REVIEW

Trimetazidine Revisited: A Comprehensive Reviewof the Pharmacological Effects and Analytical Techniquesfor the Determination of TrimetazidineA. Onay-Besikci1 and S.A. Ozkan2

1 Department of Pharmacology, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey2 Department of Analytical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey

KeywordsCardiac metabolism; Electrochemistry; GC;

HPLC; Spectrophotometry; Trimetazidine.

CorrespondenceDr A. Onay-Besikci, PhD, Department of

Pharmacology, Faculty of Pharmacy, Ankara

University, Tandogan 06100, Ankara, Turkey.

Tel.: +90-312-203-3147;

Fax: +90-312-213-1081;

E-mail: onay@pharmacy.ankara.edu.tr

doi: 10.1111/j.1527-3466.2008.00043.x

Trimetazidine (TMZ) is an effective and well-tolerated antianginal drug thatpossesses protective properties against ischemia-induced heart injury. Growinginterest in metabolic modulation in recent years urged an up-to-date review ofthe literature on TMZ. This review consists of two major sections: (1) compre-hensive and critical information about the pharmacological effects, mechanismof action, pharmacokinetics, side effects, and current usage of TMZ, and (2) de-velopments in analytical techniques for the determination of the drug in rawmaterial, pharmaceutical dosage forms, and biological samples.

Introduction

Trimetazidine (TMZ) dihydrochloride [1-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride, MW339.3; Fig. 1] is an effective, well-tolerated drug mainlyused in angina pectoris. TMZ is a dibasic compoundmarketed in a number of countries as a safe cellularantiischemic agent devoid of hemodynamic effects(Martindale 2005; The Merck Index 2001).

The neutral TMZ is a lipophilic weak base withpKa1 = 4.45 ± 0.02 and pKa2 = 9.14 ± 0.02 (Alemu 2004;Reymond el al. 1999).

The solubility of neutral TMZ is very low in aque-ous solution compared to single-protonated or double-protonated form in acidic solution. Its dihydrochloridesalt is freely soluble in water and sparingly soluble in al-cohol. In its protonated form, TMZ is slightly hygroscopic,white or almost white crystalline powder.

In addition to the vasodilatory effects on the coronaryarteries, beneficial results with TMZ treatment in patientswith ischemic heart disease and heart failure are well doc-umented. Favorable effects of TMZ are reported on otherischemic organs as well. Several mechanisms were sug-

gested to be responsible for these effects, such as a reduc-tion in reactive oxygen species, alteration in cellular lipidcomposition, and inhibition of mitochondrial fatty acidoxidation, as discussed below.

In this review, we will first focus on the pharmacolog-ical effects and mechanism of these effects. Next, we willsummarize the developments in analytical techniques forthe determination of TMZ in raw material, pharmaceuti-cal dosage forms, and biological samples.

Pharmacological Effects

Effects on the Vasculature

Initial studies reported a decrease in coronary vascularresistance and an increase in coronary blood flow withthe usage of TMZ in normal animals and in animals withexperimental coronary sclerosis (Saito 1976; Yanagisawaet al. 1979). Vasodilatory effect of the drug was then con-firmed in subcutaneous tissue of the ear chamber in rab-bits (Asano et al. 1980). In an open-chest method in anes-thetized dogs, TMZ decreased systemic blood pressureand heart rate (Imai et al. 1977; Taira et al. 1980). This,

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Trimetazidine Revisited A. Onay-Besikci and S.A. OZkan

Figure 1 The structure of TMZ.

however, is not observed in later animal or human stud-ies, and TMZ is now accepted as an agent devoid of anyhemodynamic activities (see below). When applied to iso-lated coronary artery and mesenteric vein strips, TMZ in-duced dose-dependent relaxations in prostaglandin F2-precontracted vessels (Toda et al. 1982). We also ob-served an increase in coronary flow rate in rats chroni-cally treated with TMZ (Onay-Besikci et al. 2007).

