Accepted Manuscript Canagliflozin prevents scopolamine-induced memory impairment in rats: Comparison with galantamine hydrobromide action Nadia M.S. Arafa, Elham H.A. Ali, Mohamed Kamel Hassan PII:
S0009-2797(17)30631-2
DOI:
10.1016/j.cbi.2017.08.013
Reference:
CBI 8081
To appear in:
Chemico-Biological Interactions
Received Date: 8 June 2017 Revised Date:
23 July 2017
Accepted Date: 18 August 2017
Please cite this article as: N.M.S. Arafa, E.H.A. Ali, M.K. Hassan, Canagliflozin prevents scopolamineinduced memory impairment in rats: Comparison with galantamine hydrobromide action, ChemicoBiological Interactions (2017), doi: 10.1016/j.cbi.2017.08.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
rats: comparison with galantamine hydrobromide action.
2
Nadia M. S. Arafa1; Elham H. A. Ali2; Mohamed Kamel Hassan3
3
RI PT
Canagliflozin prevents scopolamine-induced memory impairment in
SC
1- Faculty of Science, Biology Department, Jazan University, KSA & National Organization for Drug Control and Research, Department of Physiology, Egypt. 2- Zoology Department, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt. [email protected] 3- Biotechnology Program, Zoology Department, Faculty of Science, Port Said University, Port Said, Egypt. [email protected]
M AN U
*email of the corresponding author: [email protected]. Tel/Fax: +966 563725278/+966 073211052
4 5 6 7 8 9 10 11 12
Canagliflozin (CAN) is a sodium-glucose co-transporter 2 (SGLT2) inhibitor indicated to
13
improve glycemic control in adults with type 2 diabetes mellitus. There is a little
14
information about its effect on the cholinergic system that proposed mechanism for
15
memory improvement occurring by SGLT2 drugs. This study aimed to estimate the effect
16
of CAN as compared to galantamine (GAL) treatments for two weeks on scopolamine
17
hydrobromide (SCO) -induced memory dysfunction in experimental rats. Animals
18
divided into six groups; control (CON), CAN, GAL, SCO, SCO+CAN and SCO+GAL.
19
Results indicated significant decrease in body weights of the CAN groups as compared to
20
AC C
EP
TE D
Abstract
control values. Moreover, in the SCO+CAN and SCO+GAL the number of arm entry and
21
number of correct alternation in Y maze task increased, acetylcholinesterase (AChE)
22
activities decreased significantly, while
monoamines levels significantly increased
23
compared with the SCO group values. Results also recorded acetylcholine M1 receptor
24
(M1 mAChR) in SCO+CAN or SCO+GAL groups in comparison with the SCO group.
25
1
ACCEPTED MANUSCRIPT
26
scopolamine hydrobromide via cholinergic and monoamines system.
27
Key words: Canagliflozin; Scopolamine; AChE; Monoamines; M1 mAChR.
28
RI PT
The study suggested that canagliflozin might improve memory dysfunction induced by
1. Introduction
29 30 31
transporter 2 (SGLT2) targeting drug. The SGLT2 has the key role in the glucose
32
reabsorption in the kidneys and gastrointestinal tract [1]. These drugs represent a new
33
M AN U
SC
Canagliflozin (commercially known as Invokana) is sodium–dependent glucose co-
34
selective inhibitors [2]. Ipragliflozin used for reduction of body fat mass by increasing
35
fatty acid oxidation in obese rats [3]. A previous study recorded the hypolipidemic effect
36
of canagliflozin in obese diabetic rat model [4]. In addition, canagliflozin suggested as
37
AChE inhibitor [5] and a recent study discussed its effect on cerebral AChE activity in
38
obese diabetic rats [6]. Hence, the effect of canagliflozin on cholinergic system and
39
memory function are secrecy so more studies needed to evaluate its effect in memory
40
dysfunction animal models.
