m.b.b. ch , m.sc. in pharmacolog ass.lecturer, dept. of...
TRANSCRIPT
Study of some toxic effects of anticancer drug, doxorubicin and the use of some protective agents against doxorubicin induced toxicity
(Expermintal study)
Thesis Submitted For Partial Fulfillment of M.D Degree in Pharmacology
Presented By Safwa Mahmoud sorour
M.B.B. Ch. (2003 ) , M.Sc. In Pharmacology ( 2010 )
Ass.lecturer, Dept. of pharmacology
Benha Faculty of Medicine
Under Supervision of Prof. Dr. Ahmed Seleem Mohamed
Professor of Pharmacology and Head of Pharmacology Department
Benha Faculty of Medicine -Benha University
Prof. Dr. Rezk Ahmed Sanad Professor of Pharmacology
Benha Faculty of Medicine - Benha University
Prof. Dr Mary El- Komus Boutros Yacoub Ass. Prof. of Pharmacology
Benha Faculty of Medicine -Benha University
Benha Faculty of Medicine
Benha University
2014
دراسخ لجعض التأثيراد السويخ لعقبر الدوكسيريىثسيي وثعض الوىاد الت تق هي تلك السويخ )دراسخ هعوليخ(
هقدهخ هي رسبلخ
الطجيجخ /صفىه هحوىد سرور
( 3002 )هبجستير الفبرهبكىلىج –( 3002ثكبلىيىس الطت والجراحخ)
هدرس هسبعد - قسن األدويخوالعالج
كليخ طت ثنهب
خ تىطئخ للحصىل علي درجخ الدكتىراه في الفبرهبكىلىجرسبل
السبدح الوشرفىى
هحود أحود سلين أ د .
أستبذ ورئيس قسن الفبرهبكىلىج
كليخ طت ثنهب
جبهعخ ثنهب
أ د. رزق أحود سند
أستبذ الفبرهبكىلىج
كليخ طت ثنهب
جبهعخ ثنهب
أ م د. هبري القوص ثطرس يعقىة
هبكىلىجهسبعد الفبر نأستب
كليخ طت ثنهب
جبهعخ ثنهب
كليخالطت
جبهعخ ثنهب
2014
Acknowledgment
Frist of all thanks to God the most kind and the most merciful.
I would like to express my gratefulness and my great respect to Prof.
Dr.Ahmed Seleem Mohamed , Professor of Pharmacology and Head of
Pharmacology Department, Benha Faculty of Medicine, Benha
University, for his moral and scientific support and for giving me the
honor of working under his supervision and valuable guidance.
Special thanks and deepest gratitude to Prof. Dr. Rezk Ahmed
Sanad, Professor of Pharmacology, Benha Faculty of Medicine, Benha
University, for his valuable comments , suggestions and for giving me a
lot of his valuable time and enormous knowledge.
I would like to express my gratitude and appreciation to Prof. Dr
Mary El- Komus Boutros Yacoub Assisstant Professor of Pharmacology,
Benha Faculty of Medicine, Benha University, for her great scientific
and moral help and for his fruitful guidance and for encouraging me
all the time for a better performance.
I would like to express my special thanks to Dr. Ghada Mohammed
lecturer of pathology, Benha Faculty of Medicine, Benha University
for her great help in the histological part , also special thanks to Prof .Dr.
Mohammed El Shorpagy professor of clinical pathology Al Azhar
University for his great help and support.
.
CONTENTS
List of contents Page
List of abbreviations
List of Tables
List of Figures
Aim of the work
Review of literature
Materials and Methods
Results
Discussion
Summary and Conclusion
Recommendations
References
Arabic Summary
I
II,III
IV,V,VI
1
2
44
58
100
118
122
123
LIST OF ABBREVIATIONS
CPK Creatine phosphokinase
ALT Alanine transaminase
AST Aspartate transaminase
AIDS Acquired immune deficiency syndrome
AC Adriamycin
CHF Congestive heart failure
LV left-ventricle
LVEF left ventricular ejection fraction
MIBG Metaiodobenzylguanidine
SGOT Serum glutamic oxaloacetic transaminase
SGPT Serum glutamic pyruvic transaminase
CYP2D6 Cytochrome P2D6
EM Extensive metabolizers
PM Poor metabolizers
ClCr Creatinine clearance
NO Nitric oxide
cGMP Cyclic granulate mono phosphate
ROS Reactive oxygen species
GFR Glomerular filtration rate
CoA Coenzyme A
CROT Carnitine octanoyl transferase
PLC Propionyl L- carnitine
CPT Carnitine palmitoyltransferase
TMAO Trimethylamine N-oxide
H-FABP Heart fatty acid binding protein
I
LIST OF TABLES
Tab. No Title Page No
(1) Changes of T wave voltage in various groups 60
(2) Changes of heart rate in various groups 62
(3) Changes of QRS voltage in various groups 64
(4) Changes of systolic blood pressure & mean blood
pressure in various groups
66
(5) Changes of CPK & troponin I level in various
groups
71
(6) Changes of ALT & AST level in various groups 77
(7) Changes of creatinine level in various groups 83
(8)
Effect of isoprenaline on the amplitude of
spontaneous rhythmic contraction of isolated
perfused rat heart in control group
88
(9)
Effect of isoprenaline on the amplitude of
spontaneous rhythmic contraction of isolated
perfused rat heart in Dox group
90
(10)
Effect of isoprenaline on the amplitude of
spontaneous rhythmic contraction of isolated
perfused rat heart in Dox+
Neb group
92
(11)
Effect of isoprenaline on the amplitude of
spontaneous rhythmic contraction of isolated
perfused rat heart in Dox+L- car group
94
II
(12) Effect of isoprenaline on the amplitude of
spontaneous rhythmic contraction of isolated
perfused rat heart in Dox + Neb +L- car group
96
(13) Percent change of contraction of isolated perfused
rat heart in different groups after isoprenaline
different doses 2,4,8 µg
98
III
LIST OF FIGURES
Fig. No Title Page No
(1)
Proposed protective mechanism by PLC against
DOX-induced cardiomyopathy.
36
(2) Inhibition of H-FABP mRNA expression in the
heart as a novel mechanism for DOX-induced
cardiotoxicity and for L-carnitine mediated
protection against this effect
37
(3) Histogram showing changes of T wave voltage in
various groups
60
(4) Histogram showing changes of heart rate in
various groups
62
(5) Histogram showing changes of QRS voltage in
various groups.
64
(6) Histogram showing Systolic blood pressure in
various groups.
67
(7) Histogram showing mean arterial blood pressure
in various groups.
67
(8) Blood pressure and ECG measurement of control
group
68
(9) Blood pressure and ECG measurement of Dox
group
68
(10) Blood pressure and ECG measurement of Dox+
Neb group
68
(11) Blood pressure and ECG measurement of Dox+
L-Car group
69
(12) Blood pressure and ECG measurement of Dox +
Neb + L-car group
69
(13) Histogram showing changes of CPK level in
various groups.
71
(14) Histogram showing changes of troponin I level in
various groups.
72
(15) Cut section of normal myocardial tissues 73
(16) Cut section of myocardium of Dox group 73
(17) Cut section of myocardium & percardium of Dox
group
74
(18) Cut section of myocardium of Dox+ Neb group 74
(19) Cut section of myocardium of Dox +L-car group 75
(20) Cut section of myocardium of Dox+Neb +L-
carnitine group
75
(21) Histogram showing changes of ALT level in
various groups.
78
(22) Histogram showing changes of AST level in
various groups.
78
(23) Photomicrograph of a cut section in the liver of a
control rat
97
(24) Photomicrograph of a cut section in the liver
of Dox group
80
(25) Photomicrograph of a cut section in the liver
in Dox+Neb group.
80
(26) A photomicrograph of a cut section in the liver
in Dox + L- car group
81
(27) Photomicrograph of a cut section in the liver in
Dox+ Neb+L- car group
81
(28) Histogram showing creatinine level in various
groups
84
V
(28)
Cut section of normal kidney tissue 85
Cut section of kidney tissue of Dox group 85
(30) Cut section of kidney tissue of Dox+Neb group 86
(31) Cut section of kidney tissue of Dox+L- car group 86
(32) Cut section of kidney of Dox+Neb+L-car group 87
(33) Histogram showing the effect of isoprenaline on
isolated rat’s heart of control group
89
(34) A record demonstrating the effect of gradually
increasing concentrations of isoprenaline on the
isolated perfused rat's heart contractions in
control group
89
(35) Histogram showing the effect of isoprenaline
on isolated rat’s heart of Dox group.
91
(36) A record demonstrating the effect of gradually
increasing concentrations of isoprenaline on the
isolated perfused rat's heart contractions in Dox
group
91
(37) Histogram showing the effect of isoprenaline on
isolated rat’s heart of Dox +Neb group
93
(38) A record demonstrating the effect of gradually
increasing concentrations of isoprenaline on the
isolated perfused rat's heart contractions in
Dox+Neb group
93
(39) Histogram showing the effect of isoprenaline on
isolated rat’s heart of Dox+ L- car group
95
(40) A record demonstrating the effect of gradually
increasing concentrations of isoprenaline on the
isolated perfused rat's heart contractions in
Dox+L-car group
95
(41) Histogram showing the effect of isoprenaline on
isolated rat’s heart of Dox + Neb +L- car group
97
(42) A record demonstrating the effect of gradually
increasing concentrations of isoprenaline on the
isolated perfused rat's heart contractions in
Dox+Neb+L-car group
97
(43) A record demonstrating the effect of gradually
increasing concentrations of doxorubicine on the
isolated perfused rabbit's heart
99
(45) A record demonstrating the effect of gradually
increasing concentrations of doxorubicin on
isoprenaline effect on the isolated perfused
rabbit's heart
100
VII
صـدق اهلل العظيـم
" ۲۳آية –"سورة البقرة
Aim of the work
1
Aim of the work
Doxorubicin is one of the potent anticancer drugs which is
commonly indicated in many tumors. This drug has many organ toxicities
especially cardiotoxicity and this toxicity is the main limitation for its use
as anticancer drug, so this study is planned to investigate the toxicity of
this drug on some major organs including heart, liver and kidney.
Many researchers have expended great efforts aiming at preventing or
decreasing the adverse effects of doxorubicin, so the aim of this study is
to investigate the possible protective role of some chemical agents like
nebivolol and some natural agents like L-carnitine against doxorubicin
organ toxicity.
This will be achieved by investigating:
In vivo study: The effects of doxorubicin alone , doxorubicin
with nebivolol , doxorubicin with L-carnitine and doxorubicin
with nebivolol&L-carnitine on:-
1- ECG and blood pressure.
2- Cardiac enzymes as troponin I and CPK.
3- Liver enzymes as ALT and AST.
4- kidney function as creatinine.
5- histopathological examination of heart , liver and kidney.
In-vitro study:
(1) The effect of isopernaline on contractility of hearts taken from
another 5 groups of rats given the same medications as the groups of the
in-vivo experiment.
(2) The effects of doxorubicin on isolated perfused rabbit’s heart.
Review of literature
2
Doxorubicin
Doxorubicin is a drug used in cancer chemotherapy. It is an
anthracycline antibiotic , it works by intercalating DNA, with the most
serious adverse effect being life-threatening cardiotoxicity. The drug is
administered intravenously, as the hydrochloride salt. It is commonly
used in the treatment of a wide range of cancers, including some
leukemias and Hodgkin's lymphoma, as well as cancers of the bladder,
breast, stomach, lung, ovaries, thyroid, soft tissue sarcoma, multiple
myeloma, and others (Takimoto and Calvo, 2008). Commonly used
doxorubicin-containing regimens are AC (Adriamycin,
cyclophosphamide), TAC (Taxotere, AC), ABVD (Adriamycin,
bleomycin, vinblastine, dacarbazine), and FAC (5-fluorouracil,
adriamycin, cyclophosphamide) (Laginha, 2005).
Physical properties:
It is orange to red powder, soluble in water, normal saline, methanol
and stable under normal conditions. It is light and moisture sensetive
(Yang et al., 2009).
Chemistry:
7-4-amino-5-hydroxy-6-methyloxan-2-oxy-6,9,11-trihydroxy-9-4-
methoxy-8,10-dihydro-7H-tetracene-5 coated by Minotti et al., 2004
Review of literature
3
Pharmacokinetics:
Pharmacokinetic studies, determined in patients with various types of
tumors undergoing either single or multi-agent therapy have shown that
doxorubicin follows a multiphasic disposition after intravenous injection.
Doxorubicin hydrochloride is not stable in gastric acid, and animal
studies indicate that the drug undergoes little, if any, absorption from the
gastrointestinal tract. The drug is extremely irritating to tissues and,
therefore, must be administered intravenously. The initial distribution
half-life of doxorubicin is approximately 5 minutes suggests rapid tissue
uptake of doxorubicin, while its slow elimination from tissues is reflected
by a terminal half-life of 20 to 48 hours (Lewis et al ., 2006).
Binding of doxorubicin and its major metabolite, doxorubicinol to
plasma proteins is about 74 to 76% and is independent of its plasma
concentration of doxorubicin. Doxorubicin is excreted in the milk of the
lactating patient, with peak milk concentration at 24 hours after treatment
being approximately 4.4-fold greater than the corresponding plasma
concentration. Doxorubicin is detectable in the milk up to 72 hours after a
15-minute intravenous infusion (Stephen et al., 1993).
Doxorubicin does not cross the blood brain barrier. Enzymatic
reduction at the 7 position and cleavage of the daunosamine sugar yields
aglycones which are accompanied by free radical formation, the local
production of which may contribute to the cardiotoxic activity of
doxorubicin. Plasma clearance of doxorubicin ranges from 324 to 809
mL/min and is predominately by metabolism and biliary excretion.
Approximately 40% of the dose appears in the bile in 5 days, while only
5 to 12% of the drug and its metabolites appear in the urine during the
same time period. In urine, <3% of the dose was recovered as
Review of literature
4
doxorubicinol over 7 days. Systemic clearance of doxorubicin is
significantly reduced in obese women. There was a significant reduction
in clearance without any change in volume of distribution in obese
patients when compared with normal patients (Minotti et al., 2004).
Mechanism of action:
The cytotoxic effect of doxorubicin on malignant cells and its toxic
effects on various organs are thought to be related to nucleotide base
intercalation and cell membrane lipid binding activities of doxorubicin.
Intercalation inhibits nucleotide replication and action of DNA and RNA
polymerases. The interaction of doxorubicin with topoisomerase II to
form DNA-cleavable complexes appears to be an important mechanism
of doxorubicin cytocidal activity (Pommier et al ., 2010). This inhibits
the progression of the enzyme topoisomerase II, which relaxes supercoils
in DNA for transcription. Doxorubicin stabilizes the topoisomerase II
complex after it has broken the DNA chain for replication, preventing the
DNA double helix from being resealed and thereby stopping the process
of replication (Tacar et al ., 2013)
Doxorubicin cellular membrane binding may affect a variety of
cellular functions. Enzymatic electron reduction of doxorubicin by a
variety of oxidases, reductases and dehydrogenases generates highly
reactive species including the hydroxyl free radical OH•. Cells treated
with doxorubicin have been shown to manifest the characteristic
morphologic changes or apoptosis . Induced Apoptosis may be an integral
component of the cellular mechanism of action relating to therapeutic
effects, toxicities or both (Shi et al ., 2011).
Review of literature
5
Clinical pharmacology of doxorubicin:
Doxorubicin has produced significant therapeutic responses in a
number of solid tumours and haematologic malignancies, and is
commonly used in the treatment of carcinoma of the breast , the lung , the
ovary , transitional cell bladder cancer, neuroblastoma ,Wilm's tumour,
soft tissue sarcomas, osteosarcoma, acute lymphocytic - lymphoblastic
leukaemia, acute myelogenous leukaemia, non-Hodgkin's lymphoma &
Hodgkin's disease. Doxorubicin has also shown antitumour activity in
some adult and paediatric malignancies as carcinoma of the thyroid,
carcinoma of the endometrium, carcinoma of the head and neck,
carcinoma of the stomach, primary hepatocellular carcinoma, carcinoma
of the testis, carcinoma of the prostate, Ewing's sarcoma,
rhabdomyosarcoma, multiple myeloma &chronic leukaemias (Yun,
2010).
Doxorubicin is a cytotoxic drug that is usually administered to cancer
patients by the intravenous and, whenever appropriate, intravesical and
intra-arterial route also may be used. In intravenous (IV) Administration,
dosage is usually calculated on the basis of body surface area
(mg/m2).The doxorubicin dose-schedule may differ depending on the
therapeutic indication (e.g. solid tumours or acute leukaemias) as well as
on its use within a specific regimen (e.g. as a single agent or in
combination with other cytotoxics or as a part of multidisciplinary
approaches which include combination with surgery and/or radiotherapy
and/or hormonotherapy). Intravenous administration of doxorubicin
should be performed with caution. It is recommended to administer
doxorubicin IV infusion within isotonic sodium chloride or 5% glucose
solution over a period of 3 to 5 minutes. This technique is intended to
Review of literature
6
minimize the risk of thrombosis or perivenous extravasation which could
lead to severe cellulitis, vesication and tissue necrosis. A direct push
injection is not recommended due to the risk of extravasation
(Schulmeister, 2007).
Treatment of solid tumours, when doxorubicin is administered as a
single agent, the recommended dose per cycle is 60-75mg/m2every three
weeks. The drug is generally given as a single dose per cycle; however, it
is possible to give the drug dosage per cycle in divided administrations
(e.g. day 1 and 3, or days 1 and 8) (Hughes, 1986).
In the management of acute leukaemias, bone marrow aplasia is a
therapeutic achievement and intensive combination chemotherapy
schedules are employed. In this situation the recommended dose of
doxorubicin is 2.4mg/kg of body weight (approximately corresponding to
75-90mg/m2), to be administered divided over three consecutive days
(one cycle). The time and dose of the second cycle should be
determineded by both the bone marrow and peripheral blood cells status.
The interval between cycles should be however at least 10 days ( St
Marie and Camp-Sorrell, 2000).
Doxorubicin has been also used by the intra-arterial route in an
attempt to produce intense local activity with reduced general toxicity.
Since this technique is potentially hazardous and can lead to widespread
necrosis of the perfused tissue, intra-arterial administration should only
be attempted by physicians fully trained with this technique. Doxorubicin
is usually administered intravenously. The solution should be injected
over 3 to 5 minutes through the tubing of a freely-running infusion of
physiological solution, after confirmation that the needle is correctly
inserted into the vein. This technique reduces the risk of thrombosis and
Review of literature
7
perivenous extravasation of the drug that can lead to severe cellulitis and
necrosis, and ensures the washing of the vein after administration.
Injection in small veins and repeated injection in the same vein can lead
to venous sclerosis (Hughes, 1986)
Drug Interactions:
Doxorubicin is extensively metabolized by the liver. Changes in
hepatic function induced by concomitant therapies may affect
doxorubicin metabolism, pharmacokinetics, therapeutic efficacy, and/or
toxicity. Toxicities associated with doxorubicin, especially hematologic
and gastrointestinal events, may be increased when doxorubicin is used in
combination with other cytotoxic drugs. There is an increase in
cardiotoxicity when doxorubicin is co-administered with paclitaxel due to
significant decrease in doxorubicin clearance with more profound
neutropenic and stomatitis episodes. When progesterone was given
intravenously to patients with advanced malignancies at high doses (up to
10 g over 24 hours) concomitantly with a fixed Doxorubicin dose (60
mg/m2) via bolus injection, enhanced doxorubicin-induced neutropenia
and thrombocytopenia were observed. The addition of cyclosporine to
doxorubicin may result in increases in area under the curve for both
doxorubicin and doxorubicinol possibly due to a decrease in clearance of
parent drug and a decrease in metabolism of doxorubicinol. Adding
cyclosporine to doxorubicin results in more profound and prolonged
hematologic toxicity than doxorubicin alone. Coma and/or seizures have
also been described (Stephen, 1993).
Necrotizing colitis manifested by typhlitis (cecal inflammation),
bloody stools, and severe & sometimes fatal infections have been
associated when a combination of cetarabine and doxorubicin are given.
Review of literature
8
The addition of cyclophosphamide to doxorubicin treatment does not
affect exposure to doxorubicin, but may result in an increase in exposure
to doxorubicinol. Doxorubicinol only has 5% of the cytotoxic activity of
doxorubicin. Concurrent treatment with doxorubicin has been reported to
exacerbate cyclophosphamide-induced hemorrhagic cystitis. Acute
myeloid leukemia has been reported as a second malignancy after
treatment with doxorubicin and cyclophosphamide (Chen et al., 2002).
Adverse effects:
(1) Cardiotoxicity:
The most dangerous side effect of doxorubicin is heart damage. When
the cumulative dose of doxorubicin reaches 550 mg mg/m2 , the risks of
developing cardiac side effects, including dilated cardiomyopathy, CHF,
markedly increase. Doxorubicin cardiotoxicity is characterized by a
dose-dependent decline in mitochondrial oxidative phosphorylation.
Reactive oxygen species, generated by the interaction of doxorubicin with
iron, can then damage the myocytes (heart cells), causing myofibrillar
loss and cytoplasmic vacuolization. Morphologic damage has been
quantitated by pathologists from samples obtained by myocardial biopsy.
The cellular lesions are known as "myofibrillar dropout". Myofibrillar
dropout consists of swelling of the sarcoplasmic reticulum and, with more
advanced stages of damage, complete loss of myofibrils (Shi et al .,
2011).
Anthracycline-induced cardiotoxicity may be manifested by acute or
chronic event.
Acute doxorubicin cardiotoxicity , occurs during and within 2–3
days of its administration. The incidence of acute cardiotoxicity is
Review of literature
9
approximately 11% (Swain et al., 2003). The manifestations are usually
chest pain due to myopericarditis and/or palpitations due to sinus
tachycardia, paroxysmal nonsustained supraventricular tachycardia and
premature atrial and ventricular beats. The electrocardiogram may reveal
nonspecific ST-T changes, left axis deviation and decreased amplitude of
QRS complexes. The mechanisms for these acute changes are not clear
but may be due to doxorubicin-induced myocardial edema, which is
reversible . Acute left-ventricular (LV) failure is a rare manifestation of
acute cardiotoxicity, but it is also reversible with appropriate treatments
(Takemura and Fujiwara, 2007).
Chronic doxorubicin cardiotoxicity, it's incidence is much lower,
with an estimated incidence about 1.7% .It is usually evident within 30
days of administration of its last dose, but it may occur even after 6–10
years after its administration. It is manifested by a reduction in LVEF
(left ventricular ejection fraction) and/or signs and symptoms of
congestive heart failure (CHF) such as tachycardia, dyspnea, pulmonary
edema, dependent edema, cardiomegaly and hepatomegaly, oliguria,
ascites, pleural effusion, and gallop rhythm. Subacute effects such as
pericarditis/myocarditis have also been reported.Life-threatening CHF is
the most severe form of anthracycline-induced cardiomyopathy and
represents the cumulative dose-limiting toxicity of the drug (Kilickap et
al., 2007).
Age also influences the risk of developing doxorubicin
cardiomyopathy. Very young and very old individuals are more prone to
develop this complication. A history of cardiovascular disease such as
hypertension and reduced LV ejection fraction before therapy is also a
risk factor to develop this complication. The prognosis of patients who
Review of literature
10
develop congestive heart failure is poor (50% mortality in 1 year)
(Broder et al., 2008).
The histopathological changes in doxorubicin cardiomyopathy are in
the form of areas of patchy myocardial interstitial fibrosis and scattered
vacuolated cardiomyocytes (Adria cells). Adria cells are seen adjacent to
areas of fibrosis. The areas of fibrosis are usually widespread and areas of
acute myocyte damage are infrequent. There is fibroblast proliferation
and histiocyte infiltration in the areas of healed myocarditis. Partial or
total loss of myofibrils and myocyte vacuolar degeneration are essential
features of doxorubicin cardiotoxicity . With loss of myofilaments, the
remnants of Z discs are seen. There is distention of sarcoplasmic
reticulum and the T-tubules. The myocyte vacuoles coalesce and form
large membrane-bound spaces. The nucleus-chromatin disorganization
and replacement of chromatin by pale filaments are also features of
doxorubicin cardiomyopathy (Takemura and Fujiwara , 2007).
Cardiotoxicity may occur at lower doses in patients with prior
mediastinal/pericardial irradiation, concomitant use of other cardiotoxic
drugs, doxorubicin exposure at an early age, advanced age and pre-
existing heart disease is a cofactor for increased risk of doxorubicin
cardiotoxicity. In such cases, cardiac toxicity may occur at doses lower
than the recommended cumulative dose of doxorubicin. Studies have
suggested that concomitant administration of doxorubicin and calcium
channel blockers or cardiotoxic drugs, especially those with long half-
lives, e.g., trastuzumab) may increase the risk of doxorubicin
cardiotoxicity (Chatterjee et al.,2010).