TMZ completely abolished or reduced cyclic flow re-ductions by accumulating thrombus in the circumflexcoronary artery in open-chest anesthetized dogs withoutaffecting bleeding time (Belcher et al. 1993).

The effects on membrane dynamics were investigatedin human platelets. TMZ reduced cAMP content (in thepresence of Ro 15-2041, a phosphodiesterase inhibitor)and the aggregation responses to collagen and ADP. Theauthors suggested that TMZ decreased the “fluidity” ofthe outer part of the plasma membrane, the adenylyl cy-clase activity, and some steps involved in platelet activa-tion (Devynck et al. 1993).

Cardiac Effects and Mechanism of Action

In addition to its vasodilatory effects on the coronaries,TMZ has direct beneficiary effects on the ischemic heart.Earlier studies reported a depression of SA node auto-maticity, prolongation of AV conduction time, and de-pression of force contraction in addition to an increasein coronary blood flow in blood-perfused heart prepara-tions (Yanagisawa et al. 1979). A depression of contrac-tion in both cardiac and skeletal muscles and blockadeof nicotinic transmission in ganglia cells were shown infrogs with a high dose of TMZ treatment (Minota andKoketsu 1976). High dose of TMZ suppressed halothane–adrenaline arrhythmia (Komori et al. 1985). In isolatedventricular myocytes, TMZ decreased the action poten-tial duration and peak amplitude of Ca2+ current and thiseffect was suggested to protect the cardiac cells from ac-cumulation of Ca ions in ischemic hearts (Kiyosue et al.1986).

Intriguing results came from the ischemia-reperfusionstudies on isolated hearts. 31P-NMR spectroscopy, fol-lowing Langendorff perfusion of rat hearts, showed that

when added at the beginning of the perfusion, TMZ dose-dependently reduced intracellular acidosis at the end ofthe 24-min low-flow ischemia when compared to con-trol hearts and accelerated the restoration of ATP/Pi ra-tios. TMZ was suggested to decrease ischemia-inducedintracellular acidosis (Lavanchy et al. 1987). TMZ treat-ment also decreased the accumulation of Na+ and Ca2+

inside cardiac cells and reduced intracellular cell acido-sis under acidic conditions (Renaud 1988). Addition ofTMZ to the perfusate of isolated hearts significantly re-duced intracellular acidosis during low-flow ischemia anddecreased intracellular accumulation of Na+ during to-tal ischemia. Moreover, TMZ significantly improved re-covery of ventricular function during reperfusion. There-fore, the authors proposed that the beneficial effects ofTMZ were related to an improvement in ionic balance ofthe myocardium (El Banani et al. 2000). When added tonormothermic cardioplegic solution, TMZ induced a bet-ter recovery of cardiac output and coronary flow dur-ing reperfusion of ischemic rat hearts without affectingthe recovery of heart rate or aortic pressure (Rahmanet al. 1989). The effects of pretreatment with TMZ on car-diac function and on high-energy phosphate content af-ter global ischemia were determined in the Langendorffheart, the working heart, and the heart-lung preparationof the guinea pig. TMZ markedly improved the recoveryof cardiac mechanical parameters in all three models ofmyocardial ischemia (Hugtenburg et al. 1989). Beneficialeffects on biochemical parameters (CK-MB, cTnT) of TMZpretreatment and acute addition of TMZ in the perfusatewere also observed in type 1 diabetic rats (Ikizler et al.2006).

Several mechanisms were suggested to be responsiblefor the beneficial actions of the drug in both ischemicand aerobic hearts. An increase in ventricular ATP con-tent, ATP/Pi ratio, and pH were reported when isolatedrat hearts were arrested and preserved for long termin a cardioplegic solution with TMZ when compared topreservation in simple mineral solution (Aussedat et al.1993; Kay et al. 1995). Pretreatment of rabbits with TMZreduced myocardial infarct size following a 45-min pe-riod of coronary occlusion (Drake-Holland et al. 1993).Beneficial effects of TMZ on ischemic contracture werereported in isolated rat hearts, as shown by a better postis-chemic recovery of developed pressure (Allibardi et al.1998; Boucher et al. 1994).

