Project Guide: Professor Anita A. Mehta ; The Best Project Guide I have ever seen.
Special Thanks to Dr. Mukul R Jain (Senior VP, Zydus Research Center) for continuous support. Thanks to Dr. Ajay Sharma (Associate Professor, Mason Eye Institute, USA) for concept building and giving training for Langendorrf's isolated heart experiments.
Anuman- An inference for helpful in diagnosis and treatment
My phD work Title "INVESTIGATION INTO THE MECHANISM OF ACTION OF CARDIOVASCULAR EFFECTS OF CARBON MONOXIDE RELEASING MOLECULE"
1. • "All things are poison and nothing is
without poison, only the dose permits
something not to be poisonous.“
• Philippus Aureolus Paracelsus
(1493-1541)
1
All poisons are not poison
but
Dose makes them drug
…….Anita A Mehta
"The dose makes the poison"
2. 2
INVESTIGATION INTO THE MECHANISM OF
ACTION OF CARDIOVASCULAR EFFECTS OF
CARBON MONOXIDE RELEASING MOLECULE
Project Guide
Dr. Anita A Mehta
Professor & Head,
Department of Pharmacology,
L.M.College of Pharmacy,
Navarangpura,
Ahmedabad-380009, India.
Presented by
HITESH M. SONI, M.PHARM.
3. 3
Discovery of gaseous molecule-History
Pioneers in the discovery of gaseous molecule.
In the late 1200s, the Spanish alchemist Arnold of Villanova
described a poisonous gas produced by the incomplete
combustion of wood that was almost certainly CO ("Carbon
monoxide - history, sources, physiological effects, uses, Science encyclopedia Vol 1.").
4. 4
Chronological developments in the discovery of CO
Discovery of CO as gaseous molecule-History
Scientist/s Year Finding
Tenhunen et al. 1968 HO enzyme identification and role in
endogenous CO production
Maines et al.
and Yoshida et
al.
1974 Identification of inducible HO-1
Maines et al.
and Trakshel et
al.
1986 Identification of constitutive HO-2
rat liver microsomes
McCoubrey et
al.
1997 Identification of HO isoform, HO-3
5. 5
Endogenous production
Current Medicinal Chemistry, 2007, 14, 2720-2725
Introduction
byproduct
(1)HO-1- inducible and Expressed in the heart, blood
vessels, vascular endothelium, and smooth muscles.
(2)HO-2 –constitutive and Expressed in brain tissues
HO-3, similar to HO-2 but less efficient heme catalyst
(Maines MD, 1989, Durante W, 2002, Perrella MA and Yet SF, 2003).
6. 6
Introduction
Daily production of CO in the human body nearly
20-μM/hour
The predominant biological source of CO
(1) degradation of heme (> 86%) by the HO
(2) Other source- photo-oxidation, lipid peroxidation, and
xenobiotic metabolism.
(Coburn RF et al., 1965, Sjorstrand T, 1949).
7. 7
Toxic effects of CO
CO binds to Hb and prevents oxygen from binding.
Initial symptoms-dizziness, shortness of breath and
headache.
20% COHb- Symptoms of CO poisoning begins
23% COHb- lead to loss of consciousness
50-80 % COHb- death occurs
(Ryter SW & Otterbein LE, 2004, Mannaioni PF et al., 2006, Kondo A et al., 2007, Ekblom B & Huot R, 1972)
8. 8
Targets of CO toxicity
In addition to Hb,
myoglobin (Volpe JA et al., 1975),
sGC (Furchgott RF and Jothianandan D 1991),
iNOS (Stevenson TH et al., 2001),
cytochrome P-450 and cytochrome-c oxidase (Keilin D and Hartree EF,
1939, Guengerich FP, 1975),
NADPH:oxidase (Cross AR et al., 1982),
HO (Migita CT et al., 1998)
9. 9
CO gas-no control on release pattern & concentration
Tricarbonyldichlororuthenium (II) dimmer - CO-releasing
molecule-2 (CORM-2), a lipid soluble molecule -able to
deliver CO in a controlled manner and simulate the
cytoprotective action of HO-1 derived CO.
Tricarbonyldichloro (glycinato) ruthenium (II) (CORM-3), a
water-soluble form -demonstrated protection against cardiac
I/R injury.
(Józkowicz et al., 2003; Choi et al., 2003, Motterlini et al., 2002; Clark et al., 2003; Guo et al., 2004; Stein et al., 2005;
Fujimoto et al., 2004; Lavitrano et al., 2004; Akamatsu et al., 2004).
CO and CORMs
11. 11
Name, Formula Peak COHb (%) Pharmacological
properties
CORM-2,
[Ru(CO)3Cl2]2
At therapeutic doses
there is no elevation
of basal COHb levels
after i.p. or i.v.
administration
Half-life of 1 min for
transfer of CO to Hb in
vitro
hemolytic at more than
1 mg/ml
CORM-3,
[Ru(CO)3Cl
(glycinato)]
- do- Half-life of 1 min for
transfer of CO to Hb in
vitro
Hemolytic at more than
0.25 mg/ml
Characteristics of CORMs
12. 12
CO and CORMs in various pathophysiological conditions
Potential targets Bioactivity of CO
Activation of sGC,
stimulating calcium-
activated potassium
channel, modulatory role of
NO
Vascular effect
Inhibition of TNF-alpha ,
IL-1 beta and NF-kB
Anti-inflammatory effect
↑ cGMP Inhibition of platelet
aggregation
13. 13
CO and CORMs in various pathophysiological conditions
(Motterlini R et al., Curr Pharm Des, 2003.)
