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Journal of Pharmacological Sciences
©2004 The Japanese Pharmacological Society
J Pharmacol Sci 95, 19 – 26 (2004)
Doxorubicine-Congestive Heart Failure-Increased Big Endothelin-1
Plasma Concentration: Reversal by Amlodipine, Losartan, and Gastric
Pentadecapeptide BPC157 in Rat and Mouse
, Predrag Sikiric
*, Jadranka S. Hanzevacki
, Sven Seiwerth
, Dunja Rogic
, Gorana Aralica
, Pasko Konjevoda
, Lovorka Batelja
, and Alenka B. Blagaic
Department of Pharmacology, Medical Faculty, University of Zagreb, Zagreb, Croatia
Received November 14, 2003; Accepted February 16, 2004
Abstract. Overall, doxorubicine-congestive heart failure (CHF) (male Wistar rats and NMRI
mice; 6 challenges with doxorubicine (2.5 mg/kg, i.p.) throughout 15 days and then a 4-week-
rest period) is consistently deteriorating throughout next 14 days, if not reversed or ameliorated
by therapy (/kg per day): a stable gastric pentadecapeptide BPC157 (GEPPPGKPADDAGLV,
MW 1419, promisingly studied for inflammatory bowel disease (Pliva; PL 10, PLD-116,
PL 14736)) (10 m g, 10 ng), losartan (0.7 mg), amlodipine (0.07 mg), given intragastrically (i.g.)
(once daily, rats) or in drinking water (mice). Assessed were big endothelin-1 (BET-1) and
plasma enzyme levels (CK, MBCK, LDH, AST, ALT) before and after 14 days of therapy and
clinical status (hypotension, increased heart rate and respiratory rate, and ascites) every 2 days.
Controls (distilled water (5 ml /kg, i.g., once daily) or drinking water (2 ml / mouse per day) given
throughout 14 days) exhibited additionally increased BET-1 and aggravated clinical status, while
enzyme values maintained their initial increase. BPC157 (10 m g/kg) and amlodipine treatment
reversed the increased BET-1 (rats, mice), AST, ALT, CK (rats, mice), and LDH (mice) values.
BPC157 (10 ng/kg) and losartan opposed further increase of BET-1 (rats, mice). Losartan
reduces AST, ALT, CK, and LDH serum values. BPC157 (10 ng/kg) reduces AST and ALT
serum values. Clinical status of CHF-rats and -mice is accordingly improved by the BPC157
regimens and amlodipine.
Keywords: pentadecapeptide BPC157, big endothelin-1, doxorubicine-congestive heart failure,
Endothelin-1 (ET-1), a 21-amino acid peptide, is
the predominant isoform of the endothelin family. ET-1
is ubiquitously expressed as a potent vasoconstrictor
and promotor of cell proliferation. Most cardiovascular
diseases, such as arterial hypertension, atherosclerosis,
restenosis, congestive heart failure (CHF), idiopathic
cardiomyopathy, pulmonary hypertension, and renal
failure, are associated with local activation of the
endothelin system (1 – 3). ET-1 is importantly involved
in the functional and structural changes in the cardio-
vascular system (4 – 12), and its relationship with nitric
oxide (NO)-system dysfunction is well recognized (12 –
15). Measuring big endothelin-1 (BET-1) plasma levels,
the biologically less active precursor of ET-1 could
suitably assess both the severity of CHF and the effects
of therapy as well as the rate of ET-1 synthesis (4, 7,
16 – 18). Namely, while the stimulated endothelin
system has gained special prognostic interest, the value
of measuring plasma BET levels in patients with
advanced disease left ventricular ejection fraction
(LVEF) <20% is well established. Therefore, BET-1
assessment will be used for determination of doxorubi-
cine-CHF and estimation of subsequent deterioration
(4, 7, 17, 18).
