mechanisms underlying the contractile responses of
TRANSCRIPT
MECHANISMS UNDERLYING THE CONTRACTILE
RESPONSES OF INTESTINAL SMOOTH MUSCLES TO
AZITHROMYCIN USING ANIMAL MODELS
Rizwan Faisal
2014/KMU/IBMS/PhD/FS/8
PhD Thesis
Pharmacology
Institute of Basic Medical Sciences
Khyber Medical University
Peshawar
October 2020
MECHANISMS UNDERLYING THE CONTRACTILE
RESPONSES OF INTESTINAL SMOOTH MUSCLES TO
AZITHROMYCIN USING ANIMAL MODELS
A thesis submitted in the partial fulfillment of the requirement for the degree of
Doctor of Philosophy
In
Pharmacology
Rizwan Faisal
2014/KMU/IBMS/PhD/FS/8
Institute of Basic Medical Sciences
Khyber Medical University
Peshawar
October 2020
CERTIFICATE
This thesis by Mr. Rizwan Faisal titled “Mechanisms underlying the contractile
responses of intestinal smooth muscles to azithromycin using animal models” is
accepted in its present form, by the Department of Pharmacology, Institute of Basic
Medical Sciences, Khyber Medical University Peshawar, as satisfying thesis
requirements for award of degree of Doctor of Philosophy in Pharmacology.
Supervisor: ____________________
(Prof. Dr. Niaz Ali)
External Examiner: ____________________
(Dr…………………...)
Internal Examiner: ____________________
(Dr…………………...)
Director IBMS: ____________________
(Dr…………………...)
Dean Basic Medical Sciences,
Institute of Basic Medical Sciences. _____________________
(Prof. Dr. Jawad Ahmed)
i
DEDICATION
This work is dedicated to my two beautiful souls; my parents. You have made me
stronger, better and more fulfilled than I could have ever imagined. Your positivity and
immense love has made me a better version of me. I hope I have brought pride to my
family which is a source of self- contentment and harmony at my end.
ii
DECLARATION
I hereby declare that the work accomplished in this thesis is my own research effort
carried out in the department of Pharmacology, Institute of Basic Medical Sciences,
Khyber Medical University Peshawar. The thesis has been written and composed by
me.
The work in this thesis has neither been previously submitted for examination leading
to the award of a degree nor does it contains any material from the published
resources that can be considered as the violation of the international copyright law.
I also declare that I am aware of the terms ‘copyright’ and ‘plagiarism’. I will be solely
responsible for the consequences of violation to these rules (if any) found in the thesis.
The thesis has been checked for plagiarism through turnitin software.
Name: Rizwan Faisal
Signature: _____________
Date: November 16, 2021
iii
ABSTRACT
Background: Azithromycin is one of the most widely prescribed antibiotics worldwide
for various clinical conditions. So far there are controversial reports regarding its
actions on intestinal smooth muscles. The mechanisms by which Azithromycin affects
intestinal contractility are still unexplored.
Objectives:
i. To determine the effects of Azithromycin on the contractility of intestinal
smooth muscles in vitro model of rabbits and rats using Power Lab system
ii. To investigate the mechanisms of Azithromycin affecting the intestinal
contractility through receptors and voltage gated ion channels by using Power
Lab system
Methodology: Rabbit’s jejunal and rat’s ileal preparations were mounted in organ
bath containing 15 ml Tyrode’s solution, constantly aerated with carbogen gas.
Azithromycin was tested on rabbit’s jejunal and rat’s ileal preparations in
concentrations of 0.01, 0.03, 0.1, 0.3, 1, 3, 5, 10 and 15µM µg/ml and response for
each concentration was recorded for 2 min. Spasmogenic activity of Azithromycin was
observed. To explore its mechanism of action, response of Azithromycin was noted in
the presence of 0.3µM Atropine, 3µM Loratadine, 0.3µM Ondansetron, 3µM
Metaclopramide, 0.3µM Verpamil, 0.3µM Propranolol, 3µM Amiodarone and 0.3µM
Atropine, Ondansetron, Verapamil and Propranolol.
Results: Mean (%) Emax for AZM was 67.6±1.6 and 54.0±2.1 for rabbits’ jejunal and
rats’ ileal preparations, respectively. Mean (%) Emax in the presence of Atropine for
rabbits’ jejunal preparations was 2.4±0.1 and for rats’ ileal preparations it was
11.4±1.3. Similarly, mean (%) Emax was 67.9 ± 2.0 for rabbits’ jejunal preparations and
was 50.7±1.9 for rats’ ileal preparations in the presence of Loratadine. Mean (%) Emax
in the presence of Ondansetron was 27.5±0.5 for rabbits’ jejunal preparations and
34.0±2.9 for rats’ ileal preparations. In the presence of Metoclopramide, mean (%)
iv
Emax was 88.4±1.2 for rabbits’ jejunal preparations and 79.1±3.8 for rats’s ileal
preparations. Mean (%) Emax in the presence of Verapamil for rabbits’ jejunal
preparations was 13.6±1.2 and for rats’ ileal preparations it was 22.3±2.5. Mean (%)
Emax in the presence of Propranolol was 10.2±2.1 for rabbits’ jejunal preparations and
15.6±1.4 for rats’ ileal preparations. Mean (%) Emax in the presence of Amiodarone
was 68.4±1.3 for rabbits’ jejunal preparations and 58.0±3.4 for rats’ ileal preparations.
The spasmogenic response of Azithromycin was completely lost when it was given in
the presence of Atropine, Ondansetron, Verapamil and Propranolol.
Conclusion: Results reveal that the spasmogenic response of Azithromycin is mainly
mediated through muscarinic receptors. However, we found involvement of mixed
pathways including serotonergic receptors, voltage gated calcium channels and
voltage gated sodium channels.
Keywords: Azithromycin, Acetylcholine, Atropine, Voltage gated sodium channels,
Voltage gated calcium channels, Voltage gated potassium channels
5
ACKNOWLEDGMENT
Thanks to Allah, Almighty for all his blessings upon me throughout my life and who
enabled me to complete my thesis.
I am deeply obliged to my supervisor Prof. Dr. Niaz Ali who guided me during my
research and thesis. His keen interest and valuable suggestions made this work to end
in a refined manner.
It is my privilege to thank my family, my wife, who has been extremely supportive
throughout and has made countless sacrifices to help me reached to this status. My
children, Rameen Karim and Hania Karim, they had been my inspiration to achieve
greatness, without them, I would not have reached my goal where I am standing now.
My mother, brother, sister in law and nephew Alyaan Karim; deserves special thanks
for their continued support and encouragement. I have been blessed with such kind
and affectionate family who had always faith in me.
A very special thanks to my father-in-law, Prof. Dr. Zabta Khan Shinwari, who has
always been my true role model and mentor, his encouraging words and assistance in
every step of my research work has always given me strength.
Lastly, I am indebted to my endear colleague, PhD scholar, Muhammad Nabi who
untiringly assisted me in Power Lab activity. I am also very thankful to Amjad Khan,
Pharmacology laboratory technician, whose association and facilitation helped me in
performing various activities related with my research work and stood firm by my side,
which is a true asset.
RIZWAN FAISAL
1-Introduction
6
1-Introduction
7
TABLE OF CONTENTS
DEDICATION i
DECLARATION
ii
ABSTRACT
iii
ACKNOWLEDGEMENTS
v
TABLE OF CONTENTS vi
LIST OF TABLES
x
LIST OF FIGURES
xi
LIST OF ABBREVIATIONS xiv
1. INTRODUCTION
1
1.1 Neurotransmitters
2
1.1.1 Acetylcholine
2
1.1.2 Histamine
3
1.1.3 Dopamine
6
1.1.4 Serotonin
7
1.2 Voltage gated ion channels
10
1-Introduction
8
1.2.1 Sodium channels
10
1.2.2 Calcium channels
11
1.2.3 Potassium channels
12
1.3 Hormonal control of gastrointestinal motility
16
1.4 Intrinsic and extrinsic innervation of gastrointestinal motility
17
1.4.1 Parasympathetic control of gastrointestinal tract
17
1.4.2 Sympathetic control of gastrointestinal tract
18
1.4.3 Role of gut microbiota in gastrointestinal motility
18
1.5 Animal models for in vitro preparation
21
1.5.1 History of experimental animals
21
1.5.2 Purpose of animal models
21
1.5.3 Animal models for gastrointestinal disorders
22
1.5.3.1 In vitro animal models for GI motility
23
1.5.3.2 Rabbits
23
1.5.3.3 Rats
24
1.5.3.4 Advantages of in vitro techniques
25
1.6 Current prokinetic agents
25
1-Introduction
9
1.6.1 Cholinergic agonists
26
1.6.2 Serotonergic agonists
26
1.6.3 Dopaminergic antagonists
27
1.6.4 Erythromycin
28
1.6.5 Miscellaneous drugs
29
1.7 Azithromycin
29
1.7.1 Source
29
1.7.2 Structure
29
1.7.3 History
32
1.7.4 Administration
32
1.7.5 Mechanism of action
32
1.7.6 Half-life and tissue penetration
34
1.7.7 Clinical uses
34
1.7.8 Advantages of Azithromycin over Erythromycin
35
1.7.9 Advantages of Azithromycin over other antibiotics
35
1.8 Pharmacological actions of Azithromycin
35
1.8.1 Antibacterial action
35
1-Introduction
10
1.8.2 Immunomodulatory action
36
1.8.3 Anti-inflammatory action
37
1.9 Research studies of azithromycin related with smooth muscle contractility
37
1.9.1 Effect of Azithromycin on aortic strips
37
1.9.2 Effect of Azithromycin on uterine smooth muscles
38
1.9.3 Effect of Azithromycin on respiratory smooth muscles
38
1.9.4 Effect of Azithromycin on GI smooth muscles
41
1.9.4.1 Azithromycin and esophageal smooth muscle contractility
41
1.9.4.2 Effect of Azithromycin on gall bladder smooth muscles
41
1.9.4.3 Azithromycin and gastroparesis
42
1.9.4.4 Gastric motility disorders
44
1.9.4.5 Azithromycin and intestinal contractility
45
1-Introduction
11
2. MATERIAL AND METHODS
48
2.1 Animals
48
2.2 Chemicals and drugs
48
2.3 Preparation of isolated tissue
49
2.3.1 Rabbit’s jejunal and rat’s ileal preparation
49
2.4 Effects of drugs on rabbit’s jejunal and rat’s ileal preparation
52
2.4.1 Effect of azithromycin on rabbit’s jejunal and rat’s ileal preparation
52
2.4.2 Validation of curves
52
2.4.2.1 Acetylcholine
52
2.4.2.2 Atropine
52
2.4.2.3 Loratadine
53
2.4.2.4 Ondansetron
53
2.4.2.5 Metoclopramide
54
2.4.2.6 Verapamil
54
2.4.2.7 Propranolol
54
2.4.2.8 Amiodarone
54
2.4.3 Effect of Azithromycin in the presence of receptor and channel
blockers 55
1-Introduction
12
2.4.3.1 Effect of Azithromycin in the presence of Atropine
55
2.4.3.2 Effect of Azithromycin in the presence of Loratadine
55
2.4.3.3 Effect of Azithromycin in the presence of Ondansetron
55
2.4.3.4 Effect of Azithromycin in the presence of Metoclopramide
56
2.4.3.5 Effect of Azithromycin in the presence of Verapamil
56
2.4.3.6 Effect of Azithromycin in the presence of Propranolol
56
2.4.3.7 Effect of Azithromycin in the presence of Amiodarone
57
2.4.3.8 Effect of Azithromycin in the presence of Atropine, Ondansetron,
Verapamil and Propranolol
57
2.5 Statistical analysis
57
3. RESULTS
58
3.1 Validation of curves
58
3.1.1 Atropine
58
3.1.2 Loratadine
61
3.1.3 Ondansetron
64
3.1.4 Metoclopramide
67
3.1.5 Verapamil
70
1-Introduction
13
3.1.6 Propranolol
73
3.1.7 Amiodarone
76
3.2 Effect of Azithromycin on rabbit’s jejunal and rat’s ileal preparation
79
3.3 Effect of Azithromycin in the absence and presence of antagonists
82
3.3.1 Effect of Azithromycin in the absence and presence of Atropine
82
3.3.2 Effect of Azithromycin in the absence and presence of Loratadine
87
3.3.3 Effect of Azithromycin in the absence and presence of Ondansetron
92
3.3.4 Effect of Azithromycin in the absence and presence of
Metoclopramide 97
3.3.5 Effect of Azithromycin in the absence and presence of Verapamil
102
3.3.6 Effect of Azithromycin in the absence and presence of Propranolol
107
3.3.7 Effect of Azithromycin in the absence and presence of Amiodarone
112
3.3.8 Effect of Azithromycin in the absence and presence of Atropine,
Ondansetron, Verapamil and Propranolol
117
4. DISCUSSION
122
5. CONCLUSION
130
REFERENCES
131
PUBLICATION
1-Introduction
14
LIST OF TABLES
Table 3.1 Effect of Atropine on rabbits’ jejunal preparations and rats’ ileal
preparations
58
Table 3.2 Effect of Loratadine on rabbits’ jejunal preparations and rats’ ileal
preparations
61
Table 3.3 Effect of Ondansetron on rabbits’ jejunal preparations and rats’
ileal preparations
64
Table 3.4 Effect of Metaclopramide on rabbits’ jejunal preparations and
rats’ ileal preparations
67
Table 3.5 Effect of Verapamil on rabbits’ jejunal preparations and rats’ ileal
preparations
70
Table 3.6 Effect of Propranolol on rabbits’ jejunal preparations and rats’
ileal preparations
73
Table 3.7 Effect of Amiodarone on rabbits’ jejunal preparations and rats’
ileal preparations
76
Table 3.8 Effect of Azithromycin on rabbit’s jejunal preparations and rat’s
ileal preparations
79
Table 3.9 Effect of Azithromycin in the absence and presence of Atropine
on rabbit’s jejunal preparations and rat’s ileal preparations
82
Table 3.10 Effect of Azithromycin in the absence and presence of Loratadine
on rabbit’s jejunal preparations and rat’s ileal preparations
87
Table 3.11 Effect of Azithromycin in the absence and presence of
Ondansetron on rabbit’s jejunal preparations and rat’s ileal
preparations
92
Table 3.12 Effect of Azithromycin in the absence and presence of
Metaclopramide on rabbit’s jejunal preparations and rat’s ileal
preparations
97
Table 3.13 Effect of Azithromycin in the absence and presence of Verapamil
on rabbit’s jejunal preparations and rat’s ileal preparations
102
Table 3.14 Effect of Azithromycin in the absence and presence of
Propranolol on rabbit’s jejunal preparations and rat’s ileal
preparations
107
Table 3.15 Effect of Azithromycin in the absence and presence of
Amiodarone on rabbit’s jejunal preparations and rat’s ileal
preparations
112
Table 3.16 Effect of Azithromycin in the absence and presence of Atropine,
Ondansetron, Verapamil & Propranolol (AOVP) on rabbit’s
jejunal preparations and rat’s ileal preparations
117
1-Introduction
15
LIST OF FIGURES
Figure 1.1 Chemical structure of acetylcholine 4
Figure 1.2 Gq-coupled receptor signal transduction pathway 4
Figure 1.3 Chemical structure of histamine 5
Figure 1.4 Gs and Gi-coupled receptor signal transduction pathway 5
Figure 1.5 Chemical structure of dopamine and serotonin 8
Figure 1.6 Ligand gated ion channels 9
Figure 1.7 Voltage Gated Sodium Channels 13
Figure 1.8 Voltage gated calcium channel 14
Figure 1.9 Voltage-gated Potassium channel 15
Figure 1.10 Intrinsic and Extrinsic Innervation of Gut motility 19
Figure 1.11 Bacterial distribution in the GI tract 20
Figure 1.12 Chemical structure of azithromycin 30
Figure 1.13 3D structure of azithromycin 31
Figure 1.14 Mechanism of action of Azithromycin 33
Figure 2.1 Small and Large intestines of a rabbit 50
Figure 2.2 Isolated rabbit’s jejunal preparation 51
Figure 3.1a Effect of Atropine on spontaneous rabbits’ jejunal preparations 59
Figure 3.1b Effect of Atropine on spontaneous rats’ ileal preparations 60
Figure 3.2a Effect of Loratadine on spontaneous rabbits’ jejunal preparations 62
Figure 3.2b Effect of Loratadine on spontaneous rats’ ileal preparations 63
Figure 3.3a Effect of Ondansetronon spontaneous rabbits’ jejunal preparations 65
Figure 3.3b Effect of Ondansetron on spontaneous rats’ ileal preparations 66
Figure 3.4a Effect of Metoclopramide spontaneous rabbits’ jejunal
preparations
68
Figure 3.4b Effect of Metoclopramide on spontaneous rats’ ileal preparations 69
Figure 3.5a Effect of Verapamil spontaneous rabbits’ jejunal preparations 71
Figure 3.5b Effect of Verapamil on spontaneous rats’ ileal preparations 72
Figure 3.6a Effect of Propranolol spontaneous rabbits’ jejunal preparations 74
Figure 3.6b Effect of Propranolol on spontaneous rats’ ileal preparations 75
Figure 3.7a Effect of Amiodarone spontaneous rabbits’ jejunal preparations 77
Figure 3.7b Effect of Amiodarone on spontaneous rats’ ileal preparations 78
Figure 3.8a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations
80
Figure 3.8b Effect of Azithromycin on rat’s ileal preparations 81
Figure 3.9a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Atropine
83
Figure 3.9b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Atropine
84
Figure 3.9c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Atropine
85
1-Introduction
16
Figure 3.9d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Atropine
86
Figure 3.10a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Loratadine
88
Figure 3.10b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Loratadine
89
Figure 3.10c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Loratadine
90
Figure 3.10d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Loratadine
91
Figure 3.11a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Ondansetron
93
Figure 3.11b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Ondansetron
94
Figure 3.11c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Ondansetron
95
Figure 3.11d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Ondansetron
96
Figure 3.12a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Metaclopramide
98
Figure 3.12b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Metaclopramide
99
Figure 3.12c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of
Metaclopramide
100
Figure 3.12d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Metaclopramide
101
Figure 3.13a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Verapamil
103
Figure 3.13b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Verapamil
104
Figure 3.13c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Verapamil
105
Figure 3.13d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Verapamil
106
Figure 3.14a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Propranolol
108
Figure 3.14b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Propranolol
109
Figure 3.14c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Propranolol
110
Figure 3.14d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Propranolol
111
1-Introduction
17
Figure 3.15a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Amiodarone
113
Figure 3.15b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Amiodarone
114
Figure 3.15c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Amiodarone
115
Figure 3.15d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Amiodarone
116
Figure 3.16a Effect of Azithromycin on spontaneous rabbit’s jejunal
preparations in the absence and presence of Atropine,
Ondansetron, Verapamil & Propranolol
118
Figure 3.16b Effect of Azithromycin on rat’s ileal preparations in the absence
and presence of Atropine, Ondansetron, Verapamil & Propranolol
119
Figure 3.16c Comparison of Azithromycin’s effect on spontaneous rabbit’s
jejunal preparations in the absence and presence of Atropine,
Ondansetron, Verapamil & Propranolol
120
Figure 3.16d Comparison of Azithromycin’s effect on rat’s ileal preparations in
the absence and presence of Atropine, Ondansetron, Verapamil &
Propranolol
121
1-Introduction
18
LIST OF ABBREVIATIONS
ACh Acetylcholine ASMs Airway smooth muscles BA Bile Acids BAL Bronchoalveolar Lavage BALF Broncheoalveolar Lavage Fluid B-ARs Beta-adrenoceptors BOS Bronchiolitis Obliterans Syndrome Ca++ Calcium cAMP Cyclic Adenosine monophosphate CCL5 Chemokine ligand 5 CF Cystic Fibrosis CK Creatinine kinase COPD Chronic obstructive pulmonary disease Cyt P450 Cytochrome P450 DA Dopamine DAG DPB
Diacylglycerol Diffuse Pan bronchiolitis
DRC Dose Response Curve EC SR
Enterochromafin Sarcoplasmic Reticulum
ERK Extracellular regulated protein kinases ERY Erythromycin FDA Federal Drug Administration FEV1 Forced expiratory volume in one second GBCI Gallbladder Contraction Index GERD Gastroesophageal Reflux Disease GFR GGT
Glomerular filtration rate Gamma glutamyl transferase
GI Gastrointestinal GPCR Gαq
G protein coupled receptor Gq alpha sub unit
HB 5-HT
Hemoglobin Serotonin
ICC Interstitial cells of Cajal IFN-γ IP3
Interferon-gamma Inositol triphosphate
IL-1β Interleukin-1 beta Kv Potassium channel LPS Lipopolysaccharide LTx Lung Transplantation LVDCCs L-type voltage dependent calcium channels MCs Mast cells MLCK Myosin light chain kinase MMCs Migrating Motor Contractions Nav Sodium channel (voltage gated)
1-Introduction
19
NE Norepinephrine
NF-κB Nuclear factor Kappa-B NZW New Zealand white PCV Packed cell volume PKA Protein kinase A PKC PIP2
Protein kinase C Phosphatidylinositol 4,5-bisphosphate
R848 Resiquimod RCT Sp 1 STIM
Randomized control trial Specificity protein 1 Stromal interaction molecule
TEC Total erythrocyte count TLC Total leukocyte count TRs TRPC
Tracheal rings Transient receptor potential channel
VGCCs
Voltage gated calcium channels
1 INTRODUCTION
Intestinal motility has a complex function and is regulated by sensory and motor system
of the enteric nervous system (ENS) which in turn are linked and controlled by central
and autonomic nervous systems (Furness et al., 2014). ENS performs various functions
like food propulsion, regulation of blood flow and immunological defense (Fleming et
al., 2020). The intestine is made up of smooth muscles, in most areas of the tract they
are aligned in circular and longitudinal layers (Campbell, 2015). The function of these
smooth muscles is to produce tonic contractions that maintain the dimension of organ
against a load such as imposed by a bolus of food. They also produce powerful
contractions which results in shortening of muscles to push the bolus along
gastrointestinal tract (GIT) (Bitar, 2003). There are gap junctions between adjacent
smooth muscle cells which keep cells electrically coupled. When a portion of smooth
muscle is depolarized, that depolarization spreads to adjacent portions of smooth
muscle which leads to a well-coordinated series of contraction (Haffen et al., 2020).