Potential targets Bioactivity of CO
Activation of KATP channel and
p38MAPK
Cardioprotective effect
Up-regulation of HO-1 Organ transplantation
Decreased production of ROS Inhibition of smooth muscle
cell proliferation
Inhibition of mitochondrial
cytochrome c release and the
suppression of p53
Expression, Up-regulation of
HO-1
Antiapoptotic and
cytoprotective effects
14. 14
CO and myocardial ischemia-reperfusion injury
CORM-2 & 3-protected cardiac cells & isolated rat hearts
against I/R injury-activation of mitoKATP channel
Exogenous CO limited I/R injury in vivo in a mouse model of
MI- activation of p38 MAPK
Hearts from HO-1(-/-) mice- greater susceptibility to I/R injury
& overexpression of HO-1 attenuated I/R injury
(Clark JE et al., 2003), Guo Y et al., 2004, Yoshida T et al., 2001,Yet SF et al., 2001, Bak I et al., 2005).
15. 15
(i) To find out the concentration of CORM-2 required for the
marked cardioporotection in isolated rat heart.
CO as Cardioprotective agent
Low concentrations of CO (0.001–0.01%) improves
post-ischemic recovery and reduces infarct size,
High concentration of CO (0.1%)-severe ventricular
fibrillation.
Cardioprotection by CO may be highly restricted to
concentration used
. (Bak I et al.,Cell Mol Biol. 2005; 51: 453–9)
16. 16
Effect of CO and NO occurs at different levels - may be
synergistic or antagonistic-depending on concentration and
tissue type
CO – stimulates sGC when NO concentration is low
CO inhibits sGC activity when NO concentration is high
(ii) To evaluate the effect of CORM-2 in presence of L-
NAME, a NOS inhibitor, in I/R induced myocardial
injury in isolated rat heart.
(Kajimura M et al., FASEB J. 2003;17(3):506-8)
CO as Cardioprotective agent
17. 17
(iii) To observe the effect of CORM-2 in presence and
absence of coronary endothelium and its underlying
mechanism.
Exogenous CO produced an endothelial
dependent/independent vasorelaxant response in isolated
vascular smooth muscle preparations
CO as Cardioprotective agent
18. 18
(iv)To investigate the Role of KATP channels in
cardioprotective effects of CORM-2 in presence and absence
of coronary endothelium
In I/R injury, mitochondrial KATP channel was believed to be
major end effectors of preconditioning
(Garlid et al., 1997; Liu et al., 1998; Quayle and Standen, 1994; Taggart and Wray, 1998).
CO as Cardioprotective agent
20. Cardiac Parameters
Coronary endothelium disrupted - a single bolus injection
(0.2 ml, 20s) of triton X-100 (0.05% in K-H solution) in the
aortic cannula . (Stangl V et al., J Am Coll Cardiol. 1997;29(6):1390-6)
20
Heart Rate
Coronary Flow
Cardiac
Parameters
Myocardial injury
Markers
CK, LDH
Heart Rate
Coronary Flow
Cardiodynamics
LVEDP, LVDP,
dp/dt max, dp/dt
min (Biopac
MP100, USA)
Infarct Size
(TTC staining)
Cardiac
Parameters
21. 21
-30 -10 0 30
120
Global
Ischemia Reperfusion
Treatment
Time
(Minutes)
Pretreatment Duration
DMSO (0.02%) 10 min
i CORM-2 (RuCl3) 10 min
CORM-2 (10 µM) 10 min
CORM-2 (30 µM) 10 min
CORM-2 (50 µM) 10 min
CORM-2 (100 µM) 10 min
L-NAME (100 µM) 15 min (Before 5 min of iCORM-2 and
CORM-2 (50 µM))
Stabilization
0
Experimental protocol- Langendorff’s rat Heart Preparation
25. 25@p<0.05, #p<0.01, *p<0.001 Vs vehicle control.
Representative plot of LVDP and dp/dt in ischemic heart using I/R
injury model in rat.
No recovery after global ischemia
Results
26. 26
30 and 50 µM. CORM-2 improved cardiodynamics
Similar effect of CORM-2 in presence of L-NAME
@p<0.05, #p<0.01, *p<0.001 Vs vehicle control.
Representative plot of LVDP and dp/dt in CORM-2-treated heart
(Showing cardioprotection) using I/R injury model in rat.
Recovery after global ischemia in CORM-2 treated hearts
Results
27. 27
CORM-2 maximum cardioprotection at 50 μM in I/R induced isolated
rat heart.
50µM concentration was chosen for the subsequent experiments.
Concentration dependent
NO-independent effect
@p<0.05, #p<0.01, *p<0.001 Vs vehicle control.
0
5
10
15
20
25
30
35
40
45
50
55
60
65
Vehicle control
i CORM-2
CORM-2 (10 µM)
CORM-2 (50 µM)
CORM-2 (100 µM)
CORM-2 (30 µM)
L-NAME(100 µM) + iCORM-2
#
#
#
L-NAME(100 M) +CORM-2 (50 µM)
%InfarctSize
0
5
10
15
20
25
30
35
40
45
50
55
60
65
Vehicle control
i CORM-2
CORM-2 (10 µM)
CORM-2 (50 µM)
CORM-2 (100 µM)
CORM-2 (30 µM)
L-NAME(100 µM) + iCORM-2
#
#
#
L-NAME(100 M) +CORM-2 (50 µM)
%InfarctSize
Results
28. 28
CO from CORM-2
Intact Coronary Endothelium
VSMC
L-Arginine NO
NOS
sGC
GTP cGMP
Disrupted Coronary Endothelium
L-NAME
Cardioprotection by CORM-2
(-)
??