To this end, the reversal of doxorubicine-induced
CHF, still presenting a potential therapeutic problem,
*Corresponding author. FAX: +385-1-4920-050
M Lovric-Bencic et al20
was studied in advanced status of failing heart in rats
and mice by BET-1 plasma level. The focus was the
stable gastric pentadecapeptide BPC157 (GEPPPGK
PADDAGLV, M.W. 1419), currently in clinical trials
for inflammatory bowel disease (PL 10, PLD-116,
PL 14736; Pliva, Zagreb, Croatia). Initially, as a gastric
pentadecapeptide, BPC157 opposes a variety of gastro-
intestinal lesions (19 – 21). Later, it aids in healing of
different wounds (19 – 26). Also, it has an angiogenic
effect (26) and modulates NO-synthesis (21). Besides, it
reduces duration of arrhythmias during hypoxia and
reoxygenation in isolated guinea pig hearts (27). Given
before or simultaneously with doxorubicine, it markedly
reduces doxorubicine-induced cardiotoxicity (28). Like-
wise, it also inhibits N
L-NAME) blood pressure increase and reverses already
L-NAME hypertension, as well as blood
pressure decrease induced by
L-arginine application in
rats. Similarly, it also inhibits lesions of various organs
L-NAME application in birds, unlike L-
arginine (29, 30). Furthermore, BPC157 induces and
modulates NO-release from stomach slides in vitro, an
effect resistant to blockade with
L-NAME (21). Even
though endothelium-derived NO is considered to be
primarily an important determinant of vascular tone
and platelet activity, the modulation of myocardial
metabolism by NO may be one of its most important
roles critical for the regulation of tissue metabolism.
A decrease in NO production is involved in the patho-
physiological modifications that occur in heart failure
and diabetes, disease states associated with altered
cardiac metabolism that contributes to the evolution
of the disease process. In contrast, several drugs (e.g.,
angiotensin-converting enzyme inhibitors, amlodipine,
and statins) can restore or maintain endogenous
production of NO by endothelial cells, and this mecha-
nism may explain part of their therapeutic efficiency (8).
Conversely, doxorubicine, with potent cardiotoxicity,
may variably affect NO-production in vitro (31, 32).
Therefore, considering the high ET-1 values as an
implication of NO-disarrangement, which likely occurs
in doxorubicine-induced CHF, we hypothetized BPC157
interference with ET-1 production in rats with doxorubi-
cine-induced CHF (21, 29, 30). High stability (19 – 25,
33 – 35) (i.e., no degradation in human gastric juice even
for 24 h, unlike rapidly degraded human transforming
growth factor (h-TGF), and human epidermal growth
factor (h-EGF)) (36, 37) can be used without any carrier
(19 – 25, 33 – 35) and can be given by intragastrical
(i.g.) or peroral (p.o.) application in drinking water.
Together, this means that unlike limited delivery of
other peptides (38 – 41), this stable pentadecapeptide
is highly resistant to otherwise inescapable degradation
of peptides. Likewise, acting alone, without carrier, it
presents a healing potential of its own and may be
suitable for therapy of complex disturbances such as
CHF (19 – 25, 33 – 35).
Agents commonly used in CHF, but so far not investi-
gated in doxorubicine-induced CHF, amlodipine, a Ca
channel blocker, and losartan, non-peptide antagonist of
angiotensin 1 (AT1) receptors, were also used for further
comparison (42 – 54).
Materials and Methods
Male Wistar Albino rats (200 – 250 g) or NMRI mice
(20 – 25 g), randomly assigned, were used for all of the
experiments, which were approved by Local Ethic
Pentadecapeptide BPC157 (Diagen, d.o.o., Ljubljana,
Slovenia) is a partial sequence of human gastric juice
protein BPC, freely soluble in water at pH 7.0 and in
saline. It was prepared as described before (19 – 25,
33 – 35). Peptide with 99% (HPLC) purity (1-des-Gly
peptide as impurity), dissolved in saline, was used in all
of the experiments (19 – 25, 33 – 35). Doxorubicine
(Adriablastina RD; Farmitalia, Milan, Italy); amlodipine
(Amlopin; Lek, Ljubljana, Slovenia); and losartan
(Cozaar; MSD, Whitehouse Station, NJ, USA) were
For CHF, a procedure previously described by
Kawasaki and coworkers (55) regularly producing CHF
was followed. Doxorubicine regimen (total dose of
15 mg/kg, given intraperitoneally (i.p.) at six time
points, every 3rd day during 14 days) with 4 weeks of
rest was used. The control animals were sacrificed to
establish the initial values in CHF-animals. Thereafter,
all the animals were randomly assigned for subsequent
protocols for the next 14 days.
Thereafter, for the next subsequent 14 days, the
animals received medication: losartan (0.7 mg/kg),
amlodipine (0.07 mg/kg), or BPC157 (10 ng/kg and
10 m g/kg) given i.g. once daily (rats) or in drinking
water (mice, 2 ml/mouse daily) with following concen-
trations: losartan (7 m g/ml), amlodipine (70 ng/ml),
BPC157 (0.1 ng/ml and 0.1 m g/ml), while controls
received an equivolume of distilled water (rats, 5.0 ml
/kg, i.g.) or drinking water (mice). The animals were
sacrificed at 24 h after the end of the therapy period.