The receptors responsible for the motility are present on smooth muscle cells and on
the neurons in the intrinsic and extrinsic parts of the ENS (Roa et al., 2016).
1-Introduction
20
Neurotransmitters and their receptors perform a key role in the regulation of intestinal
motility. Mostly, the neurotransmitters (e.g. dopamine, acetylcholine, serotonin) found
in the ENS are similar to those present in the central nervous system (CNS) (Nezami et
al., 2010). Initially, most of studies on neurotransmitters were conducted in exploring
their role in various disorders of the CNS, “fight or flight” responses, signal
transmission through a chemical synapse and controlling blood flow throughout the
body (Mittal et al., 2017). Later on, researchers identified the significant involvement
and regulation of the neurotransmitters in the regulation of GI physiology as well
(Reynolds, 2016).
Certain receptor agonists and antagonists interacts directly to transform excitatory or
inhibitory signals of intestinal tract. In view of this complexity it is not surprising that
our understanding regarding the mechanisms of actions of different neurotransmitters
and drugs which effects the gut motility is still inadequate. However in recent past,
considerable advancements have been attained, and drug therapy for gut dysmotility is
emerging (Mittal et al., 2017).
1.1 Neurotransmitters involved in gut motility:
1.1.1 Acetylcholine:
Acetylcholine (ACh) is the main excitatory neurotransmitter of the ENS, and
muscarinic type of cholinoreceptor mediates the excitatory effects of ACh on intestinal
smooth muscles (Jonge et al., 2013). The excitatory effects of parasympathetic nerves
acting on intestinal smooth muscles is mediated by ACh indirectly through an effect on
the ENS (Ehlert et al., 2012; Gao et al., 2016). By depolarizing smooth muscles ACh
starts their contraction via stimulating muscarinic receptors.
ACh is an esterified form of choline and acetic acid. ACh has choline molecule which
is acetylated at the oxygen atom (Figure 1.1). It has highly charged
polar ammonium group (Suryanarayanan, 2014). ACh has been proved to amplify
spontaneous contractions in the small intestine of rabbits (Parthasarathy et al., 2015).
There are five different muscarinic receptor subtypes (M1-M5) (Radu et al., 2017), M3
is the subtype of receptor that is mainly found in the smooth muscles. These receptors
are excitatory in nature and are directly involved in mediation of gastrointestinal (GI)
smooth muscle’s contraction (Ehlert et al., 2012; Gao et al., 2016).
1-Introduction
21
M3-receptors are coupled to Gq-receptors and stimulation of M3/Gq‐coupled receptors
activates phospholipase C (PLC) which breaks the phospholipid constituent of the
plasma membrane, phosphatidylinositol-4,5-bisphosphate (P1P2), in to two secondary
messengers, inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG). DAG is
restricted to the membrane, where it stimulates a phospholipid and calcium sensitive
protein kinase called protein kinase C. IP3 diffuses through the cytoplasm to generate
release of Ca2+ from sarcoplasmic reticulum (SR) and it ultimately results in smooth
muscle contraction of GIT (Figure 1.2) (Montgomery et al., 2016).
ACh is preserved in storage vesicles present in the cholinergic neurons. ACh is released
into the neuromuscular junction (NMJ) after arrival of a nerve impulse. In NMJ it binds
to a receptor in post synaptic membrane of a muscle fiber, permeability of the
membrane is changed which causes the channels to open up and with the result sodium
ions (positively charged) starts their flow inside the muscle cells. When sodium
channels along endplate membrane are fully activated after successive nerve impulses,
muscle cell contracts (Jimsheleishvili et al., 2019). This is the logic for using ACh
agonists for hypomotility and for acute intestinal pseudo obstruction in which
increasing cholinergic signaling is the first line treatment.
1.1.2 Histamine:
Histamine is an amine widely present in the human body especially in the skin, lungs,
and GIT (Fabisiak et al., 2017). Histamine is a short acting neuromodulator, it regulates
the release of acetylcholine, serotonin and norepinephrine (Kapalka, 2019). Mast cells
are responsible for most of the histamine production and to a lesser extent it is also
produced by basophils, gastro enterochromoffin (EC) cells and histaminergic neurons.
Histamine is believed to affect at least 3 major functions in GIT: regulation of GI
motility, amplification of gastric acid secretion, and adjustment of mucosal ion
secretion (Fabisiak et al., 2017).
Histamine is formed by the decarboxylation of the histidine (Figure 1.3). It is an organic
molecule with four histamine receptors. H1, H2, H3 & H4, they are G protein-coupled
receptors. Histamine shows various effects because different histamine receptors are
present on different cell types and they work by different mechanisms of intracellular
signaling. H1-receptors are mainly found in mast cells and are involved in allergic
conditions. H2-receptors are found in heart, brain, parietal cells and neutrophils. H3-
1-Introduction
22
receptors are mainly present in peripheral nervous system and brain. H4-receptors are
found in blood cells (monocytes, eosinophils, mast cells) (Thangam et al., 2018). As
far as GIT is concerned, H1 and H4 subtypes of histamine receptors are mainly found in
intestinal smooth muscles (Mittal et al., 2017). Histamine is also responsible for
increase in intestinal motility. It has been revealed that overproduction of histamine by
mast cells causes diarrhea (Fabisiak et al., 2017).
Histamine has many effects on the body, one of the effects is contraction of smooth
muscle fibers in the stomach, intestines, lungs and uterus, (Bowen, 2019). Coupling of
H1-receptors are dual in nature. They are activated through Gq protein alpha sub unit
family (Gαq/11), which in turn activates phospholipase C (PLC) and ultimately
intracellular calcium (Ca++) levels are increased, resulting in smooth muscle contraction
of GIT (Figure 1.4) (Thangam et al., 2018). They are also associated to Gi/o proteins,
resulting in decrease production of cAMP (Monczor et al., 2016). Decrease in cAMP
levels result in increase in the smooth muscle contraction (Figure 1.2) (Bilington et al.,
2003; Mizuta et al., 2013; Kuo et al., 2015).
Figure 1.1: Chemical structure of Acetylcholine (Steven et al., 2011)
1-Introduction
23
Figure 1.2: Gq-coupled receptor signal transduction pathway (Zhag et al., 2016)
Figure 1.3: Chemical structure of Histamine (Parsons et al., 2006)
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24
Figure 1.4: Gs and Gi-coupled receptor signal transduction pathway
(Rosenbaum et al., 2014)
1.1.3 Dopamine:
Dopamine (DA), also called hydroxytyramine, a nitrogen-containing organic
compound (Figure 1.5). It also act as a neurotransmitter. Dopamine receptors are
involved in the regulation of motor activity and several neurological disorders.
Dopamine receptors directly regulate neurotransmission of other neurotransmitters,
release of cyclic adenosine monophosphate, cell proliferation, and differentiation
(Mishra et al., 2018).
An important factor in the regulation of GI motility is the dopaminergic mechanisms.
Enteric neurons and epithelial cells in intestine produces 50% of dopamine (Xue et al.,
2018). It is a critical neurotransmitter whose release from enteric dopaminergic neurons
and subsequent binding to DA receptors in GI smooth muscle inhibits the GI motility
mainly by activating D2 receptors (Giudicessi et al., 2018; Li et al., 2006). There are 5
sub types of DA (D1-5) which are further classified into 2 subclasses. D1 and D5 are
placed in “D1-like” DA receptors and D2, D3, and D4 are placed in “D2-like”DA
receptors.
1-Introduction
25
The D2 receptor has 7 transmembrane spanning domains. D1 like dopamine receptors
activate adenylyl cyclase while D2 like dopamine receptors inhibit adenylyl cyclase
(Xue et al., 2018). Various subtypes of DA receptors are found in the intestinal smooth
muscles but D2 is found abundantly (Ayano, 2016). Stimulation of Gs-coupled receptors
results in activation of adenylyl cyclase activity leading to an increase in cyclic AMP
(cAMP) levels which in turn activates protein kinase A (PKA). PKA phosphorylates
the myosin light chain kinase (MLCK), promoting smooth muscle relaxation (Figure
1.4) (Mizuta et al., 2013).
Metoclopramide (D2-receptor antagonist), blocks D2 receptors in the chemoreceptor
trigger zone as well as in the GI smooth muscles (Zabirowicz et al., 2019). It
antagonizes dopamine mediated relaxation on smooth muscle of GIT and exerts its
prokinetic effect. Metoclopramide increases the tone and amplitude of esophageal and
gastric contractions and thus increases gastric emptying (Mikami et al., 2016). The
resting tone of the lower esophageal sphincter is increased and at the same time relaxes
pyloric sphincter and duodenal bulb resulting in increased peristalsis (Isola and Adams,
2020).
1.1.4 Serotonin (5-HT):
Serotonin, also called 5-hydroxytryptamine (5-HT), is derived from the amino
acid tryptophan (Figure 1.5). It is a potent neurotransmitter which acts as a
vasoconstrictor (Kim et al., 2018). 5-HT as a chemical messenger is most widely
studied (McCorvy et al., 2015). 5-HT when combines with serotonergic receptors,
intestinal motility is increased (Kindig et al., 2015). 5-HT has various subtypes but 5-
HT3 & 5-HT4 are predominantly present in the intestinal smooth muscles (Mittal et al.,
2017). All 5-HT receptors are membrane-bound G-protein-coupled receptors (GPCRs)
except 5HT3 which is a ligand gated ion channel. 5-HT receptors activate intracellular
pathways by means of G proteins and produces a response. (McCorvy et al., 2015;
Giulietti et al., 2014).
5HT3-receptors belong to a group of transmembrane ion channel proteins which upon
binding with ligand, such as neurotransmitter, results in conformational change. This
allows potassium (K+), sodium (Na+) and Ca2+ions to move across the cell membrane.
Being an excitatory neurotransmitter it results in depolarization which leads to smooth
1-Introduction
26
muscle contraction (Figure 1.6) (Thompson et al., 2006). 5-HT4 receptor amplify the
peristaltic reflex pathways by exerting its action presynaptically on nerve terminals to
increase ACh release (Terry et al., 2016).
Researchers believed that 5-HT regulates the GI function because most of 5-HT is
synthesized and then stored in GIT. Moreover the agonists and antagonists alter the
intestinal behavior (Bomstein, 2012). Nearly 90–95% of the human body’s 5-HT is
synthesized and stored in EC cells of the intestinal mucosa where it controls motility,
secretion and sensation (Fuentes et al., 2016).
5-HT remained focus of clinical studies for intestinal motility disorders. 5-HT is a
regulatory neurotransmitter of enterocytes, enteric neurons and smooth muscles. It also
controls the development and long term consequences of central nervous system (CNS)
and ENS. Mucosal EC cells behave as sensory transducers to the mucosal signaling
molecule. 5-HT is secreted into the wall of GIT and starts peristaltic movements and
secretory reflexes in response to mechanical stimulation. EC cells release 5-HT which
affect both sensory motor and secretomotor neurons and also enterocytes and smooth
muscles. 5-HT has a key role in mediation of signals to CNS (Israelyan et al, 2019).
A. Dopamine
1-Introduction
27
B: Serotonin
Figure 1.5: Chemical structure of dopamine and serotonin (Steven et al., 2011)
1-Introduction
28
Figure 1.6: Ligand gated ion channels (Pacheco, 2007)
1-Introduction
29
1.2 Voltage gated ion channels involved in gut motility:
Besides neurotransmitters there are also voltage-gated ion channels (VGICs) which
control intestinal motility as well as alter the normal physiology (Radulovic et al, 2015).
VGICs are the main proteins which are permeable to ions and gated by changes in the
voltage of transmembrane. They are responsible for cell’s excitability. Muscle
contraction and synaptic vesicle exocytosis are the major cellular events that are
initiated by action potential generated by VGICs in muscles, neurons and secretory cells
(Lipscombe et al, 2014). Ion channels are often modified at expression level and
pharmacologically targeted in functional bowel disease (Fuentes et al., 2016).
VGICs open and close in reaction to electrical potential changes across the cell
membrane in which the channel is located and are structurally and functionally
homologous. Ion channels are dissimilar from other kind of membrane proteins, named
as transporters. Transporters mostly perform transmembrane transport of various
metabolites. Ion channels on the other hand are responsible for transducing ion-coded
information (Zhang et al., 2018). Ion channels are present in gut and their cell
particularity and position shows their role in normal and abnormal function in bowel
diseases. Voltage gated ion channels in smooth muscles and epithelial cells controls the
different phases of digestion like fluid secretion, absorption and motility (Fuentes et al.,
2016). A combination of the sodium, calcium, potassium and non-selective cation
channels maintain the resting potential membrane potential (Beyder et al., 2012).
VGICs are also possible targets for intervention in motility disorders of intestinal tract
(Kindig et al., 2015).
VGICs respond to altered membrane potential which results in particular ion passage.
This VGICs family include voltage-gated sodium channels (NaV), voltage-gated
potassium channels (KV) and voltage-gated calcium channels (CaV) (Moran et al.,
2015).
1.2.1 Sodium channels:
These channels are present in both the smooth muscle cells (SMC) and interstitial cells
of Cajal (ICC) (Beyder et al., 2012). NaV generate and spread the electrical signals in
cell (Moron et al., 2015). Sodium channels are made up of alpha (α) subunits that forms
1-Introduction
30
a transmembrane pore and an adjuvant beta (β) subunit. The β subunits govern channel
opening, expression and relation with cell bonding molecules with the cytoskeleton and
the extracellular molecules (Fuentes et al., 2016).
Highly sensitive sodium channels transporting sodium ions inside the cell activates and
inactivates the cells in milliseconds. They are classified in two groups according to their
sensitivity to tetrodotoxin (TTX); α-subunits that are sensitive to tetrodotoxin (TTX-S)
and those subunits that are resistant to tetrodotoxin (TTX-R) (Rocha HAC et al, 2014).
There are many isoforms of sodium channels present in the GI SMCs and ICC (Figure
1.7) (Beyder et al., 2012).
Depolarization opens the sodium channels of the axonal plasma membrane resulting in
pouring in of sodium ions. There is slow opening of potassium channels permitting
outpouring of potassium ions (K+), bringing back the resting membrane potential.
Calcium channels are voltage-dependent and are responsible for depolarization in some
cells. There are sensing regions on the sodium channel proteins having positive charge,
which upon depolarization moves towards negatively charged outer surface of
membrane. This allows transit of Na ions after opening of channel. Within a millisecond
of channel activation, the voltage perceiving region reverts back and a channel disabling
portion moves back to stop the channel and the channel proteins returns back to its
normal resting potential (Martin et al., 2015).
1.2.2 Calcium channels:
Calcium channels are of two types; L-type (CaV1) low-voltage-gated channels and T-
type (CaV3). L-type Ca2+ channels are also known as the high voltage gated channels
the while T-type Ca2+ channels are low-voltage-gated. Upon depolarization L-type
channels are slowly activated than T-type calcium channels but shows higher
communication in SMC. Calcium ions required for initiation of excitation and
contraction is made available by these channels (Beyder et al., 2012). Cytosolic calcium
is increased following calcium channel opening after membrane depolarization in
SMC. In gut, periodic motor patterns as in large and small intestine, mediators and
neurotransmitters operate on a structure in which slow waves originate in ICC and make
easier the achievement of the attainment of the stimulation threshold of calcium
channels intermittently and creation of action potential (Curro, 2016).
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31
CaV generate action potentials in a manner exactly like voltage-dependent Na+ channels.
Ca2+ channels can also modulate the shape of action potentials generated by voltage
gated NaV. The activity of calcium channels monitors a wide variety of biochemical
reactions inside the cells and they do so by influencing the concentration of calcium
inside the cell. The release of neurotransmitters at the level of synapse is the most
crucial of these biochemical reactions. Due to the wide variety of function, 16 different
calcium channel genes have been discovered. These calcium channels are different in
regard of their activation and inactivation characteristics as well as narrow
modifications in the chemical and electrical operation mediated by calcium.
Consequently the drugs that inhibit these CaV are of utmost importance in resolving a
number of ailments from heart diseases to anxiety disorders (Figure 1.8) (Freeman,
2018).
1.2.3 Potassium channels:
Half of the ion channels superfamily is made up of potassium (K+) and voltage-sensitive
potassium (KV) channels are the largest group. The wide variety of K+ channels detected
in ICCs and SMCs of the GIT reflect their rich diversity. When the intracellular
K+ concentration becomes 10-fold higher than extracellular, K+ channel open and
produces an outward K+ flux that turns the membrane potential in the negative direction
toward K+ reversal potential. As a result K+ channels are responsible for the
maintenance of resting potential, plateau current and repolarization of slow waves.
(Beyder et al., 2012). Many signaling pathways target these channels including some
kinases such as protein kinase C (PKC), protein kinase A (PKA), extracellular
regulated protein kinases (ERK) and many others (Li et al., 2014).
K+ channels are voltage gated ion channels that are numerous in number and most
variant. Almost 100 potassium channel genes are recognized so far. These genes are
divided into several groups depending upon their different gating properties. Since
channels are inactivated in minutes such as squid axon K+ channel that was studied by
Hodgkin and Hurley where others are inactivated within millisecond just like sodium
channel. Rate of action potential firing and its duration is dependent upon these
properties, thereby effecting axonal conduction and synaptic transmission. K+ channels
play an important role in generation of resting membrane potential (Figure 1.9) (Purves
et al., 2014).
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32
Figure 1.7: Voltage Gated Sodium Channels (Molnar et al., 2016)
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33
Figure 1.8: Voltage gated calcium channel (Alahamd et al., 2020)
1-Introduction
34
Figure 1.9: Voltage-gated Potassium channel (Lordfred, 2015)
1-Introduction
35
1.3 Hormonal control of gastrointestinal motility:
The GI hormones are a group of polypeptides produced by endocrine cells. The GI
hormones perform a key role in the regulation of the motor activity of the GIT. They
may exert indirect effects (neutrally mediated) or direct effects through motor activity
of smooth muscles. Motor activity of GIT is stimulated by gastrin, CCK and motilin
while secretin, vasoactive inhibitory peptide (VIP) and glucagon like peptide-1 inhibit
the motor activity.