(1)Concentration dependent (2)NO-independent
???KATPChannels (I/R)
Disrupted Coronary Endothelium
Schematic overview
29. 29
-35 -10 0 30 120
Global
Ischemia Reperfusion
-15
Time
(Minutes)
Stabilization
Pretreatment (A) Time (Min) A Pretreatment (B) Time (Min) B
- - DMSO (0.02%) -10 to 0
Triton X-100 (0.05%) -15 (for 20 sec) DMSO (0.02%) -10 to 0
Triton X-100 (0.05%) -15 (for 20 sec) i CORM-2 (RuCl3) -10 to 0
Triton X-100 (0.05%) -15 (for 20 sec) CORM-2 (50 µM) -10 to 0
- - CORM-2 (50 µM) -10 to 0
Triton X-100 (0.05%)+
Glibenclamide (10 µM)
-20 to 0 DMSO (0.02%) -10 to 0
Triton X-100 (0.05%)+
Glibenclamide (10 µM)
-20 to 0 i CORM-2 (RuCl3) -10 to 0
Triton X-100 (0.05%)+
Glibenclamide (10 µM)
-20 to 0 CORM-2 (50 µM) -10 to 0
A B
Experimental protocol- Langendorff’s rat Heart Preparation
30. 30
Basal Bradykinin (1 µM) SNP (100 µM)
0.0
2.5
5.0
7.5
10.0
12.5
Before Triton
After Triton
*
@
Coronaryflow(ml/min)
Endothelium disruption was
confirmed by comparing
coronary flow changes using
endothelium-dependent
vasodilator bradykinin (1μM,
n=4) before triton X-100
with its effects after
treatment.
Coronary flow change to
endothelium-independent
vasodilator sodium
nitroprusside (100 μM, n=4)
was unaltered in the isolated
hearts.
@p<0.05, #p<0.01, *p<0.001 Vs vehicle control.
Endothelium disruption was also
confirmed by histopathology .
Results
35. 35
KATP Channel
CO from CORM-2
Intact Endothelium
VSMC
L-Arginine NO
NOS
sGC
GTP cGMP
Disrupted Endothelium Disrupted Endothelium
L-NAME
Cardioprotection by CORM-2
(-)
Glibenclamide
(-)
Taken together
(1)Concentration dependent (2)NO-independent
(3) KATP channel activation on VSMC
37. 37
CORMs
Role of PKC isoforms,
PI3K, p38 MAPK
isoforms etc. in IR injury
needs to be explored
????????
KATP channel activation
Endothelium & NO-independent
CARDIOPROTECTION
Protecting the
heart during
I/R injury
Role of other kinases
38. Accumulating evidence suggests that p38 mitogen-activated protein
kinase (p38MAPK) activation is essentially involved in the
cytoprotective, anti-inflammatory, antiapoptotic, and anti-proliferative
effects of CO.
Proc Natl Acad Sci USA 2005; 102: 11319–11324.
Am J Physiol Lung Cell Mol Physiol 2000; 279: L1029–L1037.
Kim HP et al. (2005) suggested a critical role for the β-isoform of p38
MAPK in mediating the effects of CO on cytoprotection and Hsp70
expression because these effects were abrogated in endothelial cells by
SB 203580, a selective inhibitor of α and β isoforms, and in p38β-null
fibroblasts.
Proc Natl Acad Sci USA 2005; 102: 11319–11324.
There is little information regarding the ability of HO-1 and CO to
modulate the protein kinase C (PKC) and phosphatidylinositol 3-kinase
(PI3K) pathways.
38
Mechanism(s) of CO and cardioprotection
39. We used transition metal carbonyl compound CORM-2 that can act as
CO donor in cardiac ischemia-reperfusion injury model using isolated
rat heart preparation.
SCIO-469 (selective potent p38 MAPK alpha inhibitor)
SB-203580 (selective p38 MAPK alpha and beta dual inhibitor)
Chelerythrine (PKC inhibitor)
Wortmannin (PI3K inhibitor)
39
Role of various kinases in CORM-2-mediated cardioprotection
40. 40
Experimental protocol- Langendorff’s rat Heart Preparation
-30 -10 0 30
120
Global
Ischemia Reperfusion
Treatment
Time
(Minutes)
Pretreatment Duration
DMSO (0.02%) 10 min
i CORM-2 (RuCl3) 10 min
CORM-2 (50 µM) 10 min
SCIO-469 (1 µM) 15 min before CORM-2 (50 µM)
SB-203580 (10 µM) 15 min before CORM-2 (50 µM)
Chelerythrine (10 µM) 15 min before CORM-2 (50 µM)
Wortmannin (100 nM) 15 min before CORM-2 (50 µM)
Wortmannin (100 nM) 15 min before CORM-2 (50 µM) and continued till
reperfusion
Stabilization
0
47. 0
10
20
30
40
50
60
Vehicle Control
Vehicle Control + i CORM-2
Vehicle Control +CORM-2 (50µM)
Chelerythrine (10M)+ Vehicle
Chelerythrine (10M)+ iCORM-2
Chelerythrine (10M)+ CORM-2 (50µM)
a
b
%infarctsize
Vehicle control CORM-2 (50 µM) Chelerythrine (10 µM) +
CORM-2 (50 µM)
Vehicle control CORM-2 (50 µM) Chelerythrine (10 µM) +
CORM-2 (50 µM)
Results
47
PKC activation is also
important for CORM-2-
mediated cardio-Protective
effect
48. Results
48
Wortmannin (before ischemia)+CORM-2- Similar cardio-protection as CORM-2 alone.