The effect of TMZ in reducing reperfusion arrhyth-mias was evaluated in isolated rat hearts and in patientswith acute myocardial infarction (AMI) after thromboly-sis. TMZ successfully reduced the rate of reperfusion ar-rythmias (Di Pasquale et al. 1999; Kara et al. 2004; 2006;Papadopoulos et al. 1996). Addition of TMZ treatmentto primary angioplasty in AMI patients resulted in an

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fatty acyl CoA

carnitine

carnitine

CPT I

CAT

CPT II

fattyacids

ß-oxidation

fatty acyl CoA

trans-Δ2-enoyl CoA

3-hydroxyacyl CoA

3-ketoacyl CoA

acyl CoA dehydrogenase

enoyl CoA hydratase

3-hydroxyacyl CoA dehydrogenase

3-ketoacyl CoA thiolase (3-KAT)

acetyl CoA

Figure 2 Fatty acid transport into mitochondria and β-oxidation.

Fatty acids are transported into the cytoplasm and activated to their CoA

esters. The acyl CoA enters mitochondria as fatty acylcarnitine through

an enzyme system (carnitine palmitoyltransferase I [CPT I], carnitine acyl-

carnitine translocase [CAT], carnitine palmitoyltransferase II [CPT II]). The

intramitochondrial acyl CoA then enters β-oxidation spiral, the last step of

which is 3-keto acyl CoA thiolase (3-KAT), the enzyme that is inhibited by

TMZ. The inhibition of 3-KAT results in a reduction in mitochondrial fatty

acid oxidation. As the energy production from fatty acids will be limited,

other substrates such as glucose will be used for energy production.

earlier resolution of ST-segment elevation when com-pared to patients receiving placebo (Guler et al. 2003;Steg et al. 2001), and lower QT and corrected QT (QTc)dispersion were reported after AMI in patients receiv-ing other drugs (Kountouris et al. 2001). In AMI pa-tients treated with streptokinase, TMZ induced a sig-nificant reduction in plasma C-reactive protein (CRP)compared to the group of patients without TMZ treat-ment (Pudil et al. 2001). Hypoxia-induced arrhythmiasand worsening of electrophysiologic profile did not oc-cur in the presence of TMZ in rat cardiomyocytes (Fan-tini et al. 1997). On the other hand, TMZ had no effectin ischemia-reperfusion arrythmias in rats (Iskit and Guc1996). Long-term oral treatment with TMZ efficientlydecreased Ca2+ overload and hypertrophy in cardiomy-opathic Syrian hamster (CMH) of the strain BIO 14:6,which is a model for both cardiac and skeletal muscle ab-normalities, to the levels of normal Syrian hamsters (F1B)(D’hahan et al. 1997). The same authors later attemptedto prevent the redistribution of myosin phenotype fromV1 to V3 with TMZ and a Ca2+ channel blocker, vera-

pamil, but failed to show an association between the ef-fects of the drugs on hypertrophy and on myosin isoforms(D’hahan et al. 1998). The authors concluded that thepositive effects with long-term TMZ therapy might be re-lated to the maintenance of V3 isoform that has a lowerATPase activity and thus utilizes less energy through anunknown mechanism (D’hahan et al. 1998).

The possible effect of TMZ on lipid metabolism ledSentex et al. (1997) to investigate the effects of the drugon cardiac lipid composition and phospholipid turnover.Treatment with TMZ induced a significant decrease inphospholipid linoleic acid with a small increase in oleicand stearic acids (Sentex et al. 1997). The same grouplater investigated the influence of TMZ on complex lipidsynthesis from (2-[3]H) glycerol in ventricular myocytes,isolated rat hearts, and in vivo in the myocardium andseveral other tissues. They reported that TMZ stimu-lated phospholipid synthesis and reduced incorporationof glycerol in nonphosphorous lipids (Sentex et al. 2001).Moreover, TMZ was reported to accelerate the turnoverof phosphatidyl inositol (PI)s through the activation of

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PI synthase in cardiomyocyte membranes and, therefore,suggested to reduce the availability of inositide phos-phates (IPs) and diacylglycerol (DAG) involved in theinduction of cardiomyocyte hypertrophy resulting fromchronic α-adrenergic stimulation (Tabbi-Anneni et al.2003a). The same group later reported that TMZ treat-ment in a pressure-overload model of ischemia-reducedplasma levels of brain natriuretic peptide (BNP), whichis a marker of the severity of heart failure, prevented thedownregulation or desensitization of adrenergic receptorsand cardiac hypertrophy (Tabbi-Anneni et al. 2003b).