41
Scopolamine is a non-selective muscarinic receptor antagonist, which blocks cholinergic
42
AC C
EP
TE D
strategy for diabetes treatment. SGLT2-selective inhibitors considered safer than non-
signaling and pharmacologically interferes with memory performance in a transient
43
manner [7]. Scopolamine produces memory and cognitive impairments and subsequently
44
induces learning and memory defects including long-term and short term memory
45
dysfunction [8,9]. Animals with scopolamine-induced memory impairment have been
46
widely used to probe drugs attenuating cognitive deficits.
47
2
ACCEPTED MANUSCRIPT
48
snowdrop) and Galanthus woronowii (Amaryllidaceae) in the 1950s. It is currently
49
prescribed for Alzheimer treatment because of its activity as a moderate
50
acetylcholinesterase (AChE) inhibitor where it increases the response to ACh in the
51
synaptic cleft [10]. It is also reported as an allosteric modulator of nicotinic receptors
52
[11,12]. The three-year follow-up study by Richarz et al, [13] informed that galantamine
53
safety and tolerability during the 3-year observation period with improved cognition
54
compared with an untreated population.
55
SC
RI PT
Galantamine was first discovered and isolated from Galanthus nivalis (common
56
scopolamine induced rat model of memory dysfunction as compared to galantamine
57
administration, as a reference drug, through Y maze task and determination of AChE
58
enzyme activities as well as monoamines contents and M1 mAChR expression in
59
different brain areas.
60
TE D
M AN U
The current study, we aimed to evaluate the effect of canagliflozin administration in
61
2.1. Experimental animals:
62
EP
2. Material and methods
63
(140 ± 10.5 g) and was obtained from the animal house of National Organization for
64
AC C
This study was carried out using fifty-four young adult male rats (Wister strain) weighing
Drug Control and Research (NODCAR). The animals aged 5-6 weeks. Animals were
65
housed in plastic cages as groups of nine rats per cage. Animals were kept under
66
controlled temperature of 25±2oC and 12 hours light/12 hours dark cycle throughout the
67
experiment. A commercial pelleted diet was used during the experiment. The animals
68
were allowed to adapt to the laboratory conditions one week before the beginning of the
69
experiment. The experimental protocols and procedures was approved by Ain Shams
70
3
ACCEPTED MANUSCRIPT
71
performed according to the U.K. Animals (Scientific Procedures) Act, 1986 and
72
associated guidelines, the European Communities Council Directive of 24 November
73
1986 (86/609/EEC). All efforts made to minimise animal suffering, to reduce the number
74
of animals used, and to utilise alternatives to in vivo techniques, if available.
75
RI PT
University authorities and followed Egyptian rules for animal protection, which was
2.1.1. Drugs:
76 77
≥90%) (Sigma St. Louis, MO, USA). Canagliflozin (C24H25FO5S·1/2 H2O) was supplied
78
as "Invokana" 300 µγ tablets manufactured by Janssen pharmaceuticals, INC (LLC.
79
Gurabo, PR 00778. Titusville, NJ 08560; active gradient, manufactured in Belgium).
80
Galantamine hydrobromide (C17H21NO3·HBr) was supplied as "Reminyl" 16 mg
81
galantamine capsules, purchased from Janssen, Australia.
82
2.2. Experimental design:
83
TE D
M AN U
SC
Scopolamine hydrobromide (C17H21NO4·HBr) was supplied as a white powder (assay
Animals were treated for two weeks and divided into six main groups each of nine rats as
84
1- The control group (CON) was daily received by oral gavage 0.5 ml/100g of 0.5%
85
carboxy-methyl
(SCO),
86
intraperitoneally administered scopolamine (1mg SCO/1ml 0.9% NaCl/kg) two doses one
87
at the 1st day of the experiment and the second at 13th day before the behavioral test in
88
line with oral CMC administration as in CON group. 3-Canagliflozin (CAN) group
89
received orally 10 mg CAN/5ml CMC/kg [14]. 4-Galantamine (GAL) group orally
90
received 3 mg GAL/5ml CMC/kg [15]. 5-SCO+CAN, and 6-SCO+GAL groups were
91
treated with CAN or GAL after one hour from SCO injection at the 1st day and then
92
administered SCO at 13th day before the behavioral test with the aforementioned doses.
