Doxorubicine-Congestive Heart Failure-Increased Big Endothelin-1 Plasma Concentration: Reversal by Amlodipine, Losartan, and Gastric Pentadecapeptide BPC157 in Rat and Mouse

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 microg, 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 microg/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.

Full text

19
Journal of Pharmacological Sciences
©2004 The Japanese Pharmacological Society
Full Paper
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
Martina Lovric-Bencic
1
, Predrag Sikiric
1,
*, Jadranka S. Hanzevacki
1
, Sven Seiwerth
1
, Dunja Rogic
1
,
Vesna Kusec
1
, Gorana Aralica
1
, Pasko Konjevoda
1
, Lovorka Batelja
1
, and Alenka B. Blagaic
1
1
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,
amlodipine, losartan
Introduction
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
E-mail: sikiric@mef.hr

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
G
-nitro-L-arginine methylester
(
L-NAME) blood pressure increase and reverses already
established
L-NAME hypertension, as well as blood
pressure decrease induced by
L-arginine application in
rats. Similarly, it also inhibits lesions of various organs
induced by
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
2+
-
channel blocker, and losartan, non-peptide antagonist of
angiotensin 1 (AT1) receptors, were also used for further
comparison (42 – 54).
Materials and Methods
Animals
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
Committee.
Drugs
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
commercially purchased.
Experimental protocol
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
Biochemical analysis
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.
Histopathologic study
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).
Statistycal analyses
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.
Results
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)
rats mice
Four weeks following doxorubin regimen before
medication (Control-1)
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
#,
*2.51± 0.23
#
BPC157 (10 mg/kg, i.g.) (0.1 m g/ml, p.o.) 0.68 ± 0.13
#,
*0.54± 0.17
#,
*
Losartan (700 mg/kg, i.g.) (7 m g/ml, p.o.) 3.04 ± 0.21
#,
*1.67± 0.23
#,
*
Amlodipine (70 mg/kg, i.g.) (70 ng/ml, p.o.) 0.24 ± 0.043
#,
*0.51± 0.13
#,
*
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-
1,
#
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).
Discussion
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
doxorubin regimen
(Control-1)
173.7
±
14
230.28
±
21.13
78.2
±
13.3
63.71
±
8.7
2559.1
±
125.3
2290.42
±
486.3
2047.2
±
101.6
248
±
36.2
202.3
±
11.3
63
±
7.72
10
±
1.6%
26
±
5.4%
Medication for 2 weeks
BPC157
(10 ng
/
kg, i.g.)
(0.1 mg
/
ml, p.o.)
75.5
±
7.56
#
63.28
±
15.7
#,
*
48.6
±
5.1
#
35.57
±
3.61
#,
*
2231
±
971
338
±
21.01
936.1
±
210.9
#,
*
77.85
±
7.67
#,
*
235.4
±
68.4
77.85
±
7.67
#,
*
25
±
3.5%
#,
*
7.3
±
3.5%
#,
*
BPC157
(10
m
g
/
kg, i.g.)
(0.1
m
g
/
ml, p.o.)
294.8
±
103.3
80.7
±
1.91
#,
*
86
±
16.1
38.57
±
3.43
#,
*
2702.5
±
249.6
468
±
49.8
#,
*
2766.1
±
393.9
124.85
±
7.87
#,
*
274.8
±
60.1
124.85
±
7.87
#,
*
9.6
±
1.9%
12.3
±
5.5%
#
Losartan
(700
m
g
/
kg, i.g.)
(7
m
g
/
ml, p.o.)
251.5
±
34.1*
122.42
±
13.37
74.1
±
10.9
31.57
±
2.92
#,
*
2529.1
±
151.3
1691.85
±
98.6*
1652
±
285.5
762.28
±
170.2
#
194.8
±
34
762.28
±
170.2
12.2
±
3.7%
12.8
±
3.1%
#
Amlodipine
(70
m
g
/
kg, i.g.)
(70 ng
/
ml, p.o.)
93.6
±
6.78
#
55.85
±
2.89
#,
*
39.4
±
4.1
#,
*
21.71
±
1.52
#,
*
1686.8
±
125.8*
473.85
±
55.42
#,
*
996.8
±
57.7
#,
*
61.42
±
11.1
#,
*
127.3
±
9.27
#,
*
61.42
±
11.1
#,
*
13.4
±
2.3%
#,
*
8.3
±
1.0%
#,
*
Control-2
150.0
±
19.4
218.16
±
16.08
55.4
±
4.64
82
±
2.84
2657.77
±
270.7
2656.83
±
461
2035.0
±
94.2
808.16
±
56.4
#
204.5
±
34.46
808.16
±
56.4
#
10.2
±
6.3%
15.3
±
5.5%
#
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
BPC157
(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*
BPC157
(10 mg / kg, i.g.)