Fantini et al. (1994) reported that during hypoxiaTMZ reduced lactate dehydrogenase (LDH) leakage inrat cardiomyocytes. One very important finding of thisstudy was that TMZ resulted in an inhibition of palmi-toylcarnitine oxidation when added to the medium. Aslight increase in pyruvate oxidation with chronic treat-ment was also reported in the same study (Fantini et al.1994). The inhibitory effect of TMZ on fatty acid oxida-tion was further supported in ischemia-reperfusion stud-ies in rat hearts. Ischemia-induced increase in acyl car-nitine levels—an indicator of fatty acid utilization—wassignificantly decreased with acute TMZ treatment alongwith a lower reduction in the intracellular pH during is-chemia (El Banani et al. 1998). Also, TMZ increased glu-cose utilization in dogs undergoing open-chest left an-terior descending coronary artery (LAD) ligation, as im-aged by quantitative positron emission tomography (PET)(Mody et al. 1998). A possible direct effect on carnitinepalmitoyltransferase I (CPT I) activity was excluded byKennedy and Horowitz due to the relatively low potencyof the drug to inhibit myocardial CPT I (Kennedy andHorowitz 1998). The strongest evidence for the mecha-nism of action of TMZ was the decreased rates of fatty acidoxidation in isolated rat hearts (Kantor et al. 2000). Thedecrease in cardiac fatty acid oxidation was accompaniedby a significant decrease in the activity of the long-chainisoform of the last enzyme involved in mitochondrial β-oxidation, 3-ketoacyl coenzyme A (CoA) thiolase activity(Fig. 2) (3-KAT) (Kantor et al. 2000).

MacInnes et al. (2003) reported that TMZ did not in-hibit 3-KAT in rat mitochondria, or any component of β-oxidation, in an isolated human cardiomyocyte cell line(MacInnes et al. 2003). The discrepancy between the twostudies seems to be the result of reversible competitiveinhibition of 3-KAT by TMZ. In other words, high levelsof 3-KAT substrate, as used by MacInnes et al. (MacInneset al. 2003), overcame the 3-KAT inhibition induced byTMZ, as reported later by Lopaschuk et al. (2003). Theeffects of TMZ on function, glycolysis, and oxidation ofglucose, lactate, and palmitate were measured before andafter global no-flow ischemia in isolated working heartsfrom control and hypertrophied rats. Heart function was

significantly improved by TMZ after ischemia to valuesin untreated controls and in hypertrophied hearts. Sur-prisingly, this was associated with a reduction in glycol-ysis and only minor alterations in oxidative metabolism(Saeedi et al. 2005). Treatment with TMZ, single dose orfor 14 days, increased the uptake of radioactive glucoseinto rat brain (Nowak et al. 2006).

TMZ, when added to the perfusate before ischemia,significantly reduced neutrophil-mediated cardiac reper-fusion injury, as assessed by the recovery of developedpressure (Tritto et al. 2005).

Mitochondrial permeability transition pore (mPTP)opening is detrimental to the heart and induces car-diomyocyte death following acute ischemia. A possiblemodulatory effect of TMZ on mPTP opening was inves-tigated by Argaud et al. (2005). The authors showedthat pretreatment of rabbits by TMZ reduced mPTPopening and this was associated with a reduction inboth infarct size and apoptosis after ischemia-reperfusion(Argaud et al. 2005).