GI functions are normally regulated by ENS which in turn is linked to autonomic and
central nervous systems (Furness et al., 2014). Biological actions of the GI hormones
are mainly mediated by binding to G-protein coupled receptors (hormone specific),
located on plasma membrane of target cell. This intracellular signal transduction
pathway entails adenylate cyclases, Ras family of GTP-binding proteins and ion
channels (Bhagavan et al., 2011).
Gastrin is found in the G cells of the gastric antrum and duodenum. It is mainly
responsible for increasing gastric motility, mucosal growth of the stomach, and
secretion of hydrochloric acid (HCl) (Prosapio et al., 2020). CCK also plays a very key
role in the regulation of digestive processes. It inhibits gastric emptying and secretion,
stimulates the intestinal and gall bladder contractions. It produces its actions in
mammals by acting on Cholecystokinin A and Cholecystokinin B receptors (Le et al.,
2019). Motilin is another hormone produced by endocrine cells of the mucosa of
duodenum and jejunum. Its receptors are also a part of the G protein-coupled receptors
family. It controls the interdigestive migrating contractions. Its levels cyclically
increase after every 90-120 min throughout the interdigestive fasting period. Motilin
release disappears after meal ingestion. These cyclical plasma motilin peaks results in
powerful peristaltic contractions originating from stomach and travels to small intestine
(Ohno et al., 2010).
Secretin is secreted in the duodenum by the S cells. Secretin is found to decrease gastric
contractions and increase gastric emptying time. Secretin receptors are G protein-
coupled receptors (Brandler et al., 2020). VIP is found in the digestive, reproductive,
respiratory and central and peripheral nervous system as neuroendocrine releasing
agent and as a neurotransmitter. VIP decreases the motor activity of GIT, thus
1-Introduction
36
decreasing the GI contractility (Bhagavan et al., 2011). GLP-1 is released in response
to meal from the gut to increases insulin secretion. GLP-1 is also involved in other GI
functions such as decrease in secretion of gastric acid, gastric wall tone and GI transit.
It has also shown to decline gastric and intestinal contractions. The mechanisms by
which GI motility is affected by GLP-1 are not completely understood (Brandler et al.,
2020).
1.4 Intrinsic and extrinsic innervation of gastrointestinal motility:
Various functions of the gut are regulated by intrinsic neurons of ENS and/or by
extrinsic parasympathetic (through pelvic and vagus nerves), sympathetic and sensory
neurons (Figure 1.10). Various studies have shown that ENS can act independently to
regulate motility reflexes even in extrinsically denervated intestine. However, CNS
performs a key role in regulating motility of esophagus and stomach while pathways of
extrinsic peripheral nerve coordinate with distant parts of GIT. The intestines have
substantial degree of autonomy to perform their functions without extrinsic neural
inputs (Uesaka et al., 2016).
Intestinal smooth muscles are innervated both by sympathetic and parasympathetic.
The parasympathetic system exerts its action through muscarinic receptors while α- and
β-adrenoceptors are used by sympathetic nervous system to mediate its inhibitory
effects.
1.4.1 Parasympathetic control of the gastrointestinal tract:
Preganglionic fibers are supplied by the parasympathetic system to ENS which synapse
with Auerbach’s and Meissner’s plexus. In these plexus, fibers from the cell bodies
reach the intestine to regulate motility and mucosal secretary cells. The parasympathetic
system, through ACh release is mainly involved in maintaining intestinal contractility
normal (Peddiready, 2011). Acetylchoine is the principal neurotransmitter at post-
ganglionic neurons. Vagus nerve is the key facilitator of the parasympathetic nervous
system. GIT is innervated not only by vagal sensory fibers but also by vagal motor
fibers One of the most vital functions of the vagus nerve is to bring information from
different organs including gut to brain (Uesaka et al., 2016).
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37
1.4.2 Sympathetic control of the gastrointestinal tract:
The sympathetic innervation of the gut arises from pre-vertebral ganglia of the
abdomen. Sympathetic post-ganglionic neurons which innervate the gastric region
reside within the celiac ganglion, while the neurons which innervate the intestine reside
in the pelvic ganglion (colon) or inferior mesenteric region (colon), the superior
mesenteric ganglion (small intestine) (Browning et al., 2011). Norepinephrine is the
neurotransmitter at the post ganglionic neurons of the sympathetic nervous system. The
sympathetic nervous system primarily exerts inhibitory effects on the GI motility, it
decreases the peristalsis and increases the contractility of sphincters (Uesaka et al.,
2016).
1.4.3 Role of gut microbiota in gastrointestinal motility:
Humans have enormous microbial ecosystem which is responsible for many GI
activities like digestion, metabolism, detoxification, prevention of bacterial
attachment to gut wall and control of GI homeostasis. High microbiotal concentration
are found in the stomach and it decreases progressively in the colon (Dieterich et al.,
2018). Due to certain differences in various regions of the GIT especially in the pH,
the type of bacteria and other microbes vary drastically. As stomach has strong acidic
environment, therefore, only few bacterial species grow and survive in it while other
gut regions have diverse bacterial species (Figure 1.11). The large intestine mainly
contains anaerobic and gram negative bacteria while small intestine contains aerobic
and gram positive bacteria (Ghoshal et al., 2012).
Recently, a lot of progress has been made in evaluating the role of gut microbiota in
the regulation of GI motility (Raja et al., 2018). Various studies have also shown its
involvement in the pathophysiology of different diseases like autoimmune, GI and
endocrine disorders. Due to its multiple effects on the metabolic and immune
functions, any alteration in the gut microbiota leads to various inflammatory and non-
inflammatory diseases of GIT (Fukui et al., 2018; Ghoshal et al., 2012).
The “faecal mircrobiotal transplantation” procedure is effective in severe drug resistant
cases of Clostridium difficile infection (Valdes et al., 2018). Treatment of disorders
related with GI motility by modifying the gut microbioata through antibiotics, diet etc
are presently under investigations (Raja et al., 2018).
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38
Figure 1.10: Intrinsic and Extrinsic Innervation of Gut motility (Uesaka et al.,
2016)
1-Introduction
39
Figure 1.11: Bacterial distribution in the GI tract (Knight, et al., 2019)
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40
1.5 Animal models for in vitro preparations:
1.5.1 History of experimental animals:
Humans have been using animals in research and education since they started to look
for ways to prevent and cure ailments. Animals have played an essential role in
scientific progression throughout history. The Greek scientist, Aristotle used animals
in his research, primarily to get a deeper understanding of these living beings (Anderson
et al., 2017). Animal oriented models have been crucial instruments since the early ages
of scientific discovery. Presently they are essential in any medical and biological
studies, helping us perceive and understand the functions of genes, the causes and
pathophysiology behind different diseases as well as drugs, their efficacies and adverse
effects. The entire genome of the organism have been sequenced, authenticating the
presence of different genetic similarities between these organisms and humans (Badyal
et al., 2014).
Most of present day's drug discoveries were possible because of the use of animals in
research as are introduced in therapeutics after experimental evaluation (Badyal et al.,
2014). The use of animals made the greatest discoveries in the 19th and 20th centuries.
Over the last century, every Nobel Prize for medical research has been dependent on
animal research. Research involving use of horses won the first Nobel Prize in 1901 in
medicine which was for serum therapy. The most recent 2012 Nobel laureates also used
animals. Experiments on animals helped in rapid progress in clinical medicine.
Discovery of Penicillin by Alexander Fleming was a wonderful example of using rats
as experimental animals in 1941, which transform the treatment of bacterial infections.
Insulin was also first isolated from dogs in 1922, Domagk and Selman Waksman used
chickens in 1939 and 1952 respectively to discover antibacterial activity of prontosil
and streptomycin (Badyal et al., 2014; Sinoussi et al., 2015).
1.5.2 Purpose of animal models:
A variety of diseases affect both humans and animals, thus animals can be used for the
study of human diseases. Combining the scientific knowledge with basic biological
studies guide us towards the better perception of function of living beings and we use
this information for the betterment of humans as well as animals. Animal models are
also preferred because of their easy availability, large reserves and feasible handling.
1-Introduction
41
It must be predicted within a reasonable limit that studies involving animal models
will contribute to the information which will result in protection and enhancement of
welfare and health of humans or animals. It is important to recognize the
experimental protocols of animal model in order to delineate a research study.
Majority of the cellular processes in animals are similar to those in humans in regard
that they carry out and execute many vital functions like breathing, digestion,
movement, sight, hearing and reproduction in the same as humans (Esteves et al.,
2018).
The primary goal of developing animal models for research is to create an
experimental system in which the conditions occurring in humans are phenocopied
as accurately as possible in the laboratory animal (Esteves et al., 2018). Due to vast
anatomical and physiological parallelism present between animals and humans,
especially mammals, the researchers were instigated to explore the properties and
characteristics of therapeutic regimens in animal oriented models prior to their
application in the human population. (Sinoussi et al., 2015).
During the studies of the animal species a lot of vital information was obtained which
could not have been used without the use of these models. Moreover the society
considers the usage of a newly developed drug or surgical procedure on humans as
immoral due to its probability that it may lead to more harmful effects as compared to
beneficial effects. This is one of the main reasons that these effects are analyzed on
animals, prior to its administration in humans. These animal trials have led to the
discovery of life saving medications for humans as well as animals (Badyal et al.,
2014).
1.5.3 Animal models for GI disorders:
The use of animal models for GI diseases is of great significance because of the
limitations of evaluating human GI diseases in clinical setting. They are used very
frequently. They have been used to investigate the pathogenesis of GI related diseases,
to find the mechanism, actions and effectiveness of different drugs in various GI
disorders. For studying human diseases, rodents are most frequently used animal
models because they are economical, easily available and reproduces rapidly (Ziegler
et al., 2016).
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42
1.5.3.1 In vitro animal models for GI motility:
Newly developed drugs need trials due to the fact that the developers must compute
and assess the favourable and unfavourable effects of a compound on an organism.
Primarily, the drug is tested in vitro using isolated organs and tissues, but it must also
be assessed using a suitable animal model, prior to performing clinical based trials in
humans (Jodat et al., 2018). In vitro studies help enable us to develop new technique
and treatment rapidly (Dias et al., 2018). In vitro assays for toxicological studies is the
refinement and replacement of animal testing in order to minimize the use of traditional
animal based toxicology studies. In the recent decades, in vitro methods have been
routinely used as these can be correlated with the in vivo studies and can help in
understanding a specific in vivo response in any given species (Srivastava et al., 2018).
Many in vitro preparations like (i) Stomach of mice, rat and hamster (ii) Guinea pig and
rat’s ileum, (iii) Rabbits’ ileum, duodenum and jejunum are commonly used to study
motility patterns and responses of intestinal smooth muscle. Rabbit and rat intestines
are routinely used in experiments relating motility. Pieces of animals intestine continues
to give responses for many hours when kept in a suitable solution. Usually they are
placed in Tyrode's solution through which air is passed. Rabbit’s intestine shows
regular pendular movements i.e., continuous relaxation and contraction (Pediready,
2011).
1.5.3.2 Rabbits:
Rabbits are small sized mammals belonging to Leporidae family, which is classified in
order Lagomorpha, based in many parts of the world. (Nowland et al., 2015). They
mainly populate the forests, woods, meadows and grass lands. They are most frequently
utilized in animal oriented studies. The reason that they are used so frequently in animal
trials is because they are smaller in size and easier to handle (Mapara et al., 2012). They
are parallel in physiology to humans. The frequency of their use in research studies is
gaining a popularity not only because of their docile nature but also because of the
financial ease to populate and conserve them as compare to larger animals (Esteves et
al., 2018).
Amongst various strains of rabbits, New Zealand white (NZW) strains of rabbits are
most frequently utilized in research trials because of their more docile nature and fewer
1-Introduction
43
health related issues as compare to other species. They also have much more genetic
similarities to humans. They are utilized as models for ocular ailments as well as
disorders of skin, immune, cardiovascular and respiratory system and in vivo and in
vitro models of GI disorders (Esteves et al., 2018).
For the evaluation of effects (preferably spasmolytic) of drug/chemical/plant on
pendular movements, jejunum is usually preferred because of its strong spontaneous
contractions. Usually there is no need to wash rabbit’s jejunum most of the times as it
is relatively wider and its spontaneous contractions causes it to clear itself (Peddiready,
2011).
1.5.3.3 Rats:
Rats were the first animal species to be specifically raised for the experimental studies.
They contribute a major bulk to the percentage of the animal models used for different
biomedical researches. Around 80-90% of the animals used for biomedical researches
are mice and rats. Therefore, they are well characterized in a number of ways. The use
of laboratory mouse for experimental purposes is significantly increased over the last
few years (Hedrich, 2012).
Laboratory rats are similar to humans in terms of physiology, anatomy and genetics,
therefore, they are frequently used animal models for various studies. Humans, mice
and rats have almost 30,000 genes, about 95% are shared by these three species. Their
smaller size, shorter gestational period and rapid growth are other advantages which
make them ideal to be used as animal models (Bryda, 2013).
The Wistar, an Albino outbred rat developed in the Wistar institute in 1906 for use in
biomedical studies, is the first species of the rat (Rattus norvegicus) to be domesticated
to serve as model organism at a time when laboratories use the common house mouse
(Mus musculus) (Sengupta, 2013).
Rats are studied as experimental models for cancer (Szpirer, 2010), cardiovascular
disease (including hypertension and cardiac failure) (Riehle et al., 2019), obesity and
diabetes (Kanasaki et al., 2010), neurodegenerative diseases (Parkinson’s disease)
(Welchko et al., 2012), GI disorders (Werawatganon, 2014), inflammatory
(Ghattamaneni et al., 2019) and autoimmune diseases (Castañeda-Lopez et al., 2017).
1-Introduction
44
On the other hand rats are also preferred in certain experiments as they breed rapidly
and are easy to handle because of their small size. Rat is one of the most used animal
model for GI diseases because in comparison to humans, rats have similar anatomic
structure of body organs (Vdoviakova et al., 2015). As ileum has very weak
spontaneous contractions, therefore, usually it is used for evaluation of spasmogenic
activity (Peddiready, 2011).
1.5.3.4 Advantages of in vitro techniques:
In vitro techniques include isolation of one or multiple pieces of required tissue from a
freshly sacrificed animal and keeping the tissue(s) alive in an appropriate solution at
required temperature and pH. The purpose of in vitro technique is to evaluate the
biological response(s) of the experimental drug.
In vitro techniques have certain advantages over in vivo techniques like:
i. The biological responses of the new experimental drugs can be easily verified
on the isolated tissue.
ii. Usually less dose of the experimental drug is required to evaluate the desired
response.
iii. Multiple tissues can be obtained from single animal.
iv. The response of the experimental drug can be immediately and directly tested
as the drug does not undergo the processes of absorption, distribution,
metabolism and excretion as it goes through in vivo techniques. (Peddiready,
2011).
1.6 Current gastro-prokinetic agents:
Prokinetic are drugs which increases gastrointestinal motility by augmenting the
number of contractions in the small intestine or making them powerful, but without
disturbing their rhythm (Litou et al., 2019). Prokinetics also rectify gastric
dysrhythmias, and encourage the flow of luminal contents in the forward direction.
Usually these agents are given at least 30 min prior to meals to have more beneficial
effects (Waseem et al., 2009).
Various prokinetic agents are already in use for these conditions but they are associated
with a lot of adverse effects e.g.
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45
1.6.1 Cholinergic agonists:
Cholinergic stimulants, the original motility inducing agents, stimulates muscarinic
receptors on smooth muscle cells. Currently anti-cholinesterase have been used as well
to a limited extent. Evidence for their efficacy is inconsistent in motility disorder.
However, Bethanecol had to be used for treatment of gastroperesis, reflex and
postoperative paralytic ileus but its effectiveness in these diseases are now diminishing
with the discovery of newer agent. Neostigmine is clinically used for Ogilvie’s
syndrome and Pyridostigmine clinically used for pseudo obstruction, but its use for
these indications are now disappearing with the introduction of newer agents.
Neostigmine and pyridostigmine continue to be employed in clinical practice for
Ogilvie’s syndrome and pseudo-obstruction respectively (Quengley, 2015).
These drugs give good prokinetic effect but they also have multiple muscarinic as well
as nicotinic side effects, decreased heart rate, hypotension or even cardiac arrest,
flushing and precipitation of bronchial asthma attack may also occur. They also cause
an increase in glandular secretions like saliva, sweat, tears, gastric and tracheobronchial
gland secretions. It may also cause stimulation of ganglia by anticholinesterases
through the muscarinic receptors after administration of anticholinesterase, ACh may
result in muscle twitching and fasciculation. Due to repetitive firing, higher doses of
these anticholinesterases drug may completely block the transmission of impulses in
neuromuscular junction causing weakness and paralysis of muscle. (Quengley, 2015).
1.6.2 Serotonergic agonists:
Cisapride and tegaserod are the two serotonin agonists which were used as prokinetic
agents but later on withdrawn from the market because of serious adverse effects.
Cisapride was observed to increase the pressure in lower esophageal sphincter and also
enhance gastric emptying. Cisapride was discontinued from market due to its serious
cardiac related side effects, which included arrhythmias involving prolongation of QT
interval (Quengley, 2015).
Intestinal contractility is increased by serotonin agonists. Prucalopride, another
serotonin agonist, with high affinity and selectivity for 5-HT4 receptors. It promotes
intestinal motility. Its high affinity for the 5-HT4 receptor and low affinity for the
hERG-K+ channel clarifies why it is not arrhythmogenic. As a prokinetic agent
1-Introduction
46
prucalopride promotes intestinal motility and transit. It is effective in improving bowel
function and reducing constipation-related symptoms.
Headache, abdominal cramps, nausea, diarrhea, bloating, fatigue and dizziness are the
common adverse effects associated with its use. 5-HT4 agonist improves GIT motility,
but ventricular arrhythmias and life threatening cardiovascular conditions can be
induced by these drugs as they effect potassium ion transport in cardiac muscle (Kamel
et al., 2015).
1.6.3 Dopamine antagonists:
Metoclopramide and Domperidone, dopamine antagonists, are primarily effective in
the enhancement of gut motility. They have shown desirable effects in gastroesophageal
reflux disease (GERD), gastroparesis and dyspepsia. Previously metoclopramide was
widely used as prokinetic agent with both central and peripheral effects while
domperidone did not have CNS effects as it does not cross the blood-brain barrier and
mainly acts through peripheral (DA2) receptors (Quengley, 2015).
Metoclopramide, being a prokinetic agent, has been used to treat GERD by improving
muscle action in the GIT. It shows variable effects on motility disorders.
Metoclopramide elevates the contractile ability of esophagus and fundus of stomach. It
also increases the pressure in lower esophageal sphincter and enhances the coordination
between antrum, pylorus and duodenum. Its use for longer period of time has been
convoluted by its adverse effects on CNS as well as its tolerance, in patient towards it.
Around 25% of patients taking metoclopramide have been recorded to have
experienced its side effects, most frequently among these are extra pyramidal
manifestations such as neuroleptic syndrome and tardive dyskinesia. Due to these
adverse effects, its use is mainly limited to gastrointestinal disorders and only for a
short duration of time (Kamel et al., 2015). They also frequently cause sedation and
diarrhea (Lau et al., 2016). Prolongation of the corrected QT interval is the most
important adverse effect of domperidone (Morris et al., 2016).
Some of the mentioned adverse effects are irreversible and resulted in fixation of “black
box” to its package in the US. Metoclopramide and domperidone both may increase
the serum prolactin levels leading to galactorrhea and gynecomastia (Quengley, 2015).
1-Introduction
47
1.6.4 Erythromycin:
Erythromycin (ERY), a macrolide antibiotic, is found to be motilin agonist (Deloose et
al., 2016). Motilin, a polypeptide hormone found mainly in the stomach distally and
duodenum, enhances the pressure in lower esophageal sphincter and is responsible for
stimulating the migrating motor complex (MMC) in the antral part of the stomach. The
intravenous form of motilin is the most powerful stimulator of solid and liquid gastric
emptying. ERY increases the contractile ability of antrum, stimulates premature MMC
phase III activity and prompt gastric emptying by binding to Motilin receptors. ERY
also enhances gastric motility in patients who have undergone vagotomy and
antrectomy, possibly this may be because of its stimulatory effects on the fundus of the
stomach (Waseem, et al., 2009).