The total dose of doxorubicin administered to the individual patient
should also take into account previous or concomitant therapy with
Review of literature
11
related compounds such as daunorubicin, idarubicin, and mitoxantrone.
Although not formally tested, it is probable that the toxicity of
doxorubicin and other anthracyclines or anthracenediones is additive.
Cardiomyopathy and/or congestive heart failure may be encountered
several months or years after discontinuation of doxorubicin therapy(
Simunek et al ., 2009).
The risk of acute manifestations of doxorubicin cardiotoxicity in
pediatric patients may be as that in adults. Pediatric patients appear to be
at particular risk for developing delayed cardiac toxicity as doxorubicin-
induced cardiomyopathy impairs myocardial growth in pediatric patients
and with advance in age congestive heart failure may develop in early
adulthood. As many as 40% of pediatric patients may have subclinical
cardiac dysfunction and 5 to 10% of pediatric patients may develop
congestive heart failure on long-term follow-up. This late cardiac toxicity
may be related to the dose of doxorubicin. The longer the length of
follow-up, the greater the increase in the detection rate.Treatment of
doxorubicin-induced congestive heart failure includes the use of digitalis,
diuretics, after load reducers such as angiotensin converting enzyme
(ACE) inhibitors, low salt diet, and bed rest. Such intervention may
relieve symptoms and improve the functional status of the patient
(Ibrahim et al., 2009).
Diagnosis of doxorubicin cardiomyopathy:
The diagnosis of doxorubicin cardiomyopathy should consist of taking
appropriate history to assess the likelihood of the diagnosis. A complete
examination of the cardiovascular system to detect presence of signs of
overt heart failure, such as elevated jugular venous pressure and S3
gallop, is essential. An electrocardiogram should be obtained, which
Review of literature
12
usually demonstrates nonspecific ST-T wave changes and sometimes
low-voltage QRS complexes. A chest X-ray is also helpful to assess
cardiomegaly and signs of pulmonary venous congestion. It should be
emphasized that the presence or absence of the abnormalities by these
evaluations are nonspecific and nondiagnostic (Sawaya et al. ,2011)
Echocardiography with Doppler studies is commonly used to detect
early diastolic and systolic LV dysfunction. Exercise echocardiography
may also be useful to assess LV contractile reserve ( Tassan-Mangina et
al.,2006)
Radionuclide ventriculography has been used to assess LV systolic
and diastolic function. Like other types of cardiomyopathy, cardiac
adrenergic denervation occurs in doxorubicin cardiomyopathy. MIBG
(metaiodobenzylguanidine) nuclear imaging can be employed to assess
cardiac adrenergic denervation. Impaired glucose and fatty acid
metabolism has been observed in doxorubicin cardiomyopathy. Impaired
myocardial glucose uptake can be assessed by positron emission
tomography using fluorine-18
F-deoxyglucose (Takemura and Fujiwara ,
2007).
Antimyosin antibody study with the use of 111
In-labeled monoclonal
antimyosin antibody is used for the diagnosis of myocarditis and has also
been employed for the diagnosis of doxorubicin cardiomyopathy
(Koopman et al., 1994).
Annexin V, which has high affinity for membrane-bound
phosphatidylserine, has been used to detect apoptosis induced by
doxorubicin.The measurements of neurohormones and cardiac enzymes
have been used for the diagnosis of doxorubicin cardiotoxicity and
Review of literature
13
presence of heart failure. Plasma levels of B-type natriuretic peptide are
elevated and correlate with the severity of congestive heart failure . The
troponin T or I levels may also be increased, indicating myocardial injury
(Cardinale and Sandri . ,2010)
The changes in neurohormones and cardiac enzymes are not diagnostic
of doxorubicin cardiomyopathy and are observed in other types of
cardiomyopathy. The endomyocardial biopsy may reveal characteristic
diagnostic features of doxorubicin cardiomyopathy. The findings that
have been suggested for the diagnosis of doxorubicin cardiomyopathy are
loss of myofibrils, distention of sarcoplasmic reticulum, and
vacuolization of the cytoplasm. The endomyocardial biopsy is also used
to grade the severity of doxorubicin cardiotoxicity (Takemura and
Fujiwara , 2007). The disadvantage of this technique is that it is invasive,
and it requires considerable experience and training. Furthermore,
endomyocardial biopsies are not universally used for the diagnosis of
doxorubicin cardiomyopathy and its severity.Endomyocardial biopsy is
recognized as the most sensitive diagnostic tool to detect anthracycline-
induced cardiomyopathy; however, this invasive examination is not
practically performed on a routine basis. ECG changes such as
dysrhythmias, a reduction of the QRS voltage, or a prolongation beyond
normal limits of the systolic time interval may be indicative of
anthracycline-induced cardiomyopathy, but ECG is not a sensitive or
specific method for following anthracycline-related cardiotoxicity
(Cardinale and Sandri . ,2010)
Pediatric patients are at increased risk for developing delayed
cardiotoxicity following doxorubicin administration and therefore a
follow-up cardiac evaluation is recommended periodically to monitor for
this delayed cardiotoxicity. In adults, a 10% decline in LVEF to below
Review of literature
14
the lower limit of normal or an absolute LVEF of 45%, or a 20% decline
in LVEF at any level is indicative of deterioration in cardiac function.In
pediatric patients, deterioration in cardiac function during or after the
completion of therapy with doxorubicin is indicated by a decline in
LVEF of 10 percentile units or an LVEF below 55% (Hershman et al.,
2008).
In general, if test results indicate deterioration in cardiac function
associated with doxorubicin, the benefit of continued therapy should be
carefully evaluated against the risk of producing irreversible cardiac
damage. Acute life-threatening arrhythmias have been reported to occur
during or within a few hours after doxorubicin administration (Chatterjee
et al.,2010).
There is no specific treatment available for the management of patients
with established heart failure. Diuretics are used to relieve symptoms and
signs of pulmonary and systemic venous congestion. β-Adrenergic
blocking agents should be considered, as for treatment of other types of
systolic heart failure (Malcom et al., 2008). It has been reported that
metoprolol is safe and can be effective in doxorubicin-induced
cardiomyopathy. Angiotensin II inhibition could also be used in
doxorubicin-induced cardiomyopathy (Takemura and Fujiwara , 2007).
In patients with advanced heart failure and in those intolerant to
angiotensin II inhibition therapy, low-dose hydralazine-isosorbide
dinitrate combination treatment is often employed. In patients with
malignant arrhythmias, amiodarone and implantable cardioverter &
defibrillator should be considered. It should be appreciated that none of
the treatments employed for ischemic or idiopathic dilated
cardiomyopathy has been demonstrated to improve the prognosis of
patients with doxorubicin cardiomyopathy. Cardiac transplantation has
Review of literature
15
been reported to improve long-term prognosis of the patients in whom the
primary malignancy is cured following chemotherapy (Thomas et
al.,2002).
In addition to staying below the cumulative doses, various preventive
measures may be employed by the oncologist in order to reduce the risk
of cardiotoxicity. Cardiac monitoring is recommended at 3, 6, and 9
months.There is a major effort has been done to limit the cumulative dose
of doxorubicin to <450 mg (Swain et al., 2003).
The use of anthracycline analogues, alternative methods of drug
delivery and continuous slow infusion instead of standard infusion
protocols have also been employed to reduce the risk of dilated
cardiomyopathy as it will result in a reduced plasma level and a much
lower left ventricular peak concentration (Zhang et al. , 2012).
A number of pharmaceutical agents have been tested, usually in
experimental animal studies, to assess their potential to reduce the risk of
doxorubicin cardiotoxicity. Most of the pharmacologic agents that have
been tested to reduce or prevent doxorubicin cardiotoxicity have the
potential to reduce oxidative stress.The mercaptopropionyl glycine
(MPG), a synthetic aminothiol that exhibits antioxidant properties, has
been shown to reduce doxorubicin cardiotoxicity (van Dalen et al.,2008).
Similarly, probucol, super oxide dismutase activator, and
dexrarzoxane with documented antioxidant properties have been reported
to decrease doxorubicin cardiotoxicity (Ducroq et al. , 2010).
Because the calcium channel blocker amlodipine and the β-and α-
adrenergic blocking agent carvedilol have antioxidant properties they
Review of literature
16
have also been studied for their potential to reduce doxorubicin
cardiotoxicity ( Kalay et al.,2006)
The PDE5 inhibitor sildenafil, erythropoietin and thrombopoietin,
granulocyte colony-stimulating factor, and the endothelin receptor-
blocking agent bosentan have been used in experimental animal models
for the protection against developing doxorubicin cardiomyopathy (Bien
et al. ,2007).
The potential protective role of nitric oxide and superoxide dismutase
has been investigated in a transgenic mouse model. Lack of nitric oxide
was associated with enhanced cardiac injury, and mitochondrial injury
was attenuated by an increase in superoxide dismutase (Cole et al., 2006).
The use of liposomal preparations of doxorubicin when appropriate as
the liposomal formulations of daunorubicin and doxorubicin are less toxic
to cardiac tissue than the non-liposomal form because a lower proportion
of drug administered in the liposome form is delivered to the heart
(Lotrionte et al., 2009).
(2) Hematologic Toxicity:
As with other cytotoxic agents, doxorubicin may produce
myelosuppression. Myelosuppression requires careful monitoring. Total
and differential white blood cell, red blood cell, and platelet counts
should be assessed before and during each cycle of therapy with
doxorubicin. A dose-dependent, reversible leukopenia and/or
granulocytopenia (neutropenia) are the predominant manifestations of
doxorubicin hematologic toxicity and are the most common acute dose-
limiting toxicities of this drug (Lyman et al. , 2011).
With the recommended dose schedule, leukopenia is usually transient,
occurring within 10 to 14 days after treatment with recovery usually
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17
occurring by the 21st day. Thrombocytopenia and anemia may also occur.
Clinical consequences of severe myelosuppression include fever,
infections, sepsis/septicemia, septic shock, hemorrhage, tissue hypoxia, or
death (Elad et al., 2010).
(3) Secondary Leukemia:
The occurrence of secondary acute meloid leukemia and
myelodysplastic syndrome has been reported most commonly in patients
treated with chemotherapy regimens containing anthracyclines (including
doxorubicin) and DNA-damaging antineoplastic agents, in combination
with radiotherapy, when patients have been heavily pretreated with
cytotoxic drugs. Such cases generally have a 1–3 year latency period.
Pediatric patients are also at risk of developing secondary acute meloid
leukemia (Marie-Cécile et al., 2007).
(4) Hepatic Impairment:
Despite the widespread clinical usage of doxorubicin, the side effects
of doxorubicin form amajor concern. It has been reported that the heart is
a prefer-ential target of doxorubicin-induced toxicity.However, increasing
evidence shows that the antitumor agent also affects other organs
including the liver, kidney and brain.Almost 40% of patients suffered
liver injury after doxorubicin treatment.The main toxic effects on
hepatocytes include: arrest cell cycle of hepatocytes,oxidative stress and
disruption of electron transport. But the exact mechanism of
hepatotoxicity of doxorubicin is not fully clarified. Due to these serious
side effects, the clinical use of doxorubicin has been restricted (Deleve et
al., 2013).
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18
Since metabolism and excretion of doxorubicin occurs predominantly
by the hepatobiliary route, toxicity of recommended doses of doxorubicin
can be enhanced by hepatic impairment; therefore, prior to individual
dosing, evaluation of hepatic function is recommended using
conventional laboratory tests such as SGOT, SGPT, alkaline phosphatase,
and bilirubin (Reuben et al .,2010).
To reduce the toxic effects of doxorubicin, several pharmacologic
agents, such as antioxidants, hematopoietic cytokines and iron-chelating
agents were used.The development of doxorubicin analogues and the
production of efficacious delivery systems were also found to be effective
ways.Although most of these attempts showed beneficial effects,
some of these attempts failed to attenuate doxorubicin toxicity in clinical
trials. Therefore, it is very necessary to search for more effective
strategies against doxorubicin-induced complications while preserve or
enhance its therapeutic effects (Gokcimen et al ., 2007).
(5) Nephrotoxicity:
Nephrotoxicity is one of the limiting factors for using doxorubicin as
an anticancer chemotherapeutic.Unfortunately, the use of doxorubicin has
been limited by the occurrence of dose-dependent toxicities to vital
organs, as the heart, the kidney, and the liver (Carvalho et al., 2009).
The exact mechanism of doxorubicin -induced nephrotoxicity is not
yet completely understood. Renal doxorubicin -induced toxicity may be
part of a multiorgan damage mediated mainly through free radical
formation eventually leading to membrane lipid peroxidation .Induction
of apoptosis and modulation of nitric oxide are other mechanisms that
may be involved in toxic adverse effects associated with doxorubicin
therapy. In addition, doxorubicin may induce nephrotoxicity through its
Review of literature
19
direct renal damaging effect, as it accumulates preferentially in the
kidney (Lee and Harris , 2011)
Doxorubicin toxic effects to other organs as the heart and the liver
may modulate blood supply to the kidney and alter xenobiotic
detoxification processes, respectively, thus indirectly contributing to
doxorubicin-induced nephropathy.A number of antioxidant compounds
have been proposed as chemopreventive therapy for doxorubicin -induced
toxicity ( Granados-Principal et al. ,2010).
(6) Immunosuppressant Effects:
Administration of live or live-attenuated vaccines in patients
immunocompromised by chemotherapeutic agents including doxorubicin,
may result in serious or fatal infections. Vaccination with a live vaccine
should be avoided in patients receiving doxorubicin. Killed or inactivated
vaccines may be admiinistered; however, the response to such vaccines
may be diminished (Elad et al., 2010).
(7) Teratogenecity :
Doxorubicin can cause fetal harm when administered to a pregnant
woman. Doxorubicin was teratogenic and embryotoxic .Characteristic
malformations included esophageal and intestinal atresia, tracheo-
esophageal fistula, hypoplasia of the urinary bladder, and cardiovascular
anomalies (Autio et al., 2007).
(8) Impairment of Fertility :
Decreased fertility in female rats at the doses of 0.05 and 0.2
mg/kg/day (about 1/200 and 1/50 the recommended human dose on a
body surface area basis) when administered from 14 days before mating
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20
through late gestation period. A single i.v. dose of doxorubicin at 0.1
mg/kg (about 1/100 the recommended human dose on a body surface area
basis) was toxic to male reproductive organs, producing testicular atrophy
and oligospermia in rats. Doxorubicin is mutagenic as it induced DNA
damage in rabbit spermatozoa and dominant lethal mutations in mice.
Therefore, doxorubicin may potentially induce chromosomal damage in
human spermatozoa. Oligospermia or azoospermia were evidenced in
men treated with doxorubicin, mainly in combination therapies. Men
undergoing doxorubicin treatment should use effective contraceptive
methods. In women, doxorubicin may cause infertility during the time of
drug administration. Doxorubicin may cause amenorrhea, ovulation and
menstruation may return after termination of therapy, although premature
menopause can occur. Recovery of menses is related to age at treatment
(Morgan et al ., 2012).
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21
Nebivolol
Nebivolol is a β1 receptor blocker with nitric oxide-potentiating
vasodilator effect used in treatment of hypertension and, also for left
ventricular failure (de Boer et al., 2007).
Chemistry:
Nebivolol's molecular formula is (C22H25F2NO4•HCl) with the
following structural formula:
Nebivolol hydrochloride (Gielen et al., 2006 )
Physical properties:
Nebivolol hydrochloride is a white to almost white powder that is
soluble in methanol, dimethylsulfoxide, and N-dimethylformamide,
sparingly soluble in ethanol, propylene glycol, and polyethylene glycol,
and very slightly soluble in hexane, dichloromethane, and methylbenzene
(Gielen et al., 2006) and soluble in water (Dursun et al., 2014).
Pharmacokinetics:
Nebivolol is metabolized by a number of routes, including
glucuronidation and hydroxylation by CYP2D6. The active isomer (d-
nebivolol) has an effective half-life of about 12 hours in CYP2D6
extensive metabolizers (EM) (most people), and 19 hours in poor
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22
metabolizers and exposure to d-nebivolol is substantially increased in
poor metabolizers (PM). This has less importance than usual, however,
because the metabolites, including the hydroxyl metabolite and
glucuronides (the predominant circulating metabolites), contribute to β-
blocking activity. The absolute bioavailability has not been determined.
Mean peak plasma nebivolol concentrations occur approximately 1.5 to 4
hours post-dosing in EMs and PMs. Food does not alter the
pharmacokinetics of nebivolol. Under fed conditions, nebivolol
glucuronides are slightly reduced. Nebivolol may be administered
without regard to meals (Mangrella et. al, 1998).
The plasma protein binding of nebivolol is approximately 98%, mostly
to albumin, and is independent of nebivolol concentrations. Nebivolol is
predominantly metabolized via direct glucuronidation of parent and to a
lesser extent via N dealkylation and oxidation via cytochrome P450 2D6.
Its stereospecific metabolites contribute to the pharmacologic activity
.Elimination after a single oral administration of nebivolol, 38% of the
dose was recovered in urine and 44% in feces for EMs and 67% in urine
and 13% in feces for PMs. Essentially all nebivolol was excreted as
multiple oxidative metabolites or their corresponding glucuronide
conjugates (Prisant , 2008).
Pharmacokinetics in Special Populations
Hepatic Disease
Nebivolol peak plasma concentration increased 3-fold and the apparent
clearance decreased by 86% in patients with moderate hepatic
impairment . No formal studies have been performed in patients with
severe hepatic impairment and nebivolol should be contraindicated for
these patients (Prisant , 2008).
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23
Renal Disease
The apparent clearance of nebivolol was unchanged following a single
5 mg dose of nebivolol in patients with mild renal impairment (ClCr 50 to
80 mL/min, n=7), and it was reduced in patients with moderate renal
impairment (ClCr 30 to 50 mL/min, n=9), but clearance was reduced by
53% in patients with severe renal impairment (ClCr < 30 mL/min, n=5).
No studies have been conducted in patients on dialysis (Prisant , 2008).
Mechanism of Action:
The mechanism of action of the antihypertensive response of nebivolol
has not been definitively established. Possible factors that may be
involved include: (1) decreased heart rate, (2) decreased myocardial
contractility, (3) diminution of tonic sympathetic outflow to the periphery
from cerebral vasomotor centers, (4) suppression of renin activity and (5)
vasodilation and decreased peripheral vascular resistance (Brunton et al.,
2006).
Pharmacological actions:
β1 Selectivity
Beta blockers help patients with cardiovascular disease by blocking β
receptors, while many of the side-effects of these medications are caused
by their blockade of β2 receptors, for this reason, beta blockers that
selectively block β1 receptors (termed cardioselective or β1-selective beta
blockers) produce fewer adverse effects (for instance,
bronchoconstriction) than those drugs that non-selectively block both β1
and β2 receptors. The drug is highly cardioselective at 5 mg (Gielen et al
., 2006) .
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24
However, at doses above 10 mg, nebivolol loses its cardioselectivity and
blocks both β1 and β2 receptors. While the recommended starting dose of
nebivolol is 5 mg, sufficient control of blood pressure may require doses
up to 40 mg (De Boer et al., 2007).
Vasodilator action
Nebivolol is unique as a beta-blocker. Unlike carvedilol, it has a nitric
oxide (NO)-potentiating, vasodilator effect (Weiss, 2006). Along with
labetalol, celiprolol and carvedilol, it is one of four beta blockers to cause
dilation of blood vessels in addition to effects on the heart ( Bakris ,
2009).
Antihypertensive effect
Nebivolol lowers blood pressure by reducing peripheral vascular
resistance, and significantly increases stroke volume with preservation of
cardiac output (Kamp et al., 2003). The net hemodynamic effect of
nebivolol is the result of a balance between the depressant effects of beta-
blockade and an action that maintains cardiac output (Gielen et al.,
2006).
Antioxidant effect:
It has been proposed that nebivolol exerts endothelium-protective
effects caused by its antioxidant properties. Nebivolol protected against
ROS-induced endothelial damage. Furthermore, it has been shown to
cause vasorelaxation via a nitric oxide/cGMP-dependent pathway in
various vascular beds. In addition to its direct negative inotropic and
vasodilator effects, nebivolol has been assumed to counteract oxidative
stress. It has been demonstrated that nebivolol protects against hydroxyl
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25
radical (OH)-induced injury in right ventricular rabbit cardiac trabeculae
and in demembranized muscles from explanted human hearts (Agabiti
and Rizzoni, 2007). In urine samples of healthy human volunteers, it was
shown that nebivolol significantly decreased the levels of 8-iso-PGF2a, a
marker of oxidative stress (Weiss, 2006). Oxidative stress is associated
with the impairment of endothelium-dependent vasorelaxation caused by
the loss of nitric oxide (·NO) bioactivity in the vessel wall (Bakris ,
2009). Endothelial dysfunction, combined with enhanced levels of
reactive oxygen species (ROS), is assumed to play an important role in
the pathogenesis of cardiovascular diseases such as, atherosclerosis,
congestive heart failure and hypertension (Baldwin and Keam, 2009).
Furthermore, it is unknown whether the observed protective effect of
nebivolol during oxidative stress results from a direct scavenging action
of the molecule or whether it is mediated by a different mechanism. If
indeed nebivolol is a scavenger of reactive oxygen species, this could
imply that the molecule itself is modulated by this reaction. Such a
molecular modification could possibly lead to a change in
pharmacological activity (Gielen et al., 2006).
Indications:
Nebivolol is indicated for the treatment of hypertension, to lower blood
pressure. Nebivolol may be used alone or in combination with other
antihypertensive agents. Lowering blood pressure reduces the risk of fatal
and nonfatal cardiovascular events, primarily strokes and myocardial
infarctions (Moen & Wagstaff , 2006)
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26
Dosage and adminstration:
Hypertension
The dose of nebivolol must be individualized to the needs of the
patient. For most patients, the recommended starting dose is 5 mg once
daily, with or without food, as monotherapy or in combination with other
agents. For patients requiring further reduction in blood pressure, the dose
can be increased at 2-week intervals up to 40 mg. A more frequent dosing
regimen is unlikely to be beneficial (Chobanian et al., 2013).
Renal Impairment
In patients with severe renal impairment (ClCr less than 30 mL/min)
the recommended initial dose is 2.5 mg once daily; titrate up slowly if
needed. Nebivolol has not been studied in patients receiving dialysis
(Prisant , 2008).
Hepatic Impairment
In patients with moderate hepatic impairment, the recommended initial
dose is 2.5 mg once daily. Nebivolol has not been studied in patients with
severe hepatic impairment and therefore it is not recommended in that
population (Agabiti and Rizzoni, 2007).
Drug interaction:
When nebivolol is co-administered with CYP2D6 inhibitors as
quinidine, propafenone, fluoxetine, paroxetine the dose of nebivolol may
need to be reduced (Moen and Wagstaff , 2006).
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27
Patients receiving catecholamine-depleting drugs such as reserpine or
guanethidine should be closely monitored because the added β-blocking
action of nebivolol may produce excessive reduction of sympathetic
activity. In patients who are receiving nebivolol and clonidine,
discontinue nebivolol for several days before the gradual tapering of
clonidine (Wojciechowski and Papademetriou, 2008).
Both digitalis glycosides and β-blockers slow atrioventricular
conduction and decrease heart rate. Concomitant use of nebivolol can
increase the risk of bradycardia (Brunton et al ., 2006) .
Nebivolol can exacerbate the myocardial depressants effects of calcium
channel blockers or inhibitors of AV conduction, such as certain calcium
antagonists (particularly of the phenylalkylamine [verapamil] and
benzothiazepine [diltiazem] classes), or antiarrhythmic agents, such as
disopyramide (Olga ,2009).
Precautions:
Sudden discontinuation of nebivolol therapy in patients with
coronary artery disease may cause severe exacerbation of angina,
myocardial infarction and ventricular arrhythmias have been reported in
patients with coronary artery disease following the abrupt discontinuation
of therapy with β-blockers. Myocardial infarction and ventricular
arrhythmias may occur with or without preceding exacerbation of the
angina pectoris.Taper nebivolol over 1 to 2 weeks when possible. If the
angina worsens or acute coronary insufficiency develops, re-start
nebivolol promptly, at least temporarily (Bakris , 2009). Patients with
bronchospastic diseases should not receive β-blockers (Tafreshi and
Weinacker, 1999).
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28
β-blockers may mask some of the manifestations of hypoglycemia,
particularly tachycardia. Nonselective β-blockers may potentiate insulin-
induced hypoglycemia and delay recovery of serum glucose levels. It is
not known whether nebivolol has these effects (Poirier et al ., 2001).
β-blockers may mask clinical signs of hyperthyroidism, such as
tachycardia. Abrupt withdrawal of β-blockers may be followed by an
exacerbation of the symptoms of hyperthyroidism or may precipitate a
thyroid storm (Pessina , 2001).
β-blockers can precipitate or aggravate symptoms of arterial
insufficiency in patients with peripheral vascular disease (Bakris , 2009).
Because of significant negative inotropic and chronotropic effects in
patients treated with β-blockers and calcium channel blockers of the
verapamil and diltiazem type, monitor the ECG and blood pressure in
patients treated concomitantly with these agents (Olga ,2009).