Treatment of rats with TMZ abolished isoprenaline-induced myocardial damage, preserved the ATP levels,and decreased malondialdehyde (MDA) content in thehearts. Examinations of isoprenaline-treated cardiomy-ocytes showed that TMZ also prevented the increase indiastolic Ca2+ ions and prevented the decrease in SR Ca2+

content, sarcoplasmic reticulum (SR) Ca2+-ATPase activ-ity, and L-type Ca2+ current (Meng et al. 2006).

In rat hearts perfused without fatty acids in Langen-dorff setting and subjected to ischemia, TMZ increasedcomplex I activity, decreased O2 consumption, and was,thus, suggested to decrease oxygen free radical produc-tion, preserve mitochondrial integrity, and maintain elec-trical potential (Monteiro et al. 2004). The authors statedthat by excluding the confounding effects of fatty acids,they were able to determine the influence of TMZ on themitochondrial function of ischemic rat hearts (Monteiroet al. 2004). However, fatty acids are the preferred sourceof energy of the heart in many species, and results fromstudies where fatty acids were not included in the per-fusate should be interpreted with caution.

The function and ascorbyl free radical (AFR) releaseof rat hearts subjected to ischemia and reperfusion werecompared to palmitate or glucose as energy substrate andthe effect of TMZ was evaluated (Gambert et al. 2006).Postischemic recovery in the palmitate group was lowerthan in the glucose group, and TMZ decreased diastolictension in both groups. AFR release was higher dur-ing reperfusion of palmitate-perfused hearts, but TMZreduced AFR release in both palmitate- and glucose-perfused hearts (Gambert et al. 2006).

Short-term TMZ lowered fasting plasma glucose indiabetic rats (Cano et al. 2003). We have not been able to

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show a reduction in glycemia of diabetic rats with chronicTMZ treatment. However, we reported that the mRNAlevels of 3-KAT, the mitochondrial enzyme inhibited byTMZ, were higher in diabetic rat hearts when comparedto control rat hearts (Onay-Besikci et al. 2007). Coro-nary flow rates were also higher in nondiabetic rat heartstreated with TMZ (Onay-Besikci et al. 2007)

As TMZ acts by increasing cell tolerance to ischemiaand adenosine is one mediator of ischemic precondition-ing, a possible interaction between TMZ and adenosinelevels was investigated. TMZ administration increasedadenosine plasma levels in patients with angina pectoris.The authors suggested that the activity of TMZ couldpartially be mediated through adenosine-related precon-ditioning (Blardi et al. 2002). In relation to that, bothacute and chronic TMZ treatments were shown to pre-serve the effects of ischemic and pharmacological (car-bachol) preconditioning in anesthetized rats (Kara et al.2004; 2006). On the other hand, Minners et al. (2000)reported that TMZ limited the effect of ischemic precon-ditioning and completely reversed the DNP, CSA, and theadenosine-mediated reduction in infarct size in a modelof regional ischemia in Langendorff-perfused isolated rathearts (Minners et al. 2000).

Interestingly, chronic TMZ treatment of rats withinjury-induced heart failure reduced the upregulation ofatrial natriuretic peptide, as indicated by lower mRNAlevels, compared to untreated rats, without affecting leftventricular function or expression of fatty acid oxidationenzymes (Morgan et al. 2006).

Possible effects of TMZ on endothelial function wereinvestigated in humans and animals. Addition of TMZ in-creased NO release, blunted endothelin-1 (ET-1) release,and protected myocardial function during recovery ofischemic rat hearts (Monti et al. 2001). In patients withstable CAD, TMZ reduced plasma ET-1 levels without af-fecting plasma NO (Fragasso et al. 2002). Di Napoli et al.(2007) recently reported that the beneficial effects of TMZwere NO-mediated because TMZ treatment resulted in anincreased coronary flow and eNOS expression in bothmRNA and protein level. Also, L-NAME, a specific in-hibitor of NO synthase, prevented the beneficial effects ofTMZ on cardiac function (Di Napoli et al. 2007a). Morerecently, short-term TMZ therapy was shown to improvethe parameters related to heart rate variability and angi-nal symptoms, reduced ET-1, and increased NO in pa-tients with slow coronary artery flow (Topal et al. 2006).

