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Original Article

Studies on cerebroprotective potential of2,4,6-trisubstituted-1,3,5-pyrimidines in globalischemia/reperfusion induced cerebral infarctionin rats

Venkata Satyanarayana Murthy Bendi a, Akula Annapurna a,Vasudeva Rao Avupati b,*a Pharmacology Division, AU College of Pharmaceutical Sciences Andhra University, Visakhapatnam 530003,

Andhra Pradesh, Indiab Pharmaceutical Chemistry Division, AU College of Pharmaceutical Sciences Andhra University,

Visakhapatnam 530003, Andhra Pradesh, India

a r t i c l e i n f o

Article history:

Received 14 July 2013

Accepted 16 August 2013

Available online 30 October 2013

Keywords:

Ischemia/reperfusion

Pyrimidines

Cerebroprotection

Antioxidant

Anti-inflammatory

* Corresponding author. Dr. No: 49-9-48/2, LaE-mail address: vasudevaraoau@gmail.co

0974-6943/$ e see front matter Copyright ªhttp://dx.doi.org/10.1016/j.jopr.2013.08.030

a b s t r a c t

Background/objectives: Cerebral I/R injury is mainly characterized by oxidant production,

complement activation, leukocyteeendothelial cell adhesion, plateleteleukocyte aggregation,

increased microvascular permeability and decreased endothelium-dependent relaxation. I/R

injury can lead tomultiorgan dysfunction or death. In recent years, pyrimidines have received

muchattentionof researchersbecauseof theirvasodilator,anti-inflammatoryandantioxidant

properties. Studies on cerebroprotectivemechanism of pyrimidine derivatives on cerebral I/R

injury are limited. Hence it is worthwhile to study the role of pyrimidines as cerebroprotective

agents and evaluated for their possible inherent underlyingmechanisms.

Methods: Experimental cerebral infarction was produced by bilateral common carotid artery

occlusion (global cerebral ischemia) for 30 min followed by 4 h reperfusion in Wistar rats.

The oxidative and anti-inflammatory biomarkers were estimated and percentage infarc-

tion was determined.

Results and conclusions: Adosedependent cerebroprotective action of pyrimidines (AUCP1 and

AUCP2) in termsof limiting the infarct sizewasobserved in thepresent in vivomodelof cerebral

I/R inWistar rats. Theantioxidant role of pyrimidines (AUCP1andAUCP2) in cerebroprotection

wasconfirmedbymeasuringSOD,CAT,MDA, levels.MDA levelsweredecreased;SODandCAT

levels were increased by treatment with pyrimidines (AUCP1 and AUCP2). The cere-

broprotective actions of pyrimidines (AUCP1 and AUCP2) are partially attributed to their anti-

inflammatory effects against I/R injury in rats as evidenced by significant reduction in pro-

inflammatory markers MPO, TNF-a and significant increase in anti-inflammatory marker IL-

10. Pyrimidines (AUCP1 and AUCP2) evaluated in the present investigation has offered signif-

icant cerebroprotection against ischemia-reperfusion induced cerebral infarction in rats.

Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights

reserved.

lithanagar, Visakhapatnam 530016, Andhra Pradesh, India. Tel.: þ91 7893348681 (mobile).m (V.R. Avupati).2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 9 3 9e9 4 4940