Renal clearance of nebivolol is decreased in patients with severe renal
impairment. Nebivolol has not been studied in patients receiving dialysis
(Prisant , 2008).
Metabolism of nebivolol is decreased in patients with moderate hepatic
impairment. (Agabiti and Rizzoni, 2007).
While taking β-blockers, patients with a history of severe anaphylactic
reactions to a variety of allergens may be more reactive to repeated
accidental, diagnostic, or therapeutic challenge. Such patients may be
unresponsive to the usual doses of epinephrine used to treat allergic
reactions (Pessina , 2001). In patients with known or suspected
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29
pheochromocytoma, initiate an α-blocker prior to the use of any β blocker
(Punzi et al., 2010).
Adverse effects:
Nebivolol is associated with a number of serious risks. Nebivolol is
contraindicated in patients with severe bradycardia, heart block greater
than first degree, cardiogenic shock, decompensated cardiac failure, sick
sinus syndrome (unless a permanent pacemaker is in place), severe
hepatic impairment and in patients who are hypersensitive to any
component of the product (Gielen et al., 2006).
Nebivolol therapy is also associated with warnings regarding abrupt
cessation of therapy, cardiac failure, angina and acute myocardial
infarction, bronchospastic diseases, anesthesia and major surgery,
diabetes and hypoglycemia, thyrotoxicosis, peripheral vascular disease,
non-dihydropyridine calcium channel blockers use, as well as precautions
regarding use with CYP2D6 inhibitors, impaired renal and hepatic
function, and anaphylactic reactions. Finally, Nebivolol is associated with
other risks for example, a number of treatment-emergent adverse events
with an incidence greater than or equal to 1 percent in nebivolol -treated
patients and at a higher frequency than placebo-treated patients were
identified in clinical studies, including headache, fatigue, and
dizziness(De Boer et al., 2007)..
In clinical trials and worldwide post marketing experience, there were
reports of nebivolol overdose. The most common signs and symptoms
associated with nebivolol overdosage are bradycardia and hypotension.
Other important adverse reactions reported with nebivolol overdose
include cardiac failure, dizziness, hypoglycemia, fatigue and vomiting.
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30
Other adverse reactions associated with β-blocker overdose include
bronchospasm and heart block.Because of extensive drug binding to
plasma proteins, hemodialysis is not expected to enhance nebivolol
clearance (Germino , 2009) .
Contraindications:
Nebivolol is contraindicated in severe bradycardia, heart block greater
than first degree, patients with cardiogenic shock, decompensated cardiac
failure, sick sinus syndrome (unless a permanent pacemaker is in place),
patients with severe hepatic impairment and patients who are
hypersensitive to nebivolol (Germino , 2009).
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31
L-Carnitine
L-Carnitine is a naturally occurring compound that facilitates the
transport of fatty acids into mitochondria for beta-oxidation. Exogenous
L-carnitine is used clinically for the treatment of carnitine deficiency
disorders . it is synthesized in the liver and kidneys and stored it in the
skeletal muscles, heart, brain, and sperm. (Volek, 2008).
Carnitine has been proposed as a treatment for many conditions
because it acts as an antioxidant. Antioxidants fight harmful particles in
the body known as free radicals, which damage cells and DNA.
Antioxidants can neutralize free radicals and may reduce or help prevent
some of the damage they cause (Othman et al., 2010).
Chemistry:
Carnitine is a quaternary ammonium compound biosynthesized from
the amino acids lysine and methionine In living cells, it is required for
the transport of fatty acids from the cytosol into the mitochondria during
the breakdown of lipids for the generation of metabolic energy. It is
widely available as a nutritional supplement. Carnitine exists in two
stereoisomers: Its biologically active form is L-carnitine, whereas its
enantiomer, D-carnitine, is biologically inactive (Steiber et al., 2004).
Pharmacokinetics of L- Carnitine:
In humans, the endogenous carnitine pool, which comprises free L-
carnitine and a range of short-, medium- and long-chain esters, is
maintained by absorption of L-carnitine from dietary sources,
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32
biosynthesis within the body and extensive renal tubular reabsorption
from glomerular filtrate. In addition, carrier-mediated transport ensures
high tissue-to-plasma concentration ratios in tissues that depend critically
on fatty acid oxidation. The absorption of L-carnitine after oral
administration occurs partly via carrier-mediated transport and partly by
passive diffusion (Charle , 2006).
L-Carnitine and its short-chain esters do not bind to plasma proteins
and, although blood cells contain L-carnitine, the rate of distribution
between erythrocytes and plasma is extremely slow in whole blood. After
intravenous administration, the initial distribution volume of L-carnitine
is typically about 0.2-0.3 L/kg, which corresponds to extracellular fluid
volume (Evans and Fornasini , 2003) .
L-Carnitine is eliminated from the body mainly via urinary excretion.
Under baseline conditions, the renal clearance of L-carnitine (1-3
mL/min) is substantially less than glomerular filtration rate (GFR),
indicating extensive (98-99%) tubular reabsorption. The threshold
concentration for tubular reabsorption (above which the fractional
reabsorption begins to decline) is about 40-60 micromol/L, which is
similar to the endogenous plasma L-carnitine level. Therefore, the renal
clearance of L-carnitine increases after exogenous administration,
approaching GFR after high intravenous doses. Patients with primary
carnitine deficiency display alterations in the renal handling of L-
carnitine and/or the transport of the compound into muscle tissue.
Similarly, many forms of secondary carnitine deficiency, including some
drug-induced disorders, arise from impaired renal tubular reabsorption.
Patients with end-stage renal disease undergoing dialysis can develop a
secondary carnitine deficiency due to the unrestricted loss of L-carnitine
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33
through the dialyser, and L-carnitine has been used for treatment of some
patients during long-term haemodialysis (Charle , 2006).
Pharmacodynamics of L- Carnitine:
Role in fatty acid metabolism:
Carnitine transports long-chain acyl groups from fatty acids into the
mitochondrial matrix, so they can be broken down through β-oxidation to
acetyl CoA to obtain usable energy via the citric acid cycle. In some
organisms, such as fungi, the acetate is used in the glyoxylate cycle for
gluconeogenesis and formation of carbohydrates. Fatty acids must be
activated before binding to the carnitine molecule to form 'acylcarnitine'.
The free fatty acid in the cytosol is attached with a thioester bond to
coenzyme A (CoA). This reaction is catalyzed by the enzyme fatty acyl-
CoA synthetase and driven to completion by inorganic pyrophosphatase
(Pekala et al., 2011).
The acyl group on CoA can now be transferred to carnitine and the
resulting acylcarnitine transported into the mitochondrial matrix. This
occurs via a series of similar steps: 1- Acyl CoA is transferred to the
hydroxyl group of carnitine by carnitine acyltransferase I
(palmitoyltransferase) located on the outer mitochondrial membrane. 2-
Acylcarnitine is shuttled inside by a carnitine-acylcarnitine translocase .
3-Acylcarnitine is converted to acyl CoA by carnitine acyltransferase II
(palmitoyltransferase) located on the inner mitochondrial membrane. The
liberated carnitine returns to the cytosol (Marcovina et al., 2013).
Human genetic disorders, such as primary carnitine deficiency,
carnitine palmitoyltransferase I deficiency, carnitine palmitoyltransferase
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34
II deficiency and carnitine-acylcarnitine translocase deficiency, affect
different steps of this process (Olpin , 2005).
Carnitine acyltransferase I and peroxisomal carnitine octanoyl
transferase (CROT) undergo allosteric inhibition as a result of malonyl-
CoA, an intermediate in fatty acid biosynthesis, to prevent futile cycling
between β-oxidation and fatty acid synthesis (Volek et al., 2008).
Role in doxorubicin cardiotoxicity:
Carnitine has a protective effect against doxorubicin cardiotoxicity as
showed in the study done by by Sayed-Ahmed et al., 2000a that studied
doxorubicin cardiotoxicity in absence and presence of propionyl L-
carnitine (PLC) on palmitoyl-CoA and palmitoyl-carnitine oxidation and
on the activity of carnitine palmitoyltransferase (CPT 1). The results
showed that doxorubicin induced concentration-dependent inhibition of
substrates oxidation and inhibition of CPT I and that PLC completely
reversed this inhibition to the control values. They concluded that
doxorubicin induced its cardiotoxicity by inhibition of CPT I and beta-
oxidation of long-chain fatty acids with the consequent depletion of ATP
in cardiac tissues, and that PLC can be used as a protective agent against
doxorubicin -induced cardiotoxicity
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35
Figure ( 1 ): Proposed protective mechanism by PLC against DOX-
induced cardiomyopathy. CT, carnitin acyl-carnitine translocase;
CAT, carnitine acetyl transferase; CPT I, outer carnitine
palmitoyltransferase; CPT II, inner carnitine palmitoyltransferase;
PCC, propionyl-CoA carboxylase; ADR, adriamycin (trade name
of DOX); (−) and (+) indicate inhibition and stimulation,
respectively coated by Sayed-Ahmed et al., 2000a.
In doxorubicin cardiomyopathic rat model, Sayed-Ahmed et al.
(2001) have initiated another study to confirm their in vitro findings and
investigated the protective effects of PLC against doxorubicin induced
cardiotoxicity. Chronic administration of doxorubicin (3 mg/kg ,
intraperitonial) significantly increased creatine kinase-MB, lactate
dehydrogenase and malondialdehyde and decreased reduced glutathione.
Treatment with PLC induced complete reversal of these effects without
decreasing the antitumour activity of doxorubicin.
In another study, Sayed-Ahmed et al., 2000b investigated the effects
of doxorubicin on mRNA expression of heart fatty acid binding protein
(H-FABP) using northern blot analysis. Results showed that chronic
administration of doxorubicin caused dose-dependent and cumulative
Review of literature
36
inhibition of H-FABP gene expression and daily administration of L-
carnitine protected against this effect in cardiac tissues.
Figure ( 2 ) :Inhibition of H-FABP mRNA expression in the heart
as a novel mechanism for DOX-induced cardiotoxicity and for L-
carnitine mediated protection against this effect. FFA: free fatty
acid; CT: carnitine/acyl-carnitine translocase; FA-CoA: fatty
acyl-CoA; CAT: carnitine acetyl transferase; FA-Carnitine: fatty
acyl-carnitine; H-FABP: heart-type fatty acid binding protein;
CPT I: outer carnitine palmitoyl transferase; CPT II: inner
carnitine palmitoyl transferase; OCTN2: organic cation/carnitine
transporter; (−) and (+) indicate inhibition and stimulation,
respectively.(Sayed-Ahmed et al. (2000b).
Effect on Atherosclerosis
There may be a link between dietary consumption of carnitine and
atherosclerosis, but there is also evidence that it lowers the risk of
mortality and arrythmias after an acute myocardial infarction.When
certain species of intestinal bacteria were exposed to carnitine from food,
they produced a waste product, trimethylamine, which is transformed in
Review of literature
37
the liver to trimethylamine N-oxide (TMAO). TMAO may be associated
with atherosclerosis. The presence of large amounts of TMAO-producing
bacteria was a consequence of a long-term diet rich in meat. However,
when the authors compared the risk of cardiovascular events to the levels
of carnitine and TMAO, they found that the risk was higher in those with
higher TMAO levels, independent of the carnitine levels.Vegetarian and
vegans who ate a single meal of meat had much lower levels of TMAO in
their blood stream than did regular meat-eaters, as vegetarian and vegans
had lower levels of the intestinal bacteria that converts carnitine into
TMAO (Koeth and Robert, 2013).
Effects on bone mass:
In the course of human aging, carnitine concentration in cells
diminishes, affecting fatty acid metabolism in various tissues. Particularly
adversely affected are bones, which require continuous reconstructive and
metabolic functions of osteoblasts for maintenance of bone mass (Pekala
et al., 2011)
Antioxidant effects:
The carnitines exert antioxidant action, thereby providing a protective
effect against lipid peroxidation of phospholipid membranes and against
oxidative stress induced at the myocardial and endothelial cell level
(Othman et al., 2010).
Possible health effects
Carnitine has been proposed as a supplement to treat a variety of health
conditions including myocardial infraction or angina (Dinicolantonio et
Review of literature
38
al., 2013), heart failure, angina and diabetic neuropathy, improving
exercise performance (Pekala et al., 2011).
Uses:
Heart Conditions
Angina: carnitine can be used along with conventional treatment
for stable angina. Several clinical trials show that L-carnitine and
propionyl-L-carnitine can help reduce symptoms of angina and
improve the ability of people with angina to exercise without chest
pain (Witte and Clark , 2006).
A few studies have found that carnitine may help when used with
conventional medicines after a heart attack, but not all studies
agree. Some small studies suggest that people who take L-carnitine
supplements soon after a heart attack may be less likely to have
another heart attack, die of heart disease, have chest pain and
abnormal heart rhythms, or develop heart failure. However, other
studies have shown no benefit. Treatment with oral carnitine may
also improve muscle weakness (Xue, 2007).
Heart failure: A few small studies have suggested that carnitine
(usually propionyl-L-carnitine) can help reduce symptoms of heart
failure and improve exercise capacity in people with heart failure
(Witte & Clark , 2006).
Peripheral Vascular Disease
Decreased blood flow to the legs from atherosclerosis or hardening of
the arteries where plaque builds up in the arteries often causes an aching
or cramping pain in the legs while walking or exercising. This pain is
called intermittent claudication, and the reduced blood flow to the legs is
Review of literature
39
called peripheral vascular disease (PVD). Several studies show that
carnitine can help reduce symptoms and increase the distance that people
with intermittent claudication can walk. Most studies have used
propionyl-L-carnitine (Carrero and Grimble , 2006).
Diabetic Neuropathy
Diabetic neuropathy happens when high blood sugar levels affect
nerves in the body, especially the arms, legs, and feet, causing pain and
numbness. Some small preliminary studies suggest acetyl-L-carnitine
may help reduce pain and increase feeling in affected nerves. It is also
possible that carnitine can help nerves regenerate (Head, 2006).
Exercise Performance
Carnitine is often taken to improve exercise performance (Hiatt, 2001).
Weight Loss
Although L-carnitine has been marketed as a weight loss supplement,
there is no scientific evidence to show that it works. Some studies do
show that oral carnitine reduces fat mass, increases muscle mass, and
reduces fatigue, which may contribute to weight loss in some people
(Villani , 2000).
Alzheimer's disease and memory impairment
The evidence is mixed as to whether carnitine is useful in treating
Alzheimer's disease. Several early studies showed that acetyl-L-carnitine,
might help slow down the progression of Alzheimer's disease, relieve
depression related to senility and other forms of dementia, and improve
Review of literature
40
memory in the elderly. But larger and better-designed studies found it
didn’t help at all (Pettegrew, 2000).
Kidney Disease and Dialysis
Because the kidneys make carnitine, kidney disease could lead to low
levels of carnitine in the body (Lynch , 2008).
Male Infertility
Low sperm counts have been linked to low carnitine levels in men.
Several studies suggest that L-carnitine supplements may increase sperm
count and mobility (Sinclair, 2000).
Erectile Dysfunction
Preliminary studies suggest propionyl-L-carnitine may help improve
male sexual function. One study found that carnitine improved the
effectiveness of sidenafil in men with diabetes who had not previously
responded to sidenafil. In another study, a combination of propionyl-L-
carnitine and acetyl-L-carnitine improved the effectiveness of sidenafil in
men who had erectile dysfunction after prostate surgery (Cavallini et al.,
2005).
Peyronie's Disease
Peyronie's disease is characterized by a curvature of the penis that
leads to pain during an erection. One promising study compared acetyl-L-
carnitine to the medication tamoxifen in 48 men with this condition.
Acetyl-L-carnitine worked better than tamoxifen at reducing pain during
sex and reducing the curve of the penis. Acetyl-L-carnitine also had fewer
side effects than tamoxifen (Biagiotti, and Cavallini ,2001).
Review of literature
41
Hyperthyroidism
Some research suggests that L-carnitine may help prevent or reduce
symptoms of an overactive thyroid, such as insomnia, nervousness, heart
palpitations, and tremors. In fact, in one study, a small group of people
with hyperthyroidism saw these symptoms improve, and their body
temperature become normal, when taking carnitine. But a larger, better-
designed clinical trial is needed to see if carnitine really works. In
addition, researchers think carnitine may work by blocking the action of
thyroid hormone, which could be dangerous for people with low thyroid
levels (Benvenga et al ., 2001).
Dietary Sources
The highest concentrations of carnitine are found in red meat and dairy
products. Carnitine can be found at significantly lower levels in many
other foods including nuts and seeds (e.g. sunflower, sesame, beans, peas,
lentils, peanuts), vegetables (asparagus, beet greens, broccoli, brussels
sprouts, collard greens, garlic, mustard greens, okra, parsley, kale), fruits
( bananas), cereals (buckwheat, corn, millet, oatmeal, rice bran, rye,
whole wheat, wheat bran, wheat germ) and other foods (bee pollen,
brewer's yeast, carob). In general, 20 to 200 mg is ingested per day by
that on usual diet, whereas those on a strict vegetarian or vegan diet may
ingest as little as 1 mg/day. No advantage appears to exist in giving an
oral dose greater than 2 g at one time, since absorption studies indicate
saturation at this dose (Carrero and Grimble , 2006)..
Available Forms
Carnitine is available as a supplement in a variety of forms.
Review of literature
42
L-carnitine: the most widely available and least expensive
Acetyl-L-carnitine: Often used in studies for Alzheimer's disease
and other brain disorders
Propionyl-L-carnitine: Often used in studies for heart disease and
peripheral vascular disease
Avoid D-carnitine supplements. They interfere with the natural form of
L-carnitine and may produce unwanted side effects.In some cases, L-
carnitine may be taken by prescription or given intravenously by a health
care provider.Recommended doses of L-carnitine vary depending on the
health condition being treated. The usual dose is between 1 - 3 g per day
(Steiber et al., 2004).
Precautions:
Side effects are generally mild. High doses (5 or more grams per day)
may cause diarrhea. Other rare side effects include increased appetite,
body odor, and rash.
People with the following conditions should talk to their health care
provider before taking carnitine:Peripheral vascular disease, high blood
pressure, liver disease from alcoholism (cirrhosis), kidney disease, history
of seizures, diabetes ((Steiber et al., 2004).
Possible Interactions
In a laboratory study, L-carnitine supplements protected muscle
tissue against toxic side effects from Azalatine, a medication used to treat
HIV and AIDS. Treatment with L-carnitine may protect heart cells
against the toxic side effects of doxorubicin, a chemotherapy medication
used to treat cancer, without making the medication any less effective. In
Review of literature
43
case of isotretinoin which is a strong medication used for severe acne, can
cause liver problems, as measured by a blood test, as well as high
cholesterol and muscle pain and weakness. These symptoms are like
those seen with carnitine deficiency. Carnitine may stop thyroid hormone
from getting into cells, and theoretically may make thyroid hormone
replacement less effective. The anti-seizure medication valproic acid may
lower blood levels of carnitine. Taking L-carnitine supplements may
prevent any deficiency and may also reduce the side effects of valproic
acid. However, taking carnitine may increase the risk of seizures in
people with a history of seizures (Othman et al., 2010).
Materials and Method
44
Materials and Method
Materials:
A- Drugs and Chemicals:
CaCl2H2O(crystals): El-Nasr Pharmaceutical Cehmical Company
was dissolved in distilled water.
Creatinine acid reagent (solution): Sanbio Laboratory Texas ,
U.S.A.
Creatinine base reagent (solution): Sanbio Laboratory Texas ,
U.S.A.
Creatinine standered(solution): Sanbio Laboratory Texas , U.S.A.
cTnl Enzyme Conjugate Reagent (solution): GenWay Biotech.
for protein and antibody solutions SanDiego
Diethanolamine buffer (solution): GenWay Biotech. for protein
and antibody solutions SanDiego.
Doxorubicin HCL (solution): EIMC United Pharmaceuticals.
A.R.E.
Formaline (neutral 10% formaline): El-Gomhoria Pharmaceutial
Chemical Co. A.R.E
Glucose (crystals): El-Nasr Pharmaceutical Cehmical Company
was dissolved in distilled water.
Hematoxylin and eosin: E. Mark, Darmastadt. U.S.A.
Isoprenaline HCL (powder): Sigma,U.S.A was dissolved in
distilled water.
KCl (crystals): El-Nasr Pharmaceutical Cehmical Company was
dissolved in distilled water.
L-alanine solution GenWay Biotech. for protein and antibody
solutions SanDiego
Materials and Method
45
L-aspartate solution GenWay Biotech. for protein and antibody
solutions SanDiego
Lactate dehydrogenase solution GenWay Biotech. for protein and
antibody solutions SanDiego
L-Carnitine (powder. Eva Pharm. A.R.E) was dissolved in
distilled water.
Malate dehydrogenase solution GenWay Biotech. for protein and
antibody solutions SanDiego
NaCI (crystals): El-Nasr Pharmaceutical Cehmical Company was
dissolved in distilled water.
NaH2PO4(crystals): El-Nasr Pharmaceutical Cehmical Company
was dissolved in distilled water.
Nebivolol HCL (powder): Sigma, U.S.A was dissolved in distilled
water.
Phosphate buffer( solution) :GenWay Biotech. for protein and
antibody solutions SanDiego
Reduced nicotine adenine dinucleotide phosphate(solution):
GenWay Biotech. for protein and antibody solutions SanDiego
5- Stop solution (diluted hydrochloric acid) (solution): GenWay
Biotech. for protein and antibody solutions SanDiego
Tetramethyl-benzidine (TMB) Reagent (solution):GenWay
Biotech. for protein and antibody solutions SanDiego
B- Animals:
Rats: Adult male albino rats [from Helwan Farm], weighting 120-
200 gm were used.
Rabbits: of local strains ranging from 1-1.5 kg of both sexes were
used for experiments on isolated heart
Materials and Method
46
Ethical consideration of experimental animals:
Expermintal rats will be under complete healthy conditions all over the
experiment in the form of:
- Clean environment.
- Good ventilation
- Good nutrition in the form of free access to water and diet containing
cereals and bread.
- Number of rats in each cage is six
- The experimental rats will be under care of professional technicians and
qualified researchers.
C- Apparatus:
1- Harvard tension transducer and universal oscillography FT03
for ECG recording and measurement of blood pressure (Harvard,
UK).
3-Modified langendorff's apparatus (Harvard, UK).
4-Spectrophotometer for biochemical assessment.
D- Physiological solutions:
Ringer's solution (for isolated heart):
Chemicals Mg/l
NaCl 113.7
KCl 1.9
NaH2PO4 0.1
CaCl2H2O 0.2
Glucose 10
Materials and Method
47
Methods:
(A) In vivo experiment:
Rats were randomly divided into 5 groups (6 rats for each).
Animal groups:
1- Group (I): control group:
The control group will receive saline intraperitoneal in comparative
volume to the tested groups.
2- Group (II): Doxorubicin administered group (Dox):
This group will receive doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days (Amani et al., 2011).
3- Group (III): Doxorubicin + Nebivolol treated group (Dox + Neb):
This group will receive doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days and nebivolol in a dose of
1mg / kg / day orally daily for 12 days (Amani et al., 2011).
4- Group (IV): Doxorubicin + L-Carnitine treated group (Dox +L-
car):
This group will receive doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days (Amani et al., 2011) and L-
carnitine in a dose of 200 mg / kg body weight intraperitonially daily for
12 days ( Nathalie A. et al., 1999).
5- Group (V): Doxorubicin + Nebivolol & L-Carnitine group (Dox +
Neb +L-car):
This group will receive doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days, nebivolol in a dose of 1mg /
kg / day orally and L-Carnitine in a dose of 200 mg / kg body weight
intraperitonially daily for 12 days .
Materials and Method
48
Procedures:
(a) Cardiac investigations: (1) ECG:
Adult male albino rats, weighing about 150-200gm of each were used.
The animals were anaesthetized with urethane in a dose of 1.5- 1.75
gm/kg body weight. Half of the dose was injected intraperitoneally, to
induce rapid onset and the other half subcutaneously, to insure long
maintenance of the anaesthetic effect.
After complete anaesthesia, the rats were led on their back. ECG
records were done using needle electrodes. The four limb electrodes were
fixed to the animal's four limbs and records were done using the standard
lead II at rate of 25m/min. The use of lead II was more informative (in
rats) than other leads (Chan et al., 1987).
(2) Blood preasure measurement:
The blood pressure of rats was determined following the method of
Cangiano et al. (1978). The method can be outlined as follows:
The carotid artery was freed from its surrounding fascia for a
distance about 1.5 cm and then two loose ligatures were placed, one at
either end and around the freed artery. The artery was ligated at the end
far from the heart and the end near to the heart was temporarily occluded
with small bulldog clip. Now, half way across the artery was cut and the
arterial cannula filled with glucose and heparin solution (20.000 I.U liter
of 5% glucose solution) was inserted towards the heart. The cannula was
connected to the previously calibrated blood pressure transducer P.T and
the pressure was recorded as tracing by oscillograph 400 M.D. 4C.
palemr bioscience, Washington. A strain gauge copuler FC. 137 were
used.