BPC157 Reverses Raised CHF-Endothelin 21
BET-1 was measured in plasma by ELISA (Bio-
medica, Graz, Austria) with cross reactivity to big ET-2
and big ET-3, ET-1, 2, 3, and mouse ET-2 less than
0.1%. Serum enzymes (aspartate aminotransferase,
AST; alanin aminotransferase, ALT; creatine phospho-
kinase CK; myocardial fraction, MBCK; and lactatate
dehydrogenase, LDH) were assessed using autoanalyzer
AU-800 (Olympus, Tokyo).
Assesment of clinical parameters
Clinical signs of CHF (tail systolic blood pressure
(rats), respiratory rate (rats, mice), heart rate (rats)) were
monitored before therapy initiation and thereafter, every
2 days. In mice, only respiration rate was determined.
In rats, tail systolic blood pressure and pulse were
measured by blood pressure recorder Technical Science
Equipment 8002 (TSE, GmbH, Bazel, Switzerland),
and respiration rate was assessed. In generally, the basal
data correlated with the normal values.
For histopathologic study, organs (heart, liver, lungs,
and kidneys) were flushed with 10% buffered formaline,
fixed for 48 h, and cross dissected. After routine pro-
cessing and hematoxylin and eosin (HE) staining, the
analysis was performed on an optical microscope, using
a 40´ objective (Leitz Daplan, Wetzlar, Germany).
Statistical analysis was performed using non-para-
metric Kruskal-Wallis ANOVA and subsequent Mann-
Whitney U-test to compare groups. Values of P<0.05
and less were considered statistically significant. Also,
Spearman’s correlation test was used.
At presentation at 4 weeks following doxorubin
regimen, all rats and mice have elevated BET-1
(Table 1) and plasma enzyme values (Table 2) with
pronounced CHF (edema, dyspnea, acrocyanosis, but
still no increased heart rate and respiratory rate and
still normotensive) (Table 3). Next 2 weeks present a
worsening: further increase of BET-1 plasma values
parallel with a downhill course of CHF, with statistically
correlations: hypotension (rats, r =-0.46), dyspnea with
increased heart rate (rats, r = 0.63), and respiratory rate
(rats, r = 0.53; mice, r = 0.65)). Meanwhile, the enzyme
values maintained initial increases until the end of the
experiments, showing no correlation with CHF clinical
deterioration (Table 2).
These observed correlations indicate the effectiveness
of the therapy. Some dose regimens (BPC157-ng
(mice), losartan (mice, rats)) counteract the further
increase of BET-1 plasma values, which is otherwise
inevitable in control mice and rats. Moreover, others
(BPC157-m g (mice, rats), BPC157-ng (rats), amlodipine
(mice, rats)) even lead to reversal of the previously
increased values. These could be further seen in distinc-
tively reversed clinical deterioration by tested agents,
but the improvement seems to be at the best expressed
in the BPC157-treated animals. Although losartan and
amlodipine as antihypertensive agents did not induce
further hypotension aggravation, hypotension and
dyspnea with increased heart rate and respiratory rate
persist in these compounds-treated animals. Contrary,
hypotension was absent in BPC157-treated animals.
Likewise, dyspnea with increased heart rate and
respiratory rate were fully counteracted (Table 3).
Clinical outcome does not correlate with the course
Table 1. Elevated BET-1 plasma values at presentation at 4 weeks following doxorubin regimen in all rats and
mice and subsequent changes following amlodipine, losartan, and gastric pentadecapeptide BPC157 (daily dose)
given intragastrically (once daily, rats) or in drinking water (2 ml /mouse per day) at the end of 2-week medication
Big endothelin-1 plasma values (fmol/ml)
Four weeks following doxorubin regimen before
1.97 ± 0.07
2.28 ± 0.36
At the end of medication for 2 weeks
BPC157 (10 ng/kg, i.g.) (0.1 ng/ml, p.o.) 0.27 ± 0.14
BPC157 (10 mg/kg, i.g.) (0.1 m g/ml, p.o.) 0.68 ± 0.13
Losartan (700 mg/kg, i.g.) (7 m g/ml, p.o.) 3.04 ± 0.21
Amlodipine (70 mg/kg, i.g.) (70 ng/ml, p.o.) 0.24 ± 0.043
Control-2 8.04 ± 0.58 9.12 ± 0.34
Control-2 received an equivolume of distilled water (rats, 5 ml/kg, i.g.) or drinking water (mice). *P<0.05 vs control-
P<0.05 vs control-2. means ± S.D.