Clinical Outcome

Ischemic heart disease. Clinical studies demonstrated thatTMZ is an effective antianginal drug (as a single drug orin combination with other conventional drugs) and does

not alter hemodynamic parameters. A 6-month, double-blind, placebo-controlled study was carried out in 20patients with severe ischemic cardiomyopathy. TMZ in-creased ejection fraction, decreased cardiac volume, andimproved clinical status of patients without adverse ef-fects (Brottier et al. 1990). TMZ and nifedipine were com-pared in a double-blind, cross-over study in patients witheffort angina. TMZ did not affect rate–pressure product,while nifedipine decreased this parameter, indicating adifference in mechanism of action between the two drugs(Dalla-Volta et al. 1990). No alteration in hemodynamicparameters with TMZ treatment was confirmed by otherstudies (Ozdemir et al. 1999; Pornin et al. 1994; Szwedet al. 2001). The Trimetazidine in Angina CombinationTherapy (TACT) showed that TMZ in combination withnitrates or β-blockers improved anginal symptoms andduration of exercise (Chazov et al. 2005). Trimetazidinein Poland-Second (TRIMPOL II) reported better clinicalsymptoms such as a longer “time to 1-mm ST-segmentdepression,” prolonged time to onset of angina, maxi-mum ST-segment depression, lower number of anginaattacks, and less weekly nitrate consumption in patientswho received TMZ in addition to metoprolol (Szwed et al.2001). Moreover, there was no evidence of any develop-ment of tolerance to TMZ (Szwed et al. 2001). Other stud-ies reported prolonged ischemic threshold (Levy 1995)and few anginal symptoms (Manchanda 2003; Man-chanda and Krishnaswami 1997; Szwed et al. 2001) withTMZ. The report from a multicenter, double-blind studyshowed that the antianginal efficacies were similar be-tween TMZ and propranolol, but TMZ did not alter hemo-dynamic parameters of patients with stable angina (Detryet al. 1994).

Addition of TMZ to conventional treatment for 18months improved left ventricular ejection fraction andmaintained CRP plasma levels in patients with ischemicdilated cardiomyopathy in Villa Pini d’Abruzzo Trimetazi-dine Trial (Di Napoli et al. 2005). This study was extendedto 48 months and the results were reported recently (DiNapoli et al. 2007). The distribution of the patients inNew York Heart Association (NYHA) functional classifica-tion and exercise capacity (6-min walk test) significantlyimproved with TMZ. TMZ reduced all-cause mortalityand improved left ventricular function, as indicated withan increased ejection fraction (LVEF) and decreased end-systolic or end-diastolic volumes (Di Napoli et al. 2007).

Antiischemic efficacy and tolerability of TMZ was as-sessed in a multicenter study (TRIMPOL) in diabetic pa-tients also. The results showed a significant improve-ment in exercise tolerance, time to 1-mm ST-segment de-pression, time to onset of anginal pain, a significant de-crease in the mean frequency of anginal episodes, andin mean nitrate consumption when TMZ was added to

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conventional antianginal monotherapy with a long-acting nitrate, β-blocker, or calcium channel blocker(Szwed et al. 1999). Similarly, short-term TMZ treatmentof patients with diabetes and ischemic cardiomyopathyimproved left ventricular function (Fragasso et al. 2003;Rosano et al. 2003), glucose metabolism, and endothelialfunction (Fragasso et al. 2003).Heart failure. TMZ improved functional class and LVfunction in patients with heart failure associated with anincrease in the PCr/ATP ratio, as shown in a double-blind,cross-over study with TMZ and placebo (Fragasso et al.2006). Two-month treatment of patients with chronicventricular dysfunction (postnecrotic left ventricular dys-function + CAD) with TMZ in addition to conventionaltreatment improved resting and peak systolic wall thick-ening score index, ejection fraction, and peak VO2, with-out concomitant changes in heart rate and blood pressure(Belardinelli and Purcaro 2001).