Materials and Method
49
(3) Biochemical assessment.
Blood samples were collected from the retrobulbar sinus of rat's eye
by using heparinized capillary tubes (Schermer, 1967). Blood samples
were delivered in clean, dry test tubes and allowed to clot at room
temperature. The serum was separated by centrifugation at 2000
rounds/minute for 10 minutes.
a-Serum Tropinin I.
Serum Tropinin I activity was determined according to Bodor
method (Bodor, 1994).
Sample:
Serum sample from rats used in this study.
Reagents:
1. Antibody coated wells
2. Reference standered set
3- cTnl enzyme conjugate reagent
4- Tetramethyl-benzidine (TMB)Reagent
5- Stop solution (diluted hydrochloric acid)
Procedure:
1. Secure the desired number of coated wells in the holder
2. Dispense 100µ of standers, specimens, and controls into
appropriate wells.
3. Gently mix for 10 minutes
4. incubate at room temperature
5. Strike the wells sharply into absorbent paper to remove all residual
water droplets
6. Dispense 100µ of TMB Reagent into each well
Materials and Method
50
7. Read absorbance at 450nm with microtiter well reader within 15
minutes
Calculations:
Calculate the mean absorbance value for each set of reference
standards , controls and samples. Construct a standered curve. Using the
mean absorbance value for each sample we determine the corresponding
concentration of troponin I.
b- Serum Creatine phosphokinase :
Creatine phosphokinase activity was determined according to
Bergemeyer methods (Bergemeyer, 1974).
Principle:
Creatine phosphokinase in the serum catalyzes the removal of
phosphate moiety from p-nitrophenylphosphate. The rate of phosphate
release was measured calorimetrically. It is used as a measure for the
activity of the enzyme.
Sample:
Serum sample from rats used in this study were used.
Reagents:
1. Diethanolamine buffer solution .
2. Substrate solution contained p-nitroophenylphosphate .
Procedure:
1. The working solution was prepared by mixing 3 ml of substrate
solution to 10 ml of buffer solution. The solution was kept in
opaque plastic bottle. It is kept at 8oC for maximal of 3 days before
use.
Materials and Method
51
2. 2-0.01ml of serum was pipetted into clean, dry plastic cuvette of 1
ml volume.
3. 0.5 ml of working reagent solution was added to each cuvette then
carefully mixed and incubated for 1 minute at 37oC.
4. The absorbance of the solution was read at 405nm using
spectrophotometer. The instrument was calibrated using air as
blank.
5. The absorbance was read again after 2 and 3 minutes.
(4) Histopathological examination of the heart at end of the study :
Under ether anesthesia, sacrification of all animals was done at the
was opened and the heart was excised as a whole, preserved in formaline
and referred to histopathological examination.
(b) Hepatic investigations:
(1) Biochemical assessment:
- Serum alanine transaminase
Serum alanine transaminase activity was determined according to
Reitman and Frankel methods (Reitman and Frankel, 1957)
Principle:
Alanine transaminase content of the serum was used to catalyze the
transfer of amino group from L- alainne to α-oxoglutarate leading to
formation of L-glutamate and pyruvate.
Lactic dehydrogenase in reagent was used to catalyze the reduction of
pyrovate by NADH to L-lactate with formation of oxidized NAD.
The change in rate of absorbance as a result of the latter reaction was
determined at 340nm.
Reagents:
1. Buffer substrate solution contains.
Materials and Method
52
Phosphate buffer 80nmol/L at pH 7.4.
L-alanine 0.8mol/L.
2. Enzyme/coenzyme/α-oxoglutarate solution
Lactic dehydrogenase more than 2U/ml.
Reduced nicotine adenine dinucleotide phosphate (NADH) 0.18mmol/L.
Procedure:
1. 0.2ml of enzyme/coenzyme/α-oxoglutarate solution was pippted to
clean dry plastic cuvette of 1 ml volume.
2. 0.2 ml of serum sample was added to cuvette. The cuvette was
mixed carefully and transferred to 1 cm plastic cuvette.
3. The absorbance of the sample was read by spectrophotometer at
340 nm. The instrument was calibrated using air as blank. The
absorbance was read again after 2 and 3 minutes.
- Serum aspartate transaminase:
Serum aspartate transaminase activity was determined according to
Reitman & Frankel methods (Reitman and Frankel, 1957).
Principle
Aspartate transaminase content of the serum was used to catalyze
the transfer of amine group from L-alaine to α-oxoglutarate leading to
formation of L-glutamate and oxaloacetate.
Malate dehydrogenase in reagent was used to catalyze the
reduction of oxaloacetate by NADH to L-lactate with the formation of
oxidized NAD.
The change in rate of absorbance as a result of the latter reaction
was determined at 340nm.
Materials and Method
53
Sample:
Serum sample from rats used in this study.
Reagents:
1.Buffer substrate solution contains:
Phosphate buffer 80mmol/l, pH7.4.
L-aspartate 200mmol/l.
2.Enzyme/coenzyme/ α-oxoglutarate solution contains:
Malate dehydrogenase more than 0.6U/ml.
Lactate dehydrogenase more than 1.2 U/ml.
α oxoglutarate 12 mmol/l.
Reduced nicotine adenine dinucleotide phosphate (NADH)
0.18mmol/L.
Procedure:
1. Working solution was prepared by mixing buffer substrate solution
with enzyme/coenzyme/ α-oxoglutarate solution in 1:1/volume
ratio. 2-0.2 ml of the serum sample was put in clean dry plastic
cuvette of 1 ml volume.
2. 0.2 ml of the serum sample was put in clean dry plastic cuvette of 1
ml volume.
3. 1 ml of enzyme/ coenzyme/ α-oxoglutarate solution was added.
The tubes were carefully mixed and incubated at 37oC for 1
minute.
4. The solution was transferred to 1 ml plastic cuvette.
5. The absorbance of the sample was read by spectrophotometer at
240nm. The instrument was adjusted to zero using air as blank.
6. The absorbance was read again after 2 and 3 minutes.
Materials and Method
54
(2) Histopathological examination of the liver at the end of
the study.
Under ether anesthesia, sacrification of all animals was done at the
end of the experiments by a sharp blow on the head followed by
decapitation. Liver specimens were taken fixed in 40% formalin and
referred to hisotpatholgical examination (Drury and Wallington, 1967).
(c) Renal investigations: (1) Serum creatinine level measurement
Serum creatinine activity was determined according to Jaffe methods
(Jaffe, 1886).
Sample:
Serum sample from rats used in this study.
Reagents:
Creatinine acid reagent 3.6 mol/l.
Creatinine base reagent 240 mol/l.
Creatinine standered 5-mg /dl.
Procedure:
Set spectrophotometer cuvette temperature to 37oC
Put 1 ml of working reagent into test tubes and prewarm at37oC
for 3 minutes
Transfer 0.050 ml of standred to test tube , mix and immediately
place in cuvette tube
After exactly 20 seconds read and record absorbance (A1)
At exactly 60 seconds after reading (A1) read and record (A2)
Calculate change in absorbance A by subtracting (A1 - A2 )
Calculation:
Materials and Method
55
Serum creatinine (mg /dl( = Au ( unknown) X 5 .
As ( standered)
(2) Histopathological examination of the kidney at the end
of the study.
Under ether anesthesia, sacrification of all animals was done at the
end of the experiments by a sharp blow on the head followed by
decapitation. The abdomen will be opened and the kidney will be excised
as a whole, preserved in formaline and referred to histopathlogical
examination.
(b) In vitro experiment:
Langendorff's technique for isolated perfused heart was used. The
animal was sacrificed by cutting the throat. The chest was opened, the
heart quickly excised, and transported to a dish containing oxygenated
Ringer’s solution. The heart was thoroughly squeezed to expel any blood
from the chambers of the heart. The tissue was cleaned and fixed through
the aorta to the cannula of the Langendorff’s apparatus. The temperature
was kept constant by means of an electric automatic thermostat, a
thermometer was inserted through one side of the cannula to ensure a
constant temperature of the heart at 37°C throughout the experiment. A
hook was attached to the ventricular wall of the heart and was attached by
a thread to a side way lever which recorded the contractions of the
ventricle on slowly moving drum. Drugs were added to the cannula .The
experiment was repeated at least 6 times.
(A) The effect of isopernaline on contractility of hearts taken from
another 5 groups of rats given the same medications as the groups of the
in-vivo experiment.
(B) The effect of doxorubicin on contractility of isolated normal
mammalian heart
Materials and Method
56
Statistical Analysis:
All data were expressed as Mean + SEM. Difference between the groups
were compared by Student s T-test with P- value < 0.05 selected as the
level of statistical significance (Hill, 1971).
Statistical methods
The processing of the data required the calculation of the following
parameters:
1 - Arithmetic mean is used as a measure of the central tendency
n
XX
X Arithmetic mean
X Sum of values recorded.
n= number of observations.
2 - Standard error of the mean (S.E.) is used as measure of precision and
statistical reliability of the mean:
)1(.
2
nn
dES
d2 = Spread of differences between different values and the mean (X and
X ).
3 - Standard deviation (S.D.) is used as a measure of dispersion
1.
2
n
dDS
4 - Student's "t": test of significance of the difference between two mean
values (m1 and m2).
Materials and Method
57
2121
2
2
2
1
21
11
2 nnnn
dd
mmt
4- The statistically significance of data was analyzed by using paired
student's t-test for intra group comparison and unpaired student's t-test for
inter group comparison.
* All data are expressed as mean ± S.E.M.
* A "p" of less than 0.05 was regarded as significant.
The statistical analysis was carried out using the computer program
SPSS (Statistical program for social science).
Results
58
Results I. In vivo experiments:
(A) Cardiac results: 1- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in T wave in rats:
Administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in significant
elevation (P< 0.01) in T wave voltage from 0.3 + 0.17 m volt in control
group to 0.6 + 0.07 m volt in Dox group (Table 1 & Fig 3 ).
In Dox +Neb group giving nebivolol in a dose of 1mg / kg / day orally
for 12 days reduced T wave voltage significantly (P < 0.05) to 0.45 + 0.06
m volt compared to Dox group ( Table 1 & Fig 3 ).
In Dox+L-car group giving L- carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days reduced T wave voltage significantly
(P<0.05) to0.46+ 0.02 m volt compared to Dox group( Table 1& Fig 3 ).
In Dox+Neb+L-car group giving both nebivolol in a dose of 1mg / kg
/ day for 12 days & L-Carnitine in a dose of 200 mg / kg body weight
intraperitonially for 12 days reduced T wave voltage significantly (P <
0.05) to 0.4 + 0.04 m volt compared to Dox group (Table 1 & Fig 3 ).
Comparing the result of Dox+Neb +L- car group with that of
Dox+Neb group, adding L-carnitine to nebivolol was not significantly (P
< 0.05) more effective than nebivolol alone in reducing T wave voltage
( Table 1 & Fig 3 ).
Comparing the result of Dox+Neb+L- car group with that of Dox +L-
car group, adding nebivolol to L- carnitine was not significantly (P <
0.05) more effective than nebivolol alone in reducing T wave voltage
( Table 1 & Fig 3 ).
Results
59
Table ( 1 ): Showing changes of T wave voltage in various groups:
-Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox + Neb group.
P3 : compared with Dox + L- car group
0.3
0.6*
0.45** 0.46**
0.4**
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-car
Tw
ave v
olt
ag
e (
m v
olt
)
Figure ( 3 ): Histogram showing changes of T wave voltage in various
groups.
* Significant compared with control group
** Significant compared with Dox group
Parameter
Group
T wave voltage (m volt)
control
0.3 + 0.07
Dox
P
0.6 + 0.15
< 0.01
Dox + Neb
P1
0.45 + 0.09
< 0.05
Dox +L- car
P1
0.46 + 0.08
< 0.05
Dox +Neb+L- car
P1
P2
P3
0.4 + 0.04
< 0.05
> 0.05
> 0.05
Results
60
2- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in heart rate:
Administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in significant rise
(P< 0.01) in heart rate from 230 + 17.7 beat /minute in control group to
313.3+ 19.1 beat /minute in Dox group (Table 2 & Fig. 4 ).
In Dox +Neb group giving nebivolol in a dose of 1mg / kg / day orally
in for 12 days resulted in insignificant change in the heart rate (P < 0.05)
to 303+ 34.8 beat /minute compared to Dox group ( Table 2 & Fig 4 ).
In Dox+L-car group giving L- carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days resulted in insignificant change in the
heart rate (P < 0.05) to 310 + 13.6 beat /minute compared to Dox group
( Table 2 & Fig 4 ).
In Dox+Neb+L-car group giving both nebivolol in a dose of 1mg / kg /
day for 12 days & L-Carnitine in a dose of 200 mg / kg body weight
intraperitonially for 12 days resulted in insignificant change in the heart
rate (P < 0.05) to 312+ 18.9 beat /minute compared to Dox group
( Table 2 & Fig 4 ).
Comparing the result of Dox+Neb +L- car group with that of
Dox+Neb group, adding L-carnitine to nebivolol was not significantly (P
< 0.05) more effective than nebivolol alone in changing heart rate g the
result of Dox+Neb+L- car group with that of Dox +L-car group, adding
nebivolol to L- carnitine was not significantly (P < 0.05) more effective
than nebivolol alone in changing heart rate ( Table 2 & Fig 4 ).
Results
61
Table ( 2 ): Showing changes of heart rate in various groups:
-Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox + Neb group.
P3 : compared with Dox + L- car group
230
313.3*303.3 310 312
0
50
100
150
200
250
300
350
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-car
Hea
rt r
ate
(b
ea
t / m
inu
te)
Figure (4): Histogram showing changes of heart rate in various
groups.
* Significant compared with control group
Parameter
Group
Heart rate (beat /second)
control 230 +17.7
Dox
P
313.4 + 19.1
< 0.01
Dox + Neb
P1
303.3 + 34.8
> 0.05
Dox +L- car
P1
310 + 13.6
> 0.05
Dox +Neb+L- car
P1
P2
P3
312 + 18.9
> 0.05
> 0.05
> 0.05
Results
62
3- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in QRS voltage in rats:
Administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in insignificant
change (P < 0.05) in QRS voltage respectively from 8.3 + 0.11 m volt in
control group to 7.9 + 0.13 m volt in Dox group (Table 3 & Fig.5 ).
In Dox +Neb group giving nebivolol in a dose of 1mg / kg / day orally
for 12 days resulted in insignificant change (P < 0.05) in QRS voltage
respectively from 8.1 + 0.03 m volt to 7.9 + 0.13 m in Dox group
( Table 3 & Fig 5 ).
In Dox+L-car group giving L- carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days resulted in insignificant change (P <
0.05) in QRS voltage respectively from 8.2 + 0.15 m volt to 7.9 + 0.13 m
volt in Dox group ( Table 3 & Fig 5 ).
In Dox+Neb+L-car group giving both nebivolol in a dose of 1mg / kg /
day for 12 days & L-Carnitine in a dose of 200 mg / kg body weight
intraperitonially for 12 days resulted in insignificant change (P < 0.05) in
QRS voltage respectively from 8 + 0.07 m volt to 7.9 + 0.13 m volt in Dox
group ( Table 3 & Fig 5 ).
Comparing the result of Dox+Neb +L- car group with that of
Dox+Neb group, adding L-carnitine to nebivolol was not significantly (P
< 0.05) more effective than nebivolol alone in changing QRS voltage
( Table 3 & Fig 5 ).
Comparing the result of Dox+Neb+L- car group with that of Dox +L-
car group, adding nebivilol to L- carnitine was not significantly (P < 0.05)
more effective than nebivilol alone in changing QRS voltage ( Table 3 &
Fig 5 ).
Results
63
Table ( 3 ): Showing changes of QRS voltage in various groups :
-Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox + Neb group.
P3 : compared with Dox + L- car group
8.37.9 8.1 8.2 8
0
1
2
3
4
5
6
7
8
9
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-
car
QR
S v
olt
ag
e (
m v
olt
)
Figure ( 5 ): Histogram showing changes of QRS voltage in various
groups.
Parameter
Group
QRS voltage (m volt)
control 8.3 + 0.13
Dox P
7.9 + 0.11
> 0.05
Dox + Neb P1
8.1 + 0.13
> 0.05
Dox +L- car P1
8.2 + 0.15
> 0.05
Dox +Neb+L- car
P1
P2
P3
8 + 0.07
> 0.05
> 0.05
> 0.05
Results
64
4- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in systolic blood prssure & mean blood pressure:
Administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in significant drop
(P< 0.001) in systolic blood & mean blood pressure respectively from
121 + 4.8 mmHg and 97.7 + 2.6 mmHg in control group to 88.3+ 4
mmHg and 76.3 + 2.8 mmHg in Dox group ( Table 4 & Fig. 6,7 ).
In Dox+Neb group giving nebivolol in a dose of 1mg / kg / day
orally for 12 days resulted in significant elevation in systolic blood
prssure & mean blood pressure (P < 0.05) to 99.8 + 0.17 and 92 + 1
mmHg respectively compared to Dox group ( Table 4 & Fig. 6,7 ).
In Dox +L-car group giving L- carnitine in a dose of 200 mg / kg
body weight intraperitonially for 12 days resulted in significant
elevation in systolic blood prssure & mean blood pressure (P < 0.001) to
123.3 + 4.9 and 94.5+4.3 mmHg respectively compared to Dox group (
Table 4 & Fig. 6,7 ).
In Dox+Neb+L-car group giving both nebivolol in a dose of 1mg /
kg / day orally for 12 days & L-Carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days resulted in significant elevation in
systolic & mean blood pressure (P < 0.01) to 120 + 6.3& 96 + 6.7 mmHg
respectively compared to Dox group (Table 4 & Fig. 6, 7).
Comparing the result of Dox +Neb+L- car group with that of
Dox+Neb group, adding L-carnitine to nebivilol was significantly (P
<0.05) more effective than nebivilol alone in elevating systolic & mean
blood pressure(P< 0.05) ( Table 4 & Fig. 6,7 ).
Comparing the result of Dox+Neb+L- car group with that of Dox+L-
car group, adding nebivilol to L- carnitine was not significantly (P < 0.05)
more effective than nebivilol alone in elevating systolic blood prssure &
mean blood pressure ( Table 4 & Fig. 6,7 ).
Results
65
Table ( 4 ): Showing changes of systolic blood pressure & mean blood
pressure in various groups :
-Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox+ Neb group.
P3 : compared with Dox+L- car group
Parameter
Group
Systolic blood
pressure (mmHg)
Mean blood
pressure (mmHg)
control
121.7 + 4.8
97.7+ 2.6
Dox
P
88.3 + 4
< 0.001
76.3+2.8
< 0.001
Dox +Neb
P1
99.8 + 0.17
< 0.05
92 + 1
< 0.05
Dox +L- car
P1
123.3 + 4.9
< 0.001
94.5 + 4.3
< 0.001
Dox+Neb+L- car
P1
P2
P3
120 + 6.3
< 0.01
< 0.05
> 0.05
96 + 6.7
< 0.01
< 0.05
> 0.05
Results
66
121.7
88.3*
99.8**
123.3**120**
0
20
40
60
80
100
120
140
Control Dox Dox+Neb Dox+L-car Dox+Neb+
L-car
Sy
sto
lic
blo
od
pre
ss
ure
( m
mH
g (
Animal groups
Figure ( 6 ): Histogram showing Systolic blood pressure in various
groups.
* Significant compared with control group
** Significant compared with Dox group
97.7
76.3*
92** 94.5** 96**
0
20
40
60
80
100
120
Control Dox Dox+Neb Dox+L-car Dox+Neb+
L-car
Me
an
art
eri
al
blo
od
pre
ss
ure
(m
mH
g)
Animal groups
Figure (7): Histogram showing mean arterial blood pressure in
various groups.
* Significant compared with control group
** Significant compared with Dox group
Results
67
Fig. ( 8 ): Blood pressure and ECG measurement of control group.
(A typical trace of 6 seprate experiments)
Fig. ( 9): Blood pressure and ECG measurement of Dox group.
(A typical trace of 6 seprate experiments)
Fig. (10): Blood pressure and ECG measurement of Dox+ Neb group.
(A typical trace of 6 seprate experiments)
Results
68
Fig.(11): Blood pressure and ECG measurement of Dox+ L-Car
group. (A typical trace of 6 seprate experiments)
Fig. ( 12 ): Blood pressure and ECG measurement of Dox + Neb + L-
car group. (A typical trace of 6 seprate experiments)
Results
69
5- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in cardiac enzymes in rats:
Administration of doxorubicine in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in significant rise
(P< 0.001) in cardiac enzymes creatine phosphokinase and troponin I
from 317 + 86 u / l and 0.26 +0.17 ng / ml respectively in control group
to 1187.6 +63 u / l and 1.2+0.23 ng / ml in Dox group (Table & Fig. ).
In Dox+Neb group giving nebivolol in a dose of 1mg / kg / day orally
for 12 days reduced the cardiac enzymes creatine phosphokinase and
troponin I significantly (P < 0.01) to 869 + 28.7 u / l and 0.75+0.03 ng / ml
respectively compared to Dox group ( Table 5 & Fig. 13,14 ).
In Dox +L-car group giving L- carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days reduced the cardiac enzymes creatine
phosphokinase and troponin I significantly (P < 0.01) to 851 + 46.2 u / l
and 0.7 + 0.054 ng / ml respectively compared to Dox group ( Table 5 &
Fig. 13,14 ).
In Dox +Neb+ L-car group giving both nebivolol in a dose of 1mg / kg
/ day for 12 days & L-carnitine in a dose of 200 mg / kg body weight
intraperitonially for 12 days reduced the cardiac enzymes significantly (P<
0.01) to 755 + 43. 9 u / l & 0.63 + 0.036 ng / ml respectively compared to
Dox group ( Table 5 & Fig. 13,14 ).
Comparing the result of Dox+Neb+L-car group with that of Dox+Neb
group, adding L-carnitine to nebivilol was not significantly (P < 0.05)
more effective than nebivilol alone in reducing the cardiac enzymes
( Table 5 & Fig. 13,14 ).
Comparing the result of Dox+Neb+ L- car group with that of Dox + L-
car group, adding nebivilol to L- carnitine was not significantly (P < 0.05)
more effective than nebivilol alone in reducing the cardiac enzymes (
Table 5 & Fig. 13,14 ).
Results
70
Table ( 5 ): Showing changes of CPK & troponin I level in various
groups :
- Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox+ Neb group.
P3 : compared with Dox+ L- car group.
317
1187.6*
869**851.2**
755**
0
200
400
600
800
1000
1200
1400
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-car
Mean
CP
K (
u/l
)
Figure (13): Histogram showing changes of CPK level in various
groups.
* Significant compared with control group
** Significant compared with Dox group
Parameter
Group
CPK(u / l)
Troponin I (ng/ml )
Control 317 + 34 0.26 +0.17
Dox
P
1187.6 + 45
< 0.001
1.2 + 0.23
< 0.001
Dox + Neb
P1
869 + 28.7
< 0.01
0.75+0.03
< 0.01
Dox + L- car
P1
851.2 + 26.2
< 0.01
0.7 + 0.054
< 0.01
Dox+ Neb + L- car
P1
P
P3
755 + 33.2
< 0.001
> 0.05
> 0.05
0.63 +0.036
< 0.01
> 0.05
> 0.05
Results
71
0.26
1.2*
0.75**0.7**
0.63**
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-
car
Tro
po
nin
I (
ng
/ml)
Figure (14): Histogram showing changes of troponin I level in various
groups.
* Significant compared with control group
** Significant compared with Dox group
5- Histopathological evaluation of cut sections of the heart:
In the Dox group, there was cardiac myocyte degenerative changes
in the from of cell swelling, loss of cytoplasmic striations, with mild
interstitial fibrosis (Fig. 16,17 ).
The doxorubicin induced myocardial damage was improved in
nebivilol and l-carnitine and nebivilol & l-carnitine treated rats in
comparison to the Dox group, as the myocyte degeneration as well as the
interstitial fibrosis were decreased ( Fig. 18,19,20 )
Results
72
Fig. ( 15 ): Cut section of normal myocardial tissues (H & E 100).
Figure (16) : Cut section of myocardium of Dox group showing,
(A)eosynophylic cytoplasm , inflammatory infiltration(B) intestitial
edema congested capillaries between irregular wavy-directed
cardiomiocytes. (H x & E x 100).
Results
73
Figure (17) : Cut section of myocardium & percardium of Dox group
showing, (A)eosynophylic cytoplasm , inflammatory infiltration
(B)interstitial edema congested capillaries. (H x & E x 100).
Figure (18) : Cut section of myocardium of Dox+ Neb group showing
(A)eosynophylic cytoplasm , inflammatory infiltration(B) interstitial
edema congested capillaries (H x & E x 100).