M Lovric-Bencic et al22
of the enzyme values, but they were markedly lowered
by the given medication, an effect more pronounced in
mice than in rats (Table 2): (in mice treated with
BPC157-m g (AST, ALT, LDH, CK), BPC157-ng (AST,
ALT, LDH), amlodipine (AST, ALT, LDH, CK), and
losartan (AST, ALT, LDH, CK); and in rats given
BPC157-m g (AST, ALT, LDH, CK) or amlodipine
(AST, ALT, CK).
At presentation at 4 weeks following doxorubicine
regimen, the elevated BET-1 plasma values correlate
with pronounced CHF (edema, dyspnea, acrocyanosis,
but still no increased heart rate and respiratory rate
and still normotensive) (56 – 58). Likewise, a sharp
additional increase of BET-1 plasma values throughout
Table 2. Elevated serum enzyme values at presentation at 4 weeks following doxorubin regimen in all rats and mice and subsequent changes
following amlodipine, losartan, and gastric pentadecapeptide BPC157 (daily dose) given intragastrically (once daily, rats) or in drinking water
(2 ml / mouse per day) at the end of 2-week medication
Serum enzyme values (IU/L)
AST ALT LDH CK MBCK % CK
rats mice rats mice rats mice rats mice rats mice rats mice
Four weeks following
Medication for 2 weeks
Control-2 received an equivolume of distilled water (rats, 5 ml/kg, i.g.) or drinking water (mice). *P<0.05 vs control-1,
P<0.05 vs control-2.
mean ± S.D.
Table 3. Clinical findings at presentation at 4 weeks following doxorubin regimen in all rats and mice and subsequent changes following
amlodipine, losartan, and gastric pentadecapeptide BPC157 (daily dose) given intragastrically (once daily, rats) or in drinking water
(2 ml / mouse per day) at the end of 2-week medication
Regimen in rats for 2 weeks intragastrically once daily
Regimen in mice for 2 weeks
in drinking water
Blood pressure (mmHg), rats Respiratory rate (breaths/min), rats Heart rate (beats/min), rats Respiratory rate (breaths/ min), mice
Before therapy After therapy Before therapy After therapy Before therapy After therapy Before therapy After therapy
(10 ng/ kg, i.g.)
(0.1 ng/ ml, p.o.)
114.28 ± 3.45 108.57 ± 4.75* 89.42 ± 7.89 91.42 ± 3.77* 400 ± 4.36 335.71 ± 14.45* 131.7 ± 8.19 134.57 ± 7.36*
(10 mg / kg, i.g.)
118.33 ± 6.61 111.11 ± 8.93* 93.55 ± 4.87 84.88 ± 7.28* 393.33 ± 3.33 312.22 ± 7.41* 128.28 ± 5.21 130.57 ± 7.45*
(700 mg / kg, i.g.)
(7 mg / ml, p.o.)
117.5 ± 4.18 101.66 ± 6.05
90.0 ± 6.06 104.33 ± 4.96
393.33 ± 4.21 360 ± 16.32 127.42 ± 7.63 142.85 ± 6.51
(70 mg / kg, i.g.)
(70 ng/ kg. p.o.)
118.5 ± 5.29 98.5 ± 4.11
92.2 ± 7.59 88.4 ± 10.1* 382.22 ± 2.22 333.33 ± 15.89* 127.42 ± 5.74 137.14 ± 3.02*
Control 119.37 ± 5.62 99.37 ± 4.95
87.25 ± 5.65 101.25 ± 5.84
388.88 ± 12.29 382.0 ± 22.0 128.66 ± 7.96 152.33 ± 9.58
Control received an equivolume of distilled water (rats, 5 ml/kg, i.g.) or drinking water (mice).
P<0.05 vs before therapy, *P<0.05 vs control.
mean ± S.D.
BPC157 Reverses Raised CHF-Endothelin 23
the two subsequent weeks correlates with the downhill
course of CHF (i.e., CHF-hypotension, dyspnea with
increased heart rate and respiratory rate). Accordingly,
along with many tests used in clinical practice to monitor
the effect of various therapeutic efforts and extracardiac
neurohormonal systems such as the sympathetic system,
the renin-angiotensin-aldosteron system, and the endo-
thelial system up-regulated in heart failure contributing
to disease progression, the stimulated endothelin system
has gained special prognostic interest, and the value of
measuring plasma BET levels in patients with advanced
disease (LVEF <20%) is well established. Therefore,
our data using BET-1 is a suitable method for determin-
ing doxorubicine-CHF and estimation of subsequent
deterioration (4, 7, 16, 17).