In a review by Camilleri showed that ERY is most functional in acute gastroparesis
(Quengley, 2015). It is used as a prokinetic agent in post-operative ileus. It is also used
in the treatment of various clinical condition such as skin infections ,respiratory
infections (i.e community acquired pneumonia, Legionnaires disease), rheumatic
fever, intestinal amebiasis prophylaxis, prophylaxis of neonatal conjunctivitis and
chlamydia, syphilis and pelvic inflammatory disease (PID) (Kato et al., 2019; Marchant
et al., 2018). It is given to pregnant women for the prevention of Group B streptococcal
infection in the newborn (Gerber et al., 2017).
ERY have been noted to lead to certain adverse effects which include rash, allergic
reaction reversible deafness and decrease in appetite. It also increases the QT interval
and causes torsades de pointes. It is an enzyme inhibitor mainly inhibiting cytochrome
P450 and carries the risk of interaction with wide variety of medications. This leads to
an increase in the concentration of those drugs that are metabolized via the cytochrome
P450 system leading to the toxicity of those drugs (Akiyoshi et al., 2013). ERY may
also lead to cholestatic liver injury. In Sweden, ERY was the second most commonly
reported antibiotic to have caused drug induced injury. Cholestatic injury was reported
in 69% of cases among a collection of case reports. (Padda et al., 2011). In addition,
tachyphylaxis can also develop in patients on long term ERY therapy. This is due to the
down regulation of motilin receptors to which erythromycin binds. This can occur
within the first few days of starting therapy (Richards et al., 2009).
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1.6.5 Miscellaneous drugs:
Other drugs are Mitemcinal (Tilman et al., 2016), Atilmotin (Korimilli,et al., 2010),
Ghrelin (Sallam et al., 2010), Cholecystokinin receptor antagonists, loxiglumide
(Romero et al., 2014), Camicinal (Barshop et al., 2015).
There are a number of prokinetic agents available, among which most are associated
with a lot of adverse effects. So, there is always a search for new agent which is much
safer and effective. Azithromycin is one of the antibiotics which is prescribed
frequently and is relatively free from various side effects.
1.7 Azithromycin:
Azithromycin (AZM) is a broad spectrum, semi synthetic antibiotic which belongs to a
group of drugs known as macrolides (Rao et al., 2014; Lu et al., 2006; Martinez et al.,
2015). It was synthesized in early 1980s, it is still under investigation for the treatment
of various diseases (Jelic et al., 2016; Kagkelaris et al., 2018).
1.7.1 Source:
AZM is produced by Streptomyces species (gram-positive aerobic bacteria) (Jelic et al.,
2016).
1.7.2 Structure:
AZM is a part of the Azalide subclass of macrolides. It is obtained from ERY, a methyl-
substituted nitrogen atom is incorporated instead of carbonyl group into the lactone
ring, which makes the lactone ring 15-membered (Figure 1.12 & 1.13) (Bakheit et al.,
2014). In this way AZM not only improves its spectrum of activity than ERY but also
increases its tolerability (Davidson, 2019).
AZM has the chemical name (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-13-[(2,6-
dideoxy-3-C-methyl-3-O-methyl-α-L-ribo-hexopyranosyl)oxy]-2-ethyl-3,4,10-trihydr
oxy-,5,6,8,10,12,14-heptamethyl-11-[[3,4,6-trideoxy-3-dimethyl amino) -β-D-xylo-
hexopyranosyl]oxy]-1-oxa-6-azacyclopentadecan -15-one. Its molecular formula is
C38H72N2O12, and its molecular weight is 749.0g/mol. Azithromycin, as the dihydrate,
is a white crystalline powder with a molecular formula of C38H72N2O12•2H2O and a
molecular weight of 785.0g/mol (Adeli, 2016; Timoumi et al., 2014).
1-Introduction
49
Figure 1.12: Chemical structure of Azithromycin (Assi et al., 2017)
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50
Figure 1.13: 3D structure of Azithromycin (John, 2017)
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51
1.7.3 History:
Azithromycin is obtained from Erythromycin, the first 14-membered macrolide,
isolated from the actinomycete Streptomyces erythreus (Saccharopolyspora
erythraea). It has been in human use since 1952. Its antimicrobial spectrum is similar
to that of penicillin. It is widely used for penicillin allergic patients. Depending on the
antibiotic concentration used and on the type of microorganism, it exerts bacteriostatic
and bactericidal properties (Jelic et al., 2016).
AZM (9a-methyl-9-deoxo-9-dihydro-9a-aza-9a-homoerythromycin), the first 15-
membered macrolide antibiotic in the market, consists of nitrogen atom inserted into
the macrocyclic ring. AZM was discovered in 1980 by a team of researchers at PLIVA
Laboratories in Croatia, Yugoslavia. This team comprised of Gorjana Radobolja-
Lazarevski, Gabrijela Kobrehel, and Zrinka Tamburasev, led by Dr. Slobodan Dokic.
After twenty years, in 2000, they were given the medal of highest honor, “Heroes of
Chemistry”, awarded by the American Chemical Society for their remarkable
contribution to chemistry (Jelic et al., 2016).
In 1986, a licensing agreement was signed by the PLIVA and Pfizer (pharmaceutical
company), which gave Pfizer exclusive rights to sale AZM in the United States and
Western Europe. In 1988, PLIVA marketed AZM in Central and Eastern Europe. In
1991, Pfizer also launched AZM in other markets under Pliva's license (Tomisic, 2011).
1.7.4 Administration:
AZM is commercially available for oral as well as parenteral (IV) administration.
Orally it is available in the form of tablets and suspension. It can be taken on empty
stomach or 2 hours after food. Intravenous AZM must be infused slowly over the time
of 60 min at least.
1.7.5 Mechanism of action:
AZM is characteristically bacteriostatic drug that inhibits bacterial growth by
interfering with bacterial protein synthesis. It inhibits translation of mRNA by binding
to bacterial ribosomes at 50S (Figure 1.14) (Bakhiet et al., 2014). As bacterial growth
is halted, replication is stopped thus bacteria cannot increase in number and eventually
these bacteria die or are cleared by the immune system.
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Figure 1.14: Mechanism of action of Azithromycin
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53
1.7.6 Half-life and tissue penetration:
AZM is stable to acids in the stomach therefore, can be taken via oral route without
affecting its absorption. Absorption of AZM is enhanced on an empty stomach. Oral
administration of a single dose of AZM (500mg) results in 37% bioavailability with a
maximum concentration in serum of 0.4 mg/L. In adults, when given orally, time to
peak concentration (Tmax) is 2.1 to 3.2 hours. AZM’s high concentration in
phagocytes, facilitates its transportation to the target. Large amount of drug is released
during active phagocytosis. AZM is highly lipid soluble and also due to ion trapping,
its concentration in tissues is 50 times much higher than in the plasma. (James, 2017).
Concentration of drug in the tissue exceed the minimum inhibitory concentration (MIC)
that inhibits 90% of probable pathogens (MIC90) after a single oral dose of 500mg
(Jelic et al., 2016).
Even 12 to 24 hours after a single dose of 500mg high intracellular levels of AZM are
also present in the tissues (mean >2 mg/l). It has a prolong half-life of 30-40 hours
(Kong et al., 2017; McMullan et al., 2015). AZM is generally well-tolerated and has
been proved to be effective even with a single dose (Tribble, 2017).
1.7.7 Clinical uses:
AZM is used to treat infections like community-acquired pneumonia, respiratory
infections, like pertussis and legionellosis, pelvic inflammatory disease, sexually
transmitted infections chancroid and granuloma inguinale, bacterial enteritis, cholera,
traveler’s diarrhea, enteric fever, skin infections, middle ear infections, tonsillitis and
sinusitis (McMullan et al., 2015; Bakheit et al., 2014).
AZM distribution has been a core component of the World Health Organization’s
(WHO) trachoma control program, with over 700 million doses of AZM distributed to
adults and children aged 6 months and older. AZM distribution dramatically reduces
the prevalence of the ocular strains of Chlamydia trachomatis that leads to trachoma
(Oldenburg et al., 2018).
AZM is a pregnancy category B drug and is usually considered safe to treat various
bacterial infections in pregnant and breastfeeding ladies (Ayse, 2013).
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54
1.7.8 Advantages of AZM over erythromycin:
Once daily dosing frequency, better efficacy and relatively less adverse effects of AZM
makes it superior in some clinical conditions (Kong et al., 2017). In contrast with ERY,
AZM is more acid stable, which simplifies administration around food (McMullan et
al., 2015). AZM is one of the most commonly used antibiotics by physicians. More
than 50 million prescriptions are produced in the United States per annum, generally
for respiratory tract and sexually transmitted infections (Jurinnk, 2014). AZM is slowly
metabolized and generate inactive metabolites. Unlike ERY it has no effect on the
cytochrome P450 (cyt P450), enzymes responsible for the metabolism of many drugs
and in the synthesis of cholesterol and steroids, so it does not interrelate with other
medicines being metabolized by cyt P450 (De Oleveria et al., 2016; Kagkelaris et al.,
2018).
1.7.9 Advantages of AZM over other antibiotics:
Macrolides are regarded as one of the safest antibiotics available. Different researches
proved the safety and efficacy of Azithromycin. They confirmed its enhanced efficacy
as compare to other antibiotics in bacterial infections even against resistant strains of
bacteria. (Davidson, 2019).
Prolonged use of other antibiotic treatment affects patient compliance and also
increases overall cost. The recommended duration of therapy for antibiotics such as
amoxicillin is 7–10 days. It is preferred to advise an antibiotic like AZM, for 3 days, to
make certain better compliance of patient and lessen the chances of development of
bacterial resistance. There is a strong evidence that short term courses may be much
appropriate treatment for acute otitis media and acute bacterial sinusitis. Short-term
treatment has been advised even for acute pharyngitis (Donde et al., 2014).
1.8 Pharmacological actions of AZM:
1.8.1 Antibacterial action:
AZM is generally considered to be bacteriostatic, but may become bactericidal at higher
doses and exerts a post antibiotic effect (Jelic et al., 2016; Matzneller et al., 2013).
AZM has good activity against many gram-negative and gram-positive bacteria
including Haemophilus influenzae, Neisseria gonorrhoeae, Mycoplasma pneumoniae
1-Introduction
55
Branhamella catarrhalis, Campylobacter, Moraxella catarrhalis and Legionella sp.,
Treponema pallidum and Mycobacterium avium complex. It also shows activity against
Proteus, Escherichia, Shigella, Enterobacter, Salmonella, Yersinia etc of
Enterobacteriaceae family (McMullan et al., 2015; Jelic et al., 2016).
1.8.2 Immunomodulatory action:
In addition to their antimicrobial properties, macrolides are also found to exert
immunomodulatory effects in vitro and animal data on it is also available. Macrolides
(AZM) also possess immunomodulatory properties, which were first noticed and
described in the 1950s just after their introduction. (Zimmermann et al., 2018; Li et al.,
2017; Bulska et al., 2014). The idea of using macrolides for immunomodulatory actions
was introduced in the 1970s (Zimmermman et al., 2018). Their significant effectiveness
in treating diffuse panbronchiolitis (DPB) has helped to extend their use as
immunomodulator to a number of clinical conditions (Kanoh et al., 2010).
Treatment with AZM in acute phase of inflammation resulted in reduction in pro-
inflammatory cytokines and stimulates resolution of chronic inflammation in the late
phases. Specifically, AZM is found to exert direct action on airway epithelial cells to
keep their function and decrease mucus secretion. These characteristics of AZM have
favored its use in the treatment of a number of chronic lung diseases including cystic
fibrosis (CF), non-CF bronchiectasis, diffuse panbronchiolitis (DPB), bronchiolitis
obliterans syndrome, chronic obstructive pulmonary disease (COPD) and asthma
(Cramer et al., 2017).
In order to explore the immunomodulatory action of AZM in vivo, the effect of AZM
on survival and production of cytokine was assessed in the (lipopolysaccharide) LPS
tolerance model which was categorized by a macrophage-M2 skewed response.
Tolerance induction by LPS priming was related with diminished serum concentrations
of pro-inflammatory cytokines, interleukin-12 (IL-12p40), tumour necrosis factor-
alpha (TNF-α) and chemokine ligand 5 (CCL5). Treatment with AZM resulted in
reduced serum TNFα and CCL5. The immunomodulatory action of AZM was
confirmed by increasing survival and dropping pro-inflammatory cytokine production
(Bosnar et al., 2013). In another study AZM was also found to decrease matrix
1-Introduction
56
metalloproteinase-9 (MMP-9), an enzyme which is involved in the degradation of
extracellular matrix and interleukin-8 (IL-8) (Zimmermann et al., 2018).
1.8.3 Anti-inflammatory action:
AZM has anti-inflammatory properties (Zimmermann et al., 2018; Li et al., 2017;
Bulska et al., 2014). Cytokines and chemokines, both have proinflammatory effects.
e.g., tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6),
interleukin-8 (IL-8), interlekin-10 (IL-10) granulocyte-macrophage colony-stimulating
factor (GM-CSF), and interferon-gamma (IFN-γ) and anti-inflammatory e.g., (Kanoh
et al., 2010). AZM in a study inhibited Resiquimod (R848) induced TNF, interleukin-
1beta (IL-1β), IL-6, IL-8 and IL-10, and lipopolysaccharide (LPS) induced IL-1β and
IL-10 (Speer et al, 2018). Cigana and coworkers used three CF (IB3-1, 16HBE14o-
AS3, and 2CFSMEo−) and two isogenic non-CF (C38 and 16HBE14o-S1) airway
epithelial cell lines to check whether AZM decreased TNF-α mRNA and protein levels
or not. AZM decreased TNF-α mRNA levels and TNF-α secretion, to almost the levels
in the isogenic non-CF cells. NF-κB and specificity protein 1 (Sp1) DNA-binding
activities were also significantly reduced after treatment with AZM (Kanoh et al.,
2010).
1.9 Research studies of AZM related with smooth muscles
contractility:
AZM in addition to antibacterial, immunomodulatory and anti-inflammatory activities,
is also involved in the contractility of smooth muscles in various systems of the body
(El-Baki et al., 2015).
1.9.1 Effect of AZM on aortic strips:
A study was performed on AZM to find out its effect on the contractility of isolated
aortic spiral strips of rabbits. Aortic strips were treated with 0.3μg/ml norepinephrine
(NE) in order to produce contractions. At low doses AZM did not produce any
significant change in the contractility of aortic strips but when the dose was increased
(16μg/ml), it was able to significantly reduce the NE induced contractions. Amplitude
of the contractions was also reduced at higher doses (El-Baki et al., 2015).
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57
1.9.2 Effect of AZM on uterine smooth muscles:
In order to find the effects of AZM on the duration of pregnancy in women at risk of
labor, a study was conducted at Sainte Justine hospital, Canada. It was a retrospective
study in which those women who were admitted in obstetrics ward from January, 1st
2006 to August, 1st 2010 and received AZM were included in the study. Total 127
women were exposed to AZM during the study period and these were matched with
127 controls through pharmacy software and medical records. A time-to-event analysis
was done to compare gestational age at the time of recorded events (rupture of
membranes, delivery, or second intervention to prolong pregnancy). Results of the
study showed that there was no significant difference found between the two groups in
terms of prolongation of pregnancy and gestational age at event (Goyer et al., 2016).
1.9.3 Effect of AZM on respiratory smooth muscles:
Macrolide, in recent years there is increasing evidence about their favourable effects on
a number of chronic respiratory conditions. Historically, ERY and clarithromycin were
found to alleviate pulmonary worsening in diffuse panbronchiolitis. Long term
treatment with AZM prevented exacerbations and improved lung function in patients
colonized with pseudomonas aeruginosa. In noncystic fibrosis bronchiectasis, same
effects on prevention of exacerbations has been established. Similarly, long term use of
AZM also prevents bronchiolitis obliterans syndrome in patients undergoing lung
transplantation. Acute exacerbations are also prevented by AZM in chronic obstructive
pulmonary disease (COPD). Besides producing anti-inflammatory actions, AZM also
affects the contractility of airway smooth muscles (ASM) (Shteinberg et al., 2016).
Daenas et al., performed a study to find out the effect of AZM on ASMs. Isolated
tracheal strips precontracted with carbachol and KCl were suspended in an organ bath
and treated with different doses of AZM. AZM showed relaxant effect in a
concentration dependent manner. AZM was able to produce relaxant effect both in
calcium free solution and in the presence of verapamil, Ca2+ antagonist. The result
proposes that AZM has a concentration-dependent and direct relaxant effect on
precontracted tracheal strips that is not associated with inhibition of Ca2+ influx or
Ca2+ release from intracellular stores (Daenas et al., 2006).
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58
Wang et al conducted a study on AZM to find out its effect on ASMs. Mouse tracheal
rings (TRs) were used for this purpose. Mouse TRs were precontracted using
100μmol/L ACh, and the response to different doses of AZM was observed.
Precontractions induced by ACh were completely inhibited by AZM. The same
procedure was repeated with same protocols on human ASMs as well. AZM again was
found to relax the ASMs precontracted with ACh. The underlying mechanisms for
producing ASMs relaxation were also investigated in this study. AZM blocked L-type
voltage dependent calcium channels (LVDCC), Ca²⁺ permeant ion channels,
Muscarinic-2 (M2) receptors, and Transient receptor potential channel-3 (TRPC3)
and/or stromal interaction molecule channels (STIM/Orai), which reduced cytosolic
Ca²⁺ concentrations and led to relaxation of muscles (Wang et al., 2019).
Another study was performed by El-Baki and his colleagues to evaluate the actions of
AZM on the isolated tracheal strips. Contraction of tracheal strips were produced by
treating them with ACh followed by increasing cumulative doses of ACh without
washing the tissues. It was noted that AZM at lower dose (1μg/ml) did not produce any
changes while at higher dose (16μg/ml) it produced a dose dependent reductions on
ACh induced tracheal contractions (El-Baki et al., 2015).
Paicentini et al, evaluated the effects of AZM on the bronchial contractility
(hyperresponsiveness), lung function, and airway inflammation in asthmatic children.
Sixteen known asthmatic children were included in the study. They were divided in
two group. One group received AZM while the other group was given placebo. Lung
function and contractility were expressed as the dose response curve (DRC) of forced
expiratory volume in 1 second (FEV1) fall after hypertonic saline inhalation while for
inflammation neutrophils and lymphocytes level were monitored. No significant
change was found in lung function. In some of the AZM treated asthmatic children
there was reduction in bronchial contractility and airway neutrophilic infiltration
(Paicentini et al., 2007).
AZM is found to increase the functions of lung in lung transplant recipients (LTx). It
has been reported that gastroesophageal reflux (GER) is involved with the pathogenesis
of chronic rejection after LTx. AZM by increasing esophageal and gastric motility
improves glomerular filtration rate (GFR). Mertens et al determine the effect of AZM
on GER and gastric aspiration after LTx. Both strong and weakly acidic GER was
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59
measured with 24-h pH-impedance monitoring in 47 recipients of lung transplant in
which 12 patients were using AZM. In a separate group of 30 LTx patients gastric
aspiration was evaluated before and after taking AZM by measuring pepsin and bile
acid in bronchoalveolar lavage fluid (BALF). Results showed that recipients who were
using AZM had less number of GER events. Analysis of BALF showed less
concentration of bile acids with no significant effect on the level of pepsin. Overall
patients treated with AZM had less GER events and bile acids aspiration. These effects
might be because of improved esophageal motility and enhanced gastric emptying
(Mertens et al., 2009).
GERD is common in COPD and is usually associated with the development of acute
exacerbations. Different studies have proved the prokinetic effect of AZM, so may be
helpful in GERD. Therefore it was hypothesized by the authors that AZM may decrease
acute exacerbations in COPD by decreasing the episodes of GERD. Therefore, they
performed a retrospective review of the data collected in a prospective, randomized
controlled trial (RCT) of AZM for preventing COPD exacerbations. Participants were
broadly divided into two groups (i) those having a history of GERD (ii) those having
no history of GERD. Participants with GERD developed more frequent and rapid
exacerbations as compare to without GERD participants. AZM decreased exacerbations
both in GERD and without GERD participants, but more significantly in those without
GERD (Ramos et al., 2014).