Cardioprotective effect of TMZ during coronary arterygraft surgery was assessed by treatment of patients withthe drug for 3 weeks before the surgery and by in-cluding TMZ to cardioplegic solution. Patients pretreatedwith TMZ had a better ventricular function comparedto patients treated with placebo (Fabiani et al. 1992).TMZ reduced the severity of myocardial dysfunction afterdobutamine undergoing dobutamine-echocardiography(Lu et al. 1998). TMZ protected myocardium and im-proved cardiac function during percutaneous translumi-nal coronary angioplasty (PTCA) without altering heartrate, systemic, or intracoronary pressures (Kober et al.1992; Polonski et al. 2002). Similarly, treatment of pa-tients with CAD after PTCA-reduced sympathetic overac-tivity and augmented vagal influences improved left ven-tricular contractility and diminished the clinical picture ofischemia (Birand et al. 1997). Treatment of patients be-fore PTCA significantly suppressed the elevation of tumornecrosis factor-alpha (TNF-alpha), CRP, and NO productsmeasured just before and shortly after PTCA (Kuralayet al. 2006). This finding suggested that TMZ also pre-vented inflammatory cardiovascular events after PTCAby an unknown mechanism (Kuralay et al. 2006). Sim-ilar beneficial effects of TMZ were observed in coronaryartery ectasia patients, as assessed by treadmill exercisetest (Dogan et al. 2003).

In a group of patients with a history of PTCA or CABG,12-week treatment with TMZ in addition to β-blockersignificantly improved time to 1-mm ST-segment depres-sion, exercise test duration, total workload, and time toonset of angina. Weekly number of angina attacks andnitrate consumption were significantly reduced in theTMZ group when compared to placebo (Ruzyllo et al.2004). Opposite results were obtained in patients withsyndrome X, reporting better (Rogacka et al. 2000) and

worse (Leonardo et al. 1999) exercise tolerance with TMZtreatment.

Antiischemic Effects on Other Organs

Cyclosporine, an immunosupressive drug commonlyused in organ transplantation, is associated with nephro-toxicity. A possible protective effect of TMZ was tested incanine single-kidney model of cyclosporine-induced is-chemic renal failure. TMZ treatment resulted in improvedbiochemical and histological markers of renal functionand structure (Creagh et al. 1993; Satyanarayana andChopra 2002). TMZ also reversed cyclosporine-inducedaccumulation of Ca2+ and decrease in oxidative phospho-rylation in rat liver mitochondria in a dose-dependentmanner (Salducci et al. 1996). Somewhat similarly,Ca-induced inhibition of mitochondrial respiratory con-trol ratio (RCR) was partially restored by TMZ in rat brainmitochondria (Zini et al. 1996).

As the main target of the drug seemed to be mitochon-dria, possible binding sites of TMZ in the mitochondriawere investigated. Two populations of binding sites lo-cated on the outer and inner membranes of purified ratliver and brain mitochondria were reported (Morin et al.1998; 2000). TMZ inhibited the mitochondrial swellinginduced by Ca2+ + t-BH (Elimadi et al. 1997; Morin et al.1998), and there was strong correlation between low-affinity binding sites and the inhibitory effect of the drug(Morin et al. 1998). TMZ binding was highly affected bypH, and an endogenous cytosolic ligand—most probablya protein—was reported to displace TMZ from its bindingsites (Morin et al. 2000).

Mitochondrial alterations were investigated in a dogmodel of ischemic preconditioning pretreated with TMZor placebo (Morillas Blasco et al. 2005). TMZ did not af-fect mitochondrial damage induced by ischemic episodes,but altered the morphology of mitochondria in bothcontrol and ischemic zones. In the nonischemic zone,TMZ produced an increase in mitochondrial turnover(Morillas Blasco et al. 2005).