93
sodium
salt
(CMC).
2-Scopolamine
group
AC C
EP
cellulose
4
ACCEPTED MANUSCRIPT
94
before decapitation as represented in Figure (1).
95
2.2.1. Y maze task:
96
The Y maze is consisted of three equally arms tagged A, B and C. The angle between the
97
arms is 120°, each division of the maze is 40 X 15 X 30 cm, long X wide X high
98
respectively. The floor and sides of each arm is formed of wood. The maze employed to
99
evaluate spontaneous alternations. Each rat put at one of the sections and permitted to
100
move freely among the three divisions for 5 minutes. The number of arm entries and the
101
number of trials documented to estimate the alternation percentage. An entry calculated
102
when all the rat four limbs are within the arm. The maze cleaned with 70% alcohol and
103
allowed to dry between sessions [16].
104
2.2.2. Biochemical investigation:
M AN U
SC
RI PT
The Y maze test was begun on the day 13th of the experiment for two consecutive days
TE D
The body weights were recorded weekly. Rats were sacrificed 24 hours after the last dose
105 106 107
collected and centrifuged to separate serum, then serum was used in the same day of
108
decapitation within the first 4 hrs for determination of serum blood glucose levels. The
109
EP
(15th day) by rapid decapitation. Immediately after decapitation blood samples were
110
Laboratory, Inc., San Antonio [17]. In addition, brain tissues were dissected out from six
111
rats/group on ice, frontal cortex (Cor), hippocampus (Hippo) and midbrain (MB) areas
112
excised, cut into two halves and weighed. The first halves were used for the
113
acetylcholinesterase activity assay, which was conducted as previously described [18].
114
The second halves were homogenized in 75% methanol (HPLC grade) for the
115
AC C
blood glucose was determined colorimetrically using glucose kit from Stanbio
5
ACCEPTED MANUSCRIPT
116
HT) were evaluated as previously described [19].
117
2.2.3. Immunohistochemistry:
118
Immunohistochemical staining of M1mAChR was performed as previously described
119
[20]. Brain tissues were postfixed in 10% formalin solution, dehydration in graded
120
alcohols, cleared in xylene, and embedded in paraffin wax. At first samples incubated (30
121
min) at 37°C, then, for deparaffinization and hydrate procedures embedded in xylene and
122
different degrees of ethanol. Sections were cut at 4 mm for cortex and hippocampus using
123
a rotary microtome (MK 1110) as previously described [21]. After blocking the
124
endogenous peroxidase, the formalin-fixed, paraffin-embedded tissues were used for
125
detection of acetylcholine receptor (M1 mAChR) expression. At first samples incubated
126
(30 min) at 37°C, then, for deparaffinization and hydrate procedures embedded in xylene
127
and different degrees of ethanol. After these the samples washed in purified water. The
128
TE D
M AN U
SC
RI PT
determination of monoamines, norepinephrine (NE), dopamine (DA), and serotonin (5-
129
overnight, with anti- M1 mAChR rabbit monoclonal antibody (cat. No. ab11100) (Abcam,
130
Cambridge, UK) at 1:250 dilution. After TBS washes (three times; 10 min each), then
131
EP
antigen was retrieved by microwave and then the sections were incubated, at 4C
132
h at room temperature (dilution 1:1000) and washed in TPBS (10 min X3) All of the
133
slides were carefully examined and photos obtained for each target area using camera-
134
supported Olympus microscope. Image analysis performed using (Image Analysis –
135
Olympus - BX40F-3) program and the results of the integral staining density (sum of all
136
individual optical densities of each pixel in the measured area, "sum of color intensity")
137
for three photos from the three animals were analyzed by SPSS.