(0.1
m
g
/
ml, p.o.)
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*
Losartan
(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
#
Amlodipine
(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
dosage, Ca
2+
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
L-NAME-hyper-
tension, along with ulcerogenesis antagonization.
Similarly, it also inhibits lesions of various organs
induced by
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.
References
1 Cannan CR, Burnett JC, Lerman A. Enhanced coronary vaso-
constriction to endothelin B receptor activation in experimental
chronic heart failure. Circulation. 1996;93:646–651.
2 Cannan CR, Verghese M, Lerman A. New insight into coronary
endothelial dysfunction. Role of endotehlin. J Lab Clin Med.
1998;131:300–305.
3 Kiowski W, Sutsch G, Hunziker P, et al. Evidence for endo-
thelin-1 mediated vasoconstriction in severe chronic heart
failure. Lancet. 1995;346:732–736.
4 Berger R, Strecker K, Huelsmann M et al. Prognostic power of
neurohumoral parameters in chronic heart failure depends on
clinical stage and observation period. J Heart Lung Transpl.
2003;22:1037–1045.
5 Mulder P, Richard V, Derumeaux G, et al. Role of endogenous
endothelin in chronic heart failure. Circulation. 1997;96:1976–
1982.
6 Nakamura M, Saito S, Yoshida H, Arakawa N, Sugawara S,
Hiramori K. Effects of chronic subdepressor dose of
angiotensin II type 1 receptor antagonist on endothelium-depen-
dent vasodilatation in patients with congestive heart failure.
J Cardiovasc Pharmacol. 2002;40:411–419.
7 Pacher R, Bergler-Klein J, Globits S, et al. Plasma big endo-
thelin-1 concentrations in congestive heart failure compared to
systemic hypertension. Am J Cardiol. 1993;71:1293–1299.
8 Trochu JN, Bouhour JB, Kaley G, Hintze TH. Role of endo-
thelium-derived nitric oxide in the regulation of cardiac oxygen
metabolism: implications in health and disease. Circ Res.
2000;87:1108–1117.
9 Vasku A, Spinarova L, Goldbergova M, et al. The double
heterozygote of two endothelin-1 gene polymorphisms (G8002
and -3A/-4A) is related to big endothelin levels in chronic
heart failure. Exp Mol Pathol. 2002;73:230–233.
10 Yamauchi Y, Kurosaki E, Azuma H. Pathophysiological role of
endothelin-1 in the vascular remodeling process. Folia Pharma-
col Jpn (Nippon Yakurigaku Zasshi). 2002;120 Suppl 1:79P–
91P. (in Japanese)
11 Yoshizumi M, Kagami S, Suzaki Y, et al. Effect of endothelin-
1(1 – 31) on human mesangial cell proliferation. Jpn J Pharma-
col. 2000;84:146–155.
12 Lavallee M, Takamura M, Parent R, Thorin E. Crosstalk
between endothelin and nitric oxide in the control of vascular
tone. Heart Fail Rev. 2001;6:265–276.
13 Ozaki K, Yamamoto T, Ishibashi T, Matsubara T, Nishio M,
Aizawa Y. Regulation of endothelial nitric oxide synthase and
endothelin-1 expression by fluvastatin in human vascular
endothelial cells. Jpn J Pharmacol. 2001;85:147–154.
14 Wagner FD, Buz S, Knosalla C, Hetzer R, Hocher B. Modula-
tion of circulating endothelin-1 and big endothelin by nitric
oxide inhalation following left ventricular assist device implan-
tation. Circulation. 2003;108 Suppl 1:278–284.
15 Wittstein IS, Kass DA, Pak PH, Maughan WL, Fetics B, Hare
JM. Cardiac nitric oxide production due to angiotensin-convert-
ing enzyme inhibition decreases beta-adrenergic myocardial
contractility in patients with dilated cardiomyopathy. J Am Coll
Cardiol. 2001;38:429–435.
16 Hulsmann M, Stanek B, Frey B, et al. Exercise test, endothelin
and prognosis in heart failure. J Am Coll Cardiol. 1998;32:
1695–1700.
17 Pacher R, Stanek B, Hulsmann M, et al. Prognostic impact of
big endothelin-1 plasma concentrations compared with invasive
hemodynamic evaluation in severe heart failure. J Am Coll
Cardiol. 1996;27:633–641.
