast, which only inhibit the PDE4 family (Hatzelmann
et al., 2010).
Possibly, the broader spectrum activities of compounds 1 and 2 lead to
a different activation of intracellular signalling compartments (in-
cluding those responsible for the migration process) than roflumilast or
cilomilast. For some responses, this may result in more efficacy, for
others (e.g. responses heavily dependent on PDE4) this difference may
be less profound. Comparison of the observed activities of compounds 1
and 2 to PDE4-selective inhibitors suggests that inhibition of PDE1,
PDE3 and/or PDE7 isoforms may also be involved in reducing TGF-β-
induced remodelling responses in ASMC. For the inhibition of TGF-β
functional responses, these isoforms may contribute directly, or work
together by enhancing the functional effects associated with the in-
hibition of PDE4 isoforms. As the selective PDE inhibitors can act sy-
nergistically in reducing inflammation or AR (Maurice et al., 2014) this
contention seems to be plausible.
The mechanisms of action of the compounds from the methyl-
xanthine group include not only the inhibition of intracellular PDEs but
also the binding and blocking of adenosine receptors. It is known that
caffeine, theophylline and IBMX are antagonists of adenosine receptors
(Müller and Jacobson, 2011). From previous literature reports and
structure-activity relationship (SAR) studies it appears that in order to
be a strong antagonist of adenosine receptors (stronger than theo-
phylline), the compound should have large, complex and strongly li-
pophilic substituents, e.g. phenyl or phenylethyl, at position 8 of the
purine-2,6-dione core (Müller and Jacobson, 2011). Also, tricyclic
structures with a third ring added to xanthine are preferential (Müller
and Jacobson, 2011). Our compounds have less lipophilic benzylamine
or butoxy substituents at position 8 of the purine-2,6-dione compared to
phenyl or phenylethyl. Moreover, the obtained research results showed
that the mechanism of adenosine A1 and A2A receptor blockade did not
play a significant role in the changes associated with ASMC remodel-
ling, although additional mechanisms of action of the compounds stu-
died cannot be excluded. From the preliminary studies, it also follows
As the studies of metabolic stability carried out in the HLM model
showed favourable metabolic profiles of compounds 1 and 2 we eval-
uated their ability to modulate cell responses associated with ASMC
remodelling. ASMC were used in the study as their hyperplasia and
hypertrophy is described in AR and they are a good experimental model
to test the efficiency of possible anti-fibrotic compounds (Baroffio et al.,
2008). Our experiments also confirmed the strong and prominent pro-
fibrotic response of TGF-β in ASMC. This growth factor activates ASMC
expression of certain genes linked to the remodelling response pheno-
type (e.g. ACTA2, CNN1, COL1α1, FN1). Studies by Burgess and co-
workers have shown that roflumilast may reduce TGF-β-induced ex-
pression of CTGF and ECM proteins in ASMC (Burgess et al., 2006). Our
research confirms the potential anti-fibrotic properties of PDE4 in-
hibitors, although purine-2,6-dione derivatives appear to have prefer-
able activity. These strong, non-selective PDE inhibitors were de-
termined to be more potent in reducing FBS or TGF-β-induced ASMC
remodelling response. This was expressed by a significant limitation of
ASMC proliferation and expression of profibrotic and ECM proteins
genes. Our observations relate to control ASMC but we would expect
similar effects in asthmatic airway smooth muscle cells as the PDE in-
hibitors provide a functional antagonism response, not a mediator
blocking response. Therefore, the effect is expected to occur for other
growth stimuli or growth conditions as well. Conducted studies have
shown compounds 1 and 2 have a slightly weaker effect on TGF-β-in-
duced ASCM migration when compared to the selective PDE4 inhibitors
roflumilast and cilomilast. This may be related to the high cAMP and
PDE compartmentalization in the cells (Zuo et al., 2019a, 2019b).
8

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Fig. 6. Compound 2 reduces TGF-β1-induced extracellular matrix components in ASMC. ASMC (n = 3) were cultured in DMEM with 10% FBS for 24 h, then
serum deprived in DMEM with ITS for 72 h. Compounds 1 and 2 and reference substances (roflumilast and cilomilast) were added at increasing concentrations
(1–25 μM) 1 h before TGF-β1 (2 ng/ml) treatment. Expression of COL1α1 (A), FN1 (B) and VCAN (C) genes in comparison to 18S rRNA was quantified by qPCR, after
24 h of incubation. Values represent means ( S.E.M.) of three ASMC cultures run in duplicates. (D) Representative immunoblot and densitometry analysis of
collagen type I protein in ASMC cultured in the presence of compounds 1 and 2, roflumilast or cilomilast, and TGF-β1. ROD was measured as compared to GAPDH
level. Each bar represents the mean value ( S.E.M.) obtained from three ASMC cultures run in duplicates. *P < 0.05 - in reference to control, #P < 0.05 - in
reference to TGF-β1-treated ASMC.
that compounds 1 and 2 do not regulate the catalytic activity of ade-
nylate kinases (unpublished data).
receptors may play an important role in the inflammatory response and
airway hyperresponsiveness in asthma or COPD (Jha et al., 2015;
Mukhopadhyay et al., 2016). The hypothesis that amplification of anti-
inflammatory and anti-fibrotic properties may be achieved by si-
multaneous inhibition of selected PDEs and TRPA1 channels can un-
doubtedly be an interesting direction for further studies. Future per-
spectives also include research into the therapeutic properties of
purine-2,6-dione derivatives. At a later point, we plan to involve mouse
models of chronic allergic asthma by which verification of the com-
pounds’ effectiveness will be conducted.
In the applied experimental model, both tested compounds de-
monstrated the ability to reduce TGF-β-induced ASMC remodelling
response, although compound 2 was more potent. We observed this
activity despite the fact that both compounds showed a comparable
inhibitory effect on selected PDE isoforms. We can therefore exclude
that the superior properties of compound 2 correspond to the preferable
inhibition profile of individual PDE isoforms. The predominant activity
of compound 2 in limiting ASMC remodelling response may be asso-
ciated with stronger anti-inflammatory properties. In pharmacological
studies, compound 2 was shown to possess a greater ability to inhibit
lipopolysaccharide-induced TNF-α secretion in rats (Chłoń-Rzepa et al.,
2018a, 2018b). Another reason for the stronger activity of compound 2
is its metabolic stability. The obtained CLint values indicate that the
compounds were similar in terms of biotransformation, although in the
case of compound 2, two metabolites were formed. The direction of
metabolism we suggested indicates the oxidation of a methyl group at
the tert-butyl substituent, which is a characteristic reaction for this
moiety. Analogical phenomenon was observed for the metabolism of
terfenadine, an antihistaminic drug. The metabolite formed as a result
of the oxidation of the tert-butyl group in the parent structure, turned
out to have similar properties and became a new individual anti-
histaminic drug (Terrien et al., 1999). That is why we cannot exclude
that compound 2 metabolites also have similar properties that can
additionally enhance compound 2 activity. Finally, it is worth empha-
sizing that compound 2 possesses additional activity towards transient
receptor potential cation channel, subfamily A, member 1 (TRPA1)
(Chłoń-Rzepa et al., 2018b). Recent reports indicate that these
5. Conclusion
Currently, the state of the art in the search for new drugs is directed
at re-using known structures and improving and modifying them to
design new ones. Research on theophylline derivatives fits this trend
very well. Although methylxanthines are currently not the drugs of
choice in the treatment of asthma or COPD, nevertheless, they are an
interesting and valuable group of substances with anti-inflammatory
and anti-fibrotic properties. The new theophylline derivatives - PDE
inhibitors, may find particular application in the treatment of cases of
severe asthma and COPD resistant to standard therapy with corticos-
teroids (Barnes, 2013a). It is believed that one of the mechanisms re-
sponsible for resistance to corticosteroids in this group of patients may
be linked to histone deacetylase dysfunction through its reduced ac-
tivity and expression (Barnes, 2013b). As mentioned, theophylline and
its derivatives can activate various signalling pathways in cells and one
of them is histone deacetylase activation. Undoubtedly, this is another
important reason to search for new drugs for treatment of asthma or
9

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This work was supported by the National Science Centre, Poland,
funded grants No. UMO-2017/27/B/NZ7/01633 and No. UMO-2014/
15/B/NZ7/00885. We acknowledge the support of the European
Respiratory Society, Fellowship STRF 2017.
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