1. Introduction

tetrazolium, Nicotinamide adenine dinucleotide phosphate

Cerebrovascular diseases (CD) are the third leading cause of

death and disability worldwide and in developed countries.1

The term “cerebral-ischemia” is caused by decreased perfu-

sion of the brain due to occlusion of the blood vessels sup-

plying the brain.2 Although restoration of blood flow to an

ischemic tissue is essential to prevent irreversible tissue

injury, reperfusion may result in a local and systemic in-

flammatory response that may enhance tissue injury in

excess of that produced by ischemia alone. This results in

reduced blood flow and a major decrease in the supply of

oxygen, glucose and other nutrients to the affected tissues.3

The tissue damage after reperfusion is defined as ischemia-

reperfusion (I/R) injury, which can lead to multiorgan

dysfunction or death.4e6

Recent evidence suggests that oxidative stress and

inflammation are the two important pathophysiological

mechanisms play an important role in several models of

experimentally induced I/R injury.7,8 It appears likely that

reactive oxygen and nitrogen-derived free radicals (especially

superoxide O2��, hydroxyl �OH, perhydroxyl HO2

�, hydrogen

peroxide H2O2, nitric oxide NO�, nitronium NO2� and perox-

ynitrite ONOO�) and inflammatory cells (such as the cytokines

TNF-a, the interleukins (IL) IL-1b, IL-6, IL-10, IL-20 and trans-

forming growth factor (TGF)-b, and the chemokines IL-8,

interferon inducible protein-10 (IP-10) and monocyte chemo-

attractant protein-1 (MCP-1)) abundantly produced in

ischemic tissues may make a major contribution in the pro-

gression of injury in reperfused reoxygenated tissue.9,10 Still

there are several controversies concerning the treatment for I/

R injury that is timely reperfusion of the ischemic tissue at risk

remains the keystone of clinical practice. Recombinant tissue

plasminogen activator (rt-PA) is the only US FDA (United

States Food and Drug Administration) approved treatment,

focuses on recanalization to reduce the size of ischemic

damage.11,12 So far, numerous attempts have been made to

find the best among the various therapeutic interventions

such as ischemic preconditioning, controlled reperfusion and

antioxidant, complement or neutrophil therapy.13

Therefore, it is still essential to search for new class of

neuroprotective strategies which may perhaps significantly

prevent or limit I/R injury in humans. Currently both experi-

mental and epidemiological evidences demonstrate that 2,4,6-

trisubstituted-1,3,5-pyrimidines have received much atten-

tion of researchers because of their cerebroprotective

actions.14e17 Hence in the present investigation it was pro-

posed worthwhile to study the possible inherent mechanisms

behind their cerebroprotection by targeting oxidation and

inflammation pathways in global ischemia-reperfusion

induced cerebral infarction in rats.

Fig. 1 e Chemical structures and nomenclature of

pyrimidines (AUCP1 and AUCP2).

2. Methods

2.1. Chemicals and drugs

Thiopentone sodium, 2,3,4-tetrazolium chloride, Thio-

barbituric acid, 1,1,3,3-tetraethoxy-propane, nitroblue

reduced form, 2,4,6-trisubstituted-1,3,5-pyrimidines (AUCP1

and AUCP2) were procured from Pharmaceutical Chemistry

Research Laboratories, Andhra University as gift samples

(Fig. 1).

2.2. Animals

All experimental protocols were approved by the Institutional

Animal Ethics Committee of AU College of Pharmaceutical

Sciences, Andhra University vide proposal no: (Approval No.

516/01/A/CPCSEA) under the regulation of Committee for the

Purpose of Control and Supervision of Experiments on Ani-

mals (CPCSEA), New Delhi. Adult Wistar rats weighing

250e300 g of either sex were used which were obtained from

National Institute of Nutrition, Hyderabad, Andhra Pradesh,

India. Animals were housed in groups of 6e7 in colony cages

at an ambient temperature of 25 � 2 �C and 45e55% relative

humidity with 12 h light/dark cycle. They had free access to

pellet chow (Pranav Agro Limited) and water ad libitum.

2.3. Pyrimidines treatment

As pyrimidines (AUCP1 and AUCP2) are very sparingly soluble

in aqueous solutions, to solubilize these compounds, 99%

dimethyl sulphoxide (DMSO) was used as vehicle and

different concentrations (5 mg/kg, 10 mg/kg, 20 mg/kg and

30 mg/kg) were prepared by dissolving in 50% DMSO and

administered intraperitoneally 10 min before reperfusion. At

the end of the experiment the brainwas removed and used for

quantification of infarct size using 2,3,5-triphenyltetrazolium

chloride (TTC) staining method.