18 Wexberg P, Pacher R, Rodler S, et al. Intimal hyperplasia and
coronary flow reserve after heart transplantation: association
with big endothelin-1. J Heart Lung Transpl. 2002;21:1257–
1263.
19 Mikus D, Sikiric P, Seiwerth S, et al. Pentadecapeptide BPC 157
cream improves burn-wound healing and attenuates burn-gastric
lesions in mice. Burns. 2001;127:817–827.
20 Sikiric P, Seiwerth S, Grabarevic Z, et al. Salutary and pro-
phylactic effect of pentadecapeptide BPC 157 on acute pancre-
atitis and concomitant gastroduodenal lesions in rats. Dig Dis
Sci. 1996;41:1518–1526.
21 Sikiric P, Seiwerth S, Grabarevic Z, et al. The influence of a
novel pentadecapeptide BPC 157 on N
G
-nitro-L-arginine methyl-
ester and
L-arginine effect on stomach mucosal integrity and
blood pressure. Eur J Pharmacol. 1997;332:23–33.
22 Prkacin I, Aralica G, Perovic D, et al. Chronic cytoprotection:
pentadecapeptide BPC 157, ranitidine and propranolol prevent,
attenuate and reverse the gastric lesions appearance in chronic
alcohol drinking rats. J Physiol (Paris). 2001;95:295–301.
23 Sebecic B, Nikolic V, Sikiric P, et al. Osteogenic effect of a
gastric pentadecapeptide BPC 157, on the healing of segmental
bone defect in rabbits. A comparison with bone marrow and
autologous cortical bone implantation. Bone. 1999:24:195–202.
24 Sikiric P, Seiwerth S, Grabarevic Z, et al. The beneficial effect
of BPC 157, a 15 aminoacid peptide BPC fragment, on gastric
and duodenal lesions induced by restraint stress, cysteamine and
96% ethanol in rats. A comparative study with H2 receptor
antagonists, dopamine promotors and gut peptides. Life Sci.
1994;54:PL63–PL68.
25 Sikiric P, Seiwerth S, Mise S, et al. Corticosteroid-impairment
of healing and gastric pentadecapeptide BPC-157 creams in
burned mice. Burns. 2003;29:323–334.
26 Sikiric P, Separovic J, Anic T, et al. The effect of pentade-
capeptide BPC 157, H2-blockers, omeprazole and sucralfate on
new vessels and new granulation tissue formation. J Physiol
(Paris). 1999;93:479–485.
27 Bosnjak ZJ, Graf BM, Sikiric P, Stowe DF. Protective effects of
newly isolated gastric peptide following hypoxic and reoxygen-
ation injury in the isolated guinea pig heart. FASEB J. 1994;
8:A12.
28 Separovic J, Sikiric P, Lovric-Bencic M, Seiwerth S, Cikes I.
Pentadecapeptide BPC-157 reduces doxorubicine cardiotoxicity
in rats. Fund Clin Pharmacol. 1999;13 Suppl 1:231.
29 Grabarevic Z, Tisljar M, D aja P, et al. The influence of nitric
oxide and BPC 157 on stress induced gizzard erosions and
development of pulmonary hypertension syndrome in chicks.
Dig Dis Sci. 1997;42:216.
30 Grabarevic Z, Tisljar M, Artukovic B, et al. The influence of
BPC 157 on nitric-oxide agonists and antagonist induced lesions
in broiler chicks. J Physiol (Paris). 1997;91:139–150.
31 Aldieri E, Bergandi L, Riganti C, Costamagna C, Bosia A,
Ghigo D. Doxorubicin induces an increase of nitric oxide

BPC157 Reverses Raised CHF-Endothelin 25
synthesis in rat cardiac cells that is inhibited by iron supple-
mentation. Toxicol Appl Pharmacol. 2002;185:85–90.
32 Jung ID, Lee JS, Yun SY, et al. Doxorubicin inhibits the produc-
tion of nitric oxide by colorectal cancer cells. Arch Pharm Res.
2002:25:691–696.
33 Konjevoda P, Nasic M, Curkovic T, Sikric P, Seiwerth S,
Stambuk N. Effects of BPC 157 on the healing of corneal
lesions. In: Ohno S, Aoki K, Usui M, Uchio E, editors. Uveitis
today. Amsterdam/Lausanne / New York / Oxford/Shannon/Sin-
gapore/Tokyo: Elsevier; 1998. p. 311–314.
34 Seiwerth S, Sikiric P, Grabarevic Z, et al. BPC 157 effect on
healing. J Physiol (Paris). 1997;91:173–178.