Results
74
Figure (19) : Cut section of myocardium of Dox +L-car group
showing (A)eosynophylic cytoplasm , inflammatory infiltration
(B) interstitial edema, congested capillaries (H x & E x 100).
Figure (20 ) : Cut section of myocardium of Dox+Neb +L-carnitine
group showing interstitial edema (H x & E x 100).
Results
75
(B) Hepatic results: 1- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in liver enzymes ALT & AST level in rats: Administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in significant rise
(P< 0.001) in liver enzymes ALT & AST level from 34 + 5.77 u / l and
47 + .95 u / l respectively in control group to 89.2 + 3.08 u / l and 95 +
2.46 u / l in doxorubicin administered group ( Table 6 & Fig. 21,22 ).
In Dox + Neb group giving nebivolol in a dose of 1mg / kg / day
orally for 12 days reduced the cardiac liver enzymes ALT& AST
significantly (P < 0.01) to 45.7 + 2.08 u / l and 55 + 2.39 u / l
respectively compared to Dox group ( Table 6 & Fig. 21,22 ).
In Dox + L-Car group giving L- carnitine in a dose of 200 mg / kg
body weight intraperitonially for 12 days reduced the liver enzymes
ALT& AST significantly (P < 0.01) to 48 + 2.65 u / l and 60 + 2.86 u / l
respectively compared to Dox group ( Table 6 & Fig. 21,22 ).
In Dox +Neb + L_Car group giving both nebivolol in a dose of 1mg /
kg / day orally for 12 days & L-Carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days reduced the cardiac enzymes
significantly (P< 0.01) to 44 + 1.78 u / l & 50 + 2.86 u / l respectively
compared to Dox group ( Table 6 & Fig. 21,22 ).
Comparing the result of Dox +Neb + L_Car group with that of Dox +
L-car group, adding nebivolol to L- carnitine was not significantly (P <
0.05) more effective than nebivolol alone in reducing the liver enzymes
( Table 6 & Fig. 21,22 ).
Comparing the result of Dox +Neb + L_Car group with that of Dox +
Neb group, adding L-carnitine to nebivolol was not significantly (P <
0.05) more effective than nebivolol alone in reducing the liver enzymes
( Table 6 & Fig. 21,22 ).
Results
76
Table ( 6 ): Showing changes of ALT & AST level in various groups :
-Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox+Neb group.
P3 : compared with Dox+ L- car group.
Parameter
Group
ALT ) u \ l)
AST ) u \ l)
control
34 + 5.77
47 + 0.95
Dox
P
89.2 + 3.08
< 0. 001
95 + 2.46
< 0.0 01
Dox + Neb
P1
45.7 + 2.08
< 0.001
55 + 2.39
< 0.01
Dox + L- car
P2
48 + 2.65
< 0.001
60 + 2.86
< 0.01
Dox+ Neb + L- car
P1
P2
P3
44 + 1.78
< 0.001
>0.05
>0.05
50 + 2.86
< 0.01
> 0.05
> 0.05
Results
77
34
89.2*
45,7** 48**44**
0
10
20
30
40
50
60
70
80
90
100
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-
car
AL
T (
u/l)
Figure (21): Histogram showing changes of ALT level in various
groups.
* Significant compared with control group
** Significant compared with Dox group
47
95*
55**60**
50**
0
20
40
60
80
100
120
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-
car
AS
T (
u/l
)
Figure (22): Histogram showing changes of AST level in various
groups.
* Significant compared with control group
** Significant compared with Dox group
Results
78
2- Histopathological evaluation:
The liver of the Dox group showed marked hydropic changes,
some necro-infilammmatory foci and portal tract infilammation (fig.
24 ).
The doxorubicin induced liver damage was improved in nebivolol
and l-carnitine and nebivolol & l-carnitine treated rats in comparison to
the Dox group, as hydropic changes, necro-infilammmatory foci and
portal tract infilammation were decreased ( fig. 25, 26, 27 ).
Figure ( 23 ): A photomicrograph of a cut section in the liver of a control rat
showing normal liver architecture composed of hexagonadal or pentagonadal
lobules with central veins and peripheral hepatic triads or tetrads embedded in
connective tissue. Hepatocytes are arranged in trabecules running radiantly
from the central vein and are separated by sinusoids containing Kupffer cells.
(H x & E x 400).
Results
79
Figure (24 ): A photomicrograph of a cut section in the liver of Dox group
showing preserved liver architecture, (A) hepatocytes show marked hydropic
changes, (B)some necro-infilammmatory foci & (C) portal tract infilammation
(H x & E x 400)
Figure (25 ): A photomicrograph of a cut section in the liver in Dox+Neb
group, showing preserved liver architecture, hepatocytes show (A) hydropic
changes , no necro-infilammmatory foci (B) congested central vein (H x & E x
400)
Results
80
Figure ( 26 ): A photomicrograph of a cut section in the liver in Dox + L- car
group, showing preserved liver architecture , (A) hepatocytes show hydropic
change (B) infilammmatory foci (C) congested central vein (H x & E x 400).
Figure ( 27 ): A photomicrograph of a cut section in the liver in Dox+ Neb+L-
car group showing preserved liver architecture, (A)hepatocytes show moderate
hydropic changes , (B)few infilammmatory foci and no portal tract
infilammation. (H x & E x 400).
Results
81
(C) -Renal results:
1- Effect of treatment with nebivolol & L-Carnitine on doxorubicin
induced changes in creatinine level in rats:
Administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days resulted in significant rise
(P< 0.001) in creatinine level from 0.65 + 0.05 mg / dl in control group
to0.95 + 0 .02 mg / dl in Dox group ( Table 7 & Fig 28 ).
In Dox +Neb group giving nebivolol in a dose of 1mg / kg / day
orally for 12 days reduced the creatinine level significantly (P < 0.01) to
0.72 + 0.04 mg / dl compared to Dox group ( Table 7 & Fig 28 ).
In Dox +L- Car group giving L- carnitine in a dose of 200 mg / kg
body weight intraperitonially for 12 days reduced the creatinine level
significantly (P < 0.01) to 0.74 +0 .07 mg / dl compared to Dox group
( Table 7 & Fig 28 ).
In Dox +Neb + L- car group giving both nebivolol in a dose of 1mg /
kg / day orally for 12 days & L-Carnitine in a dose of 200 mg / kg body
weight intraperitonially for 12 days reduced the creatinine level
significantly (P< 0.001) to 0.69 +0 .02 mg / dl respectively compared to
Dox group ( Table 7 & Fig 28 ).
Comparing the result of Dox+Neb+L- car group with that of Dox+ L-
car group, adding nebivilol to L- carnitine was not significantly (P <
0.05) more effective than nebivilol alone in reducing the creatinine level
( Table 7 & Fig 28 ).
Comparing the result of Dox+Neb + L- car group with that of Dox +
Dox+Neb group, adding L-carnitine to nebivolol was not significantly (P
< 0.05) more effective than nebivolol alone in reducing the creatinine
level ( Table 7 & Fig 28 ).
Results
82
Table ( 7 ): Showing changes of creatinine level in various groups :
-Data represented as Mean ± SEM (n = 6) - Significant level at P < 0.05
P : compared with control group. P1 : compared with Dox group.
P2 : compared with Dox+Neb group.
P3 : compared with Dox+ L- car group
Parameter
Group
Creatinine (mg \dl)
control
0.65+ 0.05
Dox
P
0.95 + 0.02
< 0.001
Dox+ Neb
P1
0.72 + 0.04
< 0.01
Dox + L- car
P1
0.74 + 0.07
< 0.01
Dox+ Neb + L- car
P1
P2
P3
0.69 +0.02
< 0.001
> 0.05
> 0.05
Results
83
0.65
0.95*
0.72** 0,74**0.69**
0
0.2
0.4
0.6
0.8
1
1.2
Control Dox Dox+Neb Dox+L-car Dox+Neb+L-
car
Cre
ati
nin
e l
eve
l (m
g/d
l)
Figure (28 ): Histogram showing creatinine level in various groups.
* Significant compared with control group
** Significant compared with Dox group
2- Histopathological evaluation:
The kidney of the Dox group shows glomerulopathy characterized
by mesangeal proliferation, tubular atrophy & dilatation and congestion
of interstitial capillaries (Fig. 30 ).
The doxorubicin induced kidney damage was improved in
nebivilol and l-carnitine and nebivilol & l-carnitine treated rats in
comparison to the Dox group, as the mesangeal proliferation, tubular
atrophy & dilatation and congestion of interstitial capillaries was
decreased ( Fig. 31, 32, 33 ).
Results
84
Fig. ( 29 ): Cut section of normal kidney tissue (H & E 100)
Fig. (30): Cut section of kidney tissue of Dox group showing
glomerulopathy characterized by(A) mesangeal proliferation,
(B)tubular atrophy & (C)dilatation and congesion of interstitial
capillaries (H & E 100).
Results
85
Fig. (31): Cut section of kidney tissue of Dox+Neb group showing (A)
mesangeal proliferation and (B)some congestion of interstitial
capillaries (H & E 100).
Fig. ( 32 ): Cut section of kidney tissue of Dox+L- car group showing
(A)mesangeal proliferation (B) congesion of interstitial capillaries
(H & E 100)
Results
86
Fig. (33 ): Cut section of kidney of Dox+Neb+L-car group showing
(A) some mesangeal proliferation (H & E 100)
Results
87
II. In vitro experiments:
(A) The effect of isopernaline on contractility of hearts taken from
rats treated as the groups of the in-vivo experiment:
Effect of isoprenaline on isolated perfused rat’s hearts of
control group:
It was noticed that isoprenaline in a gradually increasing doses (2 ,
4 and 8 μg/bath) produced an increase in the force of spontaneous
contraction of isolated perfused rat’s heart in dose related manner.
This increase of the force of spontaneous contractions of the
isolated perfused rabbit’s heart was significant (P < 0.05) with the dose of
2µg/bath with percentage of increase 63.6 % compared to pre-
isoprenaline contraction.Also it was significant (P < 0.05) with the dose
of 4µg/bath compared to preisoprenaline contraction and with percentage
of increase 85.2 % .Meanwhile it was significant (P < 0.01) with the dose
of 8µg/bath compared to preisoprenaline contraction and with percentage
of increase 110.5 % ( Table 8 & Fig 34, 35 ).
Table ( 8 ): Effect of isoprenaline on the amplitude of spontaneous
rhythmic contraction of isolated perfused rat heart in control group.
Dose of
isoprenaline
µg/ml bath
Amplitude of
contraction
before
isoprenaline (cm)
Amplitude of
contraction after
isoprenaline (cm)
Percentage
changes (%)
P
2
3.24 ± 0.6
5.3 ± 0.2
63.6 %
< 0.05*
4
6 ± 0.3
85.2 %
< 0.05*
8
6.8 ± 0.26
110.5 %
< 0.01*
Data represented as mean ± SEM of six experiments.
* Significant level at (P < 0.05) compared to pre isoprenaline value.
Results
88
3.24
6*
6.8*
5.3*
0
1
2
3
4
5
6
7
8
Preisoprenaline
2µg isoprenaline
4µg isoprenaline
8µg isoprenaline
Am
pli
tud
e o
f co
ntr
acti
on
(c
m)
Figure ( 34 ): Histogram showing the effect of isoprenaline on
isolated rat’s heart of control group.
* Significant ( P < 0.05) compared to pre isoprenaline value.
Fig. ( 35 ) : A record demonstrating the effect of gradually increasing
concentrations of isoprenaline on the isolated perfused rat's heart
contractions in control group(A typical trace of 6 seprate experiments)
Results
89
Effect of isoprenaline on isolated perfused rat’s hearts of Dox
group:
It was noticed that isoprenaline in increasing doses (2 and
4μg/bath) produced a non significant increase in the force of spontaneous
contraction of isolated perfused rat’s heart (P > 0.05) .
This increase of the force of spontaneous contractions of the
isolated perfused rat’s heart was significant (P < 0.05) with the dose of
8g/bath with percentage of increase 59.1 % compared to preisoprenaline
contraction ( Table 9 & Fig 36, 37 ).
Table ( 9 ): Effect of isoprenaline on the amplitude of spontaneous
rhythmic contraction of isolated perfused rat heart in Dox group.
Dose of
isoprenaline
µg/ml bath
Amplitude of
contraction before
isoprenaline (cm)
Amplitude of
contraction after
isoprenaline (cm)
Percentage
changes (%)
P
2
1.15 ± 0.27
1.2 ± 0.28
4.3 %
> 0.05
4
1.43 ± 0.34
24.2 %
> 0.05
8
1.83 ± 0.48
59.1 %
< 0.05 *
Data represented as mean ± SEM of six experiments.
* Significant level at (P < 0.05) compared to pre isoprenaline value.
Results
90
1.15
1.43
1.83*
1.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Preisoprenaline
2µg isoprenaline
4µg isoprenaline
8µg isoprenaline
Am
pli
tud
e o
f co
ntr
acti
on
(c
m)
Figure (36): Histogram showing the effect of isoprenaline on
isolated rat’s heart of Dox group.
* Significant ( P < 0.05) compared to pre isoprenaline value.
Fig. (37 ): A record demonstrating the effect of gradually increasing
concentrations of isoprenaline on the isolated perfused rat's heart
contractions in Dox group (A typical trace of 6 seprate experiments)
Results
91
Effect of isoprenaline on isolated perfused rat’s hearts of Dox
+ Neb group:
It was noticed that isoprenaline in increasing doses (4 and 8
μg/bath) produced an increase in the force of spontaneous contraction of
isolated perfused rat’s heart in dose related manner.
This increase of the force of spontaneous contractions of the
isolated perfused rat’s heart was not significant (P > 0.05) with the dose
of 2µg/bath with percentage of increase 27.3 %. Meanwhile it was
significant (P < 0.05) with the dose of 4µg/bath with percentage of
increase 51.5 % and it was significant (P < 0.01) with the dose of
8µg/bath and with percentage of increase 118.2 % compared to
preisoprenaline contraction ( Table 10 & Fig 38 , 39 ).
Table (10): Effect of isoprenaline on the amplitude of spontaneous
rhythmic contraction of isolated perfused rat heart in Dox+
Neb group.
Dose of
isoprenaline
µg/ml bath
Amplitude of
contraction before
isoprenaline (cm)
Amplitude of
contraction after
isoprenaline (cm)
Percentage
changes (%)
P
2
1.65 ± 0.22
2.1± 0.17
27.3 %
> 0.05
4
2.5 ± 0.27
51.5 %
< 0.05 *
8
3.6 ± 0.41
118.2 %
< 0.01 *
Data represented as mean ± SEM of six experiments.
* Significant level compared to pre isoprenaline value.
Results
92
1.65
2.5*
3.6*
2.1
0
0.5
1
1.5
2
2.5
3
3.5
4
Preisoprenaline
2µg isoprenaline
4µg isoprenaline
8µg isoprenaline
Am
pli
tud
e o
f co
ntr
acti
on
(c
m)
Figure (38): Histogram showing the effect of isoprenaline on isolated
rat’s heart of Dox +Neb group. * Significant compared to pre isoprenaline value.
Fig. ( 39 ) : A record demonstrating the effect of gradually increasing
concentrations of isoprenaline on the isolated perfused rat's heart
contractions in Dox+Neb group(A typical trace of 6 seprate experiments)
Results
93
Effect of isoprenaline on isolated perfused rabbit’s hearts of
Dox+L- car group:
It was noticed that isoprenaline in increasing doses (4 and 8
μg/bath) produced an increase in the force of spontaneous contraction of
isolated perfused rabbit’s heart in dose related manner.
This increase of the force of spontaneous contractions of the
isolated perfused rabbit’s heart was not significant (P > 0.05) with the
dose of 2µg/bath with percentage of increase 25.5 %. Meanwhile it was
significant (P < 0.05) with the dose of 4µg/bath percentage of increase
36.2 % and it was significant (P < 0.05) with the dose of 8µg/bath and
with percentage of increase 58.7 % compared to preisoprenaline
contraction ( Table 11& Fig 40, 41 ).
Table (11): Effect of isoprenaline on the amplitude of spontaneous
rhythmic contraction of isolated perfused rat heart in Dox+L- car
group.
Dose of
isoprenaline
µg/ml bath
Amplitude of
contraction before
isoprenaline (cm)
Amplitude of
contraction after
isoprenaline (cm)
Percentage
changes (%)
P
2
2.35 ± 0.33
2.9 ± 0.38
25.5 %
> 0.05
4
3.2 ± 0.41
36.2 %
< 0.05 *
8
3.6 ± 0.38
58.7 %
< 0.05 *
Data represented as mean ± SEM of six experiments.
* Significant level at (P < 0.05) compared to pre isoprenaline value.
Results
94
2.35
3.2*
3.6*
2.9
0
0.5
1
1.5
2
2.5
3
3.5
4
Preisoprenaline
2µg isoprenaline
4µg isoprenaline
8µg isoprenaline
Am
pli
tud
e o
f co
ntr
acti
on
(c
m)
Figure (40 ): Histogram showing the effect of isoprenaline on isolated
rat’s heart of Dox+ L- car group
* Significant ( P < 0.05) compared to pre isoprenaline value.
Fig. ( 41 ) : A record demonstrating the effect of gradually increasing
concentrations of isoprenaline on the isolated perfused rat's heart
contractions in Dox+L-car group(A typical trace of 6 seprate experiments)
Results
95
Effect of isoprenaline on isolated perfused rat’s hearts of Dox +
Neb +L- car group.:
It was noticed that isoprenaline in increasing doses (2 , 4 and 8
μg/bath) produced an increase in the force of spontaneous contraction of
isolated perfused rat’s heart in dose related manner.
This increase of the force of spontaneous contractions of the
isolated perfused rat’s heart was significant (P < 0.01) with the dose of
2µg/bath with percentage of increase 48.6 % , also it was significant (P <
0.01) with the dose of 4µg/bath and with percentage of increase 79.4 %
and it was significant (P < 0.001) with the dose of 8µg/bath and with
percentage of increase 106.2 % compared to preisoprenaline contraction
( Table 12 & Fig 42, 43 )
Table (12): Effect of isoprenaline on the amplitude of spontaneous
rhythmic contraction of isolated perfused rat heart in Dox + Neb +L-
car group:
Dose of
isoprenaline
µg/ml bath
Amplitude of
contraction before
isoprenaline (cm)
Amplitude of
contraction after
isoprenaline (cm)
Percentage
changes (%)
P
2
2.4 ± 0.31
3.6 ± 0.46
48.6 %
< 0.01 *
4
4.37 ± 0.47
79.4 %
< 0.01 *
8
5.1 ± 0.41
106.2 %
< 0.001 *
Data represented as mean ± SEM of six experiments.
* Significant level at (P < 0.05) compared to pre isoprenaline value.
Results
96
2.4
4.37*
5.1*
3.6*
0
1
2
3
4
5
6
Preisoprenaline
2µg isoprenaline
4µg isoprenaline
8µg isoprenaline
Am
pli
tud
e o
f co
ntr
acti
on
(c
m)
Figure (42 ): Histogram showing the effect of isoprenaline on isolated
rat’s heart of Dox + Neb +L- car group. * Significant ( P < 0.01) compared to pre isoprenaline value.
Fig. ( 43 ) : A record demonstrating the effect of gradually increasing
concentrations of isoprenaline on the isolated perfused rat's heart
contractions in Dox+Neb+L-car group(A typical trace of 6 seprate
experiments)
Results
97
Table (13 ): Percent change of contraction of isolated perfused rat
heart in different groups after isoprenaline different doses 2,4,8 µg :
Dose of
isoprenaline
µg/ml bath
Control
group
Doxorubicin
group
Nebivolol
group
L-carnitine
group
Nebivolol &
L-carnitine
group
2
63.6 %
4.3 %
27.3 %
25.5 %
48.6 %
4
85.2 %
24.2 %
51.5 %
36.2 %
79.4 %
8
110.5 %
59.1 %
118.2 %
58.7 %
106.2 %
In the control group the percent change of contraction of isolated
perfused rat heart 63.6 %, 85.2 %, 110.5 % after
isoprenaline different doses 2,4,8 µg respectively but in the Dox
group the percent change was markedly decreased to 4.3 %,
24.2 %, 59.1 % and in the Dox+Neb group the percent change
was markedly improved compared to Dox group to be 27.3 %,
51.5 %, 118.2 % respectively , in the l- carnitine group the
percent change was also markedly improved compared to Dox
group to be 25.5 %, 36.2 %, 58.7 % respectively and finaly in
the Dox + Neb +L- car group the percent change showed the best
improvement compared to Dox group to be 48.6 %, 79.4 %,
106.2 % respectively
Results
98
(B) The effect of Doxorubicin on contractility of isolated rabbit’s
heart:
It was noticed that doxorubicin in a gradually increasing doses (1/2 ,
1, 2 and 4 μg / bath) produced a decrease in the force of spontaneous
contraction of isolated perfused rabbit’s heart in dose related manner
(fig. 44 )
Also it was noticed that doxorubicin decrease the positive inotropic
effect of isoprenaline in a dose of 2 μg / bath on isolated perfused
rabbit’s heart (fig. 44 )
Fig. ( 44 ) : A record demonstrating the effect of gradually increasing
concentrations of doxorubicine on the isolated perfused rabbit's
heart(A typical trace of 6 experiments)
Results
99
Fig. (45 ) : A record demonstrating the effect of gradually increasing
concentrations of doxorubicin on isoprenaline effect on the isolated
perfused rabbit's heart (A typical trace of 6 experiments)
Discussion
100
Discussion
Doxorubicin is one of the potent anticancer drugs. It is commonly
used in the treatment of a wide range of cancers, including leukemia and
Hodgkin's lymphoma, as well as cancer bladder, breast, stomach, lung,
ovaries, thyroid, soft tissue sarcoma, multiple myeloma (Takimoto and
Calvo,2008).This drug has many organ toxicities especially
cardiotoxicity and this toxicity is the main limitation for its use as
anticancer drug (Tangpong et al., 2011)
It causes disruption in basal body metabolism and shows toxic effect
especially in heart, liver and kidney tissues. It is known that doxorubicin
is considered the most toxic anthracyclines which may cause death (Sayed
et al., 2010b).
Many researchers have expanded great efforts aiming at preventing or
decreasing the adverse effects of doxorubicin. In this study, the possible
cardioprotective , hepatoprotective and nephroprotective effect of some
chemical agents like nebivolol and some natural agents like L-carnitine
was studied in a model of cardiotoxicity, hepatotoxicity, nephrotoxicity
induced by doxorubicin. Also the effect of isoprenaline on isolated rat
heart of the treated rat groups and effect of doxorubicin on isolated
rabbit heart were studied.
Animals used in the in-vivo study were rats. The choice of rats was
because they are cheap, easy to deal with and to take samples, as well as
it is easy to take sufficient amount of blood easily through
puncture of the retro bulbar sinus by capillary tube.
Parameters studied in this work were ECG, blood pressure
measurement, cardiac enzymes troponin I and serum creatine
phosphokinase , as a markers for cardiac cell injury and liver enzymes
ALT, AST as they can be used as a markers of hepatic cell injury and
Discussion
101
creatinine level as markers of renal cell function also histopathopogical
evaluation of heart , liver and kidney was done.
In the present study acute toxicity was induced in experimental
animals by administration of doxorubicin in a dose of 3 mg / kg / day
intraperitoneally every other day for 12 days (Amani et al., 2011) .The
potential organ protective effect of nebivolol was tested by concomitant
oral administration of nebivolol in a dose of 1mg / kg / day orally daily
for 12 days (Amani et al., 2011) and the potential organ protective
effect of L-carnitine was tested by concomitant administration of L-
carnitine in a dose of 200 mg / kg body weight intraperitonially daily for
12 days ( Nathalie et al., 1999).
The present study showed that doxorubicin administration caused
ECG changes as increased T wave voltage in Dox group compared to
control group, concerning heart rate and QRS voltage there was
insignificant change in Dox group compared to control group also there
was significant decrease in blood pressure indicating deterioration of
cardiac function, There was also histopathological changes including
cardiac myocyte degenerative changes in the from of cell swelling, loss of
cytoplasmic striations, with mild interstitial fibrosis accompanied by
elevation of cardiac enzymes troponin I and CPK which indicate
structural affection of the heart by doxorubicin toxicity.
The result of present study showed that treatment of rats of Dox +
Neb group with nebivolol for 12 days produced a significant reduction in
T wave voltage , significant elevation in blood pressure and significant
reduction in cardiac enzymes troponin I & CPK in comparison to Dox
group & treatment of rats of Dox+L-Car group with L-carnitine
produced also a significant reduction in T wave voltage , significant
elevation in blood pressure and significant reduction in cardiac enzymes
troponin I & CPK in comparison to Dox group . When rats of Dox+Neb+
Discussion
102
L-Car group given both nebivolol and L-carnitine, there was also
significant reduction in T wave voltage , significant elevation in blood
pressure and significant reduction in cardiac enzymes troponin I & CPK
in comparison to Dox group but without significant difference when
compared with groups Dox+Neb and Dox+L-Car group.