Wortmannin (before ischemia and during Reperfusion) +CORM-2- Abolish the
improvement in cardiac parameters
49. 0
10
20
30
40
50
60
Vehicle Control
Vehicle Control + i CORM-2
Vehicle Control +CORM-2 (50µM)
Wortmannin (100nM)+ Vehicle
Wortmannin (100nM)+ iCORM-2
Wortmannin (100nM)+ CORM-2 (50µM)
iCORM-2+Wortmannin (100nM)
CORM-2 (50 M) + Wortmannin (100nM)
Vehicle+Wortmannin (100nM)
Wortmannin Preischemic Wortmannin till reperfusion
a
a
b
%infarctsize
Vehicle control CORM-2 (50 µM) Wortmannin (100 nM) + CORM-2 (50 µM) +
CORM-2 (50 µM) Wortmannin (100 nM)
Wortmannin Preischemic Wortmannin till reperfusion
Vehicle control CORM-2 (50 µM) Wortmannin (100 nM) + CORM-2 (50 µM) +
CORM-2 (50 µM) Wortmannin (100 nM)
Wortmannin Preischemic Wortmannin till reperfusion
Results
49
Activation of PI3K during
reperfusion- Responsible for
CORM-2-induced cardio-
protection
50. Langendorff’srat heart
CORM-2 pretreatment (50 µM, 10 minutes)
Activation of
KATP Channel
before ischemia
Activation of
Beta isoform of
P38 MAPK before
ischemia
Activation of
PKC before
ischemia
Activation of
PI3K during
reperfusion
Cardioprotection
-
Glibenclamide
(KATP channel
inhibitor)
-
SCIO-469 (p 38 MAPK
alpha inhibitor)
SB203580 (p 38 MAPK
alpha and beta inhibitor)
- -
Chelerythrine
(PKC inhibitor)
Wortmannin
(PI3K inhibitor)
50
Schematic overview
52. A number of studies using HO-1 transgenic mice are supportive of HO-1–mediated
cardioprotection. Cardiac specific overexpression of human HO-1 protected against
cardiac I/R injury in the transgenic mice, as well as improved functional recovery
after reperfusion, and limited cardiomyocyte apoptosis.
Circ Res 2001;89:168–173.
Am J Physiol Heart Circ Physiol 2002;283:H688–H694.
The underlying mechanism of DXR-induced cardiotoxicity is unclear but numerous
reports suggested that DXR causes cardiomyocyte apoptosis and also damage from
ROS is likely to be the primal cause for DXR-mediated cardiotoxicity.
Cardiovasc Res 43:398–407.
Circ Res 84:257–265
Toxicol. Appl. Pharmacol. 194, 180–188.
We studied,
CORM-2 prevents doxorubicin-induced apoptosis in cardiomyocyte in vitro
52
CORM has anti-apoptotic potential
53. 53
Methodology- in vitro study
The rat embryonic ventricular myocardial cell line,
H9c2
cultured in DMEM
supplemented with 10% fetal bovine serum, 100 U/ml
of penicillin, 100 μg/ml of streptomycin and L-glutamine
(2mM)
The cells were grown in 25-cm2 flask at 37 C in 5%
CO2
subcultured once they reached 70–80% confluence
Cells used were between passages 7 and 14 and the
medium was changed every 2-3 days. All experiments
were performed in triplicate.
54. 400 200 100 50 25 10 5 2 0 0
0
10
20
30
40
50
60
70
80
90
100
110
i CORM-2 Vehicle
control
Concentration of CORM-2 (µM)
CellViability(%)
25 10 5 2.5 1.25 0.625 0 25 10 5 2.5 1.25 0.625 0
0
10
20
30
40
50
60
70
80
90
100
110
120
24 hour exposure time 48 hour exposure time
Concentration of doxorubicin (µM)
CellViability(%)
Results
54
Optimization of concentration of CORM-2 and DXR using MTT assay
1 Hour incubation
55. 0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
i CORM-2 (RuCl3)
CORM-2 (50 µM) 1 hour pre-treatment
CORM-2 (50 µM) 1 hour pre-treatment+
DXR (2.5 µM) 48 hour exposure
Vehicle control (0.1 % DMSO)
DXR (2.5 µM) 48 hour exposure
*
@
CellViability(%)
0
1
2
3
4
5
6
7
8
i CORM-2 (RuCl3)
CORM-2 (50 µM) 1 hour pre-treatment
CORM-2 (50 µM) 1 hour pre-treatment+
DXR (2.5 µM) 48 hour exposure
Vehicle control (0.1 % DMSO)
DXR (2.5 µM) 48 hour exposure
*
*
LDH(Foldchange)
Results
55
CORM-2 followed by DXR
treatment showed increase in
cell viability and decrease in
LDH levels in the culture
medium as compared to DXR
treated cells
56. Typical apoptotic nuclear morphologic changes were induced by DXR in H9C2 cells. Cells were pre-treated with i
CORM-2 (1 hour exposure), CORM-2 (50 µM, 1 hour exposure), doxorubicin (2.5 µM, 48 hour exposure) and CORM-
2 (50 µM, 1 hour exposure) plus doxorubicin (2.5 µM, 48 hour exposure). Cells were stained with Hoechst 33342 to
examine nuclear morphology. DXR alone induced morphological changes characteristic of apoptosis (chromatin
condensation and nuclear shrinkage) as indicated by the circle.
Results
56
CORM-2 (50µM) 1 hour preincubationRuCl3 (i CORM-2) 1 hour preincubationVehicle control (0.1% DMSO)
CORM-2 (50µM) 1 hour preincubation+ Doxorubicin 2.5
µM 48 hour exposureDoxorubicin 2.5 µM 48 hour exposure
CORM-2 (50µM) 1 hour preincubationRuCl3 (i CORM-2) 1 hour preincubationVehicle control (0.1% DMSO)
CORM-2 (50µM) 1 hour preincubation+ Doxorubicin 2.5
µM 48 hour exposureDoxorubicin 2.5 µM 48 hour exposure
57. 1 2 3 4 5 6 71 2 3 4 5 6 7
Results
57
Lane 1: molecular weight marker,
Lane 2: untreated,
Lane 3: Vehicle treated,
Lane 4: i CORM treated,
Lane 5: CORM-2 (50 µM, 1 hour)
treated,
Lane 6: DXR (2.5 µM, 48 hour)
treated and
Lane 7: CORM-2 (50 µM, 1 hour) +
DXR (2.5 µM, 48 hour).
DNA laddering using H9c2 cells
58. Results
58
Caspase-3 in cell lysate
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
i CORM-2 (RuCl3)
CORM-2 (50 µM) 1 hour pre-treatment
CORM-2 (50 µM) 1 hour pre-treatment+
DXR (2.5 µM) 48 hour exposure
Vehicle control (0.1 % DMSO)
DXR (2.5 µM) 48 hour exposure
*
*
Foldchangeincaspase-3activity
CORM-2 followed by
DXR treatment showed
significant decrease in
caspase-3 levels as
compared to DXR
treated cells
60. HO-1 participate more directly in protecting cells against oxidative and
nitrosative stress.