AZM decreases risk of aspiration in recipients of lung transplant and improves
bronchiolitis obliterans syndrome (BOS). The authors assumed that AZM can improve
graft and overall survival much effectively in LTx patients with BOS who suffered from
aspiration of bile acids (BA) by inhibiting the progression of BOS associated with
aspiration. The objective was to compare FEV1 (% baseline), BOS progression and
overall survival in the recipients of LTx treated with AZM for BOS, both with and
without aspiration of BA. Those LTx recipients were included in the study who were
treated with AZM for BOS. Before starting the AZM treatment, samples of
bronchoalveolar lavage (BAL) were checked for the presence of BA and neutrophilia.
Short term effect of AZM on FEV1 and BAL neutrophilia was evaluated, 3 years follow
up was done regarding progression and survival of BOS in patients with and without
BA aspiration. BA in BAL were found in 19 out of 37 participants. Aspiration of BA
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60
resulted in deterioration of outcome, in terms of FEV1, BOS progression and survival.
Resulted showed that AZM is unlikely to protect against the allograft dysfunction for
longer period caused by GER and BA aspiration. Therefore, supplementary treatment
may be required targeting aspiration in the recipients of lung transplant (Merten et al.,
2011).
1.9.4 Effect of AZM on gastrointestinal smooth muscles:
1.9.4.1 AZM and esophageal smooth muscle contractility:
Jafari et al conducted a study to determine the effects of AZM on esophageal
hypomotility. Twenty six participants were included in the study who were suffering
from hypomotility disorders of esophagus. Half of them received AZM and half were
kept as control by just giving them placebo. At the end of the study AZM improved the
contraction of esophagus while the esophageal contractions of the placebo treated group
remained unchanged (Jafari et al., 2015).
1.9.4.2 Effect of AZM on gall bladder smooth muscles:
Gallstone formation is directly related with impaired gallbladder motility. Cisapride
and ERY are effective cholecystokinetic agents but they also exist arrhythmogenic
properties, therefore not used in routine for this effect. AZM is similar to ERY in
structure but does not possess significant drug-drug interaction as seen with ERY
(Ugwu et al., 2013).
Ugwu et al studied the effects of AZM on the contractility of gall bladder and compared
its effects with those of ERY. Twenty four males, looking apparently healthy were
included in the study. They were studied in pre-prandial and post prandial states. After
an overnight fast, one of the groups received 500mg AZM and the other group 500mg
ERY, 30 minutes before the study. Length, width and height of the gall bladder were
noted in each participant just before the ingestion of a standardized liquid meal in order
to get the ellipsoid volume with real time sonography. After taking the liquid meal, the
gall bladder measurements were again` taken after every 5 min for 40 min and
gallbladder contraction index (GBCI) was calculated. Most of the times ERY proved
significantly higher GBCI values than AZM while AZM only at few points showed
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61
higher GBCI. Results are suggestive of gall bladder smooth muscle contractility
induced by AZM (Ugwu et al., 2013).
GBCIs were assessed in 24 healthy volunteers after an overnight fast by using Real-
time ultrasonography. AZM and placebo were given to participants of the two groups
respectively and GBCIs were obtained in supine position every 5 min for 40 min. AZM
significantly improved the motility of gallbladder in the first 5 to 10 min. This increase
was not sustained till the 35th min when maximum GBC1s for the placebo was noted
(Ugwu et al., 2013).
1.9.4.3 AZM and gastroparesis:
The treatment of gastroparesis (disease in which the stomach cannot empty food in a
normal way by itself) is directly related with the severity of the symptoms, but it usually
comprises dietary modifications, antiemetics and prokinetic medications. The
preliminary treatment for gastroparesis is a prokinetic agent. ERY showed very
significant effect on gastric emptying, therefore it is often used for this purpose. There
are certain limitations to ERY which include adverse reactions like QT interval
prolongation, GI disturbances, tachyphylaxis and CYP3A-associated drug interactions.
AZM, another macrolide, on the other hand has been shown less GI disturbances, less
incidence of QT interval prolongation, fewer drug interactions and a longer half-life.
Use of AZM may be useful in patients with gastric and small bowel dysmotility (Potter
et al., 2013).
Shakir et al., evaluated the effects of AZM on the antroduodenal motility and compared
it with the effects of ERY. A retrospective analysis of the manometric data of gastric
and small bowel activity of the patients who were given intravenous AZM was done.
The pressure profiles acquired during motility studies were compared to those of
patients who were treated with intravenous ERY during motility studies. Total duration
of effect, number of cycles per min, mean amplitude of contractions, and duration of
highest antral and duodenal contractions were compared for both the drugs. Intravenous
AZM produced migrating motor contractions (MMCs) in the stomach followed by
small intestinal contractions. The mean amplitude of stomach contractions were slightly
higher in patients treated with AZM. The mean amplitude of small intestinal MMCs,
frequency and duration of gastric and small intestinal contractions were almost same in
2 groups (Shakir et al., 2018).
1-Introduction
62
Larson et al., done a study to determine the effects of AZM on gastroparesis. They also
compared the effects of AZM with erythromycin (ERY). Patients already diagnosed
with gastroparesis were included in the study. They were divided into two groups. All
the medications affecting the gastric emptying were stopped before starting the study.
Patients were given a scrambled egg meal which was labelled with 18.5-37 MBq of
technetium-99m sulfur colloid followed by continuous imaging for 120 minutes, at 1
minute per frame. Gastric emptying half-time (t½) was noted (normal = 45-90 minutes).
If the stomach had not properly emptied at 75-80 minutes. Half of the patients were
given AZM while the other half was given ERY and a new post-treatment gastric
emptying t½ was noted. Both the drugs enhanced gastric emptying t½ with no
significant difference (p-value 0.30). The results of this study are suggestive of
prokinetic effect of AZM (Larson et al., 2010).
Sutera et al., studied the effects of AZM on gastric emptying time in a diabetic patient.
The patient was an 83-year-old woman with a fourty year history of type II diabetes.
She was admitted with malnutrition, persistent vomiting and severe constipation.
Different treatment regimens were given but were unsuccessful. She was given i/v
AZM (500mg/day). After getting treatment for 3 days, vomiting was settled and the
patient was able to pass normal stool, with obvious progress in her general conditions.
This study supports the prokinetic activity of the AZM (Sutera et al., 2008).
Goshal et al., did the comparison of AZM with ERY in terms of gastric emptying
acceleration in patients with gastroparesis. The authors established that AZM equally
accelerated the gastric emptying as ERY did. They further suggested that AZM may be
favored over ERY because of its less involvement in drug interactions (Cyt P450),
relatively less adverse effects and longer duration of action (Goshal et al., 2010).
Another study was performed by Reddy et al at Veterinary College and Research
Institute, Namakkal, to compare the effect of AZM with Neostigmine on GI motility in
cows with functional ileus. Fourty cows with ileus, during the study period were
presented to the institute, out of which 20 were selected. Cows were investigated for
the levels of hemoglobin (Hb), packed cell volume (PCV), total erythrocyte count
(TEC) and total leucocyte count (TLC). After collecting serum it was checked for
chloride, potassium, sodium, albumin, gamma glutamyl transferase (GGT), creatinine
kinase (CK), inorganic phosphorus and calcium. Trans abdominal and rectal ultrasound
1-Introduction
63
was also done by using 2 - 3.5 MHz transducer of MyLab Vet 40 ultrasound scanner.
After investigation the cows were divided into two groups i.e group 1 and group 2. Each
group included 10 cows. Group 1 was treated with neostigmine (0.02 mg/kg) while
animals of group 2 received AZM (1 mg/kg). All the cows were also given ringers
lactate (10 ml/kg), dextrose normal saline (10 ml/kg) and procaine penicillin (20,000
IU/kg) for three days. Efficacy of the drugs was evaluated on the basis of clinical
improvement in terms of passing dung, and resumption of water and food. Results
showed that cows treated with AZM passed dung earlier as compare to the cows who
received neostigmine (Reddy et al., 2018).
1.9.4.4 Gastric motility disorders:
Moshiree et al., performed a study to find the effects of AZM on the activity of gastric
antrum. Thirty patients were included in the study who were diagnosed to have prolong
gastric emptying t½. Before starting the study all the patients were advised not to take
any drug affecting the gastric motility at least 3 days earlier. Patients with a history of
psychiatric illness, diabetes, intestinal obstruction, eating disorders, malignancy and
allergic to macrolide antibiotics were excluded from the study. A six channel
manometric microtransducer catheter was inserted intranasally and advanced through
the pylorus into the duodenum to analyze the gastroduodenal activity. AZM was
found to stimulate gastric antral activity. AZM also increased the duration and mean
amplitude of contractions (Moshiree et al., 2010).
A similar study was conducted to evaluate the pharmacological actions of AZM on
isolated gastric fundal strips of experimental animals. Each gastric fundal strip after
mounting in an organ bath was treated with ACh to produce contractions. Then
different doses of AZM were added in a cumulative way and response to each dose
was recorded. AZM was found to increase the ACh induced contractions in a dose
dependent manner (El-Baki et al., 2015).
Blondeau et al., performed a study to check the effects of AZM on GERD. AZM was
given to one group of patients while the other group was kept devoid of it. When the
reflux parameters were compared between the patients with and without AZM, patients
with AZM were found to have significantly lowered esophageal reflux exposure.
1-Introduction
64
However, the frequency of reflux was not affected but the proximal extent of the acid
was observed less in AZM treated patients (Blondeau et al., 2007).
Broad et al., in 2013 designed a study to confirm the involvement of motilin receptors
in affecting the GI motility by AZM. It was performed on human isolated stomach.
Human stomach segments were obtained from patients going through surgery for
cancer or obesity. The stomach segments were shifted to the research laboratories
within 2 hours after resection in Krebs' solution aerated with 5% CO2 and 95% O2. The
strips were then mounted in tissue bath. Human recombinant motilin receptors in CHO
cells were used for conducting motilin binding and calcium flux experiments. AZM
was found to displace motilin binding to motilin receptors in a concentration dependent
manner. AZM and motilin concentration-dependently initiated short-term increase in
intracellular [Ca2+] in cells expressing the motilin receptor. In human stomach, AZM
induced cholinergically mediated contractions. The results of this study justifies the
gastric prokinetic action of AZM (Broad et al., 2013).
1.9.4.5 AZM and intestinal contractility:
Certain studies have been conducted to evaluate the effects of AZM on the contractility
of intestinal smooth muscles. (El-Baki et al., 2015; Ugwu et al., 2013). Some of them
have proved that AZM significantly increases the contractility of intestinal smooth
muscles while the others showed controversy regarding its effect (Broad et al., 2013;
Nguyen, 2014). So still there is a confusion whether AZM increases or decreases the
intestinal contractility.
A study was carried out by El-Baki and his co-workers to determine the
pharmacological effects of AZM on different isolated smooth muscle preparations of
experimental animals. After recording the normal contractions of rabbit’s jejunum,
AZM was given in different doses ranging from 1-16µg/ml. It was found out that AZM
increased the amplitude of jejunal contractions in all doses in a dose-dependent manner
(El-Baki et al., 2015).
Another study was conducted by Chini and his colleagues to determine the effect of
AZM on the small intestinal motility and to compare it with the effects of ERY. Twenty
one patients were included in the study. The frequency, amplitude and duration of small
intestinal contractions were recorded. Result of the study confirmed that AZM was
1-Introduction
65
found to stimulate gastro-duodenal motility. When the effects of AZM were compared
with those of ERY, AZM was found to produce gastro-duodenal contractions more
frequently (Chini et al., 2012). The results are in favour of prokinetic activity of AZM.
In another study, effects of AZM was assessed on GI motility in men. Eleven healthy
volunteers were included in a single blinded, placebo-controlled study. A 500mg dose
of AZM was given to them. Treatment with AZM resulted in a significant increase in
the GI motility as compared to placebo (Sifrim et al., 1994). Results were suggestive
of prokinetic activity of AZM.
In contrast to all the above mentioned studies which showed the prokinetic effects of
AZM, Chiragh et al., performed a study in vitro on rabbits duodenum. The results
showed that the prokinetic effect of AZM are not well-sustained. Thus, showing AZM
not to be a beneficial therapeutic agent to produce prokinetic actions (Chiragh et al.,
2006).
Not too much work is done to investigate the mechanisms through which AZM is going
to affect the intestinal contractility. In this regard El-Baki et al performed a study to
explore the mechanisms of AZM affecting intestinal contractility but they missed many
important regulators of intestinal motility which perform a key role in its regulation.
In a study El-Baki et al evaluated the effects of AZM on isolated colon of rats. AZM in
different doses (1-16μg/ml) produced an increase in the amplitude of contractions.
Doses were increased in a cumulative way and increase in response was noted in dose
dependent manner. They further investigated the involvement of muscarinic,
serotonergic and histaminergic receptors in the colonic contractions induced by AZM.
Isolated colon was first given histamine antagonist, then without washing the tissue
different doses of AZM were given. Same procedure was repeated for serotonin and
histamine receptor blocker. It was noted that blockade of muscarinic receptors was
associated with the prevention of the stimulant action of AZM on colonic contractions.
On the other hand, AZM in the presence of serotonergic and histaminergic receptors
blockade was still able to produce jejunal stimulation. The result of this study confirmed
that AZM exerts its effect on the intestinal motility via muscarinic receptors. Various
other receptors which are also involved in the intestinal motility are not explored in this
study, thus making it very limited (El-Baki et al., 2015).
1-Introduction
66
Rationale:
As far as there are controversial reports regarding the effect of AZM on the contractility
of intestinal smooth muscles and the mechanisms involved in it are still unrevealed.
Therefore, the present study is designed to find out the effect of AZM on the contraction
of intestinal smooth muscles and to know about its possible mechanisms through which
it affects the contractility. Knowing the exact mechanism(s) of action of AZM will help
the researchers in many ways for instance i) By finding the effect of AZM on intestinal
smooth muscles, new indications, contraindication and adverse effects can be identified
ii) It will help to decrease the use of multiple drugs in the same patient where antibiotics
for bacterial infections and other drugs to produce change in GI motility is required iii)
It will help to predict the possible drug interactions of azithromycin with other drugs.
Objectives:
1 To determine the effects of Azithromycin on the contractility of intestinal smooth
muscles in vitro model of rabbits and rats using Power Lab system.
2 To investigate the mechanisms of Azithromycin affecting the intestinal contractility
through receptors and voltage gated ion channels by using Power Lab system.
67
2 MATERIAL AND METHODS
2.1 Animals:
Adult healthy New Zealand rabbits were purchased from the local market. Body weight
of the rabbits varied from 2-2.5kg. Wistar rats were purchased from the National
Institute of Health Laboratory, Islamabad. Body weight of the rats varied from 180-
230g. Both male and female rabbits and rats were included in the study. All the rabbits
and rats were carefully evaluated to make sure that none of them are pregnant or
apparently in a diseased state.
Animals were kept in the animal house of Institute of Basic Medical Sciences (IBMS),
Khyber Medical University, Peshawar under hygienic conditions. The animals were
transported from National Institute of Health Laboratory, to the animal house of IBMS
in laboratory iron cages, where the temperature was maintained at 20-24°C. The animals
were exposed to 12 hours light and 12 hours dark cycle (Guo et al., 2016). They were
given free access to drink water and to take food; rabbits were mostly given fresh grass
and carrots while rats were given rat chow. They received humane care under standard
protocols outlined in the "Animals Bye-Laws 2008 of the University of Malakand
(Scientific Procedures Issue- 1) (Ali et al., 2009).
The animals were acclimatized for one week, before performing the experiment. They
were kept in fasting condition for 24 hours prior to experiment. However, they were
allowed to drink water freely (Naseri et al., 2008). The protocols were approved for this
study by the Ethical Committee of Khyber Medical University, Peshawar via No.
Dir/KMU-EB/MU/000745.
2.2 Chemicals and drugs:
All the chemicals and drugs used in the experiments were of analytical grade. The
chemicals and drugs used in the experiment were azithromycin dihydrate,
acetylcholine, atropine sulphate, loratadine, ondansetron, metoclopramide, verapamil,
propranolol and amiodarone (Sigma-Aldrich chemicals, St. Louis, U.S or E. Merck
grade). Fresh stock solutions for all the chemicals were prepared on the day of
experiments.
2-Material and Methods
68
2.3 Preparation of isolated tissues
2.3.1 Rabbits’ jejunal and rats’ ileal preparations
The abdomen of each rabbit and rat after scarifying was opened through a midline
incision. The abdominal viscera on the ventral abdominal wall was exposed, 1-1.5 cm
pieces of jejunum from each rabbit and the same size pieces of ileum from each rat were
isolated. All the tissues were handled very gently in order to keep the morphology and
physiology of the tissues intact. The isolated tissues were then quickly placed in a petri
dish (Nath et al., 2016; Ali et al., 2011). The petri dish contained Tyrode’s solution
which had the following composition; NaCl 8, KCl 0.2, MgCl2 0.1, CaCl2 0.2, NaHCO3
1, NaH2PO4 0.05, Glucose 1 g/L. All the chemicals were dissolved in distilled water
(Chiragh et al, 2006).
The petri dish was persistently aerated with carbogen gas (a mixture of 95% O2 and 5%
CO2) in order to keep the tissues viable (Ghayur et al., 2012). Mesentery was detached
from each tissue and each tissue was also properly cleaned from fecal content where
found. Jejunal and ileal preparations were suspended individually in a 15 milliliter (ml)
tissue bath containing Tyrode’s solution. The temperature of the tissue bath was kept at
37±1°C and aerated with carbogen gas persistently. One end of each preparation was
tied to the hook and the other was attached by a thread to the transducer, it was made
sure that lumen of the jejunum and ileum was not closed by the threads (Janbaz et al.,
2015; Chiragh et al., 2006). The transducers were linked to an amplifier to augment the
magnitude of contractions; these in turn were interpreted by a data acquisition system
to get the final results on computer system (Yasin et al., 2012).
Each tissue was kept undisturbed and allowed to equilibrate for a period of 30 min
before adding any drug. The bath fluid was replaced subsequently with normal Tyrode’s
solution before starting the experiment. (Chda et al., 2016; Janbaz et al., 2015). Those
jejunal preparations in which the spontaneous contractions were found were included
in the study while the preparations in which the spontaneous contractions were either
absent or improper were removed from the study protocols. The excluded tissues were
then replaced by the other healthy tissues having proper spontaneous contractions. To
prevent desensitization, single tissue was used once only for one concentration-
response curve (Figure 2.1).
2-Material and Methods
69
Figure 2.1: Small and Large intestines of a rabbit
2-Material and Methods
70
Figure 2.2: Isolated rabbit’s jejunal preparation
2-Material and Methods
71
2.4 Effects of drugs on rabbit’s jejunal and rat’s ileal preparations
2.4.1 Effect of AZM on rabbit’s jejunal and rat’s ileal preparations
Each rabbit’s jejunal and rat’s ileal preparation was suspended in an organ bath and
following stabilization for 30min, AZM was added cumulatively in doses of 0.01, 0.03,
0.1, 0.3, 1, 3, 5, 10, 15µM. The response to each dose was recorded for 2 min. The same
doses of AZM were considered as standard for the next series of experiments. The
contraction of isolated tissue preparations was expressed as percent of the control
response produced by ACh (3µM). Each rabbit’s jejunal and rat’s ileal preparation was
pre-treated with a validated dose of respective receptor and channel blockers. The dose
at which the receptor and channel blockers showed maximum decrease in the amplitude
of contractions was consider standard for pre-treating the relevant tissues, after which
the above mentioned doses of AZM were added. Contraction and relaxation of the
mounted tissues were detected by Force transducer (Model No: MLT 0210/A Pan Lab
S.I.) attached to Power Lab. (Model No: 4/25 T) ADInstruments, Australia. Data were
recorded at range 20 mv, Low pass 5Hz X 10 gain using input 1, rate 40/S. The
cumulative dose response was recorded in three rabbits and rats individually. Results
were described as percent response of control on y-axis while dose (µM) on x-axis.