138
AC C
sections were incubated with biotinated goat anti rabbit secondary antibody (abcam) for 1
6
ACCEPTED MANUSCRIPT
139
Results presented as the mean ± SE. The statistical analyses were conducted using a one-
140
way analysis of variance (ANOVA), and the least significant difference (LSD) was used
141
to evaluate significant differences (p
S0009-2797(17)30631-2
DOI:
10.1016/j.cbi.2017.08.013
Reference:
CBI 8081
To appear in:
Chemico-Biological Interactions
Received Date: 8 June 2017 Revised Date:
23 July 2017
Accepted Date: 18 August 2017
Please cite this article as: N.M.S. Arafa, E.H.A. Ali, M.K. Hassan, Canagliflozin prevents scopolamineinduced memory impairment in rats: Comparison with galantamine hydrobromide action, ChemicoBiological Interactions (2017), doi: 10.1016/j.cbi.2017.08.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
rats: comparison with galantamine hydrobromide action.
2
Nadia M. S. Arafa1; Elham H. A. Ali2; Mohamed Kamel Hassan3
3
RI PT
Canagliflozin prevents scopolamine-induced memory impairment in
SC
1- Faculty of Science, Biology Department, Jazan University, KSA & National Organization for Drug Control and Research, Department of Physiology, Egypt. 2- Zoology Department, Faculty of Women for Arts, Science and Education, Ain Shams University, Cairo, Egypt. [email protected] 3- Biotechnology Program, Zoology Department, Faculty of Science, Port Said University, Port Said, Egypt. [email protected]
M AN U
*email of the corresponding author: [email protected]. Tel/Fax: +966 563725278/+966 073211052
4 5 6 7 8 9 10 11 12
Canagliflozin (CAN) is a sodium-glucose co-transporter 2 (SGLT2) inhibitor indicated to
13
improve glycemic control in adults with type 2 diabetes mellitus. There is a little
14
information about its effect on the cholinergic system that proposed mechanism for
15
memory improvement occurring by SGLT2 drugs. This study aimed to estimate the effect
16
of CAN as compared to galantamine (GAL) treatments for two weeks on scopolamine
17
hydrobromide (SCO) -induced memory dysfunction in experimental rats. Animals
18
divided into six groups; control (CON), CAN, GAL, SCO, SCO+CAN and SCO+GAL.
19
Results indicated significant decrease in body weights of the CAN groups as compared to
20
AC C
EP
TE D
Abstract
control values. Moreover, in the SCO+CAN and SCO+GAL the number of arm entry and
21
number of correct alternation in Y maze task increased, acetylcholinesterase (AChE)
22
activities decreased significantly, while
monoamines levels significantly increased
23
compared with the SCO group values. Results also recorded acetylcholine M1 receptor
24
(M1 mAChR) in SCO+CAN or SCO+GAL groups in comparison with the SCO group.
25
1
ACCEPTED MANUSCRIPT
26
scopolamine hydrobromide via cholinergic and monoamines system.
27
Key words: Canagliflozin; Scopolamine; AChE; Monoamines; M1 mAChR.
28
RI PT
The study suggested that canagliflozin might improve memory dysfunction induced by
1. Introduction
29 30 31
transporter 2 (SGLT2) targeting drug. The SGLT2 has the key role in the glucose
32
reabsorption in the kidneys and gastrointestinal tract [1]. These drugs represent a new
33
M AN U
SC
Canagliflozin (commercially known as Invokana) is sodium–dependent glucose co-
34
selective inhibitors [2]. Ipragliflozin used for reduction of body fat mass by increasing
35
fatty acid oxidation in obese rats [3]. A previous study recorded the hypolipidemic effect
36
of canagliflozin in obese diabetic rat model [4]. In addition, canagliflozin suggested as
37
AChE inhibitor [5] and a recent study discussed its effect on cerebral AChE activity in
38
obese diabetic rats [6]. Hence, the effect of canagliflozin on cholinergic system and
39
memory function are secrecy so more studies needed to evaluate its effect in memory
40
dysfunction animal models.
