http://ehjcimaging.oxfordjournals.org/content/ejechocard/7/suppl_1/S85.4.full.pdf 526 The efficacy of Trimetazidine MR in improvement of left ventricular function and reduction of major cardiovascular events in patients with coronary heart disease E. Bobescu1; M. Radoi1; G. Datcu2; A. Burducea3; C. Strempel1 1Transilvania University Faculty of Medicine , Clinic of Cardiology Clinic County Hospital, Brasov, Romania; 2Gr T Popa University of Medicine and Pharm, Clinic of Cardiology University Hospital, Iasi, Romania; 3Clinic County Emergency Hospital, Clinic of Cardiology, Brasov, Romania Background: Energetic metabolism during myocardial ischemia and reperfusion is connected with cardiac function. T rimetazidine act as a specific partial inhibitor of fatty acid oxidation with indirect increase in glucose metabolism and many cardio protective mechanisms which may include diminished mitochondrial uncoupling, enhances efficiency of mitochondrial ATP production and reduced apoptosis. Aims: To evaluate the efficacy of treatment with trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary heart disease (CHD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular global and regional function. Methods: 252 pts with high risk CAD were included in a prospective study for a period of 24 months and divided in six groups in relation with type of CHD and addition of TMZ MR to optimal standard medical therapy (OSMT). Patients were evaluated clinical and para clinical (biochemical analyses, electrocardiography, echocardiography) at 1, 6, 12 and 24 months. Results: Treatment with TMZ MR in addition to OSMT was followed at 24 months of follow up by a significant improvement of left ventricular function in pts with NSTEMI (p<0.05) and UA(p<0.025) in comparison with OSMT pts. Primary endpoint was significantly reduced in all TMZ MR treatment added to OSMT pts at 24 months of follow up respectively: NSTEMI -20%, p<0.001, relative risk (RR) 0.35), UA 13.6%, p<0.001, RR 0.29 and SA-7.7%, p<0.001, RR 0.19. Ejection fraction of left ventricle as a measure of global function was significantly improved at 24 months of follow up in NSTEMI and UA TMZ MR added to OSMT pts, only 15% of NSTEMI pts (p<0.05) and 9,1% of UA pts (p<0.05) remain with an ejection fraction <40% in comparison with 35,7% of NSTEMI and 28,3% of UA OSMT pts. Conclusions: In patients with stable angina, unstable angina and non ST elevation myocardial infarction, treatment with trimetazidine MR in addition to optimal standard medical therapy was followed by a significant reduction in primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke and by a significant improvement in left ventricular function at 24 months of follow up.

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http://ehjcimaging.oxfordjournals.org/content/ejechocard/7/suppl_1/S85.4.full.pdf 526 The efficacy of Trimetazidine MR in improvement of left ventricular function and reduction of major cardiovascular events in patients with coronary heart disease E. Bobescu1; M. Radoi1; G. Datcu2; A. Burducea3; C. Strempel1 1Transilvania University Faculty of Medicine , Clinic of Cardiology Clinic County Hospital, Brasov, Romania; 2Gr T Popa University of Medicine and Pharm, Clinic of Cardiology University Hospital, Iasi, Romania; 3Clinic County Emergency Hospital, Clinic of Cardiology, Brasov, Romania Background: Energetic metabolism during myocardial ischemia and reperfusion is connected with cardiac function. T rimetazidine act as a specific partial inhibitor of fatty acid oxidation with indirect increase in glucose metabolism and many cardio protective mechanisms which may include diminished mitochondrial uncoupling, enhances efficiency of mitochondrial ATP production and reduced apoptosis. Aims: To evaluate the efficacy of treatment with trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary heart disease (CHD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular global and regional function. Methods: 252 pts with high risk CAD were included in a prospective study for a period of 24 months and divided in six groups in relation with type of CHD and addition of TMZ MR to optimal standard medical therapy (OSMT). Patients were evaluated clinical and para clinical (biochemical analyses, electrocardiography, echocardiography) at 1, 6, 12 and 24 months. Results: Treatment with TMZ MR in addition to OSMT was followed at 24 months of follow up by a significant improvement of left ventricular function in pts with NSTEMI (p<0.05) and UA(p<0.025) in comparison with OSMT pts. Primary endpoint was significantly reduced in all TMZ MR treatment added to OSMT pts at 24 months of follow up respectively: NSTEMI -20%, p<0.001, relative risk (RR) 0.35), UA 13.6%, p<0.001, RR 0.29 and SA-7.7%, p<0.001, RR 0.19. Ejection fraction of left ventricle as a measure of global function was significantly improved at 24 months of follow up in NSTEMI and UA TMZ MR added to OSMT pts, only 15% of NSTEMI pts (p<0.05) and 9,1% of UA pts (p<0.05) remain with an ejection fraction <40% in comparison with 35,7% of NSTEMI and 28,3% of UA OSMT pts. Conclusions: In patients with stable angina, unstable angina and non ST elevation myocardial infarction, treatment with trimetazidine MR in addition to optimal standard medical therapy was followed by a significant reduction in primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke and by a significant improvement in left ventricular function at 24 months of follow up.

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http://ehjcimaging.oxfordjournals.org/content/ejechocard/7/suppl_1/S85.4.full.pdf 526 The efficacy of Trimetazidine MR in improvement of left ventricular function and reduction of major cardiovascular events in patients with coronary heart disease E. Bobescu1; M. Radoi1; G. Datcu2; A. Burducea3; C. Strempel1 1Transilvania University Faculty of Medicine , Clinic of Cardiology Clinic County Hospital, Brasov, Romania; 2Gr T Popa University of Medicine and Pharm, Clinic of Cardiology University Hospital, Iasi, Romania; 3Clinic County Emergency Hospital, Clinic of Cardiology, Brasov, Romania Background: Energetic metabolism during myocardial ischemia and reperfusion is connected with cardiac function. T rimetazidine act as a specific partial inhibitor of fatty acid oxidation with indirect increase in glucose metabolism and many cardio protective mechanisms which may include diminished mitochondrial uncoupling, enhances efficiency of mitochondrial ATP production and reduced apoptosis. Aims: To evaluate the efficacy of treatment with trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary heart disease (CHD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular global and regional function. Methods: 252 pts with high risk CAD were included in a prospective study for a period of 24 months and divided in six groups in relation with type of CHD and addition of TMZ MR to optimal standard medical therapy (OSMT). Patients were evaluated clinical and para clinical (biochemical analyses, electrocardiography, echocardiography) at 1, 6, 12 and 24 months. Results: Treatment with TMZ MR in addition to OSMT was followed at 24 months of follow up by a significant improvement of left ventricular function in pts with NSTEMI (p<0.05) and UA(p<0.025) in comparison with OSMT pts. Primary endpoint was significantly reduced in all TMZ MR treatment added to OSMT pts at 24 months of follow up respectively: NSTEMI -20%, p<0.001, relative risk (RR) 0.35), UA 13.6%, p<0.001, RR 0.29 and SA-7.7%, p<0.001, RR 0.19. Ejection fraction of left ventricle as a measure of global function was significantly improved at 24 months of follow up in NSTEMI and UA TMZ MR added to OSMT pts, only 15% of NSTEMI pts (p<0.05) and 9,1% of UA pts (p<0.05) remain with an ejection fraction <40% in comparison with 35,7% of NSTEMI and 28,3% of UA OSMT pts. Conclusions: In patients with stable angina, unstable angina and non ST elevation myocardial infarction, treatment with trimetazidine MR in addition to optimal standard medical therapy was followed by a significant reduction in primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke and by a significant improvement in left ventricular function at 24 months of follow up.