35 Zoricic I, Sikiric P, Seiwerth S, et al. Pentadecapeptide BPC 157
beneficially influences the healing of colon-colon anastomosis
in rats. In: Mozsik G, Nagy L, Par A, Rainsford KD, editors.
Cell injury and protection in the gastrointestinal tract. From
basic sciences to clinical perspectives 1996. Dodrecht/Boston
/London: Kluwer Academic Publishers; 1997. p. 249–258.
36 Veljaca M, Chan K, Guglietta A. Digestion of h-EGF, h-TGF
alpha, and BPC-15 in human gastric juice. Gastroenterology.
1995;108:761.
37 Veljaca M, Krnic Z, Brajsa K, et al. The development of
PL 14736 for treatment of inflammatory bowel disease. Proceed-
ings of IUPHAR-GI Section Symposium, Honolulu, Hawaii,
July 13 – 15, 2002; O-32.
38 Aspenberg P, Forslund C. Enhanced tendon healing with GDF 5
and 6. Acta Orthop Scand. 1999;70:51–54.
39 Franklin TJ, Gregory H, Morri SW. Acceleration of wound
healing by recombinant human urogastrone (epidermal growth
factor). J Lab. 1986;108:103–108.
40 Kreuzer T, Grube R, Zarkovic N, Schaur RJ. 4-Hydroxynonenal
modifies the effects of serum growth factors on the expression
of the c-fos proto-oncogene and the proliferation of HeLa
carcinoma cells. Free Radic Biol Med. 1999;25:42–49.
41 Staresinic M, Sebecic B, Patrlj L, et al. Gastric pentadecapeptide
BPC 157 accelerates healing of transected rat Achilles tendon
and in vitro stimulates tendocytes growth. J Orth Res. 2003;
21:976–983.
42 Crozier I, Ikram H, Awan N, et al. Losartan in heart failure:
hemodynamic effects and tolerability. Circulation. 1995;91:691–
697.
43 Dahlof B, Devereux RB, Kjeldsen SE, et al. Cardiovascular
morbidity and mortality in the losartan intervention for endpoint
reduction in hypertension study (LIFE): a randomized trial
against atenolol. Lancet. 2002;359:995–1003.
44 Donmez G, Derici U, Erbas D, et al. The effects of losartan and
enalapril therapies on the levels of nitric oxide, malondialde-
hyde, and glutathione in patients with essential hypertension. Jpn
J Physiol. 2002;52:435–440.
45 Elkayam U. Calcium channel blockers in heart failure.
Cardiology. 1998;89 Suppl 1:38–46.
46 Gheorghiade M, Cody RJ, Francis GS, McKenna WJ, Young JB,
Bonow RO. Current medical therapy for advanced heart failure.
Am Heart J. 1998;35:S231–S248.
47 Iliceto S. Left ventricular dysfunction: which role for calcium
antagonists? Eur Heart J. 1997;18 Suppl A:A87–A91.
48 Kobayashi N, Yanaka H, Tojo A, Kobayashi K, Matsuoka H.
Effects of amlodipine on nitric oxide synthase mRNA expres-
sion and coronary microcirculation in prolonged nitric oxide
blockade-induced hypertensive rats. J Cardiovasc Pharmacol.
1999;34:173–181.
49 Martinez Y, Martinez S, Meaney A, Meaney E, Escalante B.
Angiotensin II type 1 receptor blockade restores nitric oxide-
dependent renalvascular rasponses in renovascular hypertension.
J Cardiovasc Pharmacol. 2002;40:381–387.
50 Mital S, Loke KE, Slater JP, Addonizio L, Gersony WM, Hintze
TH. Synergy of amlodipine and angiotensin-converting enzyme
inhibitors in regulationg myocardial oxygen consumption in
normal canine and failing human hearts. Am J Cardiol. 1999;83
(17A):92H–98H.
51 Nikolaidis LA, Doverspike A, Huerbin R, Hentosz T, Shannon
RP. Angiotensin-converting enzyme inhibitors improve coro-
nary flow reserve in dilated cardiomyopathy by bradykinin-
mediated, nitric oxide-dependent mechanism. Circulation. 2002;
105:2785–2790.
52 Pitt B. Evaluation of Losartan in the Elderly (ELITE) Trial:
clinical implications. Eur Heart J. 1997;18:1197–1199.
53 Pitt B, Segal R, Martinez FA, et al. Randomized trial of losartan
versus captopril in patients over 65 with heart failure. (Evalua-
tion of Losartan in the Elderly Study, ELITE). Lancet.
1997;349:747–752.