Meanwhile, the enzyme values maintained their
increased level until the end of the experiments, without
correlation with CHF clinical deterioration (59, 60).
Therefore, the elevated BET-1 values, and thereby
increased ET-1 synthesis rate could be clearly used for
better evaluation of worsening of doxorubicine-induced
CHF. Thus, reversal of otherwise prominently increased
BET-1 plasma values, counteraction of progressing
CHF manifestations, such as increased heart rate and
respiratory rate, hypotension, were all presented in
BPC157-treated animals. Importantly, since all together
counteracted with BPC157 regiment, these could be
indicative for a useful therapy.
Moreover, the sustainedly raised serum enzyme
values in controls were also consistently counteracted
by BPC157 regimens. Consistent with this findings are
the results obtained during hypoxia and reoxygenation
experiment-reduced arrhythmias, prolonged periods of
sinus rhythm, as well as prevention and reversal of acute
doxorubicine cardiotoxicity (27, 28).
Amlodipine and losartan have comparable effects on
BET-1 and enzyme values, but unlike normotensive-
BPC157 rats, the animals remain hypotensive like
the controls, but not more so, which is consistent with
the potential anti-hypertensive effect of (amplodipine
and losartan). Thus, presenting blockade of either
calcium channel (i.e., amlodipine), or AT1-receptors
(i.e., losartan) with BPC157, they share some other
common points. Presenting hypotension as an impli-
cation of CHF in rats, BPC157 seems to be more
successful in CHF-correction. With respect to losartan,
a more consistent lowering effect on BET-1 concentra-
tions was found in animals treated with amlodipine.
Therefore, it could be speculated that in the given
blockade seems to be more effective than
AT1 receptor blockade (61 – 64).
Although many peptides are implicated in CHF-
pathophysiology (i e., BNP, TNF-a , ANP) or therapy,
in vitro or in vivo studies, so far no peptide is reported
to be beneficial in doxorubicine-induced CHF. BPC157
is stable in human gastric juice even for 24 h (36, 37).
Importantly, no carrier was used in the previous (19 –
25, 33 – 35) and present studies. Together, this means
that unlike limited delivery of other peptides (38 – 41,
65, 66), this stable pentadecapeptide is highly resistant
to otherwise inescapable degradation of peptides.
Consistently, BPC157 is given systemically and/or
locally (19 – 25, 33 – 35) along with the positive effect
following i.g. applications. Likewise, acting alone,
without carrier (unlike other peptides), it presents a
salutary potential of its own (19 – 25, 33 – 35) and
may be suitable for therapy of a complex disturbance
such as the CHF. Besides, this gastric pentadecapeptide
likely controls functions of collagen fragments (19, 41).
Showing consistent endothelium protection and angio-
genesis promotion in healing, of special interest for its
effect in CHF may be its interaction with the NO-
system. It opposes
L-arginine NO-synthesis over-expres-
sion in gastric mucosa from rat stomach tissue homo-
genates, a finding relevant for various tissues. Interest-
ingly, BPC157 induces by itself a generation of NO
that could be not inhibited by
L-NAME (similar direct
evidence is still lacking for other growth factors (21)).
Showing NO-restoration needed for CHF-recovery,
BPC157 both prevents and reverses
tension, along with ulcerogenesis antagonization.
Similarly, it also inhibits lesions of various organs
L-NAME application in birds, unlike L-argi-
nine (29, 30). Along with reversal of hypotension in
CHF-animals, BPC157 antagonizes L-arginine-hypo-
tension (21, 29).
Since several lines of evidence indicate for NO-ET
relationship that NO impairs ET production presenting
increased BET serum values as NO-failure, the reversal
of otherwise increased BET serum values thereby likely
indicates the restored balance and NO-function. Like-
wise, increase of NO release in CHF- and hypertension-
models is fully recognized and extensively documented
for angiotensin-converting enzyme inhibitors, losartan
and other AT1 inhibitors, as well as for amlodipine
(6, 15, 42, 44, 45, 49 – 53, 61 – 64, 67 – 73). For
instance, amlodipine significantly increased nitrite pro-
duction from coronary microvessels in both normal and
failing dog hearts. Moreover, it releases NO even after
heart failure and this may be partly responsible for its
favorable effect in the treatment of heart failure (61 – 64,
71 – 73).
Thus, most likely, exerting the activity throughout
the healing process, gastric pentadecapeptide BPC157
reaches the treatment aim to induce and then maintain
healing. In addition, the toxicology studies indicate a
M Lovric-Bencic et al24
very safe profile. Therefore, these findings clearly
suggest its possible use in further therapy of CHF.
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