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Revista Română de Medicină de Laborator Vol. 8, Nr. 3, Septembrie 2007

Evaluarea stresului oxidativ şi sistemelor antioxidante inbolile cardiovasculare

Oxidative stress and antioxidant systems evaluation in cardiovasculardisease

Elena Bobescu*

Universitatea „Transilvania” Braşov, Facultatea de Medicină, Catedra de medicină internă

Rezumat

Bolile cardiovasculare sunt principala cauză de mortalitate şi morbiditate în lume. Ateroscleroza la ni-vel coronarian are ca expresie clinică boala cardiacă ischemică manifestată ca angină stabilă, angină instabilă,infarct miocardic acut sau moarte subită. Aceasta impune o mai bună cunoaştere a mecanismelor moleculare şimetabolismul celular al miocardului. În condiŃiile în care patogeneza aterosclerozei este explicată prin ipotezadisfuncŃiei endoteliale datorate raspunsului complex la agresiune, rolul stresului oxidativ şi al sistemelor antio-xidante în bolile cardiovasculare reprezintă un subiect de cercetare fundamentală şi clinică. DisfuncŃia endote-lială poate fi realizată de agenŃi virali şi microbieni expunerea la toxine, nivele înalte ale metaboliŃilor sau fac-tori mecanici. Leziunile pot fi agravate de stresurile metabolice cum sunt hiperglicemia şi hipercolesterolemia şide stresul oxidativ. Factorii majori de risc cardiovascular prin intermediul mediatorilor ce apar ca răspuns laaceşti factori de risc - oxLDL, produşi de glicare avansată, angiotensina II, TNFα - stimuleză la nivelul endoteli-ului vascular sistemele enzimatice si neeenzimatice răspunzatoare de generarea speciilor rective ale oxigenului.Biomarkerii stresului oxidativ sunt biomarkeri de depleŃie antioxidantă şi markeri specifici: produşi de peroxida-re lipidică şi produşi de degradare oxidativă a proteinelor şi a ADN. Peroxidarea lipidelor este un proces com-plex prezent din faza de iniŃiere a aterosclerozei şi se desfăşoară în multiple stadii. Metodele de determinare aperoxidării lipidelor pot fii indirecte prin determinarea pierderilor de substrat sau directe prin determinarea pe-roxizilor care se pot realiza prin evaluarea totală sau parŃială. Imposibilitatea obŃinerii unei metode ideale dedeterminare a peroxidării lipidice a impus evaluarea complexă a stresului oxidativ şi perfecŃinarea metodelor dedeterminare a activităŃii antioxidante totale a plasmei şi serului uman.

Abstract

Cardiovascular disease are the major cause of mortality and morbidity in the world. Clinic expressionof atherosclerosis is coronary heart disease – stable angina, unstable angina, myocardial infarction and suddencardiac death. All these involve a great need for research in celular myocardial metabolism and molecular

*Adresa de corespondenŃă: Elena Bobescu, Spitalul Clinic JudeŃean de UrgenŃă Braşov, Clinica de Cardiologie,Str. IndependenŃei nr 1, 500157 BraşovTel.: 0268425701, Fax:0268470318, E-mail: elena_bobescu@yahoo.com

13

Revista Română de Medicină de Laborator Vol. 8, Nr. 3, Septembrie 2007

mechanisms. The role of oxidative stress in cardiovascular disease was evaluated by both experimental and clini-cal studies. In this context the most accepted in atherosclerosis is hypothesis of endothelial disfunction becauseof multiple agression factors. Endothelial disfunction could be produced by mechanic, viral or bacterial factors,toxic exposure, high level of metabolites, turbulent blood flow, all this aggresion are followed by atherosclerosis.These atherosclerotic lesions could be agravated by metabolic stress – hyperglicemia and hypercholesterolemiaand oxidative stress. Mediators produced in response to major cardiovascular risk factors including advancesglycates end-products, LDL and ox-LDL, angiotensin II and cytokines such as TNF-α stimulate generation of re-active oxigen species at endotelial level by a variety of enzymatic and non-enzymatic sources. Oxidative stressbiomarkers are biomarkers of antioxidants depletion and specific markers: lipid peroxidation products, proteinand ADN oxidative degradation products. Lipid peroxidation is a complex process, started from the begining ofatherosclerosis. Peroxide assay could be loss of substrate and total or partial peroxide measurements. Impossi-bility to obtain an ideal assay to determinate lipid peroxidation was followed by complex evaluation of oxidativestress and development of total antioxidant activity assays.

Bolile cardiovasculare sunt principalacauză de mortalitate şi morbiditate în lume.Ateroscleroza la nivel coronarian are ca expre-sie clinică boala cardiacă ischemică manifestatăca angină stabilă, angină instabilă, infarct mio-cardic acut sau moarte subită. Aceasta impuneo mai bună cunoaştere a mecanismelor molecu-lare şi metabolismului celular al miocardului acărui afectare ischemică reprezintă patologia cucel mai mare impact asupra stării de sănătate apopulaŃiei adulte şi vârstnice5.

În condiŃiile în care patogeneza ateros-clerozei este explicată prin ipoteza disfuncŃieiendoteliale datorate răspunsului complex laagresiune, rolul stresului oxidativ şi al sisteme-lor antioxidante în bolile cardiovasculare repre-zintă un subiect de cercetare fundamentală şiclinică13 . DisfuncŃia endotelială este rezultatulagresiunii prin agenŃi virali şi microbieni (Her-pes virus, Citomegalovirus, Chlamydia pneu-monie, Helicobacter pylori), expunerea la toxi-ne (fumul de Ńigară), nivele înalte ale metaboli-Ńilor ( glucoza, homocisteina), factori mecanici- fluxul turbulent de sânge la bifurcaŃia artere-lor, accentuat de valori înalte ale tensiunii arte-riale1. Leziunile pot fi agravate de stresurile me-tabolice cum sunt hiperglicemia şi hipercoleste-rolemia şi de stresul oxidativ. Factorii majori derisc cardiovascular – dislipidemia, hipertensiu-nea, diabetul zaharat, fumatul, varsta înaintată,stresul psihic şi fizic, prin intermediul mediato-

rilor ce apar ca răspuns la aceşti factori de risc –oxLDL17, produşi de glicare avansată, angioten-sina II, TNFα, - stimuleză la nivelul endoteliu-lui vascular sistemele enzimatice si neeenzima-tice răspunzatoare de generarea speciilor rectiveale oxigenului (SRO): ciclooxigenaze, lipooxi-genaze, Citocrom P450, NAD(P)H oxidaza,xantin oxidaza, lanŃul respirator la nivel mito-condrial, NO sintetaza14,15 şi epuizează sisteme-le endogene de apărare antioxidantă – enzimati-ce: superoxid-dismutaze, catalaze, glutation pe-roxidaze si neenzimatice: glutation, α-tocoferol,ascorbat. SRO devin astfel al doilea mesagertransmiŃând semnale extracelulare care module-ază expresia genelor implicate in bolile cardio-vasculare, cum sunt cele ale moleculelor deadeziune, gene proliferative, citokine, metalo-proteinaze3-7.

Generarea SRO este o consecinŃă a me-tabolismului aerob. Stresul oxidativ este definitca situaŃia în care se produce un dezechilibruîntre producŃia de specii reactive ale oxigeniluiSRO respectiv specii reactive ale azotului(SRN) şi apărarea antioxidantă. Speciile reacti-ve ale oxigenului includ radicali liberi ai oxige-nului: superoxid(O2i), hidroxil, (OHi), peroxil

(RO2i), Alkoxil (ROi), hidroxiperoxil( HO2

i)dar şi non-radicali derivaŃi din oxigen: peroxidde hidrogen (H2O2), acidul hipocloros (HOCl),ozonul (O3), oxigen singlet (‘∆g), peroxinitrit

14

J.M.B. no 3 2006

The efficacy of Trimetazidine MR in left ventricular function improvement and

reduction of major cardiovascular events, inflammatory syndromes and oxidative stress in coronary heart

disease

Elena Bobescu MD,1, Prof. Mariana Radoi MD, PhD1, Georgeta Datcu MD,3, Antoniu Burducea MD,2

1Transylvania University-Faculty of Medicine, 2Clinic County Emergency Hospital- Clinic of Cardiology,

Brasov, 3Gr.T.Popa University of Medicine and Pharmacy Iasi

Aims: To evaluate the efficacy of treatment with

trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary heart disease (CHD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular function, reduction of inflammatory syndrome and oxidative stress.

Methods and results: 252 pts with high risk CAD were included in a prospective study for a period of 24 months and divided in six groups in relation with type of CHD and addition of TMZ MR treatment to optimal standard medical therapy (OSMT). Patients were evaluated clinical and paraclinical (biochemical analyses, electrocardiography, echocardiography) at 1, 6, 12 and 24 months. Markers of inflammatory syndrome used were C-reactive protein serum level and fibrinogen plasma level. Anti ox-LDL antibody titers and total antioxidant

J.M.B. no 3 2006

The efficacy of Trimetazidine MR in left ventricular function improvement and

reduction of major cardiovascular events, inflammatory syndromes and oxidative stress in coronary heart

disease

Elena Bobescu MD,1, Prof. Mariana Radoi MD, PhD1, Georgeta Datcu MD,3, Antoniu Burducea MD,2

1Transylvania University-Faculty of Medicine, 2Clinic County Emergency Hospital- Clinic of Cardiology,

Brasov, 3Gr.T.Popa University of Medicine and Pharmacy Iasi

Aims: To evaluate the efficacy of treatment with

trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary heart disease (CHD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular function, reduction of inflammatory syndrome and oxidative stress.