54 Wang WZ, Matsumori A, Yamada T, et al. Beneficial effects
of amlodipine in a murine model of congestive heart failure
induced by viral myocarditis. Circulation. 1997;95:245–251.
55 Kawasaki N, Lee JD, Shimizu H, Ueda T. Long term 1-carnitine
treatment prolongs the survival in rats with adriamycin-induced
heart failure. J Cardiac Failure. 1996;2:293–299.
56 Ganz WI, Sridhar KS, Ganz SS, Gonzales R, Chakko S, Serafini
A. Review of tests for monitoring doxorubicine-induced
cardiomyopathy. Oncology. 1996;53:461–470.
57 Loffler BM, Guillarmod HJ, Marie JP. Concentrations and ratios
of immunoreactive big-endothelin-1 and endothelin-1 in human,
rat and rabbit plasma. Biochem Int. 1992;27:755–761.
58 Lundblad R, Giercksky KE. Endothelin concentrations in
experimental sepsis: profiles of big endothelin and endothelin
1 – 21 in lethal peritonitis in rats. Eur J Surg. 1995;161:9–16.
59 Kaplan MM. Liver dysfunction secondary to congestive heart
failure. Practical Cardiol. 1980;6:39.
60 Kubo SH, Walter BA, John DA, Clark M, Cody RJ. Liver
function abnormalities in chronic heart failure: influence of
systemic hemodynamics. Arch Intern Med. 1987;147:1227.
61 Zhang X, Hintze TH. Amlodipine releases nitric oxide from
canine coronary microvessels: an unexpected mechanism of
action of a calcium channel-blocking agent. Circulation. 1998;
97:576–580.
62 Zhang X, Kichuk MR, Mital S, et al. Amlodipine promotes
kinin-mediated nitric oxide production in coronary micro-
vessels of failing human hearts. Am J Cardiol. 1999;84 (4A):
27L–33L.
63 Zhang X, Recchia FA, Bernstein R, Xu X, Nasjletti A, Hintze
TH. Kinin-mediated coronary nitric oxide production contributes
to the therapeutic action of angiotensin-converting enzyme and
neutral endopeptidase inhibitors and amlodipine in the treatment
in heart failure. J Pharmacol Exp Ther. 1999;288:742–751.
64 Zhang X, Xu X, Nasjletti A, Hintze TH. Amlodipine enhances
NO production induced by an ACE inhibitor through a kinin-
mediated mechanism in canine coronary microvessels. J
Cardiovasc Pharmacol. 2000;35:195–202.
65 Zarkovic N, Schaur RJ, Puhl H, Jurin M, Esterbauer H. Mutual
dependence of growth modifying effects of 4-hydroxy-nonenal

M Lovric-Bencic et al26
and fetal calf serum in vitro. Free Radic Biol Med. 1994;16:877–
884.
66 Zarkovic N, Zarkovic K, Schaur RJ, et al. 4-Hydroxynonenal as
a second messenger of free radicals and growth modifying
factor. Life Sci. 1999;65:1901–1904.
67 Costa LE, La-Padula P, Lores-Arnaiz S, et al. Long-term
angiotensin II inhibition increases mitochondrial nitric oxide
synthase and not antioxidant enzyme activities in rat heart. J
Hypertens. 2002;20:2487–2494.
68 Kalinowski L, Matys T, Chabielska E, Buczko W, Malinski
T. Angiotensin II AT 1 receptor antagonists inhibit platelet
adhesion and aggregation by nitric oxide release. Hypertension.
2002;40:521–527.
69 Loke KE, Messina EJ, Shesely EG, Kaley G, Hintze TH.
Potential role of eNOS in the therapeutic control of myocardial
oxygen consumption by ACE inhibitors and amlodipine. Cardio-
vasc Res. 2001;49:86–93.
70 Okamura K, Jin D, Takai S, Miyazaki M. Beneficial effects of
angiotensin-converting enzyme inhibition in adramycin-induced
cardiomyopathy in hamsters. Jpn J Pharmacol. 2002;88:183–
188.
71 Mohler ER 3rd, Sorensen LC, Ghali JK, et al. Role of cytokines
in the mechanism of action of amlodipine: The PRAISE Heart
Failure Trial. J Am Coll Cardiol. 1997;30:35–41.
72 Packer M, O Connor CM, Ghali JK, et al. Effect of amlodipine
on morbidity and mortality in severe chronic heart failure.
N Engl J Med. 1996;335:1107–1114.
73 Pousset F, Isnard R, Lechat P, et al. Prognostic value of plasma
endothelin-1 in patients with chronic heart failure. Eur Heart J.
1997;18:254–258.