Amlodipine and Carvedilol Exhibits Cardioprotective Effect on Cyclophosphomide Induced Cardiotoxicity in Rats.

Cardiotoxicity is a well-known side effect of several cytotoxic drugs, especially of the anthracyclines and can lead to long term morbidity. The mechanism of anthracycline induced cardiotoxicity seems to involve the formation of free radicals leading to oxidative stress. This may cause apoptosis of cardiac cells or immunologic reactions.CYP has been recorded to be carditoxic. Amlodipine and carvedilol has been exploited to test its cardioprotective activityvon CYP induced cardiotoxicity. Objective: the aim of this study was assessment of cardioprotective effect of Amlodipine and carvedilol on CYP induced cardiotoxicity in albino wistar rats by measuring the enzymatic, non-enzymatic antioxidant levels, serum enzyme levels and study of ECG alteration. Materials Methods: Albino wistar rats were allotted in to 4 groups (6 rats/group), normal control: (i.p. injection with normal saline), CYP group (200 mg/kg ip), Alodipine and CYP group (Amlo-10 mg/kg oral & CYP- 200 mg/kg ip), carvediol and CYP group (Carve-3mg/kg oral & CYP- 200 mg/kg ip) for 10 days of duration and ECG was measured using power lab software. Results: cardioprotects of amlodipine and carvediol signicantly reduced the elevated levels of serum biomarkers like CK, CK-MB, LDH, calcium when compare to CYP induced cardiotoxicity. Amlodipine and carvediol has shown signicant increase in the levels of tissue biomarkers such as SOD, GSH, catalase when compare to CYP induced cardiotoxicity. In Histopathological studies, the group treated with amlodipine + CYP and carvediol + CYP has shown intact arrangement of cardiac muscle bres, intact integrity of myocardial cell membrane, myobrillar structure with striations and continuity with adjacent myobrils. Considering improvement in the serum biomarker levels and tissue biomarker levels amlodipine and carvediol showed cardioprotective


Introduction
Cardiovascular disease (CVD) remains the principle cause of death in both developed and developing countries, accounting for roughly 20% of all worldwide deaths per year. CVD in India cause 3 million deaths per year, accounting for 25%of all mortality. the World Health Organization (WHO) predicts that deaths due to circulatory system disease are projected to double between 1985 and 2015. 1 Cyclophosphamide is one of the most widely used antitumor and immunosuppressant drug. Cyclophosphamide has wide spectrum of clinical uses and is an essential component of numerous combination chemotherapeutic regimens. Cyclophosphomide is activated by the cytochrome P450 oxidase system and it decomposes to phosphoramide mustard and acrolein. Phosphoramide mustard is linked to cyclophosphamide therapeutic effect while acrolein associated with the side effect. Acrolein interfere with the tissue antioxidant defense system and induces reactive oxygen species which causes cardiac injury, arrhythmias, congestive heart failure. 2 Amlodipine is a intrinsically long acting dihydropyridine calcium channel blocker. It is used for the treatment systolic hypertension and it is most commonly prescribed branded cardiovascular agent. 3 Carvediol is third-generation, nonselective β-adrenoceptor antagonist and it is a calcium channel blocker. 4

Materials And Methods
Experimental animals: Albino Wistar rats were purchased from Venkateshwara Enterprises Bengaluru. Animal procedure were performed in accordance with the declaration of   After 10 days of experimental duration at 11 th day rats were anaesthetized and ECG was recorded using power lab instrument. Blood samples from retroorbital venous plexus were collected and serum was separated for the estimation of serum biomarkers Creatinine, Creatinine Kinase, Creatinine Kinase-MB, LDH. Animals were sacri ced by cervical dislocation and heart was immediately isolated and used for Histopathological studies and also for tissue biochemical estimations SOD, LPO, catalase & GSH.
Recording of ECG. 6 At the end of experimental period animals were restrained by ketamine 30 mg/kg ip. The positive and negative electrodes were attached to the surface of left and right limbs and earth electrodes were attached to ECG was recorded the surface of right limb and surface of the posterior limb of the animal.
Biochemical analysis. 7 After recording of ECG, blood samples were collected from retro-orbital plexux, serum was separated for the estimation of enzyme marker. The activities of CK, CK-MB, LDH, calcium were measured by using standard kits.
Animals were sacri ced by injecting higher dose of thiopental sodium. The hearts were removed and washed immediately with saline and then xed in 4% paraformaldehyde in 0.1 M phosphate buffer. Then heart were then routinely embedded in para n and stained with Hematoxylin-Eosin. These sections were then examined under a light microscope for histological changes.

Statistical analysis:
Values were expressed as Mean±SEM from 6 animals. The results were subjected to statistical analysis by using one way ANOVA followed by Bartlett's test for to calculate signi cance. P<0.05 was considered as signi cant.  The data were expressed as Mean ± S.E.M for six rats in each group by One Way ANOVA followed by Bartlett's test for equal variances. The ECG parameters were expressed in seconds (sec) and the heart rate as Beats per Minute (BPM). Where *** a P<0.001; compared with normal control group. ** b P<0.01,*** b P<0.001; compared to toxic group. Normal rats showed normal ECG wave patterns whereas animals treated with Cyclophosphamide alone showed signi cant alterations in QRS complex, ST segment and RR interval.  The data were expressed as Mean ± S.E.M for six rats in each group. Statistical Comparisions were performed by One Way ANOVA followed by Bartlett's test for equal variances. Where *** a P<0.001; compared with normal control group ** b P<0.01, *** b P<0.001; compared to toxic group. The data were expressed as Mean ± S.E.M for six rats in each group. Statistical Comparisions were performed by One Way ANOVA followed by Bartlett's test for equal variances. Where *** a P<0.001; compared with normal control group ** b P<0.01, *** b P<0.001; compared to toxic group.

Discussion
Cyclophosphamide induced cardio toxicity is not yet fully unravelled. However, toxicity of CYP was postulated to be mediated by oxidative stress which may have deleterious effects on the heart. Moreover, it is thought to involve direct endothelial damage, with extravasation of plasma proteins, high concentration of cyclophosphamide and erythrocytes into the myocardial interstitium and muscle cells, resulting in damage of myocardial cells.
ECG studies showed signi cant alterations in group-II rats (Cyclophosphamide treated) rats as compared group-I (normal rats). There was a change in ECG pattern such as decrease in the heart rate, P wave inversion, prolongation of QT interval, QRS complex and decreased in R wave amplitude. CYP causes cardiac dysfunction through impaired mitochondrial metabolism. Bradycardia observed in CYP treated animals which may be due to release of signi cant amount of acetylcholine (Ach) which is also linked with the genesis of myocardial damage. CYP is known to increase the cellular Na + content and decreases in K + content. Hypokalemia may be associated with QT interval elongation 8 .
Rats treated with Amlodipine (10 mg/kg oral) + Cyclophosphamide (200 mg/kg ip) and Carvedilol (3 mg/kg oral)+ Cyclophosphamide (200 mg/kg ip) showed signi cant changes in the P wave, QRS complex, reduction in QT interval, RR interval and normal heart rate when compared to DOX group. This indicates the normal heart condition. This may support as cardio protective 9 .
In this study, a signi cant increase in the serum LDH, Calcium, Creatinine kinase, Creatinine kinase-MB was observed in CYP treated rats (Group II)