Methods and results: 252 pts with high risk CAD were included in a prospective study for a period of 24 months and divided in six groups in relation with type of CHD and addition of TMZ MR treatment to optimal standard medical therapy (OSMT). Patients were evaluated clinical and paraclinical (biochemical analyses, electrocardiography, echocardiography) at 1, 6, 12 and 24 months. Markers of inflammatory syndrome used were C-reactive protein serum level and fibrinogen plasma level. Anti ox-LDL antibody titers and total antioxidant

J.M.B. no 3 2006

status (TAS) serum level were used for oxidative stress evaluation. Treatment with TMZ MR in addition to OSMT was followed at 24 months of follow up by a significant improvement of left ventricular function in pts with NSTEMI (p<0.05) and UA(p<0.025) in comparison with OSMT pts. Primary endpoint was significantly reduced in all TMZ MR treatment added to OSMT pts at 24 months of follow up respectively: NSTEMI -20%, p<0.001, relative risk (RR) 0.35), UA-13.6%, p<0.001, RR 0.29 and SA-7.7%, p<0.001, RR 0.19). In NSTEMI and UA groups treated with TMZ MR in addition to OSMT was significantly reduced at 6 months of follow-up the incidence of high level of serum CRP (p<0.025), plasma fibrinogen (p<0.01), anti ox-LDL antibody titers (p<0.05), low value of serum TAS (p<0.05), in comparison with NSTEMI and AI groups with OSMT.

Conclusions: In patients with stable angina, unstable angina and non ST elevation myocardial infarction, treatment with trimetazidine MR in addition to optimal standard medical therapy was followed by a significant reduction in primary endpoint composite of cardiovascular death, acute myocardial infarction and stroke and by a significant improvement in left ventricular function at 24 months of follow up. A significant reduction in inflammatory syndrome and oxidative stress at 6 months of follow up was observed in patients with unstable angina and non ST elevation myocardial infarction with above mentioned therapy.

J.M.B no 2 – 2007

The efficacy of Trimetazidine MR in left ventricular function improvement and reduction of major cardiovascular

events, inflammatory syndromes and oxidative stress in coronary heart

disease

As.univ.dr. Elena Bobescu “Transilvania” University - Faculty of Medicine

Aims: To evaluate the efficacy of treatment with

trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary artery disease (CAD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary composite endpoint of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular function, reduction of inflammatory syndrome and oxidative stress.

Methods and results: 252 patients with high risk CAD were included in a prospective study for a period of 36months and divided in six groups in relation with type of CAD and addition of TMZ MR treatment to optimal standard medical therapy (OSMT). Clinical, biologic, electrocardiographic, echocardiographic evaluation was performed at 1, 6, 12, 24 and 36 months. C-reactive protein serum level and fibrinogen plasma level were determinate as markers of inflammatory syndrome. Anti ox-LDL antibody titers and total antioxidant status (TAS) serum level were measured for oxidative stress evaluation. Treatment with TMZ MR in addition to OSMT was followed at 36 months by a significant improvement of left ventricular function in pts with NSTEMI (p<0.05) and UA(p<0.025) in comparison with OSMT pts. Primary composite endpoint of cardiovascular death, acute myocardial infarction and stroke was significantly reduced in all TMZ MR treatment added to OSMT pts at 36 months of follow up respectively: NSTEMI –(p<0.01), UA

J.M.B no 2 – 2007

The efficacy of Trimetazidine MR in left ventricular function improvement and reduction of major cardiovascular

events, inflammatory syndromes and oxidative stress in coronary heart

disease

As.univ.dr. Elena Bobescu “Transilvania” University - Faculty of Medicine

Aims: To evaluate the efficacy of treatment with

trimetazidine modified release (TMZ MR) in addition to optimal standard medical therapy (OSMT) in patients (pts) with coronary artery disease (CAD) - stable angina (SA), unstable angina (UA) and non ST elevation myocardial infarction (NSTEMI) in reduction of primary composite endpoint of cardiovascular death, acute myocardial infarction and stroke, improvement of left ventricular function, reduction of inflammatory syndrome and oxidative stress.

Methods and results: 252 patients with high risk CAD were included in a prospective study for a period of 36months and divided in six groups in relation with type of CAD and addition of TMZ MR treatment to optimal standard medical therapy (OSMT). Clinical, biologic, electrocardiographic, echocardiographic evaluation was performed at 1, 6, 12, 24 and 36 months. C-reactive protein serum level and fibrinogen plasma level were determinate as markers of inflammatory syndrome. Anti ox-LDL antibody titers and total antioxidant status (TAS) serum level were measured for oxidative stress evaluation. Treatment with TMZ MR in addition to OSMT was followed at 36 months by a significant improvement of left ventricular function in pts with NSTEMI (p<0.05) and UA(p<0.025) in comparison with OSMT pts. Primary composite endpoint of cardiovascular death, acute myocardial infarction and stroke was significantly reduced in all TMZ MR treatment added to OSMT pts at 36 months of follow up respectively: NSTEMI –(p<0.01), UA

J.M.B no 2 – 2007

(p<0.025), and SA (p<0.025). In NSTEMI and UA groups treatment with TMZ MR in addition to OSMT was followed by significantly reduced incidence at 6 months of high level of serum CRP (p<0.025), plasma fibrinogen (p<0.01), anti ox-LDL antibody titers (p<0.05), low value of serum TAS (p<0.05), in comparison with NSTEMI and AI groups with OSMT.

Conclusions: In patients with stable angina, unstable angina and non ST elevation myocardial infarction, treatment with trimetazidine MR in addition to optimal standard medical therapy was followed by a significant reduction in primary endpoint and by a significant improvement in left ventricular function at 36 months of follow up. A significant reduction in inflammatory syndrome and oxidative stress at 6 months of follow up was observed in patients with unstable angina and non ST elevation myocardial infarction treated with trimetazidine MR in addition to optimal standard medical therapy.

Trimetazidine, Administered at the Onset of Reperfusion,Ameliorates Myocardial Dysfunction and Injury by Activationof p38 Mitogen-Activated Protein Kinase and Akt Signaling

Mahmood Khan, Sarath Meduru, Mahmoud Mostafa, Saniya Khan, Kalman Hideg,and Periannan KuppusamyDivision of Cardiovascular Medicine, Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio StateUniversity, Columbus, Ohio (M.K., S.M., M.M., S.K., P.K.); and Institute of Organic and Medicinal Chemistry, Universityof Pecs, Pecs, Hungary (K.H.)

Received December 22, 2009; accepted February 17, 2010

ABSTRACTTrimetazidine [1-(2,3,4-trimethoxybenzyl)piperazine; TMZ] is ananti-ischemic cardiac drug; however, its efficacy and mecha-nism of cardioprotection upon reperfusion are largely unknown.The objective of this study was to determine whether TMZ,given before reperfusion, could attenuate myocardial reperfu-sion injury. Ischemia/reperfusion (I/R) was induced in rat heartsby ligating the left anterior descending (LAD) coronary artery for30 min followed by 48 h of reperfusion. TMZ (5 mg/kg b.wt.)was administered 5 min before reperfusion. The study usedthree experimental groups: control (�I/R; �TMZ), I/R (�I/R;�TMZ), and TMZ (�I/R; �TMZ). Echocardiography and EPRoximetry were used to assess cardiac function and oxygen-ation, respectively. The ejection fraction, which was signifi-cantly depressed in the I/R group (62 � 5 versus 84 � 3% incontrol), was restored to 72 � 3% in the TMZ group. Myo-

cardial pO2 in the TMZ group returned to baseline levels(�20 mm Hg) within 1 h of reperfusion, whereas the I/R groupshowed a significant hyperoxygenation even after 48 h ofreperfusion. The infarct size was significantly reduced in theTMZ group (26 � 3 versus 47 � 5% in I/R). TMZ treatmentsignificantly attenuated superoxide levels in the tissue. Tis-sue homogenates showed a significant increase in p38 andp-Akt and decrease in caspase-3 levels in the TMZ group. Insummary, the results demonstrated that TMZ is cardiopro-tective when administered before reperfusion and that thisprotection appears to be mediated by activation of p38mitogen-activated protein kinase and Akt signaling. Thestudy emphasizes the importance of administering TMZ be-fore reflow to prevent reperfusion-mediated cardiac injuryand dysfunction.

Ischemic heart disease is the leading cause of mortalityamong both men and women in the United States, and in theworld. Clinical interventions such as coronary angioplasty,coronary artery bypass graft surgery, or percutaneous trans-luminal coronary angioplasty are routinely used to reintro-duction of blood flow to an ischemic region of the myocar-dium. Such interventions are unavoidably accompanied byan enzymatic cascade of reactions that result in damage tothe myocardium, termed ischemia/reperfusion (I/R) injury.