2.4.2 Validation of curves:
Validation of curves was performed after following the procedure described in section
2.4.1, for different drugs used in the experiments in order to verify the desired response.
2.4.2.1 Acetylcholine:
Each tissue after stabilization was treated with 3 µM ACh which served as control. This
procedure was repeated on three different rabbits and rats. From each rabbit and rat, a
single tissue was taken and reading of each tissue was recorded. Results were described
as percent response on y-axis while dose (µM) on x-axis (Ali et al., 2011).
2.4.2.2 Atropine:
After stabilization, each rabbit’s jejunal and rat’s ileal preparation was given 0.01 µM
Atropine and response was noted. The concentration of atropine was gradually
increased without washing the tissue. The contractile response of tissues for each dose
2-Material and Methods
72
was recorded. At a certain point no further decrease in tissue’s response was found
depite increasing the dose of atropine. It was considered as maximum response (Emax)
of Atropine. The same method was repeated on three different rabbits and rats. Results
were described as percent response on y-axis while dose (µM) on x-axis (Ali et al.,
2011).
2.4.2.3 Loratadine:
Each rabbit’s jejunal and rat’s ileal preparation after stabilization was treated with 0.01
µM Loratadine. After using Loratadine, the response of individual tissue was recorded.
The concentration of Loratadine was kept on increasing cumulatively and the response
for each dose was noted till there was no further decrease in tissue’s contractile response
despite adding more dose, It was considered as maximum response (Emax) of
Loratadine. The same procedure with same protocols was repeated on three different
rabbits and rats. Results were described as percent response on y-axis while dose (µM)
on x-axis (Ali et al., 2011; Faisal et al., 2018).
Loratadine belongs to the second generation anti-histamines. It was selected for this
study because it lacks various adverse effects including anti-muscarinic actions which
are usually associated with first generation anti-histamines. Therefore, using loratadine
will give the actual results as compared to first generation anti-histamines where the
result may be biased because of their anti-muscarinic effects (Church et al., 2013; Liu
et al., 2005).
2.4.2.4 Ondansetron:
After stabilization, each rabbit’s jejunal and rat’s ileal preparation was given 0.01 µM
Ondansetron and response of each tissue was noted for 2min. The concentration of
Ondansetron was gradually increased without washing the tissue. The contractile
response of tissues for each dose was recorded. At a certain stage no further decrease
in tissue’s response was found despite increasing the dose of Ondansetron, it was
considered as maximum response (Emax) of Ondansetron. The same method was
repeated on three different rabbits and rats and its response was recorded. Results were
described as percent response on y-axis while dose (µM) on x-axis (Ali et al., 2011;
Afzal et al., 2016).
2-Material and Methods
73
2.4.2.5 Metoclopramide:
Each tissue after stabilization was treated with 0.01 µM Metoclopramide. After using
Metoclopramide, the response of individual tissue was recorded. The concentration of
Metoclopramide was kept on increasing cumulatively and the response for each dose
was noted. At a certain point no further increase in tissue’s contractile response was
found despite adding more dose, it was considered as maximum response (Emax) of
Metoclopramide. The same procedure with same protocols was repeated on three
different rabbits and rats. Results were described as percent response on y-axis while
dose (µM) on x-axis (Ali et al., 2011; Kamel et al., 2015).
2.4.2.6 Verapamil:
After stabilization, tissues were given 0.01 µM Verapamil and response of each tissue
was noted. The concentration of Verapamil was gradually increased without washing
the tissue. The contractile of tissues for each dose was recorded. At a certain stage no
further decrease in tissue’s response was found despite of increasing the dose of
Verapamil, it was considered as maximum response (Emax) of Verapamil. The same
method was repeated on three different rabbits and rats. Results were described as
percent response on y-axis while dose (µM) on x-axis (Ali et al., 2011; Ali et al., 2017).
2.4.2.7 Propranolol:
Each tissue after stabilization was treated with 0.01 µM Propranolol. After using
Propranolol, the response of individual tissue was recorded. The concentration of
Propranolol was kept on increasing cumulatively and the response for each dose was
noted till there was no further decrease in tissue’s contractile response despite of adding
more dose, it was considered as maximum response (Emax) of Propranolol. The same
procedure with same protocols was repeated on three different rabbits and rats. Results
were described as percent response on y-axis while dose (µM) on x-axis (Ali et al.,
2011; Ali et al., 2017).
2.4.2.8 Amiodarone:
After stabilization, each tissue was given 0.01 µM Amiodarone and response of each
tissue was noted. The concentration of Amiodarone was gradually increased without
2-Material and Methods
74
washing the tissue. The contractile response of tissues for each dose was recorded. At
a certain stage no further decrease in tissue’s response was found despite increasing the
dose of amiodarone, it was considered as maximum response (Emax) of Amiodarone.
The same method was repeated on three different rabbits and rats. Results were
described as percent response on y-axis while dose (µM) on x-axis (Ali et al., 2011).
2.4.3 Effect of AZM in the presence of receptor and channel blockers:
2.4.3.1 Effect of AZM in the presence of Atropine:
To determine the effects of AZM for possible actions on muscarinic receptors, each
rabbit’s jejunal and rat’s ileal preparation was first allowed to get stabilized for 30 min.
Tyrode’s solution of the bath in which tissues were suspended was replaced by fresh
Tyrode’s solution before adding any drug. Each jejunal and ileal preparation was pre-
treated with atropine (0.3µM), a muscarinic receptor antagonist, for 30 minutes (Faisal
et al., 2018; Ehlert et al., 2012). Different cumulative doses of AZM were applied in
the same concentration as mentioned in the section 2.4.1 and responses were recorded
(each for 2 min). This procedure was repeated on three rabbits and rats. Results were
described as percent response of control on y-axis while dose (µM) on x-axis.
2.4.3.2 Effect of AZM in the presence of Loratadine:
For evaluation of possible actions of AZM on histamine receptors, each rabbit’s jejunal
and rat’s ileal tissue after stabilization was incubated with 0.3µM Loratadine, a
histamine receptor antagonist for 30 min. Different cumulative doses of AZM were
applied in the same concentration as mentioned in the section 2.4.1 and responses were
recorded (each for 2 min). This procedure was repeated three times on three rabbits and
rats. Results were described as percent response of control on y-axis while dose (µM)
on x-axis (Faisal et al., 2018).
2.4.3.3 Effect of AZM in the presence of Ondansetron:
In order to investigate the effects of AZM for possible actions on serotonin receptors,
each rabbit’s jejunal and rat’s ileal preparation was first stabilized. Tyrode’s solution
of the bath in which tissues were suspended was replaced by fresh Tyrode’s solution
before adding any drug. Each tissue was incubated with 0.3µM Ondansatron, a
serotonin receptor antagonist, for 30 min. Different cumulative doses of AZM were
1-Introduction
75
applied in the same concentration as mentioned in the section 2.4.1 and responses were
recorded (each for 2 min). This procedure was repeated three times on three rabbits and
rats. Results were described as percent response of control on y-axis while dose (µM)
on x-axis (Faisal et al., 2018).
2.4.3.4 Effect of AZM in the presence of Metoclopramide:
To explore the effects of AZM for possible actions on dopamine receptors, each rabbit’s
jejunal and rat’s ileal preparation was first allowed to get stabilized. Before adding any
drug, Tyrode’s solution of the bath was replaced by fresh Tyrode’s solution. Each tissue
was incubated with 10µM Metoclopramide, a dopamine receptor antagonist, for 30 min.
Different cumulative doses of AZM were applied in the same concentration as
mentioned in the section 2.4.1 and responses were recorded (each for 2 min). This
procedure was repeated three times on three rabbits and three rats. Results were
described as percent response of control on y-axis while dose (µM) on x-axis (Kamel
et al., 2015).
2.4.3.5 Effect of AZM in the presence of Verapamil:
For evaluation of possible actions of AZM on calcium channels, each rabbit’s jejunal
and rat’s ileal preparation after stabilization was treated with 0.3µM verapamil, a
calcium channel blocker, for 30 minutes. Different cumulative doses of AZM were
applied in the same concentration as mentioned in the section 2.4.1 and responses were
recorded (each for 2 min). This procedure was repeated on three rabbits and rats. Results
were described as percent response of control on y-axis while dose (µM) on x-axis
(Faisal et al., 2018).
2.4.3.6 Effect of AZM in the presence of Propranolol:
In order to investigate the effects of AZM for possible actions on sodium channels, each
tissue was first allowed to get stabilized. Tyrode’s solution of the bath in which tissues
were suspended was replaced by fresh Tyrode’s solution before adding any drug. Each
rabbit’s jejunal and rat’s ileal preparation was incubated with 0.3µM propranolol for 30
minutes to block the sodium channels. Different cumulative doses of AZM were applied
in the same concentration as mentioned in the section 2.4.1 and responses were recorded
(each for 2 min). This procedure was repeated on three rabbits and rats. Results were
1-Introduction
76
described as percent response of control on y-axis while dose (µM) on x-axis (Faisal et
al., 2018).
2.4.3.7 Effect of AZM in the presence of Amiodarone:
To determine the effects of AZM for possible actions on potassium channels, each
rabbit’s jejunal and rat’s ileal preparation after stabilization was incubated with 3µM
Amiodarone, a potassium channel blocker, for 30 min. Different cumulative doses of
AZM were applied in the same concentration as mentioned in the section 2.4.1 and
responses were recorded (each for 2 min). This procedure was repeated on three rabbits
and rats. Results were described as percent response of control on y-axis while dose
(µM) on x-axis (Faisal et al., 2018).
2.4.3.8 Effect of AZM in the presence of Atropine, Ondansetron, Verapamil &
Propranolol:
Each rabbit’s jejunal and rat’s ileal preparation was pretreated with Atropine (0.3µM),
Ondansetron (0.3µM), Verapamil (0.3µM) and Propranolol (0.3µM) for 30 min.
Tissues were then treated with various increasing doses of propranolol until Emax was
achieved. Different cumulative doses of AZM were applied in the same concentration
as mentioned in the section 2.4.1 and responses were recorded (each for 2 min). This
procedure was repeated on three rabbits and rats. Results were described as percent
response of control on y-axis while dose (µM) on x-axis (Ali et al., 2017; Islami et al.,
2009).
2.5 Statistical Analysis:
Intestinal responses (%) of both rabbits’ jejunal and rats’ ileal preparations were plotted
as percent of ACh maximum concentration response using Graph Pad Prism version 6.
Similarly, in the presence and absence of each antagonists for possible involvement in
intestinal tissues, intestinal responses were expressed in tables. Their Emax in presence
of antagonist was compared with Emax of the tissues without respective antagonists by
using t-test. P-value ≤ 0.05 was considered significant.
77
3 RESULTS
This study contained 80 animals in which 48 were rabbits and 32 were rats. AZM
produced the spasmogenic response in all the animals. The current study was an attempt
to find out the effects of AZM on intestinal contractility and to investigate the possible
mechanisms by which AZM is affecting it. Percent spasmogenic effects of AZM was
expressed as % of ACh maximum (3uM), which served as control.
3.1 Validation of curves:
Validation of curve was performed for various drugs to verify the desired response and
to know the dose at which the maximum response (Emax) was produced.
3.1.1 Atropine:
Atropine decreased the contractile response both in rabbits’ jejunal and rats’ ileal
preparations. Emax for atropine was 3.6±1.3 and 1.2±0.8 for rabbits’ jejunal and rats’
ileal preparations, respectively (Table 3.1, Figure 3.1a & 3.1b).
Table 3.1: Effect of Atropine (µM) in % of ACh on rabbits’ jejunal preparations and
rats’ ileal preparations (Mean±SD, n=3)
Concentrations
(µM)
Effect of atropine on rabbits’
jejunal preparation
Effect of atropine on rats’ ileal
preparation
0.01 16.8±1.8 4.9±1.1
0.03 12.9±2.1 4.6±1.3
0.1 8.7±1.4 2.8±1.1
0.3 3.6±1.3 1.2±0.8
1 3.6±1.3 1.2±0.8
3 3.6±1.3 1.2±0.8
5 3.6±1.3 1.2±0.8
10 3.6±1.3 1.2±0.8
15 3.6±1.3 1.2s±0.8
3-Results
78
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
20
Atropine (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.1a: Effect of Atropine on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
79
0.01 0.03 0.1 0.3 1 3 5 10 15
0
2
4
6
Atropine (M)
Resp
on
se i
n %
0f
Acety
lch
oli
ne (
3u
M)
Figure 3.1b: Effect of Atropine on spontaneous rats’ ileal preparations
(n=3; Mean±SD)
3-Results
80
3.1.2 Loratadine:
Loratadine decreased the contractile response both in rabbits’ jejunal and rats’ ileal
preparations. Emax for loratadine was 9.0±1.0 and 5.2±0.3 for rabbits’ jejunal and rats’
ileal preparations, respectively (Table 3.2, Figure 3.2a & 3.2b).
Table 3.2: Effect of Loratadine (µM) in % of ACh on rabbits’ jejunal preparations and
rats’ ileal preparations (Mean±SD, n=3)
Concentrations
(µM)
Effect of loratadine on rabbits’
jejunal preparation
Effect of loratadine on rats’ ileal
preparation
0.01 20.1±1.6 14.3±0.8
0.03 18.4±1.4 12.6±0.4
0.1 12.9±1.6 8.8±0.4
0.3 10.4±1.3 6.2±0.3
1 9.2±1.3 5.8±0.3
3 9.0±1.0 5.2±0.3
5 9.0±1.0 5.2±0.3
10 9.0±1.0 5.2±0.3
15 9.0±1.0 5.2±0.3
3-Results
81
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
20
25
Loratadine (M)
Resp
on
se i
n %
0f
Acety
lch
oli
ne (
3u
M)
Figure 3.2a: Effect of Loratadine on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
82
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
Loratadine (M)
Resp
on
se i
n %
0f
Acety
lch
oli
ne (
3u
M)
Figure 3.2b: Effect of Loratadine on spontaneous rats’ ileal preparations
(n=3; Mean±SD)
3-Results
83
3.1.3 Ondansetron:
Ondansetron decreased the contractile response both in rabbits’ jejunal and rats’ ileal
preparations. Emax for ondansetron was 8.6±1.3 and 4.4±0.3 for rabbits’ jejunal and
rats’ ileal preparations, respectively (Table 3.3, Figure 3.3a & 3.3b).
Table 3.3: Effect of Ondansetron (µM) in % of ACh on rabbits’ jejunal preparations and
rats’ ileal preparations (Mean±SD, n=3)
Concentrations
(µM)
Effect of ondansetron on rabbits’
jejunal preparation
Effect of ondansetron on rats’ ileal
preparation
0.01 18.2±1.6 10.3±0.8
0.03 16.4±1.4 7.3±1.2
0.1 12.7±1.6 5.8±1.4
0.3 8.6±1.3 4.4±0.3
1 8.6±1.3 4.4±0.3
3 8.6±1.3 4.4±0.3
5 8.6±1.3 4.4±0.3
10 8.6±1.3 4.4±0.3
15 8.6±1.3 4.4±0.3
3-Results
84
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
20
Ondansetron (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.3a: Effect of Ondansetron on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
85
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
Ondansetron (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.3b: Effect of Ondansetron on spontaneous rats’ ileal preparations
(n=3; Mean±SD)
3-Results
86
3.1.4 Metoclopramide:
Metoclopramide increased the contractile response both in rabbits’ jejunal and rats’
ileal preparations. Emax for metoclopramide was 66.4±1.8 and 58.4±1.6 for rabbits’
jejunal and rats’ ileal preparations, respectively (Table 3.4, Figure 3.4a & 3.4b).
Table 3.4: Effect of Metoclopramide (µM) in % of ACh on rabbits’ jejunal preparations and
rats’ ileal preparations (Mean±SD, n=3)
Concentrations (µM)
Effect of metoclopramide on rabbits’ jejunal preparation
Effect of metoclopramide on rats’ ileal preparation
0.01 18.9±1.2 2.9±0.3
0.03 26.9±0.6 8.6±1.4
0.1 32.5±0.9 16.8±0.8
0.3 45.5±1.1 28.2±1.8
1 48.6±1.3 38.4±1.3
3 56.4±0.8 44.6±1.4
5 58.4±1.6 50.2±1.4
10 66.4±1.8 58.4±1.6
15 66.4±1.8 58.4±1.6
3-Results
87
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
80
Metoclopramide (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.4a: Effect of Metoclopramide on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
88
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
80
Metoclopramide (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.4b: Effect of Metoclopramide on spontaneous rats’ ileal
preparations (n=3; Mean±SD)
3-Results
89
3.1.5 Verapamil:
Verapamil decreased the contractile response both in rabbits’ jejunal and rats’ ileal
preparations. Emax for verapamil was 6.2±0.8 and 3.8±0.2 for rabbits’ jejunal and rats’
ileal preparations, respectively (Table 3.5, Figure 3.5a & 3.5b).
Table 3.5: Effect of Verapamil (µM) in % of ACh on rabbits’ jejunal preparations and rats’
ileal preparations (Mean±SD, n=3)
Concentrations (µM)
Effect of verapamil on rabbits’ jejunal preparation
Effect of verapamil on rats’ ileal preparation
0.01 16.4±1.4 9.8±1.2
0.03 14.2±1.2 8.2±0.8
0.1 12.7±1.0 5.7±0.4
0.3 6.2±0.8 3.8±0.2
1 6.2±0.8 3.8±0.2
3 6.2±0.8 3.8±0.2
5 6.2±0.8 3.8±0.2
10 6.2±0.8 3.8±0.2
15 6.2±0.8 3.8±0.2
3-Results
90
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
20
Verapamil (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.5a: Effect of Verapamil on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
91
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
Verapamil (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.5b: Effect of Verapamil on spontaneous rats’ ileal preparations
(n=3; Mean±SD)
3-Results
92
3.1.6 Propranolol:
Propranolol decreased the contractile response both in rabbits’ jejunal and rats’ ileal
preparations. Emax for propranolol was 5.6±0.8 and 3.2±0.3 for rabbits’ jejunal and
rats’ ileal preparations, respectively (Table 3.6, Figure 3.6a & 3.6b).
Table 3.6: Effect of Propranolol (µM) in % of ACh on rabbits’ jejunal preparations and rats’
ileal preparations (Mean±SD, n=3)
Concentrations
(µM)
Effect of propranolol on rabbits’
jejunal preparation
Effect of propranolol on rats’ ileal
preparation
0.01 18.6±0.6 9.7±0.8
0.03 15.4±1.3 6.8±0.2
0.1 10.8±1.1 4.8±0.4
0.3 5.6±0.8 3.2±0.3
1 5.6±0.8 3.2±0.3
3 5.6±0.8 3.2±0.3
5 5.6±0.8 3.2±0.3
10 5.6±0.8 3.2±0.3
15 5.6±0.8 3.2±0.3
3-Results
93
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
20
Propranolol (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.6a: Effect of Propranolol on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
94
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
Propranolol (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.6b: Effect of Propranolol on spontaneous rats’ ileal preparations
(n=3; Mean±SD)
3-Results
95
3.1.7 Amiodarone:
Amiodarone decreased the contractile response both in rabbits’ jejunal and rats’ ileal
preparations. Emax for amiodarone was 10.0±0.2 and 7.1±0.4 for rabbits’ jejunal and
rats’ ileal preparations, respectively (Table 3.7, Figure 3.7a & 3.7b).
Table 3.7: Effect of Amiodarone (µM) in % of ACh on rabbits’ jejunal preparations and
rats’ ileal preparations (Mean±SD, n=3)
Concentrations
(µM)
Effect of Amiodarone on rabbits’
jejunal preparation
Effect of Amiodarone on rats’ ileal
preparation
0.01 18.1±1.2 14.3±0.8
0.03 17.8±1.4 12.6±0.6
0.1 15.9±0.8 10.8±1.2
0.3 14.4±0.6 8.6±0.5
1 13.2±0.6 8.0±0.5
3 10.0±0.2 7.1±0.4
5 10.0±0.2 7.1±0.4
10 10.0±0.2 7.1±0.4
15 10.0±0.2 7.1±0.4
3-Results
96
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
20
Amiodarone (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.7a: Effect of Amiodarone on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
97
0.01 0.03 0.1 0.3 1 3 5 10 15
0
5
10
15
Amiodarone (M)
Res
pon
se i
n %
0f
Ace
tylc
holi
ne
(3u
M)
Figure 3.7b: Effect of Amiodarone on spontaneous rats’ ileal preparations
(n=3; Mean±SD)
3-Results
98
3.2 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations:
AZM produced significant spasmogenic response both in rabbits’ jejunal and rats’ ileal
preparations. Mean (%) Emax for AZM was 67.6±1.6 and 54.0±2.1 for rabbits’ jejunal
and rats’ ileal preparations, respectively (Table 3.8, Figure 3.8a & 3.8b).