41
Scopolamine is a non-selective muscarinic receptor antagonist, which blocks cholinergic
42
AC C
EP
TE D
strategy for diabetes treatment. SGLT2-selective inhibitors considered safer than non-
signaling and pharmacologically interferes with memory performance in a transient
43
manner [7]. Scopolamine produces memory and cognitive impairments and subsequently
44
induces learning and memory defects including long-term and short term memory
45
dysfunction [8,9]. Animals with scopolamine-induced memory impairment have been
46
widely used to probe drugs attenuating cognitive deficits.
47
2
ACCEPTED MANUSCRIPT
48
snowdrop) and Galanthus woronowii (Amaryllidaceae) in the 1950s. It is currently
49
prescribed for Alzheimer treatment because of its activity as a moderate
50
acetylcholinesterase (AChE) inhibitor where it increases the response to ACh in the
51
synaptic cleft [10]. It is also reported as an allosteric modulator of nicotinic receptors
52
[11,12]. The three-year follow-up study by Richarz et al, [13] informed that galantamine
53
safety and tolerability during the 3-year observation period with improved cognition
54
compared with an untreated population.
55
SC
RI PT
Galantamine was first discovered and isolated from Galanthus nivalis (common
56
scopolamine induced rat model of memory dysfunction as compared to galantamine
57
administration, as a reference drug, through Y maze task and determination of AChE
58
enzyme activities as well as monoamines contents and M1 mAChR expression in
59
different brain areas.
60
TE D
M AN U
The current study, we aimed to evaluate the effect of canagliflozin administration in
61
2.1. Experimental animals:
62
EP
2. Material and methods
63
(140 ± 10.5 g) and was obtained from the animal house of National Organization for
64
AC C
This study was carried out using fifty-four young adult male rats (Wister strain) weighing
Drug Control and Research (NODCAR). The animals aged 5-6 weeks. Animals were
65
housed in plastic cages as groups of nine rats per cage. Animals were kept under
66
controlled temperature of 25±2oC and 12 hours light/12 hours dark cycle throughout the
67
experiment. A commercial pelleted diet was used during the experiment. The animals
68
were allowed to adapt to the laboratory conditions one week before the beginning of the
69
experiment. The experimental protocols and procedures was approved by Ain Shams
70
3
ACCEPTED MANUSCRIPT
71
performed according to the U.K. Animals (Scientific Procedures) Act, 1986 and
72
associated guidelines, the European Communities Council Directive of 24 November
73
1986 (86/609/EEC). All efforts made to minimise animal suffering, to reduce the number
74
of animals used, and to utilise alternatives to in vivo techniques, if available.
75
RI PT
University authorities and followed Egyptian rules for animal protection, which was
2.1.1. Drugs:
76 77
≥90%) (Sigma St. Louis, MO, USA). Canagliflozin (C24H25FO5S·1/2 H2O) was supplied
78
as "Invokana" 300 µγ tablets manufactured by Janssen pharmaceuticals, INC (LLC.
79
Gurabo, PR 00778. Titusville, NJ 08560; active gradient, manufactured in Belgium).
80
Galantamine hydrobromide (C17H21NO3·HBr) was supplied as "Reminyl" 16 mg
81
galantamine capsules, purchased from Janssen, Australia.
82
2.2. Experimental design:
83
TE D
M AN U
SC
Scopolamine hydrobromide (C17H21NO4·HBr) was supplied as a white powder (assay
Animals were treated for two weeks and divided into six main groups each of nine rats as
84
1- The control group (CON) was daily received by oral gavage 0.5 ml/100g of 0.5%
85
carboxy-methyl
(SCO),
86
intraperitoneally administered scopolamine (1mg SCO/1ml 0.9% NaCl/kg) two doses one
87
at the 1st day of the experiment and the second at 13th day before the behavioral test in
88
line with oral CMC administration as in CON group. 3-Canagliflozin (CAN) group
89
received orally 10 mg CAN/5ml CMC/kg [14]. 4-Galantamine (GAL) group orally
90
received 3 mg GAL/5ml CMC/kg [15]. 5-SCO+CAN, and 6-SCO+GAL groups were
91
treated with CAN or GAL after one hour from SCO injection at the 1st day and then
92
administered SCO at 13th day before the behavioral test with the aforementioned doses.