Although the etiology of I/R injury is intricate, oxidativestress occurs due to an imbalance between free-radical pro-duction and the heart’s ability to prevent the damage causedby free radicals. Numerous studies have shown that thegeneration of reactive oxygen species (ROS) in the oxygen-deprived tissue plays a crucial role in the cellular oxidativedamage that happens during I/R (Zweier et al., 1989; Ambro-sio et al., 1993; Griendling and FitzGerald, 2003). The gen-eration of free radicals that occurs during I/R has been re-ported by several groups (Bolli et al., 1988; Zweier et al.,1989) and has revealed that ROS production peaks withinthe first few minutes of reperfusion. Free-radical scavengers(e.g., antioxidants) have been shown to protect the heart fromoxidative damage resulting from the formation of ROS dur-ing an I/R episode (Ambrosio et al., 1987b).

This work was supported by the National Institutes of Health NationalInstitute of Biomedical Imaging and Bioengineering [Grants EB006153,EB004031] and the American Heart Association [Grant SDG 0930181N].

Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.

doi:10.1124/jpet.109.165175.

ABBREVIATIONS: I/R, ischemia/reperfusion; ROS, reactive oxygen species; TMZ, trimetazidine; MAPK, mitogen-activated protein kinase; PBS,phosphate-buffered saline; DHE, dihydroethidium; TTC, triphenyltetrazolium chloride; DAF-FM, 4-amino-5-methylamino-2�,7�-difluorofluorescein;TTC, triphenyltetrazolium chloride; DEPMPO, 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide; EPR, electron paramagnetic resonance; LAD,left anterior descending; HE, hydroxyethidine; CK, creatine kinase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling;p-, phosphorylated; ERK, extracellular signal-regulated kinase.

0022-3565/10/3332-421–429$20.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 333, No. 2Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 165175/3582029JPET 333:421–429, 2010 Printed in U.S.A.

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tion of ERK1/2, suggesting that TMZ has no effect on ERK1/2activation in I/R injury (Kutala et al., 2006). It should benoted that caspase-3 level, although was significantly de-creased by TMZ, was still substantially elevated in the TMZgroup. However, the absence of any significant change in thecytochrome c level would imply that the mitochondrial func-tion was preserved in TMZ-treated I/R hearts.

Trimetazidine has been in clinical use for more than 20years. It is an effective treatment for stable angina and apotential drug for treating systolic dysfunction in cardiac

failure patients. Both in vitro and in vivo studies have dem-onstrated that, during ischemia, TMZ limits intracellularacidosis, inhibits sodium and calcium accumulation, main-tains intracellular ATP levels, reduces CK release, preservesmitochondrial function, and inhibits neutrophil infiltration(Kantor et al., 2000; Tritto et al., 2005). TMZ has a positiveinfluence on I/R injury, endothelial dysfunction, and progno-sis in patients with coronary artery disease. The presentstudy revealed that TMZ, in addition to its above-mentionedtherapeutic potential, could attenuate reperfusion-induced

Fig. 7. Evaluation of cellular apoptosis by TUNEL as-say. Effect of TMZ on the apoptosis in heart tissuessubjected to I/R. A, apoptosis (green) was imaged ininfarct heart tissues at 48 h after reperfusion. B, in-creased TUNEL-positive nuclei were seen in the I/Rgroup, whereas TMZ-treated hearts exhibited a signifi-cant decrease in TUNEL-positive nuclei (mean � S.D.;n � 4 hearts/group). �, p � 0.05 versus I/R group.C, overlay of TUNEL-positive (green) and 4,6-di-amidino-2-phenylindole (DAPI; blue) images from ex-panded regions as indicated.

Casp. 3

Bcl-2

p-ERK1/2

TotalERK

TotalAkt

p-Akt

GAPDH

Control I/R TMZ

p-p38

p38

Control I/R TMZ

Cyt

. c).

u .a( 83p-

p).

u .a(

0

25

50

75

100 *

p-A

kt (

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.)0

255075

100 *

Cas

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0

25

75

100 *

0

25

50

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Fig. 8. Western blot analysis. Western blot analysisresults for p38, p-p38, Akt, p-Akt, ERK1/2, p-ERK1/2,Bcl-2, and cytochrome c in heart tissue homogenatesobtained 48 h after ischemia/reperfusion injury (datarepresent mean � S.D.; n � 4 hearts/group). �, p � 0.05versus I/R group. The results show that TMZ treatmentsignificantly enhanced the expression of p-p38 and p-Akt, and decreased caspase-3 expression.

428 Khan et al.

ROS generation by its inhibitory as well as radical-scaveng-ing properties. The ROS-scavenging ability of TMZ in thepresent study is further augmented by its ROS-inhibitingfunctions such as the inhibition of neutrophil-mediated ROSproduction (Duilio et al., 2001; Tritto et al., 2005). Our futurestudies will evaluate the potential of the antioxidant-conju-gated forms of TMZ, which have been developed previously(Kutala et al., 2006), for protection against I/R-mediatedcardiac dysfunction and injury under in vivo settings.

In conclusion, the present study underlines the potentialclinical significance of administering TMZ before the onset ofreperfusion, which may be valuable for patients undergoingcoronary angioplasty or percutaneous coronary interventions.

Acknowledgments

We thank Brian K. Rivera for critical evaluation of the manuscript.

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Preserved high energy phosphate metabolic reserve in globally “stunned” heartsdespite reduction of basal ATP content and contractility. J Mol Cell Cardiol19:953–964.

Ambrosio G, Zweier JL, Duilio C, Kuppusamy P, Santoro G, Elia PP, Tritto I, CirilloP, Condorelli M, and Chiariello M (1993) Evidence that mitochondrial respirationis a source of potentially toxic oxygen free radicals in intact rabbit hearts subjectedto ischemia and reflow. J Biol Chem 268:18532–18541.

Ambrosio G, Zweier JL, Jacobus WE, Weisfeldt ML, and Flaherty JT (1987b) Im-provement of postischemic myocardial function and metabolism induced by ad-ministration of deferoxamine at the time of reflow: the role of iron in the patho-genesis of reperfusion injury. Circulation 76:906–915.

Argaud L, Gomez L, Gateau-Roesch O, Couture-Lepetit E, Loufouat J, Robert D, andOvize M (2005) Trimetazidine inhibits mitochondrial permeability transition poreopening and prevents lethal ischemia-reperfusion injury. J Mol Cell Cardiol 39:893–899.

Armstrong SC (2004) Protein kinase activation and myocardial ischemia/reperfusioninjury. Cardiovasc Res 61:427–436.

Bolli R and Marban E (1999) Molecular and cellular mechanisms of myocardialstunning. Physiol Rev 79:609–634.

Bolli R, Patel BS, Jeroudi MO, Lai EK, and McCay PB (1988) Demonstration of freeradical generation in “stunned” myocardium of intact dogs with the use of the spintrap alpha-phenyl N-tert-butyl nitrone. J Clin Invest 82:476–485.

Duilio C, Ambrosio G, Kuppusamy P, DiPaula A, Becker LC, and Zweier JL (2001)Neutrophils are primary source of O2 radicals during reperfusion after prolongedmyocardial ischemia. Am J Physiol Heart Circ Physiol 280:H2649–H2657.

Fliss H and Gattinger D (1996) Apoptosis in ischemic and reperfused rat myocar-dium. Circ Res 79:949–956.

Gambert S, Vergely C, Filomenko R, Moreau D, Bettaieb A, Opie LH, and RochetteL (2006) Adverse effects of free fatty acid associated with increased oxidativestress in postischemic isolated rat hearts. Mol Cell Biochem 283:147–152.

Gottlieb RA, Burleson KO, Kloner RA, Babior BM, and Engler RL (1994) Reperfusioninjury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 94:1621–1628.

Griendling KK and FitzGerald GA (2003) Oxidative stress and cardiovascular injury:Part I: basic mechanisms and in vivo monitoring of ROS. Circulation 108:1912–1916.

Guarnieri C and Muscari C (1993) Effect of trimetazidine on mitochondrial functionand oxidative damage during reperfusion of ischemic hypertrophied rat myocar-dium. Pharmacology 46:324–331.

Harpey C, Clauser P, Labrid C, Freyria JL, and Poirier JP (1989) Trimetazidine, acellular anti-ischemic agent. Cardiovasc Drug Rev 6:292–312.

Kalai T, Khan M, Balog M, Kutala VK, Kuppusamy P, and Hideg K (2006) Structure-activity studies on the protection of trimetazidine derivatives modified with ni-troxides and their precursors from myocardial ischemia-reperfusion injury. BioorgMed Chem 14:5510–5516.