Table 3.8: Effect of Azithromycin (in % of ACh) on rabbits’ jejunal preparations and
rats’ ileal preparations (Mean±SD, n=3)
Concentrations (µM) Effect of AZM on rabbits’ jejunal
preparations
Effect of AZM on rats’ ileal
preparations
0.01 33.9±2.8 4.0±2.8
0.03 38.6±1.9 9.7±4.2
0.1 45.3±1.6 11.4±3.7
0.3 47.7±3.8 15.5±3.7
1 50.5±3.7 26.9±0.1
3 52.5±4.9 35.1±1.4
5 65.9±1.3 44.9±2.8
10 67.7±1.6 54±2.1
15 67.7±1.6 54±2.1
3-Results
99
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of A
cetylc
holi
ne (
3u
M)
Figure 3.8a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations (n=3; Mean±SD)
3-Results
100
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Figure 3.8b: Effect of Azithromycin on rats’ ileal preparations (n=3;
Mean±SD)
3-Results
101
3.3 Effect of AZM in the absence and presence of antagonists:
3.3.1 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Atropine:
The spasmogenic response produced by AZM in the presence of Atropine was
significantly reduced both in rabbits’ jejunal and rats’ ileal preparations. Mean (%)
Emax for AZM in the absence and presence of Atropine for rabbits’ jejunal preparations
was 68.3±1.3 and 2.4±0.1, respectively, while for rats’ ileal preparations it was
57.8±1.8 and 11.4±1.3, respectively (Table 3.9, Figure 3.9a & 3.9b). When the mean
(%) Emax was compared in the absence and presence of Atropine, it was found
significant with p˂0.0001 for rabbits’ jejunal preparations and p˂0.0001 for rats’ ileal
preparations (Figure 3.9c & 3.9d).
Table 3.9: Effect of Azithromycin (µM) in % of ACh in the absence and presence of
Atropine (0.3µM) on rabbits’ jejunal preparations and rats’ ileal preparations
(Mean±SD, n=3)
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
Concentrations
(µM)
Rabbits’ Jejunum Rats’ Ileum
Effect of Azithromycin in
the absence of
Atropine
Effect of Azithromycin in
the presence of
Atropine (0.3µM)
Effect of Azithromycin in
the absence of
Atropine
Effect of Azithromycin
in the presence
of Atropine (0.3µM)
0.01 25.8±0.9 3.1±1.5*** 2.4±0 0±0**
0.03 29.5±1.1 3.1±1.5*** 5.6±2.8 2.4±4.2
0.1 40.5±0.8 2.3±0.1*** 9.7±2.4 4±3.7
0.3 48.1±1.4 2.3±0.1*** 13.8±1.4 4±3.7**
1 57.0±1.2 2.4±0*** 22.1±4.9 6.5±3.7**
3 63.3±1.1 2.4±0.1*** 35.9±1.4 10.6±1.3***
5 67.8±1.0 2.4±0.1*** 45.8±1.3 11.4±1.3***
10 68.3±1.3 2.4±0.1*** 57.8±1.8 11.4±1.3***
15 68.3±1.3 2.4±0.1*** 57.8±1.8 11.4±1.3***
3-Results
102
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Atropine
With Atropine (0.3µM)
Figure 3.9a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Atropine (n=3; Mean±SD)
3-Results
103
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Atropine
With Atropine (0.3µM)
Figure 3.9b: Effect of Azithromycin on rats’ ileal preparations in the
absence and presence of Atropine (n=3; Mean±SD)
3-Results
104
Without Atropine With Atropine
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.9c: Comparison of Azithromycin’s effect on spontaneous rabbits’
jejunal preparations in the absence and presence of Atropine (n=3;
Mean±SD)
3-Results
105
Without Atropine With Atropine
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.9d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Atropine (n=3; Mean±SD)
3-Results
106
3.3.2 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Loratadine:
The response of AZM in the presence of Loratadine was unaffected. Mean (%) Emax
for AZM was 68.8±0.6 for rabbits’ jejunal preparations in the absence of Loratadine
while it was 67.9 ± 2.0 in the presence of Loratadine. Similarly, mean (%) Emax for
rats’ ileal preparations was 52.4±2.8 and 50.7±1.9 in the absence and presence of
Loratadine respectively (Table 3.10, Figure 3.10a & 3.10b). Insignificant difference
(p=0.4 each) was found in rabbits’ jejunal and rats’ ileal preparation when the mean
(%) Emax for AZM was compared in the presence and absence of Loratadine (Figure
3.10c and 3.10d).
Table 3.10: Effect of Azithromycin (µM) in % of ACh in the absence and presence of
Loratadine (3µM) on rabbits’ jejunal preparations and rats’ ileal preparations (Mean±SD,
n=3)
Concentrations
(µM)
Rabbits’ Jejunum Rats’ Ileum
Effect of
Azithromycin in
the absence of Loratadine
Effect of
Azithromycin
in the presence of Loratadine
(3µM)
Effect of
Azithromycin in
the absence of Loratadine
Effect of
Azithromycin
in the presence of
Loratadine
(3µM)
0.01 31.3±3.6 22.9±1.0** 3.2±1.4 4.8±4.2
0.03 37.6±2.6 26.1±1.2*** 7.3±0 8.1±3.7
0.1 43.2±5.3 36.0±1.4 10.6±1.3 12.2±2.4
0.3 50.7±2.7 44.1±3.9 14.7±2.2 11.9±0.4
1 56.5±1.6 65.6±3.3** 26.4±2.1 19.5±4.2
3 59±1.7 66.1±3.0** 36.9±0.1 31.9±4.2
5 65.5±1.3 67.5±1.9 45.7±3.1 44.3±2.1
10 68.8±0.6 67.9±2 52.4±2.8 48.1±1.3
15 68.8±0.6 67.9±2 52.4±2.8 48.1±1.3
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
3-Results
107
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Loratadine
With Loratadine (3µM)
Figure 3.10a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Loratadine (n=3; Mean±SD)
3-Results
108
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Loratadine
With Loratadine (3µM)
Figure 3.10b: Effect of Azithromycin on rats’ ileal preparations in the
absence and presence of Loratadine (n=3; Mean±SD)
3-Results
109
Without Loratadine With Loratadine
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p = 0.4
Figure 3.10c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of Loratadine
(n=3; Mean±SD)
3-Results
110
Without Loratadine With Loratadine
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p = 0.4
Figure 3.10d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Loratadine (n=3; Mean±SD)
3-Results
111
3.3.3 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Ondansetron:
AZM was unable to produce its full spasmogenic response in the presence of
Ondansetron. Mean (%) Emax for AZM in the absence and presence of Ondansetron
was 68.3±1.0 and 27.5±0.5 for rabbits’ jejunal preparations, and 55.6±1.5 and 34.0±2.9
for rats’ ileal preparations, respectively, (Table 3.11, Figure 3.11a & 3.11b). When the
mean (%) Emax for AZM in the absence and presence of Ondansetron was compared,
a significant difference of p˂0.0001 and p=0.0003 was found for rabbits’ jejunal and
rats’ ileal preparation respectively (Figure 3.11c & 3.11d).
Table 3.11: Effect of Azithromycin (µM) in % of ACh in the absence and presence of
Ondansetron (0.3µM) on rabbits’ jejunal preparations and rats’ ileal preparations
(Mean±SD, n=3)
Concentrations
(µM)
Rabbits’ Jejunum Rats’ Ileum
Effect of
Azithromycin in the absence
of Ondansetron
Effect of
Azithromycin in the presence of
Ondansetron
(0.3µM)
Effect of
Azithromycin in the absence
of Ondansetron
Effect of
Azithromycin in the presence
of Ondansetron
(0.3µM)
0.01 27.8±1.7 12.4±0*** 3.2±1.4 5.6±2.8
0.03 35.8±1.5 14±1.4*** 5.6±2.8 6.5±1.3
0.1 43.2±1.3 14±1.4*** 9.7±2.4 7.3±2.4
0.3 49.4±0.6 15.7±1.3*** 13.8±1.4 9.4±2.4*
1 53.8±1.3 17.3±0*** 27.2±0.4 12.1±4.1*
3 58.7±0.4 17.9±1.1*** 35.2±1.3 20.6±4.2**
5 64.3±1.5 21.6±1.1*** 45.9±1.0 31.2±1.4***
10 68.3±1.0 27.5±0.5*** 55.6±1.0 34±2.9***
15 68.3±1.0 27.5±0.5*** 55.6±1.0 34±2.9***
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
3-Results
112
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
With Ondansetron (0.3µM)
Without Ondansetron
Figure 3.11a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Ondansetron (n=3; Mean±SD)
3-Results
113
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Ondansetron
With Ondansetron (0.3µM)
Figure 3.11b: Effect of Azithromycin on rats’ ileal preparations in the
absence and presence of Ondansetron (n=3; Mean±SD)
3-Results
114
Without Ondansetron With Ondansetron
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.11c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of Ondansetron
(n=3; Mean±SD)
3-Results
115
Without Ondansetron With Ondansetron
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p = 0.0003
Figure 3.11d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Ondansetron (n=3; Mean±SD)
3-Results
116
3.3.4 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Metoclopramide:
The response of AZM in the presence of Metoclopramide was unaffected. Mean (%)
Emax for AZM was 68.7±2.2 for rabbits’ jejunal preparations in the absence of
Metoclopramide while it was 70.3 ± 1.9 in the presence of Metoclopramide. Similarly,
mean (%) Emax for rats’ ileal preparations was 64.4±1.9 and 68.8±2.9 in the absence
and presence of Metoclopramide respectively (Table 3.12, Figure 3.12a & 3.12b).
Insignificant difference was found in rabbits’ jejunal (p=0.4) and rats’ ileal preparation
(p=0.09) when the mean (%) Emax for AZM was compared in the presence and
absence of Metoclopramide (Figure 3.12c and 3.12d).
Table 3.12: Effect of Azithromycin (µM) in % of ACh in the absence and presence of
Metoclopramide (10µM) on rabbits’ jejunal preparations and rats’ ileal (Mean±SD, n=3)
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
Concentrations (µM)
Rabbit’s Jejunum Rat’s Ileum
Effect of
Azithromycin in
the absence of
metoclopramide
Effect of
Azithromycin in
the presence of
metoclopramide (10µM)
Effect of
Azithromycin
in the absence of
metoclopramide
Effect of
Azithromycin
in the presence
of metoclopramide
(10µM)
0.01 37±1.1 38.4±1.9 29.2±0.7 28.6±1.3
0.03 42.5±2 43.7±1.2 33.7±1.1 35.2±1.5|
0.1 43.7±1.1 47.6±0.8 35.5±1 36.8±0.2
0.3 50.5±1.9 51.3±3.7 41.3±0.9 44.2±3.8
1 54.4±2.1 56.2±2.9 45.1±0.8 48.9±0.4
3 61.7±1.2 64.1±2.1 52.4±1.8 54.2±0.2
5 62.1±1.7 65.1±1.4 60.4±2.2 67.8±5.1
10 68.7±2.2 70.3±1.9 64.4±1.9 68.8±2.9
15 68.7±2.2 70.3±1.9 64.4±1.9 68.8±2.9
3-Results
117
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Metoclopramide
With Metoclopramide (10µM)
Figure 3.12a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Metoclopramide (n=3;
Mean±SD)
3-Results
118
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Metoclopramide (10µM)
With Metoclopramide (10µM)
Figure 3.12b: Effect of Azithromycin on rats’ ileal preparations` in the
absence and presence of Metoclopramide (n=3; Mean±SD)
3-Results
119
Without Metoclopramide With Metoclopramide
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p=0.4
Figure 3.12c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of
Metoclopramide (n=3; Mean±SD)
3-Results
120
Without Metoclopramide With Metoclopramide
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p=0.09
Figure 3.12d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Metoclopramide (n=3;
Mean±SD)
3-Results
121
3.3.5 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Verapamil:
The spasmogenic response produced by AZM in the presence of Verapamil was
significantly reduced both in rabbits’ jejunal and rats’ ileal preparations. Mean (%)
Emax for AZM in the presence of Verapamil for rabbits’ jejunal preparations was
13.6±1.2 and for rats’ ileal preparations it was 22.3±2.5 (Table 3.13, Figure 3.13a &
3.13b). When the mean (%) Emax for AZM in the absence and presence of Verapamil
was compared, a significant difference of p˂0.0001 and p=0.0003 was found for
rabbits’ jejunal and rats’ ileal preparations respectively (Figure 3.13c & 3.13d).
Table 3.13: Effect of Azithromycin (µM) in % of ACh in the absence and presence of Verapamil
(0.3µM) on rabbits’ jejunal preparations and rats’ ileal preparations (Mean±SD, n=3)
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
Concentrations
(µM)
Rabbit’s Jejunum Rat’s Ileum
Effect of
Azithromycin in the absence
of verapamil
Effect of
Azithromycin in the presence
of verapamil
(0.3µM)
Effect of
Azithromycin in the absence of
verapamil
Effect of
Azithromycin in the presence of
verapamil
(0.3µM)
0.01 43.4±1 2.4±0*** 5.5±2.7 2.4±0
0.03 45.7±1.2 2.4±0*** 6.5±3.7 2.4±0
0.1 48.9±0.5 2.4±0*** 9.9±2.7 2.4±0**
0.3 52.2±0.8 4.3±0.5*** 17.2±4.3 3.2±1.4**
1 56±0.7 8.1±0.5*** 20.7±5.3 5.7±1.4***
3 59±0.4 8.4±0.2*** 34.4±0.2 12.3±2.5***
5 63.3±0.8 9.7±0.7*** 43.3±2.4 18.1±2.8***
10 68.9±0.4 13.6±1.2*** 53.2±3.7 22.3±2.5***
15 68.9±0.4 13.6±1.2*** 53.2±3.7 22.3±2.5***
3-Results
122
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M) With Verapamil (0.3uM)
Without Verapamil
Figure 3.13a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Verapamil (n=3; Mean±SD)
3-Results
123
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Verapamil
With Verapamil (0.3µM)
Figure 3.13b: Effect of Azithromycin on rats’ ileal preparations in the
absence and presence of Verapamil (n=3; Mean±SD)
3-Results
124
Without Verapamil With Verapamil
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.13c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of Verapamil
(n=3; Mean±SD)
3-Results
125
Without Verapamil With Verapamil
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p = 0.0003
Figure 3.13d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Verapamil (n=3; Mean±SD)
3-Results
126
3.3.6 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Propranolol:
The amplitude of contraction in rabbit’s jejunum and rat’s ileum was markedly reduced
when AZM was given in the presence of Propranolol. Mean (%) Emax in the presence
of Propranolol was found to be 10.2±2.1 for rabbits’ jejunal preparations and 15.6±1.4
for rat’s ileal preparations (Table 3.14, Figure 3.14a & 3.14b). A significant difference
of p˂0.0001 for rabbit’s jejunal preparations and p˂0.0001 for rat’s ileal preparations
was found when the mean (%) Emax of AZM in the absence and presence of
Propranolol was compared (Figure 3.14c & 3.14d).
Table 3.14: Effect of Azithromycin (µM) in % of ACh in the absence and presence of
Propranolol (0.3µM) on rabbits’ jejunal preparations and rats’ ileal preparations
(Mean±SD, n=3)
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
Concentrations
(µM)
Rabbit’s Jejunum Rat’s Ileum
Effect of Azithromycin
in the absence
of propranolol
Effect of Azithromycin
in the presence
of propranolol
(0.3µM)
Effect of Azithromycin
in the absence
of propranolol
Effect of Azithromycin
in the presence
of propranolol
(0.3µM)
0.01 32.8±0.5 9.0±2.8*** 3.2±1.4 3.2±1.4
0.03 37.9±1.5 9.0±2.8*** 4.8±2.4 3.2±1.4
0.1 47±1.6 9.8±2.5*** 9.7±2.4 5.7±2.8
0.3 51.6±1.3 9.8±2.5*** 12.2±2.4 6.5±1.4**
1 53.9±1.3 9.9±2.5*** 24±2.4 5.7±1.4***
3 57.7±1.4 9.9±2.5*** 35.2±1.3 8.2±1.3***
5 66.6±2.1 10.2±2.1*** 45.1±2.5 9.8±2.4***
10 68.2±1.1 10.2±2.1*** 54.8±1.1 15.6±1.4***
15 68.2±1.1 10.2±2.1*** 54.8±1.1 15.6±1.4***
3-Results
127
Figure 3.14a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Propranolol (n=3; Mean±SD)
3-Results
128
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Propranolol
With Propranolol (0.3µM)
Figure 3.14b: Effect of Azithromycin on rats’ ileal preparations in the
absence and presence of Propranolol (n=3; Mean±SD)
3-Results
129
Without Propranolol With Propranolol
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.14c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of Propranolol
(n=3; Mean±SD)
3-Results
130
Without Propranolol With Propranolol
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.14d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Propranolol (n=3; Mean±SD)
3-Results
131
3.3.7 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Amiodarone:
The response of AZM on rabbits’ jejunal and rats’ ileal contraction remained
undisturbed in the presence of Amiodarone. Mean (%) Emax for AZM in the absence
of Amiodarone was 69.8±0.5 and 62.9±3.0 for rabbits’ jejunal and rats’ ileal
preparations, respectively, while in the presence of Amiodarone, mean (%) Emax was
68.4±1.3 for rabbits’ jejunal preparations and 58.0±3.4 for rats’ ileal preparations
(Table 3.15, Figure 3.15a & 3.15b). Mean (%) Emax for AZM was compared in the
presence of Amiodarone, it showed an insignificant difference with p=0.1 for rabbits’
jejunal preparations and p=0.1 for rats’ ileal preparations (Figure 3.15c & 3.15d).
Table 3.15: Effect of Azithromycin (µM) in % of ACh in the absence and presence of
Amiodarone (3µM) on rabbits’ jejunal preparations and rats’ ileal preparations (Mean±SD,
n=3)
Concentrations (µM)
Rabbit’s Jejunum Rat’s Ileum
Effect of
Azithromycin in
the absence of Amiodarone
Effect of
Azithromycin in
the presence of Amiodarone
(3µM)
Effect of
Azithromycin in
the absence of Amiodarone
Effect of
Azithromycin
in the presence of Amiodarone
(3µM)
0.01 35.3±2.8 30.9±1.3 4±2.8 4.1±1.3
0.03 44.5±4.9 37.5±1.4 5.6±2.8 4.1±1.3
0.1 55.6±7.6 48.8±1.7 11.3±3.8 6.5±1.3
0.3 60.9±5.5 58.5±2.2 15.5±3.7 10.5±1.3
1 64.6±2.3 60.8±1.2 23.6±5.6 22.2±2.2
3 67±2.5 62.5±3.9 35.1±1.4 38.7±7.4
5 68.4±1 65.9±1.8 44.9±2.8 49.3±2.0
10 69.8±0.5 68.4±1.3 62.9±3 58.0±3.4
15 69.8±0.5 68.4±1.3 62.9±3 58.0±3.4
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
3-Results
132
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Amiodarone
With Amiodarone (3µM)
Figure 3.15a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Amiodarone (n=3; Mean±SD)
3-Results
133
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without Amiodarone
With Amiodarone (3µM)
Figure 3.15b: Effect of Azithromycin on rats’ ileal preparations in the
absence and presence of Amiodarone (n=3; Mean±SD)
3-Results
134
Without Amiodarone With Amiodarone
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p = 0.17
Figure 3.15c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of Amiodarone
(n=3; Mean ± SD)
3-Results
135
Without Amiodarone With Amiodarone
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p = 0.16
Figure 3.15d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Amiodarone (n=3; Mean±SD)
3-Results
136
3.3.8 Effect of AZM on rabbits’ jejunal and rats’ ileal preparations in the
absence and presence of Atropine, Ondansetron, Verapamil &
Propranolol:
The spasmogenic response of AZM was completely lost in the presence of Atropine,
Ondansetron, Verapamil and Propranolol (AOVP), both in rabbits’ jejunal and rats’
ileal preparations (Table 3.16, Figure 3.16a & 3.16b). When the mean (%) Emax for
AZM in the absence and presence of AOVP was compared, a significant difference of
p˂0.0001 and p˂0.0001 was found for rabbits’ jejunal and rats’ ileal preparation,
respectively (Figure 3.16c & 3.16d).