2.4. Experimental protocols

2.4.1. Experimental induction of global cerebral infarctionCerebral infarction was induced by bilateral common carotid

artery (BCA) occlusion method described by Iwasaki et al.18

2.4.2. Measurement of percentage cerebral infarct volumePyrimidines (AUCP1 andAUCP2)were administered by 15 days

pre-treatment at doses of 5, 10, 20 and 30 mg/kg intraperito-

neally. Rats were randomly divided into eleven groups

(Table 1). After predetermined time point of I/R, the brains

were quickly removed and sliced into coronal sections of

2mm thickness. Each slice was immersed in a 1.0% solution of

Table 1 e Experimental design for the determination ofinfarct size.

Group(N ¼ 6)

Treatment

Group 1 Served as Sham control (without I/R)

Group 2 Rats received 0.2 mL of saline and served as ischemia-

reperfusion control (I/R control)

Group 3 Rats received 0.2 mL of 50% DMSO 10 min before

reperfusion and served as Vehicle control

Group 4 Rats received AUCP1 (5 mg/kg) 10 min before

reperfusion

Group 5 Rats received AUCP1 (10 mg/kg) 10 min before

reperfusion

Group 6 Rats received AUCP1 (20 mg/kg)10 min before

reperfusion

Group 7 Rats received AUCP1 (30 mg/kg) 10 min before

reperfusion

Group 8 Rats received AUCP2 (5 mg/kg) 10 min before

reperfusion

Group 9 Rats received AUCP2 (10 mg/kg) 10 min before

reperfusion

Group 10 Rats received AUCP2 (20 mg/kg)10 min before

reperfusion

Group 11 Rats received AUCP2 (30 mg/kg) 10 min before

reperfusion

Table 2 e Experimental design for the estimation ofbiochemical parameters.

Group(N ¼ 6)

Treatment

Group 1 Served as Sham control (without I/R)

Group 2 Rats received 0.2 mL of saline and served as ischemia-

reperfusion control (I/R control)

Group 3 Rats received 0.2 mL of 50% DMSO 10 min before

reperfusion and served as Vehicle control

Group 4 Rats received AUCP1 (20 mg/kg) 10 min before

reperfusion

Group 5 Rats received AUCP2 (20 mg/kg) 10 min before

reperfusion

Table 3 e Effect of AUCP1 and AUCP2 on percentageinfarct size in cerebral I/R in rats.

Group (N ¼ 6) Percentage of cerebral infarction

Sham control 2.53 � 0.36

I/R control 48.34 � 0.84

Vehicle control 48.32 � 0.36

AUCP1 (5 mg/kg) 33.15 � 0.85

AUCP1 (10 mg/kg) 26.56 � 0.64

AUCP1 (20 mg/kg) 20.36 � 0.72

AUCP1 (30 mg/kg) 16.68 � 0.56

AUCP2 (5 mg/kg) 31.52 � 0.65

AUCP2 (10 mg/kg) 24.81 � 0.56

AUCP2 (20 mg/kg) 17.68 � 0.61

AUCP2 (30 mg/kg) 12.32 � 0.67

P < 0.005, all values expressed in mean � SEM (n ¼ 6). Pyrimidines

(AUCP1 and AUCP2), I/R indicates ischemia-reperfusion.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 9 3 9e9 4 4 941

2,3,5-triphenyltetrazolium chloride (TTC) for 30 min. Necrotic

infarcted tissue was unstained and viable tissue was stained

dark red, further separated, weighed and percentage of

infarction was determined.19

2.4.3. Estimation of biochemical parametersThe stained tissue was not suitable for estimating oxidative

and inflammatory biomarkers; hence a separate group of an-

imals were used for estimating the levels of these biochemical

parameters (Table 2). The brain tissue of each animal was

removed after completion of 4 h reperfusion and used for the

estimation of superoxide dismutase (SOD), catalase (CAT),

myeloperoxidase (MPO), tumor necrosis factor-a (TNF-a) and

interleukin-10 (IL-10).