Kantor PF, Lucien A, Kozak R, and Lopaschuk GD (2000) The antianginal drugtrimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucoseoxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase.Circ Res 86:580–588.

Khan M, Kutala VK, Vikram DS, Wisel S, Chacko SM, Kuppusamy ML, Mohan IK,Zweier JL, Kwiatkowski P, and Kuppusamy P (2007a) Skeletal myoblasts trans-planted in the ischemic myocardium enhance in situ oxygenation and recovery ofcontractile function. Am J Physiol Heart Circ Physiol 293:H2129–H2139.

Khan M, Meduru S, Mohan IK, Kuppusamy ML, Wisel S, Kulkarni A, Rivera BK,Hamlin RL, and Kuppusamy P (2009a) Hyperbaric oxygenation enhances trans-planted cell graft and functional recovery in the infarct heart. J Mol Cell Cardiol47:275–287.

Khan M, Mohan IK, Kutala VK, Kotha SR, Parinandi NL, Hamlin RL, and Kup-

pusamy P (2009b) Sulfaphenazole protects heart against ischemia-reperfusioninjury and cardiac dysfunction by overexpression of iNOS, leading to enhancementof nitric oxide bioavailability and tissue oxygenation. Antioxid Redox Signal 11:725–738.

Khan M, Mohan IK, Kutala VK, Kumbala D, and Kuppusamy P (2007b) Cardiopro-tection by sulfaphenazole, a cytochrome P450 inhibitor: mitigation of ischemia-reperfusion injury by scavenging of reactive oxygen species. J Pharmacol Exp Ther323:813–821.

Khan M, Varadharaj S, Ganesan LP, Shobha JC, Naidu MU, Parinandi NL, Tridan-dapani S, Kutala VK, and Kuppusamy P (2006) C-phycocyanin protects againstischemia-reperfusion injury of heart through involvement of p38 MAPK and ERKsignaling. Am J Physiol Heart Circ Physiol 290:H2136–H2145.

Kutala VK, Khan M, Mandal R, Ganesan LP, Tridandapani S, Kalai T, Hideg K, andKuppusamy P (2006) Attenuation of myocardial ischemia-reperfusion injury bytrimetazidine derivatives functionalized with antioxidant properties. J PharmacolExp Ther 317:921–928.

Kutala VK, Parinandi NL, Pandian RP, and Kuppusamy P (2004) Simultaneousmeasurement of oxygenation in intracellular and extracellular compartments oflung microvascular endothelial cells. Antioxid Redox Signal 6:597–603.

Liu HR, Gao F, Tao L, Yan WL, Gao E, Christopher TA, Lopez BL, Hu A, and Ma XL(2004) Antiapoptotic mechanisms of benidipine in the ischemic/reperfused heart.Br J Pharmacol 142:627–634.

Ma XL, Kumar S, Gao F, Louden CS, Lopez BL, Christopher TA, Wang C, Lee JC,Feuerstein GZ, and Yue TL (1999) Inhibition of p38 mitogen-activated proteinkinase decreases cardiomyocyte apoptosis and improves cardiac function aftermyocardial ischemia and reperfusion. Circulation 99:1685–1691.

McClellan KJ and Plosker GL (1999) Trimetazidine. A review of its use in stableangina pectoris and other coronary conditions. Drugs 58:143–157.

Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, and Davidson BL (1998) Super-oxide production in vascular smooth muscle contributes to oxidative stress andimpaired relaxation in atherosclerosis. Circ Res 82:1298–1305.

Mohan IK, Khan M, Wisel S, Selvendiran K, Sridhar A, Carnes CA, Bognar B, KalaiT, Hideg K, and Kuppusamy P (2009) Cardioprotection by HO-4038, a novelverapamil derivative, targeted against ischemia and reperfusion-mediated acutemyocardial infarction. Am J Physiol Heart Circ Physiol 296:H140–H151.

Monteiro P, Duarte AI, Goncalves LM, Moreno A, and Providencia LA (2004) Pro-tective effect of trimetazidine on myocardial mitochondrial function in an ex-vivomodel of global myocardial ischemia. Eur J Pharmacol 503:123–128.

Omura T, Yoshiyama M, Shimada T, Shimizu N, Kim S, Iwao H, Takeuchi K, andYoshikawa J (1999) Activation of mitogen-activated protein kinases in in vivoischemia/reperfused myocardium in rats. J Mol Cell Cardiol 31:1269–1279.

Pandian RP, Parinandi NL, Ilangovan G, Zweier JL, and Kuppusamy P (2003) Novelparticulate spin probe for targeted determination of oxygen in cells and tissues.Free Radic Biol Med 35:1138–1148.

Pantos C, Bescond-Jacquet A, Tzeis S, Paizis I, Mourouzis I, Moraitis P, Malliopou-lou V, Politi ED, Karageorgiou H, Varonos D, et al. (2005) Trimetazidine protectsisolated rat hearts against ischemia-reperfusion injury in an experimental timing-dependent manner. Basic Res Cardiol 100:154–160.

Paolocci N, Biondi R, Bettini M, Lee CI, Berlowitz CO, Rossi R, Xia Y, Ambrosio G,L’Abbate A, Kass DA, et al. (2001) Oxygen radical-mediated reduction in basal andagonist-evoked NO release in isolated rat heart. J Mol Cell Cardiol 33:671–679.

Selvendiran K, Tong L, Vishwanath S, Bratasz A, Trigg NJ, Kutala VK, Hideg K,and Kuppusamy P (2007) EF24 induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by increasing PTEN expression. J Biol Chem282:28609–28618.

Takada Y, Hashimoto M, Kasahara J, Aihara K, and Fukunaga K (2004) Cytopro-tective effect of sodium orthovanadate on ischemia/reperfusion-induced injury inthe rat heart involves Akt activation and inhibition of fodrin breakdown andapoptosis. J Pharmacol Exp Ther 311:1249–1255.

Toth A, Halmosi R, Kovacs K, Deres P, Kalai T, Hideg K, Toth K, and Sumegi B(2003) Akt activation induced by an antioxidant compound during ischemia-reperfusion. Free Radic Biol Med 35:1051–1063.

Tritto I, Wang P, Kuppusamy P, Giraldez R, Zweier JL, and Ambrosio G (2005) Theanti-anginal drug trimetazidine reduces neutrophil-mediated cardiac reperfusioninjury. J Cardiovasc Pharmacol 46:89–98.

Wisel S, Khan M, Kuppusamy ML, Mohan IK, Chacko SM, Rivera BK, Sun BC,Hideg K, and Kuppusamy P (2009) Pharmacological preconditioning of mesenchy-mal stem cells with trimetazidine (1-[2,3,4-trimethoxybenzyl]piperazine) protectshypoxic cells against oxidative stress and enhances recovery of myocardial func-tion in infarcted heart through Bcl-2 expression. J Pharmacol Exp Ther 329:543–550.

Zhao X, He G, Chen YR, Pandian RP, Kuppusamy P, and Zweier JL (2005) Endo-thelium-derived nitric oxide regulates postischemic myocardial oxygenation andoxygen consumption by modulation of mitochondrial electron transport. Circula-tion 111:2966–2972.

Zweier JL, Kuppusamy P, Williams R, Rayburn BK, Smith D, Weisfeldt ML, andFlaherty JT (1989) Measurement and characterization of postischemic free radicalgeneration in the isolated perfused heart. J Biol Chem 264:18890–18895.

Address correspondence to: Dr. Periannan Kuppusamy, Davis Heart andLung Research Institute, Room 114, The Ohio State University, 420 West 12thAve., Columbus, OH 43210. E-mail: kuppusamy.1@osu.edu

Trimetazidine Ameliorates Cardiac Reperfusion Injury 429

CARDIOVASCULAR MEDICINE

Long term cardioprotective action of trimetazidine andpotential effect on the inflammatory process in patients withischaemic dilated cardiomyopathyP Di Napoli, A A Taccardi, A Barsotti. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr Pericle Di Napoli,Dipartimento diCardiologia-UTIC, Casa diCura Villa Pini d’Abruzzo,Via dei Frentani 228,66100 Chieti, Italy;dinapoli@unich.it

Accepted 2 June 2004. . . . . . . . . . . . . . . . . . . . . . .

Heart 2005;91:161–165. doi: 10.1136/hrt.2003.031310

Objective: To investigate the long term effects of trimetazidine in patients with dilated ischaemiccardiomyopathy. The effects of trimetazidine on left ventricular function as well as its tolerability profile andpotential anti-inflammatory effects were studied.Design: 61 patients were randomly assigned either to receive trimetazidine (20 mg thrice daily) inaddition to their conventional treatment or to continue their usual drug treatment for 18 months. Patientswere evaluated at baseline and at 6, 12, and 18 months with a clinical examination, echocardiography,and biochemical analysis (C reactive protein).Results: Trimetazidine added to the usual treatment significantly improved the patients’ functional status(assessed by New York Heart Association functional class). The functional improvement of trimetazidinetreated patients was associated with a significant increase in left ventricular ejection fraction (30 (6)%, 32(8)%, 38 (7)%, and 37 (6)% v 31 (8)%, 30 (7)%, 28 (6)%, and 26 (9)% in control patients at baseline and at6, 12, and 18 months, respectively) and with a significant effect on ventricular remodelling. C reactiveprotein plasma concentrations remained stable throughout the study in patients receiving trimetazidine(2.5 (1.0), 2.7 (2.0), 2.7 (3.0), and 3.0 (2.0) mg/l at baseline and at 6, 12, and 18 months, respectively)but increased significantly in the control group (2.4 (1.0), 3.4 (1.2), 6.0 (4.0), and 7.0 (5.0) mg/l,respectively). No significant adverse event or changes in clinical or biochemical parameters were detected.Conclusion: Treatment with trimetazidine added to the usual treatment for up to 18 months was welltolerated and induced a functional improvement in patients with dilated cardiomyopathy. Trimetazidinetreatment was associated with a significant improvement of left ventricular function and the remodellingprocess. Results also suggest that the inflammatory response was limited in patients treated withtrimetazidine.

Trimetazidine (1-[2,3,4-trimethoxybenzyl] piperazinedihydrochloride) is the first of a novel antianginal drugclass, the selective inhibitors of mitochondrial long chain

3-ketoacyl coenzyme A thiolase. Trimetazidine is widely usedfor the prophylactic treatment of episodes of angina pectorisat recommended daily doses ranging from 40–60 mg.The anti-ischaemic properties of trimetazidine are un-

related to changes in the myocardial oxygen supply to demandratio, as heart rate, blood pressure, and pressure–rate product donot change significantly either at rest or during exercise.1 2

Trimetazidine acts by reducing fatty acid oxidation and bystimulating glucose oxidation.3 4 Subsequently the coupling ofglycolysis with glucose oxidation is improved, ATP production isincreased, and the deleterious consequences of acidosis andintracellular calcium ion overload that characterise the ischae-mic or hypoxic cell are limited.The antianginal properties of trimetazidine were previously

shown in acute and chronic experimental conditions inwhich the cardioprotective effects were related to the positiveeffects on energy metabolism, hydroionic balance, coronarymicrocirculation, and oxidative stress.5 6 Few data areavailable regarding the efficacy of trimetazidine in patientswith dilated cardiomyopathy.7 8 However, long term data(. 1 year) on the safety and usefulness of this treatment insuch patients have not been fully explored; nor has its effectson the inflammatory process, an independent negativeprognostic factor in patients with coronary artery disease.9–11

The aim of this randomised study in patients withischaemic dilated cardiomyopathy already receiving conven-tional treatment was to investigate: firstly, the long term

effects of trimetazidine (20 mg thrice daily); secondly, itstolerability profile; and thirdly, its effects on the inflamma-tory process. Patients were followed up for up to 18 monthsafter their inclusion in the study. Efficacy was assessed byevaluating the patients’ functional status (New York HeartAssociation (NYHA) functional class) and left ventricular(LV) function (echographic changes in the LV ejectionfraction (LVEF) and volumes). The effect of trimetazidineon the inflammatory process was evaluated by determining Creactive protein plasma concentrations.

METHODSGeneral methodsThis randomised pilot study was conducted at a single site(Villa Pini, Centre for Study and Treatment of CongestiveHeart Failure, Chieti, Italy). The number of patients waschosen on the basis of previous studies with trimetazidineperformed with a similar design.7 The study was conductedin an open label design for patient convenience. Echo-cardiograms (for primary outcome) were recorded and ratedblindly by two independent investigators.The research protocol was approved by the local appointed

ethics committee. All patients provided written informedconsent before participation. The enrolment period took placefrom January 1997 through June 1997 and the last patientvisit occurred in January 1999.

Abbreviations: ACE, angiotensin converting enzyme; LV, leftventricular; LVEF, left ventricular ejection fraction; NYHA, New YorkHeart Association

161

www.heartjnl.com

CARDIOVASCULAR MEDICINE

Long term cardioprotective action of trimetazidine andpotential effect on the inflammatory process in patients withischaemic dilated cardiomyopathyP Di Napoli, A A Taccardi, A Barsotti. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

See end of article forauthors’ affiliations. . . . . . . . . . . . . . . . . . . . . . .

Correspondence to:Dr Pericle Di Napoli,Dipartimento diCardiologia-UTIC, Casa diCura Villa Pini d’Abruzzo,Via dei Frentani 228,66100 Chieti, Italy;dinapoli@unich.it

Accepted 2 June 2004. . . . . . . . . . . . . . . . . . . . . . .

Heart 2005;91:161–165. doi: 10.1136/hrt.2003.031310

Objective: To investigate the long term effects of trimetazidine in patients with dilated ischaemiccardiomyopathy. The effects of trimetazidine on left ventricular function as well as its tolerability profile andpotential anti-inflammatory effects were studied.Design: 61 patients were randomly assigned either to receive trimetazidine (20 mg thrice daily) inaddition to their conventional treatment or to continue their usual drug treatment for 18 months. Patientswere evaluated at baseline and at 6, 12, and 18 months with a clinical examination, echocardiography,and biochemical analysis (C reactive protein).Results: Trimetazidine added to the usual treatment significantly improved the patients’ functional status(assessed by New York Heart Association functional class). The functional improvement of trimetazidinetreated patients was associated with a significant increase in left ventricular ejection fraction (30 (6)%, 32(8)%, 38 (7)%, and 37 (6)% v 31 (8)%, 30 (7)%, 28 (6)%, and 26 (9)% in control patients at baseline and at6, 12, and 18 months, respectively) and with a significant effect on ventricular remodelling. C reactiveprotein plasma concentrations remained stable throughout the study in patients receiving trimetazidine(2.5 (1.0), 2.7 (2.0), 2.7 (3.0), and 3.0 (2.0) mg/l at baseline and at 6, 12, and 18 months, respectively)but increased significantly in the control group (2.4 (1.0), 3.4 (1.2), 6.0 (4.0), and 7.0 (5.0) mg/l,respectively). No significant adverse event or changes in clinical or biochemical parameters were detected.Conclusion: Treatment with trimetazidine added to the usual treatment for up to 18 months was welltolerated and induced a functional improvement in patients with dilated cardiomyopathy. Trimetazidinetreatment was associated with a significant improvement of left ventricular function and the remodellingprocess. Results also suggest that the inflammatory response was limited in patients treated withtrimetazidine.

Trimetazidine (1-[2,3,4-trimethoxybenzyl] piperazinedihydrochloride) is the first of a novel antianginal drugclass, the selective inhibitors of mitochondrial long chain

3-ketoacyl coenzyme A thiolase. Trimetazidine is widely usedfor the prophylactic treatment of episodes of angina pectorisat recommended daily doses ranging from 40–60 mg.The anti-ischaemic properties of trimetazidine are un-

related to changes in the myocardial oxygen supply to demandratio, as heart rate, blood pressure, and pressure–rate product donot change significantly either at rest or during exercise.1 2

Trimetazidine acts by reducing fatty acid oxidation and bystimulating glucose oxidation.3 4 Subsequently the coupling ofglycolysis with glucose oxidation is improved, ATP production isincreased, and the deleterious consequences of acidosis andintracellular calcium ion overload that characterise the ischae-mic or hypoxic cell are limited.The antianginal properties of trimetazidine were previously

shown in acute and chronic experimental conditions inwhich the cardioprotective effects were related to the positiveeffects on energy metabolism, hydroionic balance, coronarymicrocirculation, and oxidative stress.5 6 Few data areavailable regarding the efficacy of trimetazidine in patientswith dilated cardiomyopathy.7 8 However, long term data(. 1 year) on the safety and usefulness of this treatment insuch patients have not been fully explored; nor has its effectson the inflammatory process, an independent negativeprognostic factor in patients with coronary artery disease.9–11

The aim of this randomised study in patients withischaemic dilated cardiomyopathy already receiving conven-tional treatment was to investigate: firstly, the long term

effects of trimetazidine (20 mg thrice daily); secondly, itstolerability profile; and thirdly, its effects on the inflamma-tory process. Patients were followed up for up to 18 monthsafter their inclusion in the study. Efficacy was assessed byevaluating the patients’ functional status (New York HeartAssociation (NYHA) functional class) and left ventricular(LV) function (echographic changes in the LV ejectionfraction (LVEF) and volumes). The effect of trimetazidineon the inflammatory process was evaluated by determining Creactive protein plasma concentrations.

METHODSGeneral methodsThis randomised pilot study was conducted at a single site(Villa Pini, Centre for Study and Treatment of CongestiveHeart Failure, Chieti, Italy). The number of patients waschosen on the basis of previous studies with trimetazidineperformed with a similar design.7 The study was conductedin an open label design for patient convenience. Echo-cardiograms (for primary outcome) were recorded and ratedblindly by two independent investigators.The research protocol was approved by the local appointed

ethics committee. All patients provided written informedconsent before participation. The enrolment period took placefrom January 1997 through June 1997 and the last patientvisit occurred in January 1999.

Abbreviations: ACE, angiotensin converting enzyme; LV, leftventricular; LVEF, left ventricular ejection fraction; NYHA, New YorkHeart Association

161

www.heartjnl.com

Compared with control patients maintained with theirusual treatment, trimetazidine treated patients’ functionalimprovement was associated with a significant increase inLVEF and with a significant effect on ventricular remodel-ling. These effects were significant starting from 12 monthsand were maintained after 18 months of treatment. Duringthe evaluation period, C reactive protein plasma concentra-tions remained stable with trimetazidine treatment and were,in contrast, significantly increased in control patients.Trimetazidine treatment was not modified or discontinuedduring the evaluation period. No significant adverse eventrelated to the study treatments occurred during the study.Thus, patients with ischaemic dilated cardiomyopathytolerated trimetazidine well during up to 18 months oftreatment.The clinical and echocardiographic data obtained in this

study are in agreement with findings of Brottier et al,7 whoreported improved LVEF and cardiac volumes in patientswith severe ischaemic cardiomyopathy after six months’treatment with trimetazidine. Recently, Fragasso et al12 andRosano et al13 reported relevant improvement of clinical statusand LVEF in diabetic patients with ischaemic dilatedcardiomyopathy after only six months of treatment withtrimetazidine; the apparent discrepancy with our data, inwhich LVEF improved significantly after 12 months’ treat-ment, may result from the differences in patients’ character-istics and, particularly, in the low prevalence of diabetesamong our patients (about 20%).It is suggested that the effects observed here with

trimetazidine are related to the peculiar mechanism of actionof the drug. Trimetazidine is one of a new group of pharma-cological compounds whose myocardial anti-ischaemic effectis achieved independently from the changes in the oxygensupply to demand ratio.1 3 4 The anti-ischaemic effect oftrimetazidine is obtained at the cellular level, by shifting theenergy substrate reference from fatty acid oxidation toglucose oxidation.In experimental conditions, trimetazidine exerts a cardio-

protective effect by rapidly restoring the phosphorylationprocesses, protecting cardiac cells against intracellular acido-sis, preventing the intracellular accumulation of sodium andcalcium ions, and by reducing oxidative damage.14 15 All theseproperties observed experimentally with trimetazidine maycontribute to protecting the myocardial cell against necrosisand apoptosis. These two steps are considered fundamentalin regulating LV modelling and the progressive decline of thecontractile function that occurs during the normal evolutionof dilated cardiomyopathy.16

The preservation of LVEF observed in trimetazidine treatedpatients may be explained, at least partly, by the effects of thedrug in maintaining the integrity of cell membranes as well

as mitochondrial structure and function.17 In addition, theincrease of glucose oxidation induced by trimetazidine mayin turn enhance the resynthesis of glycolytic ATP andimprove contractility and microvascular function. Lastly, itis also possible that, after trimetazidine treatment, chroni-cally hibernating cells increase their energy metabolism,become effective in producing contractile activity, and thuscontribute to improving the contractile response and tolimiting further decline of LV function. Data on patients withischaemic cardiomyopathy confirmed that trimetazidinetreatment improved the contractile response to a challengeof dobutamine, without inducing any haemodynamicchanges.8 This amelioration of contractility is also evidentduring dobutamine induced ischaemic dysfunction inpatients with coronary artery disease and normal LV systolicfunction at rest.18

In addition to these effects, we observed that C reactiveprotein plasma concentrations remained stable throughoutthe study in patients receiving trimetazidine, whereas theyincreased significantly with time in patients receiving theirstandard treatment. C reactive protein is a biological markercommonly assessed in medical practice to evaluate thesystemic inflammatory process. A proinflammatory state isrecognised in chronic heart failure and the degree of immuneactivation corresponds to disease severity and prognosis. PeakC reactive protein concentration measured during myocardialinfarction has been shown to be a strong predictor of globaland heart failure mortality during the following year.19 Inpatients with heart failure, higher C reactive proteinconcentrations have also been related to higher rates ofhospital readmission and mortality.20

Although our data need to be confirmed with furtherspecific investigations, they suggest that the inflammatoryprocess may have been limited in the patient sample treatedwith trimetazidine during the 18 month evaluation period. Ifthe relevance of improvement of LV function and clinicalstatus appears prevalent, potential anti-inflammatory effectshave been previously reported with trimetazidine in experi-mental conditions.21 In a rabbit model of ischaemia andreperfusion, the authors reported that trimetazidine reducedneutrophil cell accumulation in the reperfused post-ischaemicmyocardium. In the clinical setting, whether the effect oftrimetazidine on C reactive protein concentrations is relatedto its direct action on the inflammatory process or resultsfrom its anti-ischaemic properties needs to be elucidated.Furthermore, the reduced inflammatory process may berelated to the better metabolic status of either myocytic orendothelial cells; this, in turn, provides a better explanationof their physiological functions (contraction and coronarycirculation, respectively), less cellular damage and, lastly,less tissue reaction and inflammation.In conclusion, treatment with trimetazidine added to the

usual treatment for up to 18 months was well tolerated andinduced functional improvement in patients with ischaemicdilated cardiomyopathy. Trimetazidine treatment was asso-ciated with a significant improvement of LV function and theremodelling process. Results also suggest that the inflamma-tory response was limited in patients treated with trimeta-zidine. The effects of trimetazidine on energy metabolism,hydroionic balance, coronary microcirculation, and oxidativestress contribute positively to its beneficial effects on LVremodelling and long term cardioprotection observed in thisstudy.

Authors’ affiliations. . . . . . . . . . . . . . . . . . . . .

P Di Napoli, A A Taccardi, Department of Cardiology, Intensive CareUnit, Casa di Cura Villa Pini d’Abruzzo, Chieti, ItalyA Barsotti, Department of Internal Medicine, University of Genoa,Genoa, Italy

10

7.5

5.0

2.5

0

Control

CRP

(mg/

l)

Basal 6 months 12 months 18 months

Trimetazidine

Figure 3 Changes in C reactive protein (CRP) plasma concentrations.Data are mean (SD). ***p,0.001 v control.

164 Di Napoli, Taccardi, Barsotti

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Trimetazidine Suppresses Angioplasty Related Inflammation 203

203

Tohoku J. Exp. Med., 2006, 208, 203-212

Received June 1, 2005; revision accepted for publication December 23, 2005.Correspondence: Filiz Kuralay, Department of Biochemistry, Dokuz Eylul University, School of Medicine,

Balçova, Izmir 35340, Turkey.e-mail: filiz.kuralay@deu.edu.tr

Suppression of Angioplasty-Related Inflammation by Pre-Procedural Treatment with Trimetazidine

FILIZ KURALAY, EMEL ALTEKIN, AYTEN SAYIN YAZLAR, 1 BANU ONVURAL and OZHAN GOLDELI

1

Departments of Biochemistry, and 1Cardiology, Dokuz Eylul University, School of Medicine, Izmir, Turkey

KURALAY, F., ALTEKIN, E., YAZLAR, A.S., ONVURAL, B. and GOLDELI, O. Suppression of Angioplasty-Related Inflammation by Pre-Procedural Treatment with Trimetazidine. Tohoku J. Exp. Med., 2006, 208 (3), 203-212 ── Percutaneous transluminal coronary angioplasty (PTCA) has been recognized as a reliable treatment procedure for acute reversible ischemia and reperfusion. Ischemic reperfusion cycle in PTCA leads to the sys-temic inflammation and extensive tissue injury by the production of reactive oxygen spe-cies including nitric oxide (NO) radicals. In patients with coronary artery disease, undergoing PTCA, the effects of trimetazidine (TMZ), a piperazine-derivative anti-anginal drug, were studied on several indirect markers of systemic inflammatory response: tumor necrosis factor-α (TNF-α ), C-reactive protein (CRP) and NO products (nitrite and nitrate). Patients (n = 11 each group) were untreated or pre-treated with TMZ (20 mg per orally three times a day), begun three days prior to PTCA, and marker levels were measured before the start of TMZ therapy (baseline), just before PTCA (0 hr), and 4, 24, and 48 hrs after PTCA. The baseline levels of markers were not significantly different between the untreated and pre-treated patients. In contrast, all parameters were lower in the TMZ-treated group than those in the matched control group in the pre- and post-angioplasty periods. Interestingly, in the TMZ group, CRP and nitrite levels were significantly lower than in the control group at each time point of the pre- and post-angioplasty periods, but the TNF-α levels were significantly decreased only in the post-angioplasty period. Pre-procedural treatment with oral TMZ for three days significantly suppressed the elevation of inflammatory markers before and shortly after PTCA. We suggest the usefulness of TMZ in preventing inflammatory cardiovascular events after PTCA. ──── trimetazi-dine; percutaneous transluminal coronary angioplasty; C-reactive protein; nitric oxide; tumor necrosis factor-α .© 2006 Tohoku University Medical Press

Trimetazidine (1-[2,3,4-trimethoxy-benzyl] piperazine HCl; TMZ) has been found to have cytoprotective effects on ischemic cardiac tissue. During ischemic episodes, TMZ maintains cellu-lar ATP levels, limits intracellular acidosis and

accumulation of inorganic phosphate, Na+ and Ca++ in cardiac tissue. These changes are inde-pendent of oxygen supply or demand alterations (Demaison et al. 1995; Kay et al. 1995; Cross 2000; Kantor et al. 2000). Trimetazidine selec-

Trimetazidine Suppresses Angioplasty Related Inflammation 203

203

Tohoku J. Exp. Med., 2006, 208, 203-212

Received June 1, 2005; revision accepted for publication December 23, 2005.Correspondence: Filiz Kuralay, Department of Biochemistry, Dokuz Eylul University, School of Medicine,

Balçova, Izmir 35340, Turkey.e-mail: filiz.kuralay@deu.edu.tr

Suppression of Angioplasty-Related Inflammation by Pre-Procedural Treatment with Trimetazidine

FILIZ KURALAY, EMEL ALTEKIN, AYTEN SAYIN YAZLAR, 1 BANU ONVURAL and OZHAN GOLDELI

1

Departments of Biochemistry, and 1Cardiology, Dokuz Eylul University, School of Medicine, Izmir, Turkey

KURALAY, F., ALTEKIN, E., YAZLAR, A.S., ONVURAL, B. and GOLDELI, O. Suppression of Angioplasty-Related Inflammation by Pre-Procedural Treatment with Trimetazidine. Tohoku J. Exp. Med., 2006, 208 (3), 203-212 ── Percutaneous transluminal coronary angioplasty (PTCA) has been recognized as a reliable treatment procedure for acute reversible ischemia and reperfusion. Ischemic reperfusion cycle in PTCA leads to the sys-temic inflammation and extensive tissue injury by the production of reactive oxygen spe-cies including nitric oxide (NO) radicals. In patients with coronary artery disease, undergoing PTCA, the effects of trimetazidine (TMZ), a piperazine-derivative anti-anginal drug, were studied on several indirect markers of systemic inflammatory response: tumor necrosis factor-α (TNF-α ), C-reactive protein (CRP) and NO products (nitrite and nitrate). Patients (n = 11 each group) were untreated or pre-treated with TMZ (20 mg per orally three times a day), begun three days prior to PTCA, and marker levels were measured before the start of TMZ therapy (baseline), just before PTCA (0 hr), and 4, 24, and 48 hrs after PTCA. The baseline levels of markers were not significantly different between the untreated and pre-treated patients. In contrast, all parameters were lower in the TMZ-treated group than those in the matched control group in the pre- and post-angioplasty periods. Interestingly, in the TMZ group, CRP and nitrite levels were significantly lower than in the control group at each time point of the pre- and post-angioplasty periods, but the TNF-α levels were significantly decreased only in the post-angioplasty period. Pre-procedural treatment with oral TMZ for three days significantly suppressed the elevation of inflammatory markers before and shortly after PTCA. We suggest the usefulness of TMZ in preventing inflammatory cardiovascular events after PTCA. ──── trimetazi-dine; percutaneous transluminal coronary angioplasty; C-reactive protein; nitric oxide; tumor necrosis factor-α .© 2006 Tohoku University Medical Press

Trimetazidine (1-[2,3,4-trimethoxy-benzyl] piperazine HCl; TMZ) has been found to have cytoprotective effects on ischemic cardiac tissue. During ischemic episodes, TMZ maintains cellu-lar ATP levels, limits intracellular acidosis and

accumulation of inorganic phosphate, Na+ and Ca++ in cardiac tissue. These changes are inde-pendent of oxygen supply or demand alterations (Demaison et al. 1995; Kay et al. 1995; Cross 2000; Kantor et al. 2000). Trimetazidine selec-

Trimetazidine Suppresses Angioplasty Related Inflammation 205

Pepine 2004). Because serum NO levels can be heavily influenced by dietary nitrogen intake, we fixed the dietary nitrogen intake to 1 g/kg/day of protein per meal during the week prior to PTCA.

Patients (n = 11 each group) were untreated or pre-treated with TMZ (20 mg per orally three times a day), begun three days prior to PTCA. Blood samples were collected from all cases just before starting TMZ (base-line), just before the PTCA procedure (0 hrs) and 4 hrs, 24 hrs, and 48 hrs after PTCA. Blood samples were drawn through antecubital vein, and sera were separated and stored at –70°C until analysis.

CRP measurementCRP was measured by particle-enhanced immuno-

nephelometry (N Latex CRP mono, Behring Diagnostics, Marburg, Germany) by using a 1 :400 sample dilution; a range of 2.4-220 mg/ml was covered (normal values < 5 mg/ml). The intra- and interassay coefficients of varia-tion were < 1.5% and < 2.7%, respectively.

TNF-α measurementA human TNF-α cytoscreen ELISA kit (Biosource,

Cat No: KRC 3012, Camarillo, CA, USA) was used according to manufacturer’s instructions and the result-ing yellow to blue colour intensity was recorded at 450 nm by a Sorin-Biomedica microplate reader. The intra- and interassay coefficients of variations were < 4.2% and < 7.5%, respectively.

Measurement of NO productsNO was determined by quantitating the stable NO

oxidation products, nitrite and nitrate. First, nitrite con-centrations were measured by the Griess reaction (Green et al. 1982). Then, samples were incubated with nitrate reductase (0.1 U/ml, Boehringer Mannheim Gmbh, Mannheim, Germany) for 30 min, and nitrate was reduced to nitrite (Bories and Bories 1995). The nitrate level was determined by subtracting the nitrite concentra-tion from total (nitrite + nitrate) concentrations. The intra- and interassay coefficients of variations were < 5% and < 7%, respectively.

All chemicals were purchased from Sigma (Steinheim, Germany).

Statistical analysisData were analyzed by using with Mann-Whitney’s

U-test and Pearson correlation analysis in SPSS for MS Windows Release 10.0® (Redmond, WA, USA), and p <

0.05 was accepted as the level of statistical significance.

RESULTS

Demographic data about cardiovascular risk factors and medications of the patient groups are shown in Table 1. The medications are listed (statins, ACE inhibitors), because these may heav-ily influence the levels of inflammatory markers. No significant differences were found between the groups regarding risk factors or medications used. The coronary artery characteristics of the patients before PTCA are shown in Table 2 in order to describe the type of ischemic heart disease. There were no significant differences between the male and female patients regarding risk factors, coro-nary artery characteristics or biochemical data.

Pre- and post-PTCA biochemical data are shown in Table 3. The baseline levels of markers were not significantly different between the untreated and pre-treated patients. In contrast, all parameters were lower in the TMZ group than those in the matched control group in the pre- and post-angioplasty periods. Interestingly, in the TMZ group, CRP and nitrite levels were signifi-cantly lower than in the control group at each time point in the pre- and post-angioplasty periods. TNF-α levels were significantly lower than the control group only in the post-angioplasty period. Additionally, total NO levels were significantly lower at 0, 24 and 48 hrs, while nitrate levels were significantly lower in the TMZ group at only 48 hrs.

When the time course levels of the biochemi-cal parameters in untreated and pre-treated patients were compared; significantly lower levels of CRP were detected at all measurement points in the pre-treated group. The peak levels for CRP were reached at 48 hrs in both groups ( p < 0.0001; for each) (Fig. 1). TNF-α levels at 4, 24, and 48 hrs were significantly lower ( p < 0.0001 at all measurement points) in patients pre-treated with TMZ (Table 3). In both groups, TNF-α reached its peak at 24 hrs (Fig. 2).

NO activity peaked at 24 and 48 hrs in the pre-treated and untreated groups, respectively. Significant positive correlations were found between TNF-α and CRP (p < 0.001; r = 0.620),