Streptozotocin-induced insulin deficiency decreasedmitochondrial affinity for ADP, the index of creatine ki-nase functional activity, and decreased n-3 polyunsatu-rated fatty acids in rat heart mitochondria. When TMZwas added into the diet, the decrease in creatine kinaseactivity and the increase in n-6/n-3 ratio in mitochondrialmembranes were partially prevented (Ovide-Bordeauxet al. 2005). This study supported that TMZ, althoughpartially, maintained mitochondrial homeostasis ininsulin deficiency.

Cytoprotective effects of TMZ in the setting of ischemiawere also tested during renal transplantation, wheredelayed graft function is a major problem and increases

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the posttransplant morbidity and hospital costs. The ad-dition of TMZ to cold-storage solutions significantly im-proved both the histological data and the function ofkidney during reperfusion after transplantation (Baumertet al. 1999, 2004; Faure et al. 2003; Hauet et al. 1997a,1997b, 1998b, 1998c). This effect was suggested to be re-lated to lower renal vascular resistance, interstitial edema(Hauet et al. 1997a, 1997b, 1998d), and less lipid peroxi-dation (Hauet et al. 1997b, 1998a) with TMZ. It was laterreported that there was also a greater excretion of citratein TMZ-supplemented groups, and an extensive reduc-tion in apical brush border of proximal tubular cells wasnoted in TMZ-free groups (Hauet et al. 2000a). Whenadded to storage solutions, TMZ reduced interstitial andperitubular inflammation and the numbers of infiltrat-ing mononuclear CD45+ and CD3+ T cells of pig kidneys(Goujon et al. 2000; Hauet et al. 2000b).

The protective effect of TMZ after prolonged ischemiain lung transplantation was investigated in rats (Inci et al.2001). Recipient treatment with TMZ protected tissueamounts of ATP, provided better oxygenation, and re-duced lipid peroxidation (Inci et al. 2001).

Ischemia-induced elevation of MDA levels was sig-nificantly reduced after TMZ pretreatment in rat kid-neys (Grekas et al. 1996). In a rat model of re-nal ischemia-reperfusion, clamping of the left renalpedicle and subsequent 24-h reperfusion increasedthe levels of creatinine and tissue MDA, inhibitedglutathione peroxidase (GSH-Px) activity, and inducedsevere acute tubular necrosis. Pretreatment of rats re-sulted in a decrease in the levels of creatinine and tissueMDA, lower tubular necrosis, and a significant increase inthe activity of the GSH-Px compared to the control group(Ozden et al. 1998). In the same model, pretreatment ofrats lowered lipid peroxidation and improved renal func-tion (as assessed by serum creatinine, blood urea nitro-gen, and creatinine and urea clearance) and morphology(Kaur et al. 2003). It was, thus, concluded that by its an-tioxidant properties, TMZ was able to protect the kidneysfrom ischemia-reperfusion-induced injury.

Deleterious effects of ischemia and reperfusion such asmembrane leakage, decrease in ATP content, bile flow,NAD(P)H level, activities of hepatic enzymes, mitochon-drial membrane potential, and generation of mitochon-drial permeability transition were observed in rat liver(Elimadi et al. 1998; Settaf et al. 1999). Pretreatment ofrats with TMZ prevented these alterations (Elimadi et al.1998; Settaf et al. 1999). Also, pretreatment with TMZbefore hepatic flow occlusion for partial hepatectomy re-duced liver injury and improved liver regeneration andsurvival rate (Kaya et al. 2003). Similar beneficial effectswere reported with the addition of TMZ into the storagesolution of the liver (Ben Mosbah et al. 2006).

Pretreatment with TMZ for 5 days protected ischemicrat livers, as shown by the hepatic enzymes in the bloodand reduction in centrilobular necrosis of hepatocytesfrom injury, after a period of warm ischemia and reper-fusion (Tsimoyiannis et al. 1993).

The protective effect of TMZ from ischemia-reperfusioninjury was investigated in the intestines in rats (Tetiket al. 1999). Ischemia-reperfusion increased MDA lev-els, myeloperoxidase (MPO) activity, and induced mu-cosal damage in the sham-operated group. TMZ pretreat-ment lowered MDA levels and MPO activity and reducedhistopathological damage. Accