Full text of DOI:10.1038/sj.bjp.0704539

British Journal of Pharmacology (2002) 135, 961 ± 968
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Attenuation of cardiac ®brosis by pirfenidone and amiloride in
DOCA-salt hypertensive rats
2
1
1
1Stevo Mirkovic, Anne-Marie L. Seymour, Andrew Fenning, Anna Strachan,
3Solomon B. Margolin, Stephen M. Taylor & *,1Lindsay Brown
1
1Department of Physiology and Pharmacology, School of Biomedical Sciences, The University of Queensland, Australia;
3
2Department of Biological Sciences, University of Hull, Hull and Marnac Inc., Dallas, Texas, U.S.A.
1
This study has administered pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) or amiloride to
attenuate the remodelling and associated functional changes, especially an increased cardiac sti€ness,
in DOCA-salt hypertensive rats.
2
In control rats, the elimination half-life of pirfenidone following a single intravenous dose of
200 mg kg71 was 37 min while oral bioavailability at this dose was 25.7%. Plasma pirfenidone
concentrations in control rats averaged 1.9+0.1 mg ml71 over 24 h after 14 days' administration as a
0.4% mixture in food.
3
Pirfenidone (approximately 250 ± 300 mg kg71 day71 as 0.4% in food) and amiloride (1 mg kg71
day71 sc) were administered for 2 weeks starting 2 weeks post-surgery. Pirfenidone but not amiloride
attenuated ventricular hypertrophy (2.69+0.09, UNX 2.01+0.05. DOCA-salt 3.11+0.09 mg kg71
body wt) without lowering systolic blood pressure.
4
Collagen deposition was signi®cantly increased in the interstitium after 2 weeks and further
increased with scarring of the left ventricle after 4 weeks; pirfenidone and amiloride reversed the
increases and prevented further increases. This accumulation of collagen was accompanied by an
increase in diastolic sti€ness constant; both amiloride and pirfenidone reversed this increase.
5
Noradrenaline potency (positive chronotropy) was decreased in right atria (neg log EC50: control
6.92+0.06; DOCA-salt 6.64+0.08); pirfenidone but not amiloride reversed this change. Noradrena-
line was a more potent vasoconstrictor in thoracic aortic rings (neg log EC50: control 6.91+0.10;
DOCA-salt 7.90+0.07); pirfenidone treatment did not change noradrenaline potency.
6 Thus, pirfenidone and amiloride reverse and prevent cardiac remodelling and the increased
cardiac sti€ness without reversing the increased vascular responses to noradrenaline.
British Journal of Pharmacology (2002) 135, 961 ± 968
Keywords: DOCA-salt hypertension; hypertrophy; ®brosis; pirfenidone; amiloride
Abbreviations: DOCA, deoxycorticosterone acetate; UNX, uninephrectomized
Introduction
Cardiac remodelling as in chronic hypertension involves
myocyte hypertrophy as well as ®brosis, an increased and
non-uniform deposition of extracellular matrix proteins,
especially collagens (Weber et al., 1993; 1994). Although
hypertension-induced left ventricular hypertrophy has been
implicated as the single most important prognostic indicator
for adverse cardiovascular events in humans (Levy et al.,
1990), changes in the quality of the interstitial space are also
critical. The extracellular matrix connects myocytes, aligns
contractile elements, prevents overextending and disruption
of myocytes, transmits force and provides tensile strength to
prevent rupture. Fibrosis occurs in many models of
hypertension leading to an increased diastolic sti€ness, a
reduction in cardiac function (Weber et al., 1993; 1994) and
an increased risk of arrhythmias (Assayag et al., 1997). If
®brosis rather than myocyte hypertrophy is the critical factor
in impaired cardiovascular function, then reversal of cardiac
®brosis by itself may return cardiac function towards normal.
Since collagen deposition is a dynamic process (Sun &
Weber, 2000), appropriate pharmacological intervention
could selectively reverse existing ®brosis and prevent further
®brosis and thereby improve function, even if the increased
systolic blood pressure was unchanged.
Pirfenidone and amiloride are candidate drugs for the
attenuation of cardiac ®brosis based on the relevant
literature. Pirfenidone prevented and reversed lung ®brosis
and attenuated pulmonary dysfunction following bleomycin
treatment in hamsters (Iyer et al., 1995; Schelegle et al., 1997)
by suppressing in¯ammatory events (Iyer et al., 2000) and
down-regulating lung procollagen I over-expression (Iyer et
al., 1999a). Pirfenidone decreased collagen production and
deposition in a rat model of hepatic ®brosis (Tada et al.,
2001). In addition, this compound reversed cardiac and renal
®brosis and attenuated the increase in diastolic sti€ness of
diabetic hearts from streptozotocin-treated rats without
normalizing cardiac contractility or renal function (Miric et
al., 2001). Amiloride selectively prevented cardiac collagen
accumulation in aldosterone-salt hypertensive rats (Campbell
et al., 1993). However, the more clinically relevant reversal of
cardiac ®brosis by amiloride in hypertension has not been
studied, although the ACE inhibitor, lisinopril, has been
*Author for correspondence:

962
S. Mirkovic et al
Pirfenidone in hypertension
shown to reverse cardiac ®brosis in humans (Brilla et al.,
2000).
used to access the kidneys, and the left renal vessels and
ureter were ligated. The left kidney was removed and weighed
and the skin wound sutured. Uninephrectomized rats were
given either no further treatment (UNX rats) or 1% NaCl in
the drinking water with subcutaneous injections of deoxy-
corticosterone acetate (DOCA; 25 mg in 0.4 ml dimethylfor-
mamide every fourth day) (DOCA-salt rats) (Dallemagne et
al., 2000). Experiments were performed 2 or 4 weeks after
surgery. After 2 weeks, rats were given either pirfenidone
(0.4% in powdered rat food) or amiloride (1 mg kg71 day71
sc) for a further 2 weeks.
The aim of this study was to determine the consequences of
pirfenidone and amiloride treatment on collagen deposition
and cardiac function in the deoxycorticosterone acetate
(DOCA)-salt hypertensive rat heart. This model develops
ventricular hypertrophy with cardiac ®brosis (Young et al.,
1995) following increased expression of collagen I and III
mRNA (Robert et al., 1994; Brown et al., 1999).
Methods
Food and water intake and body weights were measured
daily for all rats. Systolic blood pressure was measured in
unanaesthetized rats using a tail-cu€ method. Rats were
killed with pentobarbitone (200 mg kg71 ip). Blood was
taken from the abdominal vena cava, centrifuged and the
plasma frozen. Plasma aldosterone concentrations were
measured by a commercial radioimmunoassay kit.
Pharmacokinetic studies
Blood sample collection after intravenous dosing Wistar rats
(150 ± 200 g) were anaesthetized using Zoletil1 i.p. (zolaze-
pam 25 mg kg71 and tiletamine 25 mg kg71) with xylazine
(10 mg kg71) and catheterized via the femoral vein. Catheters
were ¯ushed with heparinized saline at the time of placement,
four days later and on the day of experimentation (7th day).
Pirfenidone (200 mg kg71 suspension in 1 ml normal saline)
was infused intravenously over 30 s. Blood samples of less
than 0.2 ml were collected in heparinized tubes up to 4 h
after pirfenidone was infused, and the same volume of saline
was injected into the catheter to maintain blood volume
throughout the trial.
Isolated Langendorff heart preparation
Rats were anaesthetized with sodium pentobarbitone
(100 mg kg71 ip) and heparin (2000 iu) was administered
via the femoral vein. After allowing 2 min for the heparin to
fully circulate, the heart was excised and placed in cooled
(08C) crystalloid perfusate (Krebs-Henseleit solution of the
following composition in mM: NaCl 118, KCl 4.7, MgSO4
1.2, KH2PO4 1.2, CaCl2 2.3, NaHCO3 25.0, glucose 11.0).
The heart was then attached to the cannula (with the tip of
the cannula positioned immediately above the coronary ostia
of the aortic stump) and perfused in a non-recirculating
Langendor€ fashion at 100 cm of hydrostatic pressure as
previously described (Smolenski et al., 1998). The bu€er
temperature was maintained at 358C. The apex of the heart
was pierced to facilitate thebesian drainage and paced at
250 b.p.m.
A balloon catheter was inserted in the left ventricle via the
mitral ori®ce for measurement of left ventricular developed
pressure (Smolenski et al., 1998). The catheter was connected
via a three-way tap to a micrometer syringe and to a Statham
P23 pressure transducer. The outer diameter of the catheter
was similar to the mitral annulus to prevent ejection of the
balloon during the systolic phase. After a 10 min stabilization
period, steady-state left ventricular pressure was recorded
from isovolumetrically beating hearts. Increments in balloon
volume were applied to the heart until left ventricular end-
diastolic pressure reached approximately 30 mmHg. At the
end of the experiment, the atria and right ventricle were
dissected away and the weight of the left ventricle plus
septum was recorded.
Blood sample collection after oral dosing Pirfenidone
(200 mg kg71 suspension in 1 ml normal saline) was
administered through
a stomach tube. Blood samples
(0.2 ml) were collected from a small incision in the tail vein
for up to 4 h following a single oral dosage and every four
hours for 24 h following chronic dosage. Rats were killed as
above following collection of the last blood sample, and the
pH of the stomach contents was measured using pH paper.
Additionally, rats were fed an unrestricted diet of pulverized
rat chow containing 0.4% pirfenidone for 14 days. All blood
samples were collected in heparinized microfuge tubes, and
centrifuged at 11,0006g for 3 min before plasma was
collected from each sample and stored at 7208C until HPLC
analysis for pirfenidone concentration as described below.
HPLC analysis of plasma pirfenidone concentration
A
Waters Novapak C18 (Picotag) reverse phase column was
used with 50% acetonitrile in HPLC grade water as the
mobile phase at 1 ml min71; pirfenidone was measured using
a Beckman DU690 UV spectrometer at 280 nm. Elution of
pirfenidone occurred at approximately 3.1 min after injection
onto the column; a standard curve was constructed and
unknown concentrations estimated from this standard curve.
Myocardial diastolic sti€ness was calculated as the diastolic
sti€ness constant (k, dimensionless), the slope of the linear
relation between tangent elastic modulus (E, dyne cm72) and
stress (s, dyne cm72) (Brilla et al., 1991; Brown et al., 1999).
To assess contractile function, maximal+dP/dt values were
calculated at a diastolic pressure of 10 mmHg.
DOCA-salt hypertensive rats
Male Wistar rats (8 ± 10 weeks old) were obtained from the
Central Animal Breeding House of The University of
Queensland. All experimental protocols were approved by
the Animal Experimentation Ethics Committee of The
University of Queensland under the guidelines of the
National Medical and Health Research Council of Australia.
Uninephrectomy was performed on all treated rats. The rats
were anaesthetized as above; a lateral abdominal incision was
Isolated cardiac muscles and thoracic aortic rings
The heart was removed under anaesthesia. The right atria and
papillary muscles from the left ventricle were removed and
suspended in organ baths at a resting tension of 5 ± 10 mN
British Journal of Pharmacology vol 135 (4)

S. Mirkovic et al
Pirfenidone in hypertension
963
adjusted to give the maximal twitch response. Tissues were
bathed in a modi®ed Tyrodes solution (in mM): NaCl 136.9,
KCl 5.4, MgCl2 1.05, CaCl2 1.8, NaHCO3 22.6, NaH2PO4
0.42, glucose 5.5, ascorbic acid 0.28, sodium edetate 0.05,
bubbled with 95%O2/5%CO2 and stimulated at 1 Hz at 358C
as previously described (Brown et al., 1991b). Cumulative
concentration-response curves were measured for noradrena-
line and, following washout and re-equilibration, to calcium
chloride. At the end of the experiment, papillary muscle
dimensions were measured under the loading conditions of the
experiment; all tissues were blotted and weighed.
Thoracic aortic rings (approximately 4 mm in length) were
suspended with a resting tension of 10 mN (Brown et al.,
1991a) and contracted twice with isotonic KCl (100 mM). The
presence of endothelium was demonstrated by addition of
acetylcholine (10 mM). Cumulative contraction responses to
noradrenaline were measured.
concentrations of 1.9+0.1 mg/ml over 24 h (Figure 1B).
Following the same protocol, plasma pirfenidone concentra-
tions in DOCA-salt rats were 1.3+0.3 mg/ml (n=5).
DOCA-salt treatment produced marked physiological
changes including hypertension, hypertrophy of the left
ventricle and remnant kidney (Table 1). Despite signi®cant
increases in water intake and decreases in food intake (Figure
2), no change in body weight was observed. Amiloride or
pirfenidone administration did not modify these parameters
in UNX or DOCA-salt hypertensive rats; pirfenidone,
however, attenuated ventricular and renal hypertrophy
(Figure 2; Table 1). Daily intake of pirfenidone averaged
287+12 mg kg71 in UNX rats and 256+18 mg kg71 in
DOCA-salt rats (Figure 2).
DOCA-salt hypertensive rats showed an increase in
interstitial collagen deposition after 2 weeks which was
further increased after 4 weeks (Figure 3A); perivascular
collagen showed similar changes. Myocardial scarring
increased only after 4 weeks (Figure 3B); administration of
pirfenidone or amiloride normalised collagen deposition thus
reversing the existing collagen deposition as well as
preventing new collagen deposition. In the isolated Langen-
dor€ preparation, DOCA-salt hearts showed an increased
diastolic sti€ness constant; treatment with pirfenidone or
Collagen distribution
Collagen distribution was determined by image analysis of
picrosirius red-stained sections of the hearts (Miric et al.,
2001). In brief, transverse sections stored in 4% paraformal-
dehyde were dehydrated and embedded in wax and then sliced
into 5 mm sections. These were stained with picrosirius red
(0.1% Sirius Red F3BA in picric acid). Slides were left in 0.2%
phosphomolybdic acid for 5 min, washed, left in picrosirius
red for 90 min, then in 1 mM HCl for 2 min and 70% ethanol
for 45 s. The stained sections were analysed with an Image Pro
plus analysis program using an Olympus BH2 microscope
with results expressed as a percentage of red area in each
screen. At least four areas were examined in each heart.
A
Data analysis
All results are given as mean+s.e.m. The negative log EC50 of
the increase in either force of contraction in mN or rate of
contraction in beats/min was determined from the concentra-
tion giving half-maximal responses in individual concentration-
response curves. These results were analysed by two-way
analysis of variance followed by the Duncan test to determine
di€erences between treatment groups and by paired or unpaired
t-tests as appropriate; P50.05 was considered signi®cant.
B
Drugs
Deoxycorticosterone acetate, amiloride and noradrenaline
were purchased from Sigma Chemical Company, St Louis,
MO, U.S.A. Pirfenidone was provided by Marnac Inc.,
Dallas, TX, U.S.A. Noradrenaline was dissolved in distilled
water; deoxycorticosterone acetate was dissolved in dimethyl-
formamide with mild heating.
Results
Following a single intravenous injection, pirfenidone showed
an elimination half-life of 37 min (Figure 1A). Comparison of
plasma concentrations following intravenous and oral
administration indicated an oral bioavailability of 25.7% in
the rat. Oral administration of pirfenidone as a 0.4% mixture
in powdered food for 14 days gave constant plasma
Figure 1 Plasma pirfenidone concentrations in control rats after a
single dose (A) and throughout 24 h when pirfenidone added to food
(B) for oral dosage (open diamonds) or intravenous dosage (®lled
diamonds); for (B), 0 h was 0900 h after 2 weeks' administration of
pirfenidone in food; n=4 for all groups.