2.4.3.1. Estimation of SOD levels. SOD levels were determined

by the method developed by Kakar et al.20

2.4.3.2. Estimation of CAT levels. CAT levels were determined

by the method developed by Aebi et al21

2.4.3.3. Estimation of MDA levels. MDA levels were deter-

mined by the method developed by Ohkawa et al22

2.4.3.4. Estimation of MPO levels. MPO levelswere determined

by the method developed by Mullane et al23

2.4.3.5. Estimation of TNF-a levels. TNF-a levels were deter-

mined by using AssayMax Rat Tumor Necrosis Factor-alpha

(TNF-alpha) ELISA Kit (Catalog No. ERT2010-1).24

2.4.3.6. Estimation of IL-10 levels. IL-10 levels were deter-

mined by using AssayMax Rat Interleukin-10 (IL-10) ELISA Kit

(Catalog No. ERI3010-1).25

2.5. Statistical analysis

Statistical analysis was performed using Prism software

(Version 6.02).

3. Results and discussion

3.1. Effect of pyrimidines (AUCP1 and AUCP2) onpercentage cerebral infarction

Results of percentage of infarct size are shown in Table 3 and

Figs. 2 and 3. Cerebral Infarct size was found to be

48.34 � 0.84% in rats subjected to cerebral I/R injury. Signifi-

cant cerebral damage was observed in I/R control group ani-

mals when compared to sham operated group. Pyrimidines

(AUCP1 and AUCP2) treatment offered dose dependent cere-

broprotection in terms of significant reduction in cerebral

infarct size when compared to I/R control group. AUCP2 has

offered more degree of cerebroprotection when compared to

AUCP1.

3.2. Effect of pyrimidines (AUCP1 and AUCP2) on theSOD levels

Results of tissue SOD levels are shown in Table 4 and Fig. 4.

Results shown in the above mentioned figure indicate that

Fig. 2 e Effect of AUCP1 on percentage infarct size in

cerebral I/R in rats.

Fig. 3 e Effect of AUCP2 on percentage infarct size in

cerebral I/R in rats.

Fig. 4 e Effect of AUCP1 and AUCP2 on SOD (U/mg protein)

levels in infarcted tissue in cerebral I/R in rats.

j o u rn a l o f p h a rma c y r e s e a r c h 6 ( 2 0 1 3 ) 9 3 9e9 4 4942

the cerebral ischemia and reperfusion significantly

decreased antioxidant enzyme (SOD) levels in the injured

brain tissue of rats as compared with the sham control

group.

3.3. Effect of pyrimidines (AUCP1 and AUCP2) on theCAT levels

Results of tissue SOD levels are shown in Table 4 and Fig. 5.

Results shown in the abovementioned figure indicate that the

cerebral ischemia and reperfusion significantly decreased

antioxidant enzyme (CAT) levels in the injured brain tissue of

rats as compared with the sham control group.

Table 4 e Effect of AUCP1 and AUCP2 on oxidative and inflam

Biomarker Normal control Sham control I/R con

SOD (U/mg protein) 9.31 � 0.19 8.94 � 0.20 4.99 �CAT (U/mg protein) 113.50 � 1.05 105.64 � 0.88 38.71 �MDA (nmol/g wet tissue) 156.52 � 0.34 159.65 � 0.38 518.22 �MPO (U/g tissue) 3.545 � 0.19 4.653 � 0.20 64.52 �TNF-a (ng/mg of tissue) 0.11 � 0.002 0.10 � 0.006 0.28 �IL-10 (ng/mg of tissue) 1.66 � 0.08 1.58 � 0.04 0.91 �

P < 0.005, all values expressed in mean � SEM (n ¼ 6). Pyrimidines (AUC

dismutase), CAT (catalase), MDA (malondialdehyde), MPO (myeloperoxid

3.4. Effect of pyrimidines (AUCP1 and AUCP2) on theMDA levels

Results of tissue MDA levels are presented in Table 4 and

Fig. 6. Results shown in the above mentioned figure indicate

that the cerebral ischemia and reperfusion significantly

increased lipid peroxidation (MDA) levels in the injured

brain tissue of rats as compared with the sham control

group.