In the Dox group there was cardiac myocyte degenerative changes in
the from of cell swelling, loss of cytoplasmic striations, with mild
interstitial fibrosis. The doxorubicin induced myocardial damage was
improved in groups treated with nebivolol, L-carnitine and in group
treated with nebivolol & L-carnitine in comparison to the Dox group, as
the present work showed marked decrease of the area of hydropic
degeneration and area of inflammation and decrease in inflammatory
infiltrate in cardiac sections of nebivolol and in L-Carnitine treated group
As regards effect of isoprenaline on isolated perfused rat heart of the
treated rats groups, isoprenaline produced concentration-dependent
increase in force of contraction in control group. The positive inotropic
effect induced by isoprenaline was significantly reduced (P<0.05) in
hearts from the Dox group compared to control rats (p<0.05). Both
Dox+Neb and Dox+L-car groups showed significant improvement in
power of cardiac contractility in response to increasing doses of
isoprenaline compared to doxorubicin group (p<0.05).There were
insignificant difference between the Dox+Neb and Dox+L-car groups in
the percent change of contraction when compared to each other.
Calcium homeostasis disturbances related to the appearance of
oxidative stress could explain why the response to isoprenaline was
attenuated in the doxrubicin group. The dysfunction of sarcoplasmic
reticulum could induce changes in intracellular calcium concentrations
(Hideo et al ., 1991) leading to impairment of cardiac contractility and/or
Discussion
103
relaxation. Moreover, it has been reported that anthracycline treatment
could induce reduction of ß-adrenergic receptor density on myocardial
membrane (Nagami et al.,1997).
The present work runs in consistence with Ibrahim et al., 2009 that
demonstrated occurrence of marked cardiotoxicity and nephrotoxicity with
doxorubicin intake as they found increased cardiac enzymes troponin I,
CPK and increased heart rate. Ibrahim et al., 2009 also found decreased
QRS voltage which isn't in agreement with our result as we found non
significant change in QRS voltage in Dox group compared to control this
may be due to difference between the two studies in doses and duration.
These results are also in agreement with Nathalie et al., 1999, Atiar et
al., 2007, Shi , et al ., 2011, Amani et al., 2011 & Imbaby et al., 2014
that demonstrated the toxic effect of doxorubicin in rat models that found
increased heart rate ,decreased blood pressure, histopathological changes
and elevation of cardiac enzymes troponin I and CPK and this is also in
agreement with Jessica et al., 2011 who studied doxorubicin toxicity in
humans.
This is also in agreement with Paolo et al., 2004 who confirmed that
doxorubicin induces oxidative stress, mitochondrial dysfunction, and
histopathological lesions in the cardiac tissue in doxorubicin induced
mitochondrial mediated cardiomyopathy.
Doxorubicin causes inhibition of nucleic acid and protein synthesis ,
release of vasoactive amines , altered adrenergic function and decreased
expression of cardiac-specific genes are other proposed mechanisms . It is
possible that more than one mechanism is operative (Monti et al., 1995)
There is also down regulation of α-actin, myosin light and heavy
chains, troponin-I, and desmin proteins by doxorubicin has been
suggested as a potential mechanism of its cardiotoxicity. Decreased
expression of the contractile proteins is associated with myofibrillar loss
Discussion
104
and reduced myocardial contractile function. Down regulation of
sarcoplasmic reticular ATPase may cause abnormal myocardial diastolic
function (Aries et al.,2004).
It has been suggested that doxorubicin can inactivate extracellular
signal-regulated kinase (ERK) (Lou et al., 2005).There is evidence that
doxorubicin induces apoptosis of cardiomyocytes. Formation of hydrogen
peroxide and superoxide has been implicated in doxorubicin-induced
cardiomyocyte toxicity (Zhang et al. , 2012).
Among the proposed mechanisms of cardiac injury, doxorubicin-
induced generation of reactive oxygen species (ROS) that can cause the
following
Doxorubicin causes myofilament apoptosis causing myocyte cell
death from the doxorubicin-induced generation of ROS, which, in turn,
activate a multiplicity of signaling pathways that determine cell fate. A
key pathway involves activation of the tumor suppressor protein p53
(Sano et al., 2007) p53-dependent apoptosis involves the transcriptional
activation or inhibition of certain target gene pathways such as mitogen-
activated protein kinases (MAPK).
Doxorubicin causes suppression of myofilament protein synthesis
through depletion of cardiac progenitor cells (CPCs) is also postulated to
contribute to doxorubicin-induced cardiac injury, De Angelis et al., 2010
reported that doxorubicin exposure significantly reduced the population
of CPCs, raising the possibility that CPC death may represent a primary
event responsible for impaired myocyte turnover and the onset of
ventricular dysfunction. Interestingly, delivery of CPCs into doxorubicin-
induced failing hearts caused regeneration of cardiomyocytes, leading to
improved LV performance and overall survival.
Discussion
105
Ultrastructural changes to myocytes by ROS which cause alterations
in calcium homeostasis leading to systolic (contractile) and diastolic
dysfunction (Kemi et al., 2008). Doxorubicin stimulates calcium release
and inhibits sarcoplasmic reticulum calcium reuptake, resulting in
cytosolic calcium overload (Saeki et al ., 2002). This calcium overload
may contribute to impaired contractile function by (1) promoting release
of the proapoptotic factor cytochrome c ( Logue et al., 2005) and/or (2)
activating the cysteine protease calpain . Calpains initiate turnover of
both regulatory and structural myofibrillar proteins through cleavage and
release of large polypeptide fragments (Gustafsson and Gottlieb, 2003).
Doxorubicin causes alterations in cardiac energy metabolism; the
heart requires ATP to sustain contraction and relaxation. As such,
deficiencies in cellular homeostasis are important factors in the
development of cardiomyopathy (Ingwall and Weiss , 2004),doxorubicin
reduces cardiac energy reserves by lowering ATP and phosphocreatine
levels as well as the phosphocreatine/ATP ratio. In the normal heart,
AMP-activated protein kinase (AMPK) plays a crucial role in protecting
cardiac cells from perturbations in energy homeostasis via activation of
catabolic pathways to generate ATP(Neumann et al., 2003). Doxorubicin
reduces the level of both AMPK protein and its basic activation state,
which leads to decreased phosphorylation of anti-acetyl-CoA carboxylase
(ACC), an AMPK downstream target. Lack of ACC inhibition results in
impairment of fatty acid oxidation; thus, interventions that increase
AMPK expression and/or normalize myocyte metabolism may be an
effective strategy to offset cardiotoxicity. The mechanisms underlying
inhibition of AMPK are not clear; alterations in gene expression and
upstream signaling require further investigation. Other important factors
activated in response to metabolic perturbations include hypoxia-
Discussion
106
inducible factor-1 and peroxisome proliferator-activated receptor gamma
coactivator 1-α (Tokarska-Schlattner et al ., 2005).
Doxorubicin increased oxidative stress appears to induce toxic
damage to the mitochondria of cardiomyocytes. Several mitochondrial
enzymes such as NADH dehydrogenase, cytochrome P-450 reductase and
xanthine oxidase are involved in generating oxygen free radicals (reactive
oxygen species) (Takemura and Fujiwara , 2007). Doxorubicin also
increases superoxide formation by increasing endothelial nitric oxide
synthase which promotes intracellular hydrogen peroxide formation
(Zhang et al. ,2012).
These results are in agreement with the results of other experimental
studies demonstrating the cardioprotective and the antioxidant effects of
nebivolol as the study done by Amani et al., 2011 & Imbaby et al.,
2014, they demonstrated that oxidative damage plays an important role in
the pathogenesis of doxorubicin cardiotoxicity, as they found significant
elevation in the level of MDA in doxorubicin cardiotoxicity which is
decreased significantly in nebivolol group .
Nebivolol was used to protect against different drug toxicity, it was
tried in rat model of anthracycline induced cardiotoxicity and
nephrotoxicity (Amani et al., 2011) and was used with curcumin against
doxorubicin-induced cardiac toxicity in rats ( Imbaby et al., 2014 )and it
was found that the oral administration of curcumin and/or nebivolol
attenuated doxorubicin cardiotoxicity as manifested by increasing
survival rate, improvement in body weight, heart index, and ECG
parameters, increase in ventricular isoprenaline responses, and
improvement in cardiac enzymes.
A study by Filomena de Nigrisa et al., 2008 reported a beneficial
effect of BBs on anthracycline treated hearts. In particular, the use of
Discussion
107
nebivolol or carvedilol with anthracyclines have reduced the release of
glutathione (GSSG) and reduced glutathione (GSH). Since nebivolol
predominantly affects nitric oxide (NO) pathway, the common protective
pathway of nebivolol could be the beta blocker properties coupled to
antioxidant and NO release. Previous studies showed that patients treated
with β1-selective antagonists without intrinsic sympathomimetic activity
(ISA) had more adrenoceptors than control subjects and that the adenylate
cyclase activation by the β-adrenoceptor agonist isoprenaline is also
enhanced in this situation (Trochu et al., 1999)
The intrinsic sympathomimetic activity is therefore an important
criterion for the therapeutical usefulness of BBs in heart failure patients.
The cardiac effects of BBs are varying in human cardiac tissue. The
cardiodepressant effects of beta-blocker may not correlate with its β1-
selectivity but result from the combined effects of β1-selectivity, intrinsic
sympathomimetic properties and inverse agonist (Klara et al ., 2001).
The prominent cardioprotective effects of the third-generation beta-
blocker nebivolol against anthracycline-induced cardiotoxicity, using the
model of an isolated perfused rat heart, were examined by De Nigris et
al., 2008 who used Sprague-Dawley rats were treated with doxorubicin in
combination with nebivolol or carvedilol. The cotreatment of beta-
blockers and anthracyclines had reduced the release of GSSG and GSH.
A more significant reduction was shown with nebivolol than with
carvedilol. Also, significant reductions of CPK and troponin I activities
were observed when the hearts were treated with nebivolol. At the same
time, glutathione peroxidase (GSHPx), Manganese superoxide dismutase
(MnSOD), and nitrite/nitrate release were increased after the co-
treatment. Three cardiac parameters have been used to evaluate the
cardiac toxicity of both anthracyclines and beta-blocker-anthracycline
combinations. The left ventricular pressure developed under a constant
Discussion
108
perfusion pressure (LVDP), then the variation rates of this parameter
were observed during contractility (LV/dt)max and relaxation LV(dP/
dt)min. Nebivolol in combination with anthracyclines had exerted the
most significant protection of heart tissue.
It has been demonstrated that nebivolol affected vasodilatation
through NO production, stimulated NO release, enhanced NO
bioavailability and prevented NO deactivation (Maffei et al .,2007).
A study by Angelo et al., 2006 demonstrated that nebivolol stimulates
NO production in the heart. This action of nebivolol is exerted via a
signaling pathway starting from the activation of β3-adrenergic receptors
and leading to overexpression of inducible NO synthase.
Nebivolol, a last-generation beta blocker, which is a selective β1-
adrenergic receptor antagonist, it leads to (1) an increase in the renal NO
excretion (2) a significant increase in the renal plasma flow and
glomerular filtration rate (GFR),(3) suppression of the rennin angiotensin
aldosterone system and inhibition of angiotensin II (Greven and
Gabriels , 2000) and reduces endothelin-1,and (iv) has an antioxidant
effect (Mason et al., 2006).
Patrianakos et al. , 2005 compared the efficacy of nebivolol versus
carvedilol on left ventricular (LV) function and exercise capacity in
patients with non ischemic dilated cardiomyopathy (NIDC). Both
nebivolol and carvedilol appear relatively safe, with beneficial effects on
LV systolic and diastolic function as well as exercise capacity in patients
with non ischemic dilated cardiomyopathy (NIDC) after 12 months'
treatment. However, carvedilol exhibits more favorable effects on LV
function than does nebivolol.
The effects of L-carnitine and propionyl-L- carnitine (PLC) on
doxorubicin induced cardiomyopathy have extensively been investigated.
Discussion
109
Several experimental and clinical studies have reported that doxorubicin
induces its cardiomyopathy by inhibition of beta-oxidation of long-chain
fatty acids with the consequent depletion of cardiac ATP and that L-
carnitine supplementation protects the myocardium against this toxicity
(Sayed-Ahmed et al., 2000a and Waldner et al., 2006).
In early experiments, Alberts et al., 1978 investigated the protective
effects of carnitine against acute (high-dose) and chronic (intermittent,
low dose) doxorubicin toxicity in rat and mice. They found that carnitine
was able to decrease both acute and chronic doxorubicin linked lethality
in normal rats without decreasing its antineoplastic activity or raising its
bone marrow toxicity.
Paterna et al., 1984 investigated whether L-carnitine could prevent
doxorubicin induced cardiomyopathies in rabbits by promoting free fatty
acid utilization for energy production and neutralizing long-chain free
fatty acid toxicity occurring after ischemia.The results showed that L-
carnitine reduced the frequency of cardiomyopathies and increased
survival rate. Histopathological examination of myocardial tissues under
light and electron microscopes revealed a marked decrease in
mitochondrial lesions. Also, the possible protective effects of L-carnitine
against doxorubicin induced cardiotoxicity were studied by Neri et al.,
1986.
Rat heart slices were incubated with doxorubicin (14 mg/ml), L-
carnitine (600 mg/ml), and doxorubicin plus L-carnitine for 60 min. They
measured cellular oxygen uptake, ATP, intracellular Ca2+
concentration.
L-carnitine significantly reduced doxorubicin induced cardiac metabolic
damage. The oxygen uptake inhibition decreased from 38% to 7%, the
decrease in ATP concentration was reduced from 78% to 27% and the
Discussion
110
inhibition of protein synthesis from 11% to 6%. Therefore, according to
Neri et al., 1986, L-carnitine appears to be a useful drug in the prevention
of doxorubicin induced cardiotoxicity. The protective effects of L-
carnitine against doxorubicin induced cardiotoxicity in rats were also
studied by McFalls et al. (1986). Cardiomyopathy was induced by
weekly intravenous injection of doxorubicin (2 mg/kg) over a period of
6–7 weeks.
Using doxorubicin cardiomyopathic rat model, Shug (1987) designed
an experiment to find an explanation for the protective effect of L-
carnitine against doxorubicin induced cardiotoxicity. After 6 weeks of
intravenous administration of doxorubicin , rats treated without carnitine
had a lower ejection fraction and left ventricular pressure than control or
rats treated with doxorubicin plus L-carnitine. Total carnitine in plasma
was significantly higher in rats treated with doxorubicin than control and
doxorubicin plus carnitine. They suggested that the increase in carnitine
ester in plasma of doxorubicin treated rats was due to doxorubicin
induced inhibition of long-chain fatty acid oxidation. Myocardial
carnitine concentration was not altered by doxorubicin, but plasma
carnitine concentration was increased resembling those of
cardiomyopathies in adults.
In isolated rat heart myocytes and mitochondria, Sayed-Ahmed et al.,
2000a studied the effects of different concentrations of doxorubicin in
absence and presence of proprionl- L -carnitine (PLC) on palmitoyl-CoA
and palmitoyl-carnitine oxidation and on the activity of carnitine
palmitoyltransferase (CPT 1). The results showed that doxorubicin -
induced concentration-dependent inhibition of substrates oxidation and
inhibition of CPT I and that PLC completely reversed this inhibition to
the control values. They concluded that doxorubicin induced its
Discussion
111
cardiotoxicity by inhibition of CPT I and beta-oxidation of long-chain
fatty acids with the consequent depletion of ATP in cardiac tissues, and
that PLC can be used as a protective agent against doxorubicin -induced
cardiotoxicity .
In DOX cardiomyopathic rat model, Sayed-Ahmed et al., 2001 have
initiated another study to confirm their in vitro findings and investigated
the protective effects of PLC against DOX-induced cardiotoxicity.
Chronic administration of DOX (3 mg/kg, I.P) significantly increased
creatine kinase-MB( CK-MB), lactate dehydrogenase (LDH) and
malondialdehyde and decreased reduced glutathione. Treatment with PLC
induced complete reversal of these effects without decreasing the
antitumour activity of DOX. In another study, Sayed-Ahmed et al.,
2000b investigated the effects of DOX on mRNA expression of heart
fatty acid binding protein (H-FABP) using Northern blot analysis. Results
showed that chronic administration of DOX caused dose -dependent and
cumulative inhibition of H-FABP gene expression and that daily
administration of L-carnitine protected against this effect in cardiac
tissues. Authors concluded that DOX-induces its cardiotoxicity by
inhibition of gene expression of H-FABP .
More recently, Sayed-Ahmed et al., 2010b reported that chronic
DOX therapy decreased the expression of H-FABP and OCTN2 mRNA
expression and increased the expression of apoptotic genes in cardiac
tissues that should be viewed as a mechanism during development of
DOX-induced cardiomyopathy.
Waldner et al., 2006 examined the effects of L-carnitine with a view
to reducing cardiotoxicity of DOX-containing chemotherapy in 20
patients with Non-Hodgkin lymphoma. Patients were scheduled to
Discussion
112
receive 3 g L-carnitine before each chemotherapy cycle, followed by 1 g
L-carnitine/day during the following 21 days. Carnitine-treated patients
showed a rise in plasma carnitine which led to an increase of relative
mRNA levels from CPT 1 and OCTN2. They concluded that biochemical
and molecular analyses indicated a stimulation of oxidative metabolism
in white blood cells through carnitine uptake.
These results are in agreement with Nathalie et al., 1999 and
Maccari and Ramacci , 1981 that demonstrated the protective role of L-
carnitine in doxorubicin cardiotoxicity as evident by improvement in
ECG changes in T wave voltage and the improvement in cardiac enzymes
troponin I & CPK in L-carnitine treated group .
Zeidan et al., 2002 evaluated the heart and liver responses after
doxorubicin toxic aggression, with and without exogenous L-carnitine
protection. Results showed that doxorubicin induced long term cardiac
subcellular pathology included loss, disruption and disassembly of
myofibrils, and mitochondrial swelling and condensation. On the other
hand, the doxorubicin induced subcellular hepatic alterations consisted of
polymorphic mitochondria, cytoplasmic vacuolization and accumulation
of lipid droplets. However, these alterations were of less severity in
carnitine treated group in both heart and liver, suggesting that carnitine
could be used as a possible protective agent against doxorubicin induced
toxicity.
As regard liver function data presented in this study demonstrated that
doxorubicin caused significant elevation in serum liver enzymes
including ALT & AST compared to control group. These elevations of
ALT and AST are due to hepatocellular damage and leakage of these
enzymes and appearance in serum.
Discussion
113
Administration of nebivolol in combination with doxorubicin in
Dox+Neb group caused significant reduction in liver enzymes when
compared to Dox group. These results suggested that nebivolol might
have protective effect against doxorubicin induced liver damage.
This is supported in our work by the improvement occurring in the
histopathological picture of liver cut section of nebivolol treated group
which showed less hydropic changes, less portal tract inflammation and
less congestion in portal vein.
Damage, at the cellular level by oxidants as doxorubicin, is attenuated
by antioxidant enzyme such as superoxide dismutase (SOD),glutathione
peroxidase (GSHPx), catalase (CAT) and glutathione reductase (GR)
(Koc et al ., 2005) . Superoxide dismutase is one of the major enzymatic
antioxidant mechanisms against superoxide radical, prevents liver toxicity
induced by doxorubicin (Yagmurca et al.,2007).Catalase and GSHPx
catalyze dismutation of the superoxide anion (O2−) into hydrogen
peroxide (H2O2) which then converting H2O2 to water thus providing
protection against reactive oxygen species (Sayed-Ahmed et al .,
2010a).The reduction in activity of these enzymes may be caused by the
increase in free radical production during doxorubicin metabolism
(Brookins Danz et al., 2009 and Fisher-Wellman et al., 2009) .
Kolodziejczyt et al., 2011 study showed decrease in the gene
expression levels of GSHPx, CAT and GR in liver tissue with the
cumulative dose of doxorubicin with decrease in their activity in the
serum. These data showed that doxorubicin not only increase the free
radical formation but also decrease its ability to detoxify reactive oxygen
species. The formation of superoxide radicals together with nitric oxide
(NO) might form peroxynitrite induced by doxorubicin causes tissue
damage. This was in agreement with Injac et al., 2009. In contrast to this
Discussion
114
study, Kalender et al 2005 found that administration of doxorubicin (5
mg/week for 6 weeks) increased the levels of SOD and the activity of
both GSHPx and catalase enzymes.
Administration of nebivolol in combination with doxorubicin in Dox
+ Neb group cause significant reduction in liver enzymes when compared
to Dox group , the action of nebivolol as antioxidant might play this
protective role as discussed above in it´s role in doxorubicin
cardiomyopathy .
Also administration of L-carnitine in combination with doxorubicin
cause significant reduction in liver enzymes when compared to
doxorubicin group. These results suggested that L-carnitine may also
have protective effect against doxorubicin induced liver damage. This
protective effect may be due to stabilization of hepatocyte membranes by
L-carnitine with the consequent decrease in the leakage of liver enzymes.
L-carnitine have been found to offer protection against doxorubicin
induced liver damage (Yapar et al ., 2007) .
Data presented by Kolodziejczyt et al., 2011 demonstrate that
doxorubicin increased serum liver enzymes including ALT, ALP and
total bilirubin. These elevations of ALT and ALP are due to
hepatocellular damage. These results were previously reported in
doxorubicin induced hepatoxicity model done by Andreadou et al., 2007
and Cosan et al., 2008. This study established that doxorubicin
significantly decrease the total carnitine in liver tissues. This could be a
secondary effect following inhibition of endogenous carnitine
biosynthesis and/or decreased carnitine transport in doxorubicin induced
hepatocytes damage. This hypothesis is consistent with data presented by
Laub et al., 1986 which showed that biosynthesis of carnitine is
Discussion
115
decreased in pediatric patients receiving valproic acid. The level of
carnitine in hepatocytes is controlled by the specific carnitine transporter
(OCTN-2) and endogenous synthesis (Hwu et al., 2007 and Fujita et al.,
200). Decreased expression of OCTN-2 has been reported in the acute
hepatitis (Chang et al 2005). Also, OCTN-2 located on hepatocyte
membranes might be destroyed when exposed to ROS induced by
doxorubicin
L-carnitine have been found to protect against doxorubicin induced
liver damage (Yapar et al., 2007). It was used to prevent the toxic effect
of cisplatin-induced nephrotoxicity by normalized kidney function, where
oxidative stress and lipid peroxidation play a major role in this toxicity.
In addition, L-carnitine attenuated the reduced GSH levels, (El-Awady et
al., 2011) in which there were no toxic effects of it on approved doses.
Sayed-Ahmed et al., 2010 reported that L-carnitine administration in
combination with doxorubicin significantly increase antioxidant enzyme
and decrease the lipid peroxidation. The increase in GSH level lead to
increase in the activity of GSHPx in which the former acts as a cofactor
for later.These results are consistent with previous studies reported that
L-carnitine had similar non-enzymatic free-radical scavenging and anti-
lipid peroxidation activities (Kolodziejczyk et al., 2011).
As regard kidney function data presented in this study demonstrated
that doxorubicin caused significant elevation in creatinine level in Dox
group compared to the control group. Administration of nebivolol or L-
carnitine in combination with doxorubicin caused significant reduction in
serum creatinine level in Dox+Neb group and Dox+L-car group
compared to Dox group. These results suggested that nebivolol and L-
carnitine may have protective effect against doxorubicin induced kidney
damage.
Discussion
116
Also, the cut sections of kidney of the Dox group showed
glomerulopathy evident by increase in serum creatinine associated with
mesangeal proliferation, swelling and vacuolation of epithelial cells,
moderate tubular atrophy and dilatation and congestion of interstitial
capillaries .
The doxorubicin induced kidney damage was improved in Dox+Neb
group and Dox+ L-car groups in comparison to the Dox group, as the
mesangeal proliferation, tubular atrophy & dilatation and congesion of
interstitial capillaries was markedly decreased.
These results are consistent with previous studies reported by other
investigators Lebrecht et al., 2004 that studied doxorubicin induced
cardiotoxicity and nephrotoxicity in normal rats. Coadministration of
nebivolol with doxorubicin was able to ameliorate up to almost reverse
doxorubicin induced myocardial injury, glomerular filtration disturbance
and renal tubular damage .According to the reported results for
doxorubicin induced toxicity, almost all organs can be attacked and
damaged via the formation of free oxygen radicals and some of the organ
specific pathways. Several mechanisms have been proposed to account
for doxorubicin cardiotoxicity, e.g., free radical stress, calcium
overloading, and mitochondrial dysfunction but the exact mechanism of
doxorubicin induced nephrotoxicity is not yet known. However, it has
been suggested by many investigators that cellular damage induced by
doxorubicin is mediated by the formation of an iron anthracycline free
radical, which in turn causes severe damage to the kidney cell plasma
membrane (Washio et al .,1994)
Moreover, nephrotoxicity and oxidative stress induced by doxorubicin
in rats were investigated by Boonsanit et al., 2006 who found that L-
carnitine can prevent renal impairment functionally, biochemically as
evident by the improvement in serum creatinine and histopathologically
Discussion
117
as evident by decreased mesangeal proliferation, swelling and
vacuolation of epithelial cells, moderate tubular atrophy and dilatation
and congestion of interstitial capillaries with a corresponding reduction of
oxidative stress.