93
sodium
salt
(CMC).
2-Scopolamine
group
AC C
EP
cellulose
4
ACCEPTED MANUSCRIPT
94
before decapitation as represented in Figure (1).
95
2.2.1. Y maze task:
96
The Y maze is consisted of three equally arms tagged A, B and C. The angle between the
97
arms is 120°, each division of the maze is 40 X 15 X 30 cm, long X wide X high
98
respectively. The floor and sides of each arm is formed of wood. The maze employed to
99
evaluate spontaneous alternations. Each rat put at one of the sections and permitted to
100
move freely among the three divisions for 5 minutes. The number of arm entries and the
101
number of trials documented to estimate the alternation percentage. An entry calculated
102
when all the rat four limbs are within the arm. The maze cleaned with 70% alcohol and
103
allowed to dry between sessions [16].
104
2.2.2. Biochemical investigation:
M AN U
SC
RI PT
The Y maze test was begun on the day 13th of the experiment for two consecutive days
TE D
The body weights were recorded weekly. Rats were sacrificed 24 hours after the last dose
105 106 107
collected and centrifuged to separate serum, then serum was used in the same day of
108
decapitation within the first 4 hrs for determination of serum blood glucose levels. The
109
EP
(15th day) by rapid decapitation. Immediately after decapitation blood samples were
110
Laboratory, Inc., San Antonio [17]. In addition, brain tissues were dissected out from six
111
rats/group on ice, frontal cortex (Cor), hippocampus (Hippo) and midbrain (MB) areas
112
excised, cut into two halves and weighed. The first halves were used for the
113
acetylcholinesterase activity assay, which was conducted as previously described [18].
114
The second halves were homogenized in 75% methanol (HPLC grade) for the
115
AC C
blood glucose was determined colorimetrically using glucose kit from Stanbio
5
ACCEPTED MANUSCRIPT
116
HT) were evaluated as previously described [19].
117
2.2.3. Immunohistochemistry:
118
Immunohistochemical staining of M1mAChR was performed as previously described
119
[20]. Brain tissues were postfixed in 10% formalin solution, dehydration in graded
120
alcohols, cleared in xylene, and embedded in paraffin wax. At first samples incubated (30
121
min) at 37°C, then, for deparaffinization and hydrate procedures embedded in xylene and
122
different degrees of ethanol. Sections were cut at 4 mm for cortex and hippocampus using
123
a rotary microtome (MK 1110) as previously described [21]. After blocking the
124
endogenous peroxidase, the formalin-fixed, paraffin-embedded tissues were used for
125
detection of acetylcholine receptor (M1 mAChR) expression. At first samples incubated
126
(30 min) at 37°C, then, for deparaffinization and hydrate procedures embedded in xylene
127
and different degrees of ethanol. After these the samples washed in purified water. The
128
TE D
M AN U
SC
RI PT
determination of monoamines, norepinephrine (NE), dopamine (DA), and serotonin (5-
129
overnight, with anti- M1 mAChR rabbit monoclonal antibody (cat. No. ab11100) (Abcam,
130
Cambridge, UK) at 1:250 dilution. After TBS washes (three times; 10 min each), then
131
EP
antigen was retrieved by microwave and then the sections were incubated, at 4C
132
h at room temperature (dilution 1:1000) and washed in TPBS (10 min X3) All of the
133
slides were carefully examined and photos obtained for each target area using camera-
134
supported Olympus microscope. Image analysis performed using (Image Analysis –
135
Olympus - BX40F-3) program and the results of the integral staining density (sum of all
136
individual optical densities of each pixel in the measured area, "sum of color intensity")
137
for three photos from the three animals were analyzed by SPSS.
138
AC C
sections were incubated with biotinated goat anti rabbit secondary antibody (abcam) for 1
6
ACCEPTED MANUSCRIPT
139
Results presented as the mean ± SE. The statistical analyses were conducted using a one-
140
way analysis of variance (ANOVA), and the least significant difference (LSD) was used
141
to evaluate significant differences (p