3.5. Effect of pyrimidines (AUCP1 and AUCP2) on theMPO levels

Results of tissueMPO levels are presented in Table 4 and Fig. 7.

In comparison with I/R control group pyrimidines (AUCP1 and

AUCP2) treatment significantly reduced the MPO levels and

thereby contributed to its anti-inflammatory activity. When

compared to AUCP1, AUCP2 exhibited more degree of

cerebroprotection.

3.6. Effect of pyrimidines (AUCP1 and AUCP2) on theTNF-a levels

Results of tissue TNF-a level are presented in Table 4 and

Fig. 8. In comparison with I/R control group pyrimidines

(AUCP1 and AUCP2) treatment significantly reduced the TNF-a

levels and thereby contributed to its anti-inflammatory ac-

tivity. When compared to AUCP1, AUCP2 exhibited more de-

gree of cerebroprotection.

matory biomarkers.

trol Vehicle control AUCP1 (20 mg/kg) AUCP2 (20 mg/kg)

0.19 4.89 � 0.37 6.74 � 0.15 8.18 � 0.31

1.80 37.65 � 0.53 66.96 � 0.94 88.02 � 1.77

1.03 513.05 � 1.90 451.31 � 3.87 365.90 � 5.24

0.30 64.57 � 0.54 11.27 � 0.32 8.708 � 0.35

0.01 0.29 � 0.02 0.09 � 0.01 0.07 � 0.009

0.03 0.72 � 0.05 1.39 � 0.05 1.44 � 0.08

P1 and AUCP2), I/R indicates ischemia-reperfusion, SOD (superoxide

ase), TNF-a (tumor necrosis factor-alpha), IL-10 (interleukin-10).

Fig. 5 e Effect of AUCP1 and AUCP2 on CAT (U/mg protein)

levels in infarcted tissue in cerebral I/R in rats.

Fig. 6 e Effect of AUCP1 and AUCP2 on MDA (nmol/g wet

tissue) levels in infarcted tissue in cerebral I/R in rats.

Fig. 8 e Effect of AUCP1 and AUCP2 on TNF-a (ng/mg of

Tissue) levels in infarcted tissue in cerebral I/R in rats.

Fig. 9 e Effect of AUCP1 and AUCP2 on IL-10 (ng/mg of

Tissue) levels in infarcted tissue in cerebral I/R in rats.

j o u r n a l o f p h a rm a c y r e s e a r c h 6 ( 2 0 1 3 ) 9 3 9e9 4 4 943

3.7. Effect of pyrimidines (AUCP1 and AUCP2) on the IL-10 levels

Results of tissue IL-10 levels are presented in Table 4 and Fig. 9.

In comparison with I/R control group pyrimidines (AUCP1 and

AUCP2) treatment significantly enhanced the IL-10 levels and

thereby contributed to its endogenous anti-inflammatory ac-

tivity. When compared to AUCP1, AUCP2 exhibited more de-

gree of cerebroprotection.

4. Conclusions

In summary, AUCP2 has offered more degree of cere-

broprotection when compared to AUCP1. The probable

Fig. 7 e Effect of AUCP1 and AUCP2 on MPO (U/g Tissue)

levels in infarcted tissue in cerebral I/R in rats.

mechanisms involved in the cerebroprotective activity of py-

rimidines (AUCP1 and AUCP2) might be due to their antioxi-

dant and anti-inflammatory properties.

Conflicts of interest

All authors have none to declare.

Acknowledgments

One of the authors (Venkata Satyanarayana Murthy Bendi) is

thankful to the Principal, Andhra University College of Phar-

maceutical Sciences, Visakhapatnam for providing required

help in carrying out the pharmacological activities.

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