Our results are in agreement with previous studies reported that L-
carnitine had similar non-enzymatic free radical scavenging and anti-lipid
peroxidation activities (Kolodziejczyt et al., 2011) and that it could be
used to prevent the toxic effect of cisplatin-induced nephrotoxicity by
normalizing kidney function, where oxidative stress and lipid
peroxidation play a major role in this toxicity.
Summary and Conclusion
118
Summary and Conclusion
Doxorubicin is a widely used anthracycline antibiotic in cancer
chemotherapy, the most serious adverse effect being life-threatening heart
damage. It is commonly used in the treatment of a wide range of cancers,
including some leukemias and Hodgkin's lymphoma, as well as cancers
of the bladder, breast, stomach, lung, ovaries, thyroid, soft tissue
sarcoma, multiple myeloma, and others . Commonly used doxorubicin-
containing regimens are AC (Adriamycin, cyclophosphamide), TAC
(Taxotere, AC), ABVD (Adriamycin, bleomycin, vinblastine,
dacarbazine), and FAC (5-fluorouracil, adriamycin, cyclophosphamide) .
Nebivolol is a β1 receptor blocker with nitric oxide-potentiating
vasodilator effect used in treatment of hypertension and, also for left
ventricular failure .
L-Carnitine is a natural substance that helps the body turn fat into
energy. Our body makes it in the liver and kidneys and stores it in the
skeletal muscles, heart, brain, and sperm. Usually, our body can make all
the carnitine it needs. Some people, however, may not have enough
carnitine because their bodies cannot make enough or can’t transport it
into tissues so it can be used .
The present study was carried out to find out and compare the
protective effects of nebivolol and L-carnitine against doxorubicin
cardiotoxicity , hepatotoxicity and nephrotoxicity, also the in vitro studies
were done to investigate the effect of doxorubicin on contractility of
isolated normal mammalian heart and the effect of increasing doses of
isopernaline on contractility of hearts taken from another 5 groups of rats
given the same medications as the groups of the in-vivo experiment.
Summary and Conclusion
119
Rats were randomly divided into 5 groups (6 rats for each group).
control group received saline intraperitoneal in comparative volume to
the tested groups, Dox group received doxorubicin intraperitoneally every
other day for 12 days, Dox + Neb group received doxorubicin
intraperitoneally every other day for 12 days and nebivolol orally daily
for 12 days , Dox +L-car group received doxorubicin intraperitoneally
every other day for 12 days and L-Carnitine intraperitonially daily for 12
days and Dox + Neb +L-car group received doxorubicin intraperitoneally
every other day for 12 days nebivolol orally and L-Carnitine
intraperitonially daily for 12 days.
At the end of the study period, ECG, blood pressure, troponin I
,CPK, creatinine ,ALT, AST were measured and histopathololgical
examination of heart , liver and kidney was done .
It was found that doxorubicin administration caused significant
increase in T wave voltage, significant reduction in blood pressure and
significant elevation in troponin I ,CPK, ALT, AST, creatinine serum
levels and prominent histopathological changes compared to control
group . This study revealed that nebivolol and L-carnitine produced a
significant reduction in T wave voltage, significant elevation in blood
pressure and significant reduction in troponin I ,CPK, , ALT, AST,
creatinine serum levels and improved markedly histopathological changes
compared to Dox group .
Comparing the result of Dox+Neb +L- car group with that of Dox+
neb group and Dox + L-car group, adding L-carnitine to nebivolol was
not significantly more effective than nebivolol or L-carnitine alone in
reducing T wave voltage .
Comparing the result of Dox +Neb+L- car group with that of Dox+
neb group, adding L-carnitine to nebivolol was significantly more
effective than nebivolol alone in elevating systolic & mean blood
Summary and Conclusion
120
pressure and comparing the result of Dox+Neb+L- car group with that of
Dox + L-car group, adding nebivolol to L- carnitine was not significantly
more effective than nebivolol alone in elevating systolic blood pressure &
mean blood pressure also was found that comparing the result of
Dox+Neb+L- car group with that of Dox+Neb group and Dox + L-car
group adding L-carnitine to nebivolol was not significantly more
effective than nebivolol or L- carnitine alone in reducing the cardiac
enzymes.
Comparing the result of Dox +Neb + L_Car group with that of Dox +
Neb group and Dox + L-car group, adding nebivolol to L- carnitine was
not significantly more effective than nebivolol alone in reducing the liver
enzymes. Comparing the result of Dox+Neb + L- car group with that of
Dox + Neb group and Dox + L-car group, adding L-carnitine to nebivolol
was not significantly more effective than nebivolol alone in reducing the
creatinine level .
In-vitro study, It was noticed that isoprenaline in increasing doses
(2, 4 and 8 μg/bath) produced an increase in the force of spontaneous
contraction of isolated perfused rat’s heart in dose related manner.
In Dox group it was noticed that isoprenaline in increasing doses
(2 , 4 μg/bath) produced an non significant increase in the force of
spontaneous contraction of isolated perfused rabbit’s heart .This increase
of the force of spontaneous contractions of the isolated perfused rabbit’s
heart was significant with the dose of 8µg/bath. with percentage of
increase 59.1 % compared to preisoprenaline contraction
In Dox + Neb group it was noticed that isoprenaline in increasing
doses (4 and 8 μg/bath) produced an increase in the force of spontaneous
contraction of isolated perfused rat’s heart in dose related manner.
This increase of the force of spontaneous contractions of the
isolated perfused rat’s heart was not significant with the dose of 2µg/bath.
Summary and Conclusion
121
In Dox + L-car group it was noticed that isoprenaline in increasing
doses (4 and 8 μg/bath) produced an increase in the force of spontaneous
contraction of isolated perfused rabbit’s heart in dose related manner.
This increase of the force of spontaneous contractions of the
isolated perfused rabbit’s heart was not significant (P > 0.05) with the
dose of 2µg/bath .
In Dox + Neb +L- car group it was noticed that isoprenaline in
increasing doses (2 , 4 and 8 μg/bath) produced an increase in the force
of spontaneous contraction of isolated perfused rat’s heart in dose related
manner.
It was noticed that doxorubicin in a gradually increasing doses (1/2
, 1, 2 and 4 μg / bath) produced a decrease in the force of spontaneous
contraction of isolated perfused rabbit’s heart in dose related manner.
Also it was noticed that doxorubicin decrease the positive inotropic
effect of isoprenaline in a dose of 2 μg / bath on isolated perfused rabbit’s
heart .
In conclusion, doxorubicin can induce cardiotoxicity,
hepatotoxicity and nephrotoxicity which can be ameliorated by using
nebivolol and L-carnitine which are effective and may be recommended
for protection against doxorubicin cardiotoxicity, hepatotoxicity and
nephrotoxicity .
Recommendation
122
On the lights of the results of this study we recommended the
following :
1-Doxorubicin has sever toxicity on three major organs heart ,
liver, kidney and it is better to avoid it's use and less toxic anthracycline
are better used.
2- β-blockers nebivolol may have a role in treatment of
doxorubicin toxicity to reduce progression of this toxicity.
3- L-carnitine as a natural agent with less adverse effects can be
used with doxorubicin administration to limit the development of
doxorubicin induced toxicity without affecting it’s anticancer activity .
4- Further studies may be needed to show the effect of free radical
scavengers as vit C and E on doxorubicin toxicity.
References
123
REFERENCES
Agabiti E. and Rizzoni D. (2007): "Metabolic profile of
nebivolol,beta-adrenoceptor antagonist with unique characteristics". J.
of drugs ; (8) 1097–107.
Alberts D.S., Yel-Mei P., Moon T.E. and Bressler R.
(1978):Carnitine prevention of adriamycin toxicity in mice.
J.Biomedicine ;29:256–258.
Amani N. , Mostafa M. and Mostafa S. (2011): Animal study of
Anthracycline-induced Cardiotoxicity and Nephrotoxicity and
Evaluation of Protective Agents. J. of cancer science and therapy ; (3)
096-103.
Andreadou I. , Sigala F. and Iliodromitis E. (2007): Acute
doxorubicin cardiotoxicity is successfully treated with the
phytochemical oleuropein through suppression of oxidative and
nitrosative stress. J. of Molecular and Cellular Cardiology ;42(3):549–
558.
Angelo Maffei, Alba Di Pardo, Rosa Carangi, Pierluigi Cariullo,
Roberta, Poulet, Maria T. Genrila, Carmine Vacchione and
Giuseppe Lembo (2007) : Nebivolol induces Nitric oxide release in
the heart through inducible Nitric oxide release synthetase activation.J.
of hypertersion ; (50) 652-56
Aries P., Paradis P., Lefevre C., Schwartz R. and Nemer M. (2004):
Essential role of GATA-4 in cell survival and drug-induced
cardiotoxicity. J. of Cancer Res.;(101)6975–6980.
References
124
Atiar M., Syed W. and Michael S. (2007): Ewer Anthracycline-
induced cardiotoxicity and the cardiac-sparing effect of liposomal
formulation. Int. J. of nanomedicine ; (4) 567–583.
Autio K., Rassnick M. and Guaus S. (2007): Chemotherapy during
pregnancy: a review of the literature J. Veterinary and Comparative
Oncology ; 5( 2) 61–75.
Bakris G. (2009): "An in-depth analysis of vasodilation in the
management of hypertension: focus on adrenergic blockade". J.
Cardiovasc. Pharmacol.; (5) 379–87.
Baldwin C. and Keam S. (2009): Nebivolol in the Treatment of
Hypertension in the US. Am. J. Cardiovasc. Drugs ; (4): 253-260.
Benvenga S., Ruggieri R., Russo A., Lapa D., Campenni A. and
Trimarchi F. (2001): Usefulness of L-carnitine, a naturally occurring
peripheral antagonist of thyroid hormone action, in iatrogenic
hyperthyroidism: a randomized, double-blind placebo-controlled
clinical trial. J. Clin. Endocrinol. Metab.; (8):3579-3594.
Bergmeyer H., Gawehn K. and Grassl M. (1974): Methods of
Enzymatic Analysis. Volume I, 2nd ed., Academic Press, Inc.,New
York ; P.515-516.
Biagiotti G. and Cavallini G. (2001): Acetyl-L-carnitine vs tamoxifen
in the oral therapy of Peyronie's disease: a preliminary report. BJU
Int.J. of drugs; (1)63-67.
Bien S., Riad A., Ritter C., Gratz M., Olshausen F., Westermann D.,
Grube M., Kreig T., Ciecholewski S., Felix S., Staudt A., Schultheiss
References
125
H., Ewart R., Volker U., Tschope C. and Kroemer H. (2007): The
endothelin receptor blocker bosentan inhibits doxorubicin-induced
cardiomyopathy.J.Cancer Res.; (67)10428–10435.
Bodor G. (1994):Cardiac troponin-I: a highly specific biochemical
marker for myocardial infarction. J. Clin. Immunoassay ; 17: 40 - 45.
Boonsanit D., Kanchanapangka S. and Buranakarl C. (2006): L-
carnitine ameliorates doxorubicin-induced nephritic syndrome in rats.
J. Nephrology;11(4):313–320.
Broder H., Gottlieb R. and Lepor N. (2008): Chemotherapy and
cardiotoxicity. Rev. Cardiovasc. Med.; (9)75–83.
Brookins E., Skramsted J., Henry N., Bennett J. and Keller R.
(2009): Resveratrol prevents doxorubicin cardiotoxicity through
mitochondrial stabilization and the Sirt1 pathway. J. Free Radical
Biology and Medicine. ; 46(12):1589–1597.
Brunton L., Lazo J. and Parker K. (2006): Therapy of hypertension.
Goodman & Gilman’s.Text book Pharmacological Basis of
Therapeutics. 11th ed. Chapter 10 : 682–698.
Cangiano J., Carmen R. and Mauuel M. (1978): Effect of
antihypertensive treatment on systolic blood pressure and rennin in
hypertensive rats. Br. J. Pharmacol. ; 5(1):1-8.
Cardinale D. and Sandri M. (2010): Role of biomarkers in
chemotherapy-induced cardiotoxicity. J.of Cardiovasc. Dis.; (53)121-9
Carrero J, and Grimble R. (2006): Does nutrition have a role in
peripheral vascular disease? .Br. J. Nutr. ;(2):217-29.
References
126
Carvalho C., Santos R., Cardoso S., Correia S., Oliveira P., Santos
M. and Moreira P. (2009): Doxorubicin: the good, the bad and the ugly
effect . J.Curr. Med. Chem.; (25)3267-3285.
Cavallini G., Modenini F. And Vitali G. (2005): Acetyl-L-carnitine plus
propionyl-L-carnitine improve efficacy of sildenafil in treatment of
erectile dysfunction after bilateral nerve-sparing radical retropubic
prostatectomy. J. of Urology; (66)1080-1085.
Chan M., Soter D. and Ko W. (1987): Effect of morphine on
cardiovascular responses to acute myocardial infarction in rats. Br J
Pharmacol. 90(3):537-43.
Chang B., Nishikawa M., Nishiguchi S. and Inoue M. (2005): L-
carnitine inhibits hepatocarcinogenesis via protection of mitochondria.
International J. of Cancer ; 113(5):719–729.
Charles J. (2006): Kinetics, Pharmacokinetics, and Regulation of
l-Carnitine and Acetyl-l-carnitine Metabolism. J. of New york
academy of sience ; (1033) 30-41.
Chatterjee K., Zhang J., Honbo N. and Karliner J. (2010): Doxorubicin
cardiomyopathy. J.of Cardiology; (115)155–162.
Chen X., Ji Z. and Chen Y. (2002): TTD: Therapeutic Target Database.
Nucleic Acids Res. ; (1)412-415.
Chobanian A., Bakris G., Black H. (2013): Seventh Report of the Joint
National Committee on Prevention, Detection, Evaluation, and Treatment
of High Blood Pressure. J. of Hypertension; (42)1206-1252.
References
127
Cosan D., Basaran A., Gunes H., Degirmenci I. and Aral E. (2008):
The effect of doxorubicin on rats that received toxic and carcinogenic
benzo(a)pyrene. J. of Cancer Res.;46(3):367–372
De Angelis A., Piegari E., Cappetta D., Marino L., Filippelli A., Berrino
L., Ferreira-Martins J., Zheng H., Hosoda T., Rota M., Urbanek K.,
Kajstura J., Leri A., Rossi F. and Anversa P. (2010): Anthracycline
cardiomyopathy is mediated by depletion of the cardiac stem cell pool
and is rescued by restoration of progenitor cell function. J. of Circulation;
(121)276–292.
De Boer R., Voors A.and van Veldhuisen D. (2007): "Nebivolol: third-
generation beta-blockade". J. Expert. Opin. Pharmacother.; (10) 1539–
1550.
De Nigris F., Rienzo M., Schiano C., Fiorito C. and Casamassimi A.
(2008): Prominent cardioprotective effects of third generation beta
blocker nebivolol against anthracycline-induced cardiotoxicity using the
model of isolated perfused rat heart. Eur. J. Cancer ; (44) 334-340.
DeLeve L. (2013):Cancer chemotherapy.Text book Drug-induced liver
disease. 3rd ed. pp. 541-568.
Dury R. and Wallington E. (1967): Carleton Histological Technique, 4th
ed. Oxford University Press, Oxford, P. 129- 134.
Ducroq J., Maati H., Guilbot S., Dilly S., Laemmel E., Pons-Himbert
C., Faivre J., Bois P., Stücker O. and Grand M. (2010): Dexrazoxane
protects heart from acute doxorubicin-induced QT prolongation: a key
role for IKs.Br. J. Pharmacol . ; (159) 93-101.
References
128
Dursun S ., Çuhadar S., Köseoğlu M., Atay A. and Aktaş S. (2014 ):The
anti-inflammatory and antioxidant effects of pravastatin and nebivolol in
rat aorta. J. Anadolu Kardiyol Derg.;14(3):229-33.
Elad S., Zadik Y., Hewson I. and Hovan A. (2010): Viral Infections and
Supportive Care in Cancer . J. of Cancer ; (8) 993–1006.
El-Awady E., Moustafa Y., Abo-Elmatty D. and Radwan A. (2011):
Cisplatin-induced cardiotoxicity: mechanisms and cardioprotective
strategies. European J.of Pharmacology ;650(1):335–341.
Evans A. and Fornasini G. (2003) : Pharmacokinetics of L-carnitine. J.
Clin Pharmacokinet. ; 42(11):941-67.
Filomena de Nigris ., Rienzo M., Schiano C., Fiorito C. and
Casamassimi A. (2008): Prominent cardioprotective effects of third
generation beta blocker nebivolol against anthracycline-induced
cardiotoxicity using the model of isolated perfused rat heart. Eur. J.
Cancer ; (44) 334-340.
Fisher R. (1958): Statistical Methods for Research Workers . Thirteenth
revised edition . Oliver and Boyed , Edinburgh. P 15 – 67.
Fisher-Wellman K., Bell H., and Bloomer R.(2009): Oxidative stress
and antioxidant defense mechanisms linked to exercise during
cardiopulmonary and metabolic disorders. J. Oxidative Medicine and
Cellular Longevity; 2(1):43–51.
Fujita M., Nakanishi T. and Shibue Y. (2009): Hepatic uptake of γ-
butyrobetaine, a precursor of carnitine biosynthesis, in rats. American J.
of Physiology; 297(4): 681–686.
Germino F. (2009): Efficacy and tolerability of nebivolol monotherapy
by baseline systolic blood pressure: a retrospective analysis of pooled
data from two multicenter, 12-week, randomized, double-blind, placebo-
References
129
controlled, parallel-group, dose-ranging studies in patients with mild to
moderate essential hypertension. J. Clin. Ther.; (31)1946-1956.
Gielen W., Cleophas T. and Agrawal R. (2006): "Nebivolol: a review of
its clinical and pharmacological characteristics". Int. J. Clin. Pharmacol.
Ther.; (8) 344–57.
Gokcimen A., Cim H., Tola D., Bayram A., Kocak F., Ozguner S. and
Ayata F. (2007): Protective effect of N-acetylcysteine, caffeic acid and
vitamin E on doxorubicin hepatotoxicity. J. of Hum. Exp. Toxicol.; (26)
519-525.
Granados-Principal S., Quiles J., Ramirez-Tortosa C., Sanchez-Rovira
P. and Ramirez-Tortosa M. (2010): New advances in molecular
mechanisms and the prevention of adriamycine toxicity by antioxidant
nutrients . J. Food Chem. Toxicol. ; (6)1425-38.
Greven J. and Gabriels G. (2000): Effect of nebivolol, a novel beta 1-
selective adrenoceptor antagonist with vasodilating properties, on kidney
function. J. of drugs; (50) 973-979.
Gustafsson A. and Gottlieb R. (2003): Mechanisms of apoptosis in the
heart. J. Clin. Immunol.; (23)447–459.
Head K. (2006): Peripheral neuropathy: pathogenic mechanisms and
alternative therapies. J.Altern. Med. Rev. ; (4)294-329.
Hershman D., McBride R. and Eisenberger A. (2008): Doxorubicin,
cardiac risk factors, and cardiac toxicity in elderly patients with diffuse
B-cell non-Hodgkin's lymphoma. J. Clin.Oncol.; (26)3159-3165.
References
130
Hiatt W., Regensteiner J., Creager M., Hirsch A., Cooke J. and Olin J.,
(2001): Propionyl-L-carnitine improves exercise performance and
functional status in patients with claudication. Am. J. Med.; (8)616-622.
Hideo K., Shiho F., Yukihiro K., Akira K. and Hiroyuki S. (1991):
Alterations of intracellular calcium homeostasis and myocardial
energetics in acute adriamycin-induced heart failure. J. Cardiovasc.
Pharmacol.; (18) 437-444.
Hill B. (1971): Principles of medical statistics 9th
ed. London: limited
publication, 147-283.
Hughes C. (1986): Giving cancer drugs IV: some guidelines.Am. J. Nurs.
; 86(1):34–3
Hwu W., Chien Y., Tang N., Law L., Lin C. and Lee N. (2007):
Deficiency of the carnitine transporter (OCTN2) with partial N-
acetylglutamate synthase (NAGS) deficiency. J. of Inherited Metabolic
Disease ; 30(5):p. 816.
Ibrahim M., Ashour O., Ibrahim Y., El-Bitar H., Gomaa W. and
Abdel-Rahim S. (2009): Angiotensin-converting enzyme inhibition and
angiotensin AT(1)-receptor antagonism equally improve doxorubicin-
induced cardiotoxicity and nephrotoxicity. The official J. of the Italian
Pharmacological Society (5)373-381.
Imbaby S., Ewais M., Essawy S. and Farag N. (2014): Cardioprotective
effects of curcumin and nebivolol against doxorubicin-induced cardiac
toxicity in rats. J. of Hum Exp Toxicol. 115:79-82.
References
131
Ingwall J. and Weiss R. (2004): Is the failing heart energy starved? On
using chemical energy to support cardiac function. J. Circ. Res.; (95)135–
145.
Injac R. , Perse M. and Cerne M. (2009): Protective effects of fullerenol
C60(OH)24 against doxorubicin-induced cardiotoxicity and hepatotoxicity
in rats with colorectal cancer. J. Biomaterials ;30(6):1184–1196.
Jaffe M. (1886): "Über den Niederschlag, welchen Pikrinsäre in
normalem Harn erzeugt und über eine neue Reaction des Kreatinins". J.
Zeitschrift für physiologische Chemie. ; 10 (5): 391–400.
Jessica M., Aarif K., John R., Mackey M., Mark J., , Pamela S.,
Douglas M., Lee W. and Jones S. (2011): Modulation of Anthracycline-
Induced Cardiotoxicity by Aerobic Exercise in Breast Cancer Current
Evidence and Underlying Mechanisms . J. Basic Science for Clinicians (
124) 642-650
Kalay N., Basar E., Ozdogru I., Er O., Cetinkaya Y., Dogan A., Inanc
T., Oguzhan A., Eryol N., Topsakal R. and Ergin A. (2006): Protective
effects of carvedilol against anthracycline-induced cardiomyopathy. J.
Am. Coll. Cardiol.; (48)2258–2262.
Kalender Y., Yel M. and Kalender S. (2005):Doxorubicin hepatotoxicity
and hepatic free radical metabolism in rats: the effects of vitamin E and
catechin. J. of Toxicology ;209(1):39–45.
Kamp O., Sieswerda G. and Visser C.(2003):"Comparison of effects on
systolic and diastolic left ventricular function of nebivolol versus atenolol
in patients with uncomplicated essential hypertension". Am. J. Cardiol.;
(3) 344–8.
References
132
Kemi O., Ellingsen O., Smith G. and Wisloff U. (2008): Exercise-
induced changes in calcium handling in left ventricular cardiomyocytes.
J. Front Biosci.; (13)356–368.
Kilickap S., Barista I., Akgul E., Aytemir K., Aksoy S. and Tekuzman G.
(2007): Early and late arrhythmogenic effects of doxorubicin. South.
Med. J. ;(100) 262-265.
Kim Y., Ma AG., Kitta K., Fitch S., Ikeda T., Ihara Y., Simon A., Evans
T. and Suzuki Y.(2003): Anthracycline-induced suppression of GATA-4
transcription factor: implication in the regulation of cardiac myocyte
apoptosis. J.Mol. Pharmacol.; (63)368–377.
Klara B., Andreas B., Birgit B., Mehlhorn U. and Robert H. (2001):
Nebivolol, bucindolol, metoprolol and carvedilol are devoid of intrinsic
sympathomimetic activity in human myocardium. Br. J. Pharmacol.;
(133) 1330-1338.
Koc A., Duru M., Ciralik H., Akcan R. and Sogut S.(2005): Protective
agent, erdosteine, against cisplatin-induced hepatic oxidant injury in rats.
J. Molecular and Cellular Biochemistry;278(1-2):79–84.
Koeth R. and Robert S. (2013): "Intestinal microbiota metabolism of L-
carnitine, a nutrient in red meat, promotes atherosclerosis". J. Nature
Medicine ; (5) 576–85.
Kolodziejczyk J., Saluk-Juszczak. J, Wachowicz S. (2011): L-carnitine
protects plasma components against oxidative alterations. J. Nutrition ;
(6)693–699.
Koopman G., Reutelingsperger C., Kuijten G., Keehnen R., Pals S. and
van Oers M. (1994): Annexin V for flow cytometric detection of
References
133
phosphatidylserine expression on B cells undergoing apoptosis. J. Blood;
(84)1415–1420.
Laginha K. (2005): "Determination of Levels Doxorubicin in Whole
Tumor and Tumor Nuclei in Murine Breast Cancer Tumors." J.Clinical
Cancer Research; (11)19-24.
Laub M., Paetzke-Brunner I. and Jaeger G. (1986) :Serum carnitine
during valproic acid therapy. J.Epilepsia.; 27(5):559–562
Lee V., Harris D. and Robert Z. (2011) : Adriamycin nephropathy: A
model of focal segmental glomerulosclerosis. J.Nephrology; (16)30–38
Lebrecht D., Setzer B., Rohrbach R. and Walker U.(2004):
Mitochondrial DNA and its respiratory chain products are defective in
doxorubicin nephrosis. J.Nephrol. Dial. Transplant.; (19)329-336.
Lewis A., Taylor R., Hall B., Gonzalez M., Willis S. and Stratford P.
(2006): Pharmacokinetic and safety study of doxorubicin-eluting beads in
a porcine model of hepatic arterial embolization . J. Vasc. Interv.
Radiol.; (8)1335-43.
Logue S., Gustafsson A., Samali A. and Gottlieb R. (2005):
Ischemia/reperfusion injury at the intersection with cell death. J. Mol.
Cell Cardiol.; (38)21–33.
Lotrionte M., Palazzoni G., Natali R. And Comerci S. (2009):
Appraising cardiotoxicity associated with liposomal doxorubicin by
means of tissue Doppler echocardiography end-points: rationale and
design of the LITE. Med. J.; (100) 262-265.
References
134
Lou H., Danelisen I. and Singal P. (2005): Involvement of mitogen-
activated protein kinases in Adriamycin-induced cardiomyopathy. Am. J.
Physiol . J. Heart Circ. Physiol.;(288) 1925–1930.
Lyman G., Kuderer N. and Crawford J. (2011): Predicting individual
risk of neutropenic complications in patients receiving cancer
chemotherapy. J. Cancer, 117(9):1917-1927.
Lynch K., Feldman H., Berlin J., Flory J., Rowan C. and Brunelli S.
(2008): Effects of L-carnitine on dialysis-related hypotension and muscle
cramps: a meta-analysis. Am J Kidney Dis., 52(5):962-71.
Maccari F. and Ramacci M. (1981): Antagonism of doxorubicin
cardiotoxicity by carnitine is specific of the L-diasteroisomer. J.
Biomedicine; (2)65-67.
Maffei A., Di Pardo A., Carangi R., Carullo P. and Poulet R. (2007):
Nebivolol Induces Nitric Oxide Release in the Heart through Inducible
Nitric Oxide Synthase Activation. J. Hypertension; (50) 652-656.
Malaguarnera M., Cammalleri L., Gargante M., Vacante M., Colonna
V. and Motta M. (2007): L-Carnitine treatment reduces severity of
physical and mental fatigue and increases cognitive functions in
centenarians: a randomized and controlled clinical trial. J. Biomedicine;
(6) 1738-1744.
Malcom J., Arnold O., Howlett J., Ducharme A., Ezekowitz J., Gardner
M., Giannetti N., Haddad H., Heckman G., Isaac D., Jong P., Liu P.,
Mann E., McKelvie R., Moe G., Svendsen A., Tsuyuki R., O’Halloran
K., Ross H., Sequeira E. and White M. (2008): Canadian Cardiovascular
Society Consensus Conference Guidelines on Heart Failure – 2008
Update: best practices for the transition of care of heart failure patients,
References
135
and the recognition, investigation and treatment of cardiomyopathies.
Can. J. Cardiol.; (24)21–40.
Mangrella M., Rossi F., Fici F. and Rossi F. (1998): Pharmacology of
nebivolol. J. Pharmacol Res. 38(6):419-31.
Marcovina S., Sirtori C., Peracino A., Gheorghiade M., Borum P.,
Remuzzi G. and Ardehali H. (2013): "Translating the basic knowledge of
mitochondrial functions to metabolic therapy: Role of L-carnitine". J. of
Translational Research . (2): 73–84.
Marie-Cécile L., Thierry L., Akthar S., Marie A., Brigitte L., Danièle S.,
Agnès C., Jean-Michel C., Chantal B., Alain B., Florent de V., Gilles .V
and Catherine A (2007): Mitoxantrone, Radiotherapy Granulocyte C
Stimulating Factor: Risk Factors for Leukemia and Myelodysplastic
Syndrome After Breast Cancer J. Cancer ; (21) 1074-1081.
Mason R., Kubant R., Jacob R., Walter M. and Boychuk B. (2006):
Effect of nebivolol on endothelial NO and peroxynitrite release in
hypertensive animals: Role of antioxidant activity. J. Cardiovasc.
Pharmacol.; (48) 862-869.
McFalls E., Paulson D., Gilbert E. and Shug A. (1986): L. Carnitine
protection against adriamycin-induced cardiomyopathy in rats.J. Life
Sci.;38:497–505.
Mehmet G., Kayaa S., Metin O., Ozgur G., Hasan A., Esma G.,
Mahmut A., Nihat K., Mustafa D., Mikail Y., Veli B., Idris A., Ali E.
and Yat Y. (2013): Protective effects of nebivolol against anthracycline-
induced cardiomyopathy: A randomized control study. International J. of
Cardiology; (167) 2306–2310
References
136
Minotti G., Menna P., Salvatorelli E., Cairo G. and Gianni L. (2004):
"Anthracyclines: molecular advances and pharmacologic developments in
antitumor activity and cardiotoxicity". J.Pharmacol. Rev.; (56) 185–229.
Moen M. and Wagstaff A. (2006): Nebivolol A review of its use in the
management of hypertension and chronic heart failure. J.Drugs ;
(66)1389–1409.
Monti E., Prosperi E., Supino R. and Bottiroli G. (1995): Free radical-
dependent DNA lesions are involved in the delayed cardiotoxicity
induced by Adriamycin in the rat. J.Anticancer Res.; (15)193–197.
Morgan S., Anderson A., Gourley C., Wallace H. and Spears N. (2012):
How do chemotherapeutic agents damage the ovary? J. Human
Reproduction Update ; 18(5) 525-535.
Nagami K., Yoshikawa T., Suzuki M., Wainai Y. and Anzai T. (1997):
Abnormal beta-adrenergic transmembrane signaling in rabbits with
adriamycin-induced cardiomyopathy.Circ. J.; (61) 249-255.
Nathalie A., Jean P., Pierre J., and Thierry L. (1999): L-carnitine
prevents doxorubicin-induced apoptosis of cardiac myocytes: role of
inhibition of ceramide generation ; The FASEB J. (13): 25-43.
Neri B., Cini-Neri G., Bartalucci S. and Bandinelli M.
(1986):Protective effect of L-carnitine on cardiac metabolic damage
induced by doxorubicin in vitro. J.Anticancer Res.;6:659–662.
Neumann D., Schlattner U. and Wallimann T. (2003): A molecular
approach to the concerted action of kinases involved in energy
homoeostasis. J.Biochem. Soc. Trans.; (31)169–174.
References
137
Olga Hilas (2009): Nebivolol (Bystolic), a Novel Beta Blocker for
Hypertension. J.Pharmacy and Therapeutics; (34)188-192.
Olpin S. (2005): "Fatty acid oxidation defects as a cause of
neuromyopathic disease in infants and adults". J.Clin. Lab.; (51) 289–
306.
Othman A. , Mohamed M. , Zeinab K. , Salim S. and Yosef A. (2010):
Doxorubicine toxicity can be amilorated during antioxidant L- Carnitine
supplementation . J. Oxid Med Cell Longev. ; 3(6): 428–433.
Paolo S. , Paola A. and Silvano G. (2004):The mechanism of
doxorubicin (DOX)-induced cardiotoxicity . J. Integrat. Cancer Ther.;
(59) 257-261.
Paterna S., Furitano G., Scaffidi L., Barbarino C., Campisi D., Parisi
D. and Carreca I. (1984): Effects of L-carnitine on adriamycin-induced
cardiomyopathy in rabbit. Int. J. Tissue React.;6:91–95.
Patrianakos A., Parthenakis F., Mavrakis H., Diakakis G. and
Chlouverakis G. (2005): Comparative Efficacy of Nebivolol Versus
Carvedilol on Left Ventricular Function and Exercise Capacity in Patients
With Nonischemic Dilated Cardiomyopathy. A 12-month study. Am.
Heart J.; (99)150: 985.
Pekala J., Patkowska-Sokoła B., Bodkowski R., Jamroz D.,
Nowakowski P., Lochyński S. and Librowski T. (2011): "L-carnitine--
metabolic functions and meaning in humans life". J.Current drug
metabolism; (12) 667–678.
Pessina A. (2001): "Metabolic effects and safety profile of nebivolol". J.
Cardiovasc. Pharmacol.; (38) 33–5.
References
138
Pettegrew J., Levine J. and McClure R.(2000): Acetyl-L-carnitine
physical-chemical, metabolic, and therapeutic properties: relevance for its
mode of action in Alzheimer's disease and geriatric depression. J.Mol.
Psychiatry; (5)616-632.
Poirier L., Cléroux J., Nadeau A. and Lacourcière Y. (2001): "Effects
of nebivolol and atenolol on insulin sensitivity and haemodynamics in
hypertensive patients". J. Hypertens.; (19) 1429–35.
Prisant L. (2008): Nebivolol: pharmacologic profile of an ultraselective,
vasodilatory B blocker. J.Clin. Pharmacol.; (48)225–39.
Punzi H., Lewin A., Lukić T., Goodin T. and Wei C. (2010): Efficacy
and safety of nebivolol in Hispanics with stage I-II hypertension: a
randomized placebo-controlled trial. J.Ther. Adv. Cardiovasc. Dis.; (4)
349-357.
Reitman S. and Frankel S. (1957): Colorimetric method for the
determination of serum glutamic oxalacetic and glutamic pyruvic
transaminases . American Jornal of Clinical Pathology ; (28):56- 63.
Reuben A., Koch D.and Lee W. (2010): Acute Liver Failure Study
Group. Drug-induced acute liver failure: results of a U.S. multicenter,
prospective study. J. Hepatology ; (52)2065-76.
Saeki K., Obi I., Ogiku N., Shigekawa M., Imagawa T. and Matsumoto
T. (2002): Doxorubicin directly binds to the cardiac-type ryanodine
receptor. J. Life Sci.; (70)2377–2389.
Sano M., Minamino T., Toko H., Miyauchi H., Orimo M., Qin Y.,
Akazawa H., Tateno K., Kayama Y., Harada M., Shimizu I., Asahara
T., Hamada H., Tomita S., Molkentin J., Zou Y. and Komuro I. (2007):
References
139
p53-induced inhibition of HIF-1 causes cardiac dysfunction during
pressure overload. J.Cardiol.; (446)444–448.
Sawaya H., Sebag I. and Plana J. (2011): Early Detection and
Prediction of Cardiotoxicity in Chemotherapy-Treated Patients. Am. J
.Cardiol.; (11)23-29.
Sayed-Ahmed M. ( 2010): Role of carnitine in cancer chemotherapy-
induced multiple organ toxicity. Saudi Pharm. J.; (18) 195-206.
Sayed-Ahmed M., Aleisa A. and Al-Rejaie S. (2010a ):Thymoquinone
attenuates diethylnitrosamine induction of hepatic carcinogenesis through
antioxidant signaling. J. Oxidative Medicine and Cellular Longevity;
3(4):254 –261.
Sayed-Ahmed M., Al-Shabanah O., Hafez M., Aleisa A. and Al-Rejaie
S. (2010 b): Inhibition of gene expression of heart fatty acid binding
protein and organic cation/carnitine transporter in doxorubicin
cardiomyopathic rat model.Eur. J. Pharmacol.;640:143–149.
Sayed-Ahmed M., Rishk A., Soloma S. and Abdel-aleem S. (2000 a):
Protection by L-carnitine against the inhibition of gene expression of
heart fatty acid binding protein by chronic administration of doxorubicin.
J. Egypt. Natl. Cancer Inst.; (12):275–281.
Sayed-Ahmed M., Salman T., Gaballah H., Abou-El-Naga S. and
Raffaella, Nicolai R. (2001): Propionyl-L-carnitine as protector against
adriamycin-induced cardiomyopathy. Pharmacol. J. Res.; (43):513–520.
Sayed-Ahmed M., Shouman S., Rezk B., Khalifa M., Osman A. and El-
Merzabani M. (2000 b):Propionyl-L-carnitine as potential protective
agent against adriamycin-induced impairment of fatty acid beta-oxidation
in isolated heart mitochondria. J.Pharmacol. Res.; (41):143–150.
References
140
Schemer S . ( 1967):The blood morphology of laboratory animals, 3rd
edition (Davis–F A Company, Pheladephia, U S A) chapter 5 . P. 14-21.
Schulmeister L. (2007): A new agent for treating anthracycline
extravasation.Clin. J. Oncol .Nurs.; 11(3):387-395.
Shi Y., Moon M., Dawood S., McManus B., Liu P. and Herz P. (2011):
Mechanisms and management of doxorubicin cardiotoxicity. J.
Prog.Cardiovasc. Dis.; (49)330-52.
Shug A. (1987):Protection from adriamycin-induced cardiomyopathy in
rats. J. cardiol.;76:46–52.
Simunek T., Sterba M., Popelova O., Adamcova M., Hrdina R .and
Gersl V. (2009): Anthracycline-induced cardiotoxicity: overview of
studies examining the roles of oxidative stress and free cellular iron. J.
Pharmacol Rep.; (61)154–171.
Sinclair S. (2000): Male infertility: nutritional and environmental
considerations.Alt Med Rev.; (5)28-38.
Steiber A., Kerner J. and Hoppel C. (2004): "Carnitine: a nutritional,
biosynthetic, and functional perspective". J.Mol. Aspects Med.; (25) 455–
73.
Stephen C., Piscitelli P., Keith A., Rodvold P., Daniel A., Rushing M.,
Duane A. and Marshfield W. (1993): Pharmacokinetics and
pharmacodynamics of doxorubicin in patients with small cell lung cancer.
J.Clinical Pharmacology and Therapeutics; (53) 555–561.
St Marie B. and Camp-Scroll R.(2000):Clinical manual for the oncology
practice nurse. J. Of drugs; (8)133-134.
References
141
Swain S., Whaley F. and Ewer M. (2003): Congestive heart failure in
patients treated with doxorubicin. A retrospective analysis of three trials.
J. Cancer ; (97)2869–2879.
Tacar O., Sriamornsak P. and Dass C. (2013): "Doxorubicin: an update
on anticancer molecular action, toxicity and novel drug delivery
systems." J. of Pharmacy and Pharmacology; (65 ) 157–70.
Tafreshi M. and Weinacker A. (1999): "Beta-adrenergic-blocking agents
in bronchospastic diseases: a therapeutic dilemma". J.Pharmacotherapy ;
(19) 974–8.
Takemura G. and Fujiwara H. (2007): Doxorubicin-induced
cardiomyopathy from the cardiotoxic mechanisms to management.
J.Prog. Cardiovasc. Dis.; (49)330–352.
Takimoto C. and Calvo E. (2008): "Principles of Oncologic
Pharmacotherapy in Cancer Management ": A Multidisciplinary
Approach. J. Cancer;(11 ) 35-39
Tangpong J., Miriyala S., Noel T., Sinthupibulyakit C., Jungsuwadee P.
and Clair S. (2011): Doxorubicin-induced central nervous system
toxicity and protection by xan- thone derivative of Garcinia Mangostana.
J. Neuroscience ;(175)292–299.
Tassan-Mangina S., Codorean D. and Metivier M. (2006): Tissue
Doppler imaging and conventional echocardiography after anthracycline
treatment in adults: early and late alterations of left ventricular function
during a prospective study. Eur. J .Echocardiology ; (7)141–146.
Tokarska-Schlattner M., Zaugg M., da Silva R., Lucchinetti E., Schaub
M., Wallimann T. and Schlattner U. (2005): Acute toxicity of
doxorubicin on isolated perfused heart: response of kinases regulating
energy supply. Am. J. Physiol.; (289 ) 37–47.
References
142
Trochu J., Erfanian M., Khandoudi N., Baron O. and Bril A. (1999):
Carvedilol produces contractile effects different from those of bucindolol
in human atria.J.Circulation; (100 ) 421-439.
Van Dalen E., Caron H., Dickinson H. and Kremer L. (2008):
"Cardioprotective interventions for cancer patients receiving
anthracyclines". Cochrane Database Syst .Rev.; (2)404–407
Villani R., Gannon J., Self M. and Rich P. (2000): L-carnitine
supplementation combined with aerobic training does not promote weight
loss in moderately obese women. Int. J .Sport Nutr. Exerc. Metab.;
(10)199-207.
Volek J., Judelson D., Silvestre R., Yamamoto L., Spiering B., Hatfield
D., Vingren J., Quann E., Anderson J., Maresh C. and Kraemer W.
(2008): Effects of carnitine supplementation on flow-mediated dilation
and vascular inflammatory responses to a high-fat meal in healthy young
adults.Am. J .Cardiol.; (102)1413-1417.
Waldner R., Laschan C., Lohninger A., Gessner M., Tüchler H.,
Huemer M., Spiegel W. and Karlic H. (2006):Effects of doxorubicin-
containing chemotherapy and a combination with L-carnitine on
oxidative metabolism in patients with non Hodgkin lymphoma. . Cancer
Res. Clin. Oncol.;132:121–128.
Washio M., Nanishi F., Okuda S., Onoyama K. and Fujishima M.
(1994): Alpha tocopherol improves focal glomerulosclerosis in rats with
Adriamycin-induced progressive renal failure. J. Nephron; (68)347-352.
Weiss R. (2006): "Nebivolol: a novel beta-blocker with nitric oxide-
induced vasodilatation". J.Vasc. Health Risk Manag.;( 2 ) 303–8.
References
143
Witte K. and Clark A.(2006): Micronutrients and their supplementation
in chronic cardiac failure. An update beyond theoretical perspectives.
Heart Fail. Rev.; (11)65-74.
Wojciechowski D. and Papademetriou V. (2008): Beta-blockers in the
management of hypertension: Focus on nebivolol. Expert Rev Cardiovasc
Ther., 6:471–479.
Xue Y., Wang L., Liu H., Qi X., Wang X. and Ren H. (2007): L-
carnitine as an adjunct therapy to percutaneous coronary intervention for
non-ST elevation myocardial infarction. J.Cardiovasc. Drugs Ther.;
(21)445-8.
Yagmurca M., Bas O. and Mollaoglu H. (2007): Protective effects of
erdosteine on doxorubicin-induced hepatotoxicity in rats. Archives of
Medical Research.;38(4):380–385.
Yang Y., Hua C. and Dong C. (2009): Synthesis, self-assembly, and in
vitro doxorubicin release behavior of dendron-like/linear/dendron-like
poly(epsilon-caprolactone)-b-poly(ethylene-glycol)-b-poly(epsilon-
caprolactone) triblock copolymers. J.Biomacromolecules; (10)2310-2318.
Yapar K., Kart A. and Karapehlivan M. (2007 ): Hepatoprotective effect
of l-carnitine against acute acetaminophen toxicity in mice. J.
Experimental and Toxicologic Pathology ;59(2):121–128.
Zeidan Q., Strauss M., Porras N. and Anselmi G. (2002):Differential
long-term subcellular responses in heart and liver to adriamycin stress.
Exogenous L-carnitine cardiac and hepatic protection. J. Submicrosc.
Cytol. Pathol.;34:315–321.
Zhang S., Liu X., Bawa-Khalfe T., Lu L., Lyu Y., Liu L. and Yeh E.
(2012): "Identification of the molecular basis of doxorubicin-induced
cardiotoxicity."J. Nature Medicine; (18) 1639–42.
Arabic summary
1
الملخص العربي
ررا زاث اعخخدت عالس سض اعرسغايعخبس عماز ادوعيسيبعي اعد أ اعما
.يت وامب اىبد اى اعماز عدة اراز صابيت أا حأريسة ع األععاء األظاظ
ان اعديرد ر اضر ر فر عرالس اعرسغا ررا فرا اظرخخدبيت اخ حغرد ر ر األراز اضا
.ابر غا ع حه اشى حمي ظي را اعمازعخ يخاط اظخخد ع طاق اظع
ع غسيرك لرك عرخمبالث يعخبس ايبيفيي األ ي اعخخد ف عالس ازحفاع ظغػ اد
عي ادي لد صد أ حأريس عا ريس اظع الأبيخا ف امب شيا اوعيد ايخسصي ذ اخ
ألوعد ا لد يخيظ اظخخدا ف الاي ظي بعط األ ي اخ حعس اضعر عر غسيرك شيرا
. ظ ر األ ي عماز ادوعيسيبعي ف اضع األوعد
فر اضعر تظرخخساس عر عرسق ارد اخر حعراعد اي وسيخي اا اطبيعي أيعا فا
فر صعر اتعرا فر اىبرد اىر يرخ حخصير فر اععرالث امرب اطال يخ حصريع
را اداء ايعا حأريس عا ألوعد ا لد يخيظ اظرخخدا فر الاير ر .اخ اغيااث اي
.ظي بعط األ ي
امرب ع ظيت اي وسيخي ايبيفيي أريس الائ اغخ اخ دزاظتس را ابغذ لد أص
غيارراث اخضررازن رر ررالي عمرراز ادوعيسيبعرري حضسيبيررا باظررطت اغدرررت اىبررد اىرر
امرب اخرسبي ا اىسيراحي فظرف وييرص اصيراث اصياث ظغػ اد زظ امب زاظت
.امب اىبد اى ازالر يي باعب ى ع زاظت عخصيا األععاءلياض اىسياح اىبد
Arabic summary
2
رع ادوعيسيبعي حأريس ورهادوعيسيبعي حأريس بغذ يجد اصسلر ادزاظت أيعا فا
صسعرراث حررأريس ورررهعرر اتمبرراض اخمررائ ععرر امررب اعررصي رر األزررب اتيصبسيرراي
امرب اعرصي ر افنرسا فر ضعراث اتمباض اخمائ ععر اتيصبسياي ع خخف
. ادزاظت
ضعراث عر ٳ فنسا اخضازن لد لعج ٳف اغيا باعبت دزاظت اخ أصسيج ا صع
واخا:و ضع ى ظخ فنسا خعايت
ادوعيسيبعري ضرت بضسعراث ر ااععماز(، اضعرت ازايرت ) حعاش بأات ) اضعت
رر ضررت بضسعرراثااع(، اضعررت ازازررت )ضررك وررش عمررت اعرردة فرر اغشرراء ابسحرر 3بضسعررت
ايبيفيرري باتظررافت اررضررك وررش عمررت اعرردة فرر اغشرراء ابسحرر 3ادوعيسيبعرري بضسعررت
)اعاضرررت بضسعررراث ررر ييرررا عررر غسيرررك افررر(، اضعرررت اسابعرررت ) ك ورررشضررر 1بضسعرررت
اي وررسيخي باتظررافت اررضررك وررش عمررت اعرردة فرر اغشرراء ابسحرر 3ادوعيسيبعرري بضسعررت
)اعاضرت بضسعراث اضعرت اخاعرت (ضك وش عمت اعدة ف اغشاء ابسحر 222بضسعت
ايبيفيري باتظرافت ارضك ورش عمرت اعردة فر اغشراء ابسحر 3يسيبعي بضسعت ادوع
ضررك وررش عمررت اعرردة فرر 222اي وررسيخي بضسعررت ضررك وررش ييررا عرر غسيررك افرر 1بضسعررت
.ي 12اعالس د أ رث و ر ضعاث (اغشاء ابسح
فرط اصيراث عرعصائيت ٳا حأريس ذ تت وسيخي ايبيفيي اي عماز لد صد أ
، امررب اخررسبي ا اىسيرراحي فظررف وييررص اصيرراث اىبررد عررب اىسيرراحيي باعررب ىرر
Arabic summary
3
فر حمير عردد ز راورا عمراز ايبيفيري اي ورسيخي أظسث ادزاظت أيعرا أ ور ر
.امب اىبد اىعبت ا اصا اخي اخغيساث
اتمباض اخمائ ععر امرب اعرصي لادوعيسيبعي ث أأيعا فا ر ادزاظت صد
ر ععر امرب اعرصي حرأريس اتيصبسيراي عر اتمبراض اخمرائ لر ر األزب وره
ععر امرب تيصبسياي ع اتمباض اخمرائحأريس صسعاث خخف ا ل األزب وره
فرر ضعرراث افنررسااأل يررت زرر صسعرراث أ رررث رفررط اعررصي رر افنررسا فرر ضعرراث
حغعرر ذ ترر اي وررسيخي ايبيفييبرراعاضررت حغعرر فرر اضعرراث خررأريسرررا ا ادزاظررت
. عصائيتٳ
ظرخخدا فر ٳيىر فعاي ذ حأريس ايبيفيي اي وسيخي ازعم يعخخش ر ادزاظت أ
عرر حرأريس رارا ادوعيسيبعري اغدررت باظر ت امب اىبرد اىر ظيت الايت
فررط اصيرراث امررب حررسبي ا اىسيرراحي فظررف وييررص اصيرراث اىبررد عررب اىسيرراحيي
.باعب ى