Table 3.16: Effect of Azithromycin (µM) in % of ACh in the absence and presence of atropine,
ondansetron, verapamil & propranolol (AOVP) (0.3µM) on rabbits’ jejunal preparations
and rats’ ileal preparations (Mean±SD, n=3)
Concentrations (µM)
Rabbit’s Jejunum Rat’s Ileum
Effect of
Azithromycin
in the absence
of AOVP
Effect of
Azithromycin
in the presence
of AOVP (0.3µM)
Effect of
Azithromycin in
the absence
of AOVP
Effect of
Azithromycin
in the
presence
of AOVP
(0.3µM)
0.01 27.8±1.7 0.0±0.0*** 7.5±0.4 0.0±0.0***
0.03 35.8±1.5 0.0±0.0*** 11.5±3.8 0.0±0.0***
0.1 43.2±1.3 0.0±0.0*** 12.4±2.4 0.0±0.0***
0.3 49.4±0.6 0.0±0.0*** 16.8±2.4 0.0±0.0***
1 53.8±1.3 0.0±0.0*** 22.4±3.9 0.0±0.0***
3 58.7±0.4 0.0±0.0*** 30±3.7 0.0±0.0***
5 64.3±1.5 0.0±0.0*** 38.8±3.1 0.0±0.0***
10 68.3±1 0.0±0.0*** 45±3.2 0.0±0.0***
15 68.3±1 0.0±0.0*** 45±3.2 0.0±0.0***
p ≤ 0.05 = *, p ≤ 0.01 = **, p ≤ 0.001 = ***, Student ‘t’ test
3-Results
137
0.01 0.03 0.1 0.3 1 3 5 10 15
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
With AOVP (0.3µM)
Without AOVP
Figure 3.16a: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Atropine, Ondansetron,
Verapamil & Propranolol (n=3; Mean±SD)
3-Results
138
0.01 0.03 0.1 0.3 1 3 5 10 150
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3u
M)
Without AOVP
With AOVP (0.3µM)
Figure 3.16b: Effect of Azithromycin on spontaneous rabbits’ jejunal
preparations in the absence and presence of Atropine, Ondansetron,
Verapamil & Propranolol (n=3; Mean±SD)
3-Results
139
Without AOVP With AOVP
0
20
40
60
80
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.16c: Comparison of Azithromycin’s effect on spontaneous
rabbits’ jejunal preparations in the absence and presence of Atropine,
Ondansetron, Verapamil & Propranolol (n=3; Mean±SD)
3-Results
140
Without AOVP With AOVP
0
20
40
60
Azithromycin (M)
Resp
on
se i
n %
of
Acety
lch
oli
ne (
3
M)
p < 0.0001
Figure 3.16d: Comparison of Azithromycin’s effect on rats’ ileal
preparations in the absence and presence of Atropine, Ondansetron,
Verapamil & Propranolol (n=3; Mean±SD)
141
4 DISCUSSION
Azithromycin is one of the most widely prescribed antibiotics worldwide for various
clinical conditions. Therefore, Azithromycin remained the focus for researchers from
very start to further explore its pharmacological actions, adverse effects and drug
interactions. Very limited studies are conducted to investigate its effects on intestinal
smooth muscles and the possible mechanisms involved in it. The focus of this study
was to evaluate the effects of AZM on the contractility of intestinal smooth muscles
and to investigate the underlying mechanisms.
Results of the present study confirmed spasmogenic response by AZM, it increased
intestinal contractility by acting on muscarinic and serotonergic receptors, and voltage
gated calcium and sodium channels. Statistical comparison of Emax of AZM in the
absence and presence of respective antagonist is as follows; p˂0.0001 for rabbits’
jejunum and p˂0.0001 for rats’ ileum in the presence of Atropine; p˂0.0001 for rabbits’
jejunum and p=0.0003 for rats’ ileum in the presence of Ondansetron; p˂0.0001 for
rabbits’ jejunum and p=0.0003 for rats’ ileum in the presence of Verapamil; p˂0.0001
for rabbits’ jejunum and p˂0.0001 for rats’ ileum in the presence of Propranolol.
4.1 Effect of AZM on intestinal motility:
Spasmogenic response produced by AZM was a bit more in rabbit’s jejunum than rat’s
ileum. It may be because of more stronger and propagative contractions of jejunum as
compare to ileum (Seidl et al., 2012).
Rabbit’s jejunum was found more sensitive even to smaller doses of AZM as it has
responded well to very low doses of AZM i.e. 0.01 to 0.3µg/ml. Not like other smooth
muscles of the GIT, rabbit’s jejunum characteristically contains high electrically
excitable cells and hence even smaller doses of AZM have produced the significant
contractions (Kurle et al., 2017).
A study was performed Sifrim et al to find the effects of AZM on the contractions of
small intestine. AZM was found to increase the frequency and amplitude of small
intestinal contractions, confirming the spasmogenic action of AZM. Similar studies
4-Discussion
142
were conducted by El-Baki et al., and Chini et al., (El-Baki et al., 2015; Chini et al.,
2012). They also found the spasmogenic activity of AZM. The results of our study are
also in accordance to the results of these studies that AZM produces spasmogenic
response.
Unlike the results of our and above mentioned studies, Chiragh et al conducted a study
and noted that the prokinetic effects of AZM in vitro on rabbit’s duodenum are not well-
sustained. Thus, showing AZM not to be a beneficial therapeutic agent to produce
prokinetic actions (Chiragh et al., 2006). Nevertheless, its spasmogenic effect was
confirmed.
4.2 AZM and atropine:
Smooth muscle of small intestine contracts at regular intervals in the absence of
neuronal or hormonal stimulation; such contractions are referred to as phasic
(Weisbrodt, 2007). A spike potential must be produced for contraction which are
assumed to be generated by inward Ca2+ currents (Montgamry, et al., 2016).
ACh is the major excitatory neurotransmitter of the ENS, has been shown to increase
the amplitude of spontaneous contractions in the rabbits small intestine, through
M3 subtype of muscarinic receptor (Montgommery et al., 2016; Grassa et al., 2014).
This subtype is coupled to Gq receptors and stimulation of M3/Gq‐coupled receptors
activates phospholipase C (PLC) which cleaves triggers Ca2+ release from sarcoplasmic
reticulum (SR) via inositoltriphosphate (IP3) receptors to initiate contraction (Semenov
et al., 2018). Atropine by blocking the binding of ACh with muscarinic receptors blocks
all these events. Hence, we tried AZM in the presence of atropine to confirm the
involvement of M3 receptors as these receptors are predominant in GIT.
According to Gordienko et al., IP3 Ca2+ release is even facilitated by Ca2+ influx
through voltage operated channels. Thus, it is possible to speculate that the AZM
might add to its spasmogenic effect through increase in extracellular influx of
Ca2+ (Gordienko et al., 2008) produced by voltage gated Ca2+ channels.
Therefore, voltage gated Ca2+ channels can facilitate in ACh induced
contractions (Chokri et al., 2010). The response of AZM in the presence of
voltage gated Ca2+ channels is discussed in the section 4.6.
4-Discussion
143
However, our results are suggestive of cholinergic receptors mediated effects of AZM
because in the presence of cholinergic antagonist (atropine 0.3µM), the increase in the
intestinal contractility was significantly minimal. The spasmogenic effect of AZM is
mainly due to release of Ca2+ from sarcoplasmic reticulum which is triggered
through M3 receptors excitation. In relation to our findings, another study also
confirmed that AZM exerts its effect on the jejunal motility via involvement of
muscarinic receptors but in contrast to our results this study did not show involvement
of muscarinic receptors by AZM in the motility of ileum (El-Baki et al., 2015). Wang
et al also showed the involvement of muscarinic receptors by AZM in the contractions
of airway smooth muscles (ASMs). They pretreated the ASMs with ACh both in
animals (rabbits, guinea pigs and mice) and humans. AZM completely inhibited the
ACh-induced precontractions of ASMs (Wang et al., 2019). Thus it is suggested the
AZM predominantly produces spasmogenic response via involvement of M3 receptors.
4.3 AZM and loratadine:
H1 and H4 receptors are mainly found in the GIT (Mittal et al., 2017). H1 receptors are
a bit dominant and widely expressed in blood vessels, muscle layer, enterocytes,
immune cells and ganglion cells of the myenteric plexus in the GIT of humans. On the
other hand expression of H4 receptors is lower than H1 receptors in human stomach,
small intestine and colon (Kim et al., 2011; Dieteren et al., 2015).
Coupling of H1 receptors is dual in nature. They are coupled to Gq/11 proteins, activates
PLC and the phosphatidylinositol 4,5-bisphosphate (PIP2) pathway. H1 receptor
stimulation results in smooth muscle contraction mediated by IP3 induced mobilization
of intracellular Ca2+. They are also associated to Gi/o proteins, resulting in decrease
production of cAMP (Monczor et al., 2016). Decrease in cAMP levels result in increase
in the smooth muscle contraction (Bilington et al., 2003; Mizuta et al., 2013; Kuo et
al., 2015).
It can be speculated from the above mentioned points that AZM has no effect on the
levels or activity of adenylyl cyclase and cAMP, therefore it does not significantly
altered the intestinal motility in the presence of histamine receptor blocker. However,
there might be a possibility that it had increased intestinal contraction up to some extent
by increasing mobilization of intracellular Ca2+ mediated by IP3, as it was obvious in
4-Discussion
144
case of muscarinic receptor blocker (section 4.2). The latter idea was confirmed when
the tissue preparations were tested in the presence of all antagonists.
In a study the effects of AZM on intestinal motility was evaluated through histamine
receptors. It was noted that AZM in the presence of histamine receptors blockade was
still able to produce jejunal stimulation (El-Baki et al., 2015). The results of this study
confirmed that AZM does not exerts its effect on the intestinal motility via histamine
receptors. Same results were found in our study that AZM does not significantly involve
the H1 receptors in the spasmogenic response.
4.4 AZM and ondansetron:
5-HT acts as an important neurotransmitter in the ENS. 5-HT has various subtypes but
5-HT3 & 5-HT4 are predominantly present in the intestinal smooth muscles (Mittal et
al, 2017). Various researches have proved significant role of 5-HT in regulating GI
functions (Bomstein, 2012).
Stimulation of 5-HT3 receptors leads to different effects, including depolarization of
membrane and rise in intracellular Ca2+, modulation of neurotransmitter release, central
and peripheral neuron’s excitation, and release of 5-HT from EC cells of small intestine.
5-HT3 receptors may enhance spontaneous release by producing depolarization locally
at presynaptic terminal, leading to augmented influx of Ca2+ through VGCCs.
Moreover, 5-HT3 receptors are closely related with the ACh receptor, present in the
central and peripheral nervous systems. (Altamirano et al, 2018; Terry et al., 2017). 5-
HT receptors increase the release of ACh from the motor nerve endings. All these
events increases the amplitude of intestinal muscles contractions Serotonin antagonist
are reported to decrease the intestinal contractility by blocking all these happenings
(Terry et al., 2017).
As in the present study we found that one of the mechanisms by which AZM affects
the intestinal contractility is through muscarinic receptors (ACh) which also act by
increasing the extracellular influx of Ca2+(Gordienko et al., 2008) . 5-HT also acts
through the same mechanism. Therefore, it can be predicted that AZM in the
presence of 5-HT antagonists increases the intestinal contractility by increasing
the extracellular influx of Ca2+.
4-Discussion
145
In contrast to the results of our study where the involvement of 5-HT receptors were
found in the spasmogenic response produced by AZM, another study to investigate the
role of 5-HT receptors in the AZM induced spasmogenic response was conducted. The
effects of AZM on the contractions of jejunum were evaluated in the presence of 5-HT
antagonist. Results showed no involvement of 5-HT receptors in increasing the
contractility of small intestine as AZM was found to increase the jejunal contractions
even in the presence of 5-HT receptors blockade (El-Baki et al., 2015).
4.5 AZM and metoclopramide:
Various subtypes of DA receptors are found in the intestinal smooth muscles but D2 is
found abundantly (Ayano, 2016). Both inhibitory and excitatory effects of DA are
observed in GIT but usually inhibitors effects are more frequent. Release of DA from
enteric dopaminergic neurons and subsequent binding to DA receptors in GI smooth
muscles inhibits the GI motility mainly by activating D2 receptors (Giudicessi et al.,
2018). D2 receptors also inhibit the release of ACh from intrinsic cholinergic nerve
terminals (Tonini et al., 2004).
AZM is not found to act through dopamine receptors because there is no significant
difference in the amplitude of contractions between the two groups. In the presence of
Metoclopramide (10µM) the AZM increases the intestinal contractility though not
statistically significant. It might be because of the effects of AZM on Metoclopramide
induced release of ACh as discussed in section 4.2 (Tonini et al., 2004).
4.6 AZM and verapamil:
The Cav 1.2 L-type Ca2+ channel is the dominant voltage-activated Ca2+ channel in
heart and smooth muscles. A rise in intracellular calcium is the trigger for Gl smooth
muscles contractions. Under normal conditions, membrane depolarization triggers an
influx of calcium and this calcium serves as an activator source for contractions. A
number of studies have found that GI smooth muscles do not show spontaneous
contractile activity during exposure to calcium-free solution (Evans et al., 2011). In the
cytoplasm of SMC, Ca2+ binds to calmodulin and the calcium-calmodulin complex
combines with the catalytic subunit of myosin light chain (MLC) kinase to form a
receptor leading to the phosphorylation of serine (Ser) at position 19 in the MLC.
Phosphorylation of Ser-19 of MLC then allows myosin ATPase to be activated by actin
4-Discussion
146
and muscle contraction takes place. When cytoplasmic (Ca2+) is reduced, MLC
phosphatase dephosphorylates MLC20, thereby deactivating actomyosin ATPase and
causing relaxation. Ca2+ is provided by both extracellular and intracellular sources (Zhu
et al., 2001).
Thus it can be predicted that the spasmogenic response of AZM is mostly dependent on
Ca2+. Verapamil, a calcium channel blocker, decreases the influx of Ca2+ by blocking
calcium channels. AZM in the presence of verapamil is unable to increase the inward
flux of Ca2+ which is basically responsible for increase in intestinal contraction.
A study performed by Wang et al., has reported the inhibition of VGCCs by AZM
(Wang et al., 2019) in smooth muscles. Zhaug and his co-workers also found the
involvement of AZM in the inhibition of VGCCs (Zhaug et al., 2014). In another study
performed by Silu and his colleagues, AZM was found to decrease the mucus secretion
in ASMs by inhibiting the Ca2+ entry into submucus gland cells (SMGCs) (Silu et al,
2010). Our results are also indicative of the fact that AZM inhibits VGCCs, as when
the effect of AZM was evaluated in the presence of calcium channel blocker, a very
minimal increase in the amplitude of intestinal smooth muscle contraction was found.
El-Baki et al., performed a study to evaluate the effects of AZM on rabbit’s jejunum.
They also investigated the role of calcium channels in the spasmogenic response of
AZM. They found that calcium channels play a significant role in the actions of AZM
as stimulant to contractions of jejunum (El-Baki et al., 2015). The results of this study
is in accordance with the results of our study as we also found the involvement of
VGCCs in jejunal and ileal contractions.
The effects of AZM were also assessed in vitro on isolated colon of rats. AZM was
found to increase colonic contractions. For further investigation of the underlying
mechanisms by which AZM affects the colonic contractility, first tissue was treated
with calcium channel blockers and then the response of AZM was noted. Colonic
contractions induced by AZM were declined in the presence of calcium channel blocker
(El-Baki et al., 2015). This showed the involvement of calcium channels in AZM
induced contractions. The association of VGCCs with intestinal contractility is
common both in this as well as in our study.
4-Discussion
147
In contrast to the results of our study, Daenas et al., conducted a study to evaluate the
effect of AZM on precontracted ASMs. AZM was found to possess dose-dependent,
relaxant effect on precontracted ASMs which is not mediated via inhibition of
Ca2+ release or Ca2+ influx. These results are dissimilar from the results of our study
which showed partially Ca2+ mediated contraction of intestinal smooth muscles by
AZM (Daenas et al., 2006).
4.7 AZM and propranolol:
The involvement of voltage-gated sodium channels (NaV) in the regulation of resting
membrane potential are evident from the Intracellular recordings of human small
intestine smooth muscle strips (Nashatian et al., 2015). NaV channels play a key role
in initiation of action potentials. During membrane depolarization, Na+ appears to
gather in areas under the plasma membrane, producing local concentration gradients
that effect cell Ca2+ handling mechanisms (Soomer et al., 2017).
AZM failed to increase the intestinal contractions in the presence of NaV channel
blocker, confirming to affect the process of depolarization. During depolarization, the
membrane potential quickly moves from negative to positive. The NaV channels open
in reaction to an early change in voltage. As the Na+ rush back into the cell, the interior
of the cell become positive, ultimately changing the membrane potential from negative
to positive. When the interior of the cell becomes more positively charged,
depolarization of the cell is complete, and the channels close again (Molnar et al.,
2015). Thus involvement of Na+ channels cannot be ruled out in the AZM triggered
tissue contractions.
4.8 AZM and amiodarone:
Potassium channels comprise half of the ion channel superfamily, and voltage-sensitive
potassium channels (KV) are the largest group. Wide diversity of potassium channels is
detected in ICCs and SMCs of the GIT. The intracellular K+ concentration is 10 times
higher than extracellular (Beyder et al., 2012).
Following depolarization, the NaV channels close again that had been open while the
cell was undergoing depolarization. The potassium channels now open due to increased
positive charge within the cell. K+ begins to move out of the cell and the potential within
4-Discussion
148
the cell declines and approaches its resting potential (Molnar et al., 2015). The effect
of AZM in the presence AZM was not found to act through KV channels, thus not
interfering in the process of repolarization.
AZM effect was completely blocked in the presence of all these antagonists suggesting
the involvement of mixed pathways in the spasmogenic response produced by AZM.
Though it is evident that the maximum spasmogenic response is mediated through
muscarinic receptors of the intestine.
149
5 CONCLUSION
Maximum spasmogenic response of Azithromycin is through involvement of triggering
of muscarinic receptors as Azithromycin effect was primarily blocked in the presence
of atropine, a standard anticholinergic drug. Moreover, our results suggest that
azithromycin increases the contractility of rabbits’ jejunal and rats’ ileal preparations
by involving multiple receptors and voltage gated channels. Decreased contractility of
rabbits’ jejunal and rats’ ileal preparations in the presence of atropine, ondansetron,
verapamil and propranolol confirmed the core involvement of muscarinic receptors
following mixed pathways involving serotonergic receptors, voltage gated calcium and
sodium channels in the production of spasmogenic response by azithromycin.
Recommendations:
1). Azithromycin is frequently prescribed by clinicians to pregnant ladies for treating
various infections. Uterus is also made up of smooth muscles and more likely it will
also respond to Azithromycin in the same manner (increase in contractility) as other
smooth muscles of the body respond, Azithromycin may lead to abortion or premature
labour if used in pregnancy. Therefore, results of our study strongly suggest to evaluate
the effects of Azithromycin on uterine smooth muscles as well for safe clinical practice.
2). Azithromycin by increasing the intestinal contractility may significantly affect the
absorption of drugs which requires more time for absorption. Hence, our studies open
a new window of research to basic scientists and clinicians to study further the possible
drug-drug interactions of Azithromycin and other primary drug therapies that requires
Azithromycin in combination.
Limitations:
The limitations of the study are that no in vivo activity was performed, all the activities
performed through Power Lab. were in vitro. Power Lab. has very sensitive transducers,
which needs specific temperature and constant pressure during the procedure. A small
change either in the temperature or pressure may affect the results.
150
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