CORM-2 has been shown to reduce the production of ROS and nitric
oxide (NO) derived from up-regulation of inducible-nitric oxide
synthase (iNOS) in RAW 264.7 macrophages stimulated with
lipopolisaccharide (LPS) .
Biochem. Pharmacol. 71, 307-318.
Both CORM-2 and CORM-3 are able to reduce NO production in
different cellular systems such as microglial cells and murine
macrophages without affecting iNOS expression.
Br. J. Pharmacol. 145, 800-810.
60
Role of CORM in oxidative stress and apoptosis
61. Groups
(1) Vehicle Control (0.5% DMSO, 10 ml/kg, i.p.) + Saline
(10 ml/kg, i.p.)
(2) CORM-2 (30 mg/kg, i.p.) + Saline (10 ml/kg, i.p.)
(3) Vehicle Control (0.5% DMSO, 10 ml/kg, i.p.) + DXR
(20 mg/kg, i.p.)
(4) i CORM-2 (RuCl3, 30 mg/kg, i.p.) + DXR (20 mg/kg,
i.p.)
(5) CORM-2 (3 mg/kg, i.p.) + DXR (20 mg/kg, i.p.)
(6) CORM-2 (10 mg/kg, i.p.) + DXR (20 mg/kg, i.p.)
(7) CORM-2 (30 mg/kg, i.p.) + DXR (20 mg/kg, i.p.)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Day 10 Day 11
Single bolus injection of
Doxorubicin (DXR) (20 mg/kg, i.p.)
Study
Termination
Parameters analyzed on day 11
•Body weight
• Heart weight
• Biochemistry
CK
LDH
AST
ALT
TAS (Heart tissue)
MDA (Heart tissue)
• Haematology
RBC
HB
Reticulocyte
Haematocrit
• Histopathology of heart (H & E staining) n=4
• DNA isolation from heart for DNA ladder n=4
• RNA isolation & gene expression by RT-PCR n=4
HO-1 (Taqman probe)
iNOS (SYBR green method)
HIF-1 alpha (SYBR green method)
VEGF (SYBR green method)
n=8
n
=
12
61
Study design
Balb/c mice
62. Effects of single dose of doxorubicin (5,10,15 and 20 mg/kg, i.p.) on CK and mortality
after 72 hours of administration (n=5)
Vehicle DXR (5
mg/kg, i.p.)
DXR (10
mg/kg, i.p.)
DXR (15
mg/kg, i.p.)
DXR (20 mg/kg,
i.p.)
CK
(IU/L)
346±42.5 555±78.9 668±88.5 793±81.3 1327±234.9
Mortality 0 0 0 0 1/5
Effects of different doses of CORM-2 (3, 10, 30, 50 and 100 mg/kg, i.p.) once daily for 10
days on COHb and mortality (n=5)
Vehicle i CORM-
2 (100
mg/kg,
i.p.)
CORM-2
(3 mg/kg,
i.p.)
CORM-2
(10
mg/kg,
i.p.)
CORM-2
(30
mg/kg,
i.p.)
CORM-2
(50
mg/kg,
i.p.)
CORM-2
(100
mg/kg,
i.p.)
COHb(%) 0.23±0.56 0.26±0.37 0.46±0.53 0.69±0.68 0.82±0.71 1.72±0.69 2.15±1.13
Mortality 0 0 0 0 0 1/5 3/5
Results
62
Pilot experiment
63. 0
10
20
30
40
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
Bodyweight(g)
0
25
50
75
100
125
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
Heartweight(mg)
Results
63
DXR- decrease body weight and heart weight
CORM-2 (30 mpk, i.p.)+DXR- Normalize the body weight and
heart weight
64. 0
500
1000
1500
2000
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
SerumCKlevels(IU/L)
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
SerumLDHlevels(IU/L)
Results
64
DXR- increase in myocardial injury markers, CK and LDH
CORM-2 (30 mpk, i.p.)+DXR- Significantly decrease in myocardial
injury marker s
65. 0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
Totalantioxidantstatus(nmol/mgofprotein)
0
1
2
3
4
5
6
7
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
b
MDA(mmol/mgofprotein)
Results
65
DXR- decrease TAS and increase in MDA in Mouse heart tissue
homogenate
CORM-2 (30 mpk, i.p.)+DXR- Improvement in TAS and MDA w.r.t.
DXR.
68. 0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
Veh Con
CORM-2 (30 mpk i.p.)
Veh + DXR (20 mpk i.p.)
i CORM-2 (30 mpk i.p.) + DXR
CORM-2 ( 3 mpk i.p.) + DXR
CORM-2 ( 10 mpk i.p.) + DXR
CORM-2 ( 30 mpk i.p.) + DXR
a
b
Foldchangeincaspase-3activity
Results
68
DXR- increase
caspase-3,
apoptosis marker, in
Mouse heart tissue
homogenate
CORM-2 (30 mpk,
i.p.)+DXR-
significantly
decrease in caspase-
3 levels w.r.t. DXR.
Antiapoptotic potential in in vivo
69. 8 1 2 3 4 5 6 7
Representative DNA ladder. Effect of CORM-2 (3,10 and 30 mg/kg i.p.) pre- and concurrent treatment on the qualitative analysis
of apoptosis using DNA ladder (hallmark of apoptosis) induced by DXR (20 mg/kg, i.p., single dose) in genomic DNA isolated
from the heart of BalbC mice. DNA extracted from heart tissues then subjected for DNA laddeing by gel electrophoresis.
Results
69
Lane 1:DNA from heart tissue of vehicle control
group,
Lane 2: CORM-2 (30 mpk i.p.) treated group,
Lane 3:vehicle control+DXR group
Lane 4: iCORM-2 (30 mpk i.p.)+DXR treated group,
Lane 5: CORM-2 (3 mpk i.p.)+DXR,
Lane 6: CORM-2 (10 mpk i.p.)+ DXR ,
Lane 7: CORM-2 (30 mpk i.p.)+DXR,
Lane 8:low range molecular weight marker (1000 bp-
MBI, Fermentas).
Ladder with 1.8 % agarose gel
70. Results
70
H & E stained representative sections of heart tissue
showing changes in myocardium from various group.
(A)and (B) Shows normal appearance of myocardium of
BalbC mice ( A&B, 400 X).
It represents loss of striation, myocardial necrosis with
inflammatory cellular infiltration and fibrosis (C) and
vacuolar changes (thin arrow) in myocardium (D).
Shows similar findings as (C & D). (E & F, 400 X).
(G) Showing minimal cellular infiltration and single cell
necrosis than DXR treated mice
while H represents findings similar to vehicle control
groups (B) (G & H, 400 X)
Vehicle Control (0.5% DMSO, 10
ml/kg, i.p.) + Saline (10 ml/kg, i.p.)
CORM-2 (30 mg/kg, i.p.) + Saline
(10 ml/kg, i.p.)
Vehicle Control (0.5% DMSO, 10
ml/kg, i.p.) + DXR (20 mg/kg).
Vehicle Control (0.5% DMSO, 10
ml/kg, i.p.) + DXR (20 mg/kg).
iCORM-2 (RuCl3, 30 mg/kg,
i.p.) + DXR (20 mg/kg, i.p.)
CORM-2 (3 mg/kg, i.p.) +
DXR (20 mg/kg, i.p.)
CORM-2 (10 mg/kg, i.p.) +
DXR (20 mg/kg, i.p.)
CORM-2 (30 mg/kg, i.p.) +
DXR (20 mg/kg, i.p.)
74. 74
Reports on CO and thrombosis
CO inhibits platelet aggregation by stimulating the
activation of sGC . (Brune B and Ullrich V, 1987; Wagner CT et al., 1997).
CO inhibits platelet aggregation and thrombosis following
organ transplantation. (Sato K et al., 2001; Peng L et al., 2004).
Inhalation of CO prevents microvascular thrombosis and
the accumulation of fibrin. (Fujita T et al., 2001).
75. 75
Reports on CO and thrombosis
Recently,
Absence of HO-1 in aortic allograft recipient mice resulted
in 100% mortality within 4 days due to arterial thrombosis.
In contrast, recipient mice normally expressing HO-1
showed 100% graft patency and survival.
Treatment of recipients with CORM-2 (10 mg/kg i.v) after
transplantation, significantly improved survival compared
with HO-1 -/- recipients treated with inactive CORM-2.
(Chen B et al., 2009).
76. 76
In vitro antiplatelet effects of CORM-3 on
washed platelets in thrombin-induced platelet
aggregation
Ex vivo antiplatelet effects of CORM-3 and its
mechanisms using washed platelets in
thrombin-induced platelet aggregation
Models used to study effect of CORM on Thrombosis
In vivo models of thrombosis
77. 77
In vivo
models
of thrombosis
in rat
FeCl3-induced
arterial thrombosis
(Soni H et al., 2008)
Thrombus assessment
in rat Arterio-Venous
(AV) shunt model
(Wong PC et al., 1996)
Partial stasis & FeCl3
-induced venous
thrombosis model
. (Peternel L et al., 2005)
Tail-vein bleeding
time (TVBT) (Dejana E
et al., 1979)
In vivo models of thrombosis
78. 78
Carotid artery
2X3 mm strip saturated with
35%FeCl3
Thermocouple for temperature
measurement
Carotid artery
2X3 mm strip saturated with
35%FeCl3
Thermocouple for temperature
measurement
To Jugular Vein
From Carotid
12.5 cm tubing12.5 cm tubing
6 cm tubing
Silk thread 4.0
To Jugular Vein
From Carotid
To Jugular Vein
From Carotid
To Jugular Vein
From Carotid
12.5 cm tubing12.5 cm tubing
6 cm tubing
Silk thread 4.0
In vivo models of thrombosis
arterial
thrombosis
Venous
thrombosis
A V Shunt
80. 80
Groups for ex vivo and in vivo experiments
Group 1: Vehicle treated (0.5 ml/kg/min i.v. infusion for 10 minutes, 0.5% DMSO +
Saline).
Group 2: i CORM-3 (3 mg/kg/min i.v.) for 10 minutes.
Group 3: CORM-3 (3 mg/kg/min i.v.) for 10 minutes.
Group 4: ODQ (10 mg/kg i.p.) before 30 min.+ CORM-3 (3 mg/kg/min i.v.) for 10
minutes.
Group 5: L-NAME (30 mg/kg i.p.) before 30 min.+ CORM-3 (3 mg/kg/min i.v.) for
10 minutes.
Group 6: Glibenclamide (10 mg/kg i.p.) before 30 min. + CORM-3 (3 mg/kg/min
i.v.) for 10 minutes.
Group 7: SCIO-469 (1 mg/kg i.p.) before 30 min. + CORM-3 (3 mg/kg/min i.v.) for
10 minutes. (SCIO-469- p38 MAPK inhibitor)
81. 81
0
10
20
30
40
50
60
70
Vehicle treated (0.5 ml/kg/min. i.v.)
i CORM-3 (3 mg/kg/min i.v.)
ODQ (10 mg/kg i.p.)+CORM-3 (3 mg/kg, i.v.)
L-NAME (30 mg/kg i.p.)+CORM-3 (3 mg/kg, i.v.)
CORM-3 (3 mg/kg/min i.v.)
SCIO-469 (1 mg/kg i.p.)+CORM-3 (3 mg/kg, i.v.)
Glibenclamide (10 mg/kg i.p.)+CORM-3 (3 mg/kg/min i.v.)
*
@
* *
*
Clopidogrel (30 mg/kg p.o.)
%plateletaggregation
Ex vivo
Antiplatelet effect of CORM-3 is sGC and NO-dependent
Not dependent on KATP channel and p 38 MAPK
Results
90. CORMs
Activation of p38MAPK beta
before ischemia
Activation of PKC before
ischemia
Activation of PI3K during
reperfusion
KATP channel activation
Endothelium & NO-independent
caspase-3 activity in
cardiomyocyte s
HO-1 mRNA in heart tissues
iNOS mRNA in heart tissues
HIF-1 alpha mRNA in heart
tissues
VEGF mRNA in heart tissues
CO-mediated NO release in
platelets
sGC and cGMP in platelets
Inhibition of platelet PAI-1
which leads to in fibrinolytic
activity
Inhibition of PAR-1 in human
platelets
CARDIOPROTECTION
Erythropoiesis
Antiplatelet activity
Antiapoptotic
Antioxidant
Protecting
the heart
during
I/R injury
91. 91
Acknowledgements
Professor Anita Mehta (Ph.D. Guide)
Dr. M.C. Gohel (Principal, LMCP)
Dr. Mukul Jain (Zydus Research Centre)
Seniors, Colleagues, friends
My wife and my Son
Editor's Notes
HO cleaves α- meso carbon bridge of hemeyieldingequimolar amounts of biliverdin, iron, and CO.This is the first and rate-limiting step in heme catabolism and is catalyzed by two distinct isoforms of HO: (1)HO-1- ubiquitously distributed isoform that is strongly induced by biochemical and biophysical stress while (2)HO-2 is constitutively expressed and concentra
CO –invisibile-lack of odor, CO can present an especially dangerous inhalation hazard (Von Berg R., 1999). Incidence rates of accidental death by CO poisoning have been reported as high as 2,100 per year in the United States (Ball LB, 1997; Yoon SS et al., 1998). CO binds to Hb and thereby prevents oxygen from binding.Results of this are dizziness, shortness of breath and headache. All toxic symptoms cannot be explained by the formation of COHb in the blood. CO is also toxic for mitochondria in man, where it binds to cytochrome c oxidase and alters function of the respiratory electron transport chain. This effect may explain persistent symptoms after CO poisoning, when CO has been eliminated from the bloodstream (Alonso JR et al., 2003). 20% COHb- Symptoms of CO poisoning begins23% COHb- lead to loss of consciousness50-80 % COHb- death occurs(Ryter SW & Otterbein LE, 2004, Mannaioni PF et al., 2006, Kondo A et al., 2007, Ekblom B & Huot R, 1972
Delivery of CO in gas form has no control on release pattern and concentration, which may lead to undesired effects. Emerging evidence suggests that exogenously applied CO could have beneficial and therapeutic effect. (Kim et al., 2006). This is conceivably true if delivery of CO can be probably controlled to convert toxic effects in to beneficial signaling activities. Tricarbonyldichlororuthenium (II) dimmer known as CO-releasing molecule-2 (CORM-2), a lipid soluble molecule that was able to deliver CO in a controlled manner and simulate the cytoprotective action of HO-1 derived CO in biologicalSystems.(Józkowicz et al., 2003; Choi et al., 2003). Subsequently tricarbonyldichloro (glycinato) ruthenium (II) (CORM-3), a water-soluble form has been developed and demonstrated protection against cardiac I/R injury .(Motterlini et al., 2002; Clark et al., 2003; Guo et al., 2004; Stein et al., 2005; Fujimoto et al., 2004; Lavitrano et al., 2004; Akamatsu et al., 2004).
CO and CORMs in various pathophysiological conditions(2 slides):No need to give name of system. Give the mechanism for the bioactivity of CO in second column
Reports CO and myocardial ischemia-reperfusion injury:First two points need more detail mechanism.One plausible hypothesis is that CO may alleviate the ischemia-reperfusion injury by activating mitochondrialATP-sensitive potassium (mitoKATP) channels. This is supported by the finding by Clark et al. (4): that theprotective effects of CORM-3 in cardiac cells and isolated hearts were abrogated by 5-hydroxydecanoic acid. Another pathway that may potentially mediate CO donor-induced cardioprotection is the p38 MAPK signaling pathway, which has been previously implicated in the protective effects of ischemic PC (19). Activation of p38 MAPK has been shown to underlie CO-dependent alleviation of hepatic ischemia-reperfusion injury (1) and CO-dependent inhibition of apoptosis during lung ischemia-reperfusion injury (24). Further studies will be needed to address the mechanism of CORM-3-induced cardioprotection.
Low concentration of CO in perfusion buffer reduces the infarct size, improves hemodynamic and reduces ventricular fibrillation - higher concentration - severe ventricular fibrillation in isolated-perfused rat hearts. Cardioprotection by CO may be highly restricted to concentration used . (1) To find out the concentration of CORM-2 required for the marked cardioporotection in isolated rat heart.CO and NO -binds to heme proteins –gasotransmitters- many common downstream signaling pathways and functions. Functional diversity in both the molecule such as CO - stimulation of sGC when NO concentration is low where as CO inhibits sGC activity when NO concentration is high in tissue. Effect of CO and NO occurs at different levels - may be synergistic or antagonistic-depending on concentration and tissue type. (2) To evaluate the effect of CORM-2 in presence of L-NAME, a NOS inhibitor, in I/R induced myocardial injury in isolated heart.Direct role of coronary endothelium in CORM-2-induced cardioprotection is still not studied using isolated heart. In isolated rabbit aorta, exogenous CO produced an endothelial-independent vasorelaxant response, albeit with a 1,000-fold less potency than NO under the same conditions. In contrast, the vasodilation elicited by CORM-3 required intact endothelium and an accessory role for endogenous NO production. (3) To observe the effect of CORM-2 in absence of coronary endothelium and its underlying mechanism.