British Journal of Pharmacology vol 135 (4)

964
Table 1 Physiological parameters
S. Mirkovic et al
Pirfenidone in hypertension
UNX+
pirfenidone
UNX+
amiloride
DOCA-salt
2 wk
DOCA-salt
4 wk
DOCA-salt+
pirfenidone
DOCA-salt+
amiloride
Parameter
UNX
Number of rats
Body weight (g) -
final
Systolic blood
pressure (mmHg)
Heart rate (bpm)
20
411+11
(n=8)
104+5
(n=5)
394+13
(n=5)
4.4+0.3
(n=10)
419+73
(n=5)
12
425+5
(n=8)
117+1
(n=4)
404+19
(n=4)
4.0+0.2
(n=8)
12
418+12
(n=8)
115+3
(n=6)
424+21
(n=6)
3.8+0.1
(n=8)
8
16
346+15*
(n=8)
185+19*
(n=6)
407+21
(n=6)
3.1+0.4*
(n=8)
20
20
346+10*
(n=8)
162+13*
(n=7)
401+19
(n=7)
3.2+0.7
(n=8)
306+11*
(n=8)
154+3
(n=6)
397+14
(n=6)
±
333+9*
(n=16)
171+17*
(n=6)
330+15*
(n=6)
2.6+0.3*
(n=11)
137+23*
(n=5)
Plasma K+
concentration (mM)
Plasma aldosterone
(pg/ml)
392+70
(n=5)
1105+313*
(n=5)
±
166+18*
(n=5)
269+44
(n=5)
Organ weights:
Left ventricle+
septum (mg/kg)
Right ventricle
(mg/kg)
2.01+0.05
(n=16)
0.56+0.02
(n=16)
1.96+0.06
(n=8)
0.58+0.01
(n=8)
2.11+0.07
(n=8)
0.55+0.03
(n=8)
2.26+0.08
(n=8)
0.59+0.03
(n=8)
3.11+0.09*
(n=16)
0.66+0.02*
(n=16)
2.69+0.09**
(n=6)
0.67+0.03*
(n=6)
3.12+0.12*
(n=8)
0.77+0.04*
(n=8)
Remnant kidney
(mg/kg)
5.24+0.27
(n=16)
5.00+0.03
(n=8)
5.5+0.67
(n=8)
7.1+0.2
(n=8)
10.6+0.4*
(n=16)
7.5+0.8**
(n=6)
12.0+0.1*
(n=6)
Functional parameters:
Diastolic stiffness
constant
Maximal +dP/dt
(6103 mmHg/s)
18.8+0.8
(n=7)
1.4+0.02
(n=7)
16.3+1.3
(n=6)
1.4+0.04
(n=6)
19.5+1.7
(n=6)
1.3+0.05
(n=6)
±
±
29.3+1.1*
(n=8)
1.3+0.2
(n=8)
19.2+0.8**
(n=8)
0.81+0.09*
(n=8)
22.1+1.4**
(n=14)
1.2+0.1
(n=14)
*P50.05 vs UNX, **P50.05 vs DOCA-salt rats.
A
B
C
D
Figure 2 Water (A) and food (B) consumption, body weight (C) and pirfenidone consumption (D) in UNX (U, n=8; open circles),
UNX+pirfenidone (UP, n=8; open triangles), UNX+amiloride (UA, n=8; open squares), DOCA-salt (D, n=8; ®lled circles),
DOCA-salt+pirfenidone (DP, n=15; ®lled triangles), DOCA-salt+amiloride (DA, n=8; ®lled squares) rats; *P50.05 vs control.
British Journal of Pharmacology vol 135 (4)

S. Mirkovic et al
Pirfenidone in hypertension
965
A
amiloride normalized this constant (Figure 4, Table 1).
Pirfenidone, but not amiloride, signi®cantly decreased left
ventricular contractility measured as+dP/dt in the DOCA-
salt hearts (Figure 4, Table 1).
The potency of positive chronotropic responses to
noradrenaline was increased in DOCA-salt rats (neg log
EC50: control 6.92+0.06; DOCA-salt 6.64+0.08) (Figure
5A); pirfenidone but not amiloride reversed this change.
Positive inotropic responses to noradrenaline and calcium
chloride (Figure 5B) were unaltered in left ventricular
papillary muscles taken from DOCA-salt hearts compared
to UNX hearts. Pirfenidone treatment did not change
A
B
B
Figure 4 Contractility as maximal dP/dt (A) and diastolic sti€ness
(k) (B): UNX (U, n=7), UNX+pirfenidone (UP, n=6), UNX+
amiloride (UA, n=6), DOCA-salt (D2, after 2 weeks; D4, after 4
weeks; n=7), DOCA-salt+pirfenidone (DP, n=10), DOCA-salt+
amiloride (DA, n=7); *P50.05 compared to UNX, **P50.05
compared to DOCA-salt.
noradrenaline responses but increased maximal responses to
calcium chloride. No changes in responses were observed in
responses in muscles from amiloride-treated DOCA-salt rat
hearts. However, the responsiveness of isolated thoracic
aortic rings to noradrenaline was increased in DOCA-salt
rats compared to UNX controls (neg log EC50: control
6.91+0.10; DOCA-salt 7.90+0.07) (Figure 6). Neither
pirfenidone nor amiloride treatment attenuated these in-
creased vasoconstrictor responses to noradrenaline.
Figure 3 Interstitial collagen deposition (A) and scarring (B) in the
ventricles (percent of total area): UNX (U, n=7), UNX+pirfenidone
(UP, n=6), UNX+amiloride (UA, n=6), DOCA-salt (D2, after 2
weeks; D4, after 4 weeks; n=7), DOCA-salt+pirfenidone (DP,
n=10), DOCA-salt+amiloride (DA, n=7); *P50.05 compared to
UNX, **P50.05 compared to DOCA-salt.
British Journal of Pharmacology vol 135 (4)

966
S. Mirkovic et al
Pirfenidone in hypertension
A
Discussion
The DOCA-salt hypertensive rat rapidly develops cardiac
hypertrophy and ®brosis (Young et al., 1995; Doggrell &
Brown, 1998). We have recently shown, in this model, that
cardiac ®brosis can be reversed, without lowering blood
pressure or decreasing the degree of cardiac hypertrophy, by
inhibition of the renin-angiotensin system using the ACE
inhibitor, captopril, the AT1 receptor antagonist, candesar-
tan, or the aldosterone antagonist, spironolactone (Brown et
al., 1999). This study extends the range of compounds that
selectively reverse existing cardiac ®brosis and prevent further
deposition to include pirfenidone and amiloride. Neither
compound decreased the increased systolic blood pressure;
pirfenidone attenuated the degree of cardiac and renal
hypertrophy and also decreased ventricular contractility.
Both compounds decreased the diastolic sti€ness; neither
reversed the increased vascular sensitivity to noradrenaline in
these hypertensive rats. This study provides more evidence
that cardiac ®brosis can be attenuated independently of blood
pressure and ventricular hypertrophy and that this e€ect may
improve cardiac function.
B
The anti®brotic actions of pirfenidone have been shown in
di€erent species and diseases, for example bleomycin-induced
pulmonary ®brosis in hamsters (Iyer et al., 1995) with
attenuation of pulmonary functional de®cits (Schelegle et al.,
1997), in sclerosing peritonitis in rats (Suga et al., 1995), in
renal ®brosis following partial nephrectomy in rats (Shimizu et
al., 1997), in human myometrial and leiomyoma cell cultures
(Lee et al., 1998), in streptozotocin-diabetic rats (Miric et al.,
2001) and in dimethylnitrosamine-induced hepatic ®brosis in
rats (Tada et al., 2001). However, pirfenidone had no
therapeutic activity in patients with myelo®brosis with
myeloid metaplasia (Mesa et al., 2001). Reversal protocols
were used by Miric et al. (2001) as well as by Shimizu et al.
(1997) who started pirfenidone treatment two weeks post
surgery, as in the current study. Daily pirfenidone doses
ranged from 0.5% in food (250 ± 375 mg kg71 at food intakes
of 20 ± 30 g day71 as in the current study), to 200 ±
300 mg kg71 (Miric et al., 2001 and current study),
350 mg kg71 (Suga et al., 1995) or 500 mg kg71 (Shimizu et
al., 1997; Tada et al., 2001). Despite these di€erences in
protocol, the results have shown that pirfenidone consistently
prevents collagen accumulation. None of these literature
studies have measured plasma pirfenidone concentrations.
This study shows that plasma concentrations of 1.3 ±
2 mg ml71 are e€ective in attenuating ®brosis in the heart
and that constant plasma levels are achieved in rats following
administration in the powdered food.
Figure 5 Cumulative concentration-response curves for noradrena-
line in right atria (A) and noradrenaline and calcium chloride in left
ventricular papillary muscles (B) from UNX (U, n=16; open circles),
DOCA-salt (D, n=9; ®lled circles), DOCA-salt+pirfenidone (DP,
n=5; ®lled triangles), and DOCA-salt+amiloride (DA, n=6; ®lled
squares) treated rats; *P50.05 compared to UNX, **P50.05
compared to DOCA-salt values.
The mechanisms of action of pirfenidone are unclear but
this compound may increase collagen breakdown by reducing
the TGFb1-induced inhibition of the degrading enzymes, the
matrix metalloproteinases (Suga et al., 1995). Pirfenidone
also down-regulated bleomycin-induced over-expression of
procollagen I and III genes in the lungs (Iyer et al., 1999a)
and suppressed an increased TGFb1 mRNA (Iyer et al.,
1999b). Thus, suppression of the synthesis or activity of this
cytokine is the most likely mechanism of action of
pirfenidone. This is supported by results showing that
suppression of an increased TGFb1 expression by tranilast
in hypertensive transgenic rats overexpressing human renin
attenuated left ventricular hypertrophy and ®brosis without
Figure 6 Cumulative concentration-response curves for noradrena-
line in the thoracic aortic rings from UNX (U, n=16; open circles),
DOCA-salt (D, n=7; ®lled circles), DOCA-salt+pirfenidone (DP,
n=7; ®lled triangles) and DOCA-salt+amiloride (DA, n=5; ®lled
squares) treated animals; *P50.05 compared to UNX, **P50.05
compared to DOCA-salt.
British Journal of Pharmacology vol 135 (4)

S. Mirkovic et al
Pirfenidone in hypertension
967
lowering blood pressure (Pinto et al., 2000). Pirfenidone may
also show anti-in¯ammatory e€ects such as suppression of
increased vascular permeability, neutrophil recruitment and
in¯ux of in¯ammatory cells, in addition to its e€ects on
collagen turnover (Iyer et al., 2000; Corbel et al., 2001).
reverse an existing increased collagen deposition and prevent
further deposition in the heart. Thus, many compounds are
likely to modulate cardiac ®brosis, including pirfenidone,
amiloride and inhibitors of the renin-angiotensin system
(Brown et al., 1999). Further, the cross-linking of myocardial
collagen is another potential target as this parameter may
determine the degree of cardiac dilatation in heart disease
(Woodiwiss et al., 2001). Removal of collagen may carry
some risks. Excessive degradation to below normal collagen
concentrations may disrupt the collagen network and distort
cardiac architecture leading to wall thinning, chamber
dilatation and possibly wall rupture. The optimal extent of
the removal of collagen and the extent of the decrease in
cross-linking in chronic studies needs to be investigated.
Repair of the abnormal cardiac structure in hypertension
by selective regression of the increased extracellular matrix
provides an exciting possibility to return cardiac function
towards normal. Previous studies have usually demonstrated
prevention of ®brosis; however, both reversal of existing
®brosis and prevention of further collagen deposition are
clinically relevant outcomes. Since both pirfenidone and
amiloride appear to both reverse and prevent ®brosis, they
may have considerable potential in attenuating cardiac
®brosis associated with chronic hypertension and also the
functional impairment of the heart in hypertensive humans.
These anti®brotic actions may help prevent the progression of
hypertensive heart disease towards heart failure with its well-
known poor prognosis.
Amiloride prevented cardiac ®brosis in
a model of
mineralocorticoid excess (Campbell et al., 1993) similar to
our model but the current study is the ®rst demonstration of
reversal of ®brosis with amiloride together with a normal-
ization of cardiac sti€ness. Amiloride is well-known as a
sodium channel inhibitor with potassium-sparing properties.
In electrolyte-steroid cardiopathy, amiloride protected the
myocardium from the necrotizing e€ects of ¯udrocortisone
(Selye, 1968). This prevention of cell death by maintenance of
myocardial potassium concentrations is a possible mechanism
for prevention of scarring by amiloride but does not explain
the reversal of ®brosis.
Collagen synthesis, mostly in the ®broblasts, is activated by
both cell surface receptors, especially for angiotensin II
(Weber et al., 1993), and nuclear receptors such as the
mineralocorticoid receptors activated by aldosterone (Young
et al., 1995; Brilla et al., 1994) which may partially explain
the responses to the renin-angiotensin system inhibitors.
Mature collagens are degraded by matrix metalloproteinases
(MMPs), especially MMP-1 and MMP-8 (collagenases);
tissue inhibitors of metalloproteinases (TIMPs) control the
activity of the MMPs (Dollery et al., 1995). Growth factors
(for example TGFb and bradykinin) modulate both the
synthesis and degradation of collagens and the proliferation
of ®broblasts (Weber et al., 1994). This level of complexity in
the control of cardiac extracellular matrix proteins provides
several points of attack for pharmacological therapy to
This study was supported by The University of Queensland.
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(Received May 22, 2001
Revised November 15, 2001
Accepted November 29, 2001)
British Journal of Pharmacology vol 135 (4)