Low concentration of CO in perfusion buffer reduces the infarct size, improves hemodynamic and reduces ventricular fibrillation - higher concentration - severe ventricular fibrillation in isolated-perfused rat hearts. Cardioprotection by CO may be highly restricted to concentration used . (1) To find out the concentration of CORM-2 required for the marked cardioporotection in isolated rat heart.CO and NO -binds to heme proteins –gasotransmitters- many common downstream signaling pathways and functions. Functional diversity in both the molecule such as CO - stimulation of sGC when NO concentration is low where as CO inhibits sGC activity when NO concentration is high in tissue. Effect of CO and NO occurs at different levels - may be synergistic or antagonistic-depending on concentration and tissue type. (2) To evaluate the effect of CORM-2 in presence of L-NAME, a NOS inhibitor, in I/R induced myocardial injury in isolated heart.Direct role of coronary endothelium in CORM-2-induced cardioprotection is still not studied using isolated heart. In isolated rabbit aorta, exogenous CO produced an endothelial-independent vasorelaxant response, albeit with a 1,000-fold less potency than NO under the same conditions. In contrast, the vasodilation elicited by CORM-3 required intact endothelium and an accessory role for endogenous NO production. (3) To observe the effect of CORM-2 in absence of coronary endothelium and its underlying mechanism.
Direct role of coronary endothelium in CORM-2-induced cardioprotection is still not studied using isolated heart. In isolated rabbit aorta, exogenous CO produced an endothelial-independent vasorelaxant response, albeit with a 1,000-fold less potency than NO under the same conditions. In contrast, the vasodilation elicited by CORM-3 required intact endothelium and an accessory role for endogenous NO production. (3) To observe the effect of CORM-2 in absence of coronary endothelium and its underlying mechanism.
The entire molecular structure of mitochondrial KATP channel has not been completely sequenced (Hanley and Daut, 2005). Therefore, the finding solely restricts involvement of KATP channel in the mechanism of I/R injury and preconditioning, certain pharmacological inhibitors of these channels can abolish the preconditioning protective effect (Shojima et al., 2006; Gross and Auchampach, 1992).
Male wistar rats (250-300g)Heparinize with 500 IU heparin/rat and anaesthetizeHeart excised and placed in ice cold K-H buffer (K-H buffer (m mol/L): NaCl 118, KCl 3.2, MgSO4 1.2, NaHCO3 25, NaH2PO4 1.2, CaCl2 1.25 and glucose 11 at pH 7.4)Cannulated via aorta and perfused in Langendorff’s modePerfusate equilibrated with carbogen at 37 CGlobal Ischemia for 30 min. and reperfusion for 120 min.Fluid filled latex balloon inserted to left ventricle and connected with pressure transducer (Biopac-MP 100; Biopac, Santa Barbara, CA, USA)Balloon inflated to achieve LVEDP of about 10 mmHgLVDP, dp/dt max (indices of left ventricular contraction) and dp/dt min (indices of left ventricular relaxation) AcqKnowledge data acquisition software was used to collect and process
Assessment of myocardial injuryCK – BGI, Imm RepLDH - BGI, Imm Rep(Randox Laboratories Ltd, UK).Assessment of heart rate (HR) and coronary flow (CF)HR-ECG by means of two silver wire attached to the aorta and apex(BPL MK801, Banglore, India) CF -timed collection of coronary effluent in graduated measuring cylinder.Measurement of cardiodynamicsFluid-fillled latex balloon LVEDP,LVDP,dp/dt max (indices of left ventricular contraction) ,dp/dt min (indices of left ventricular relaxation).Measurement of infarct size1% TTC (Phosphate buffer, pH 7.4) at 37 C for 10 min., followed by fixation with 10% formal saline for 30 min. Sections were scanned and infarct size was measured by Image/J software and expressed as percentage of total area.
To observe the effect of CORM-2 in presence and absence of coronary endotheliumTo observe the Role of KATP channels in cardioprotective effects of CORM-2 in presence and absence of coronary endothelium
Experiment designed to identify the role of coronary endothelium and underlying mechanism.Triton X-100 0.05% for 20 seconds in aortic cannula.
Male wistar rats (250-300g)Heparinize with 500 IU heparin/rat and anaesthetizeHeart excised and placed in ice cold K-H buffer (K-H buffer (m mol/L): NaCl 118, KCl 3.2, MgSO4 1.2, NaHCO3 25, NaH2PO4 1.2, CaCl2 1.25 and glucose 11 at pH 7.4)Cannulated via aorta and perfused in Langendorff’s modePerfusate equilibrated with carbogen at 37 CGlobal Ischemia for 30 min. and reperfusion for 120 min.Fluid filled latex balloon inserted to left ventricle and connected with pressure transducer (Biopac-MP 100; Biopac, Santa Barbara, CA, USA)Balloon inflated to achieve LVEDP of about 10 mmHgLVDP, dp/dt max (indices of left ventricular contraction) and dp/dt min (indices of left ventricular relaxation) AcqKnowledge data acquisition software was used to collect and process
In vivo models of thrombosisFeCl3-induced arterial thrombosis model in ratsAnimals (n=10) were treated as per given protocol and then subjected to FeCl3-induced arterial thrombosis. FeCl3 -induced chemical injury was used as a model of arterial thrombosis as previously described.(Soni H et al., 2008).Thrombus assessment in rat Arterio-Venous (AV) shunt modelAnimals (n=10) were treated as per given protocol and then subjected to A-V shunt model of thrombosis as previously described (Wong PC et al., 1996)Partial stasis combined with FeCl3-induced vessel injury in venous thrombosis model using ratMale Wistar rats (n=10) were anesthetized with urethane (1.25g/kg, intraperitoneally). Animals (n=10) were treated as per given protocol and then subjected to Venous thrombosis as previously described(Peternel L et al., 2005)Tail-vein bleeding time (TVBT) in rat Bleeding time experiment was performed as described earlier.(Dejana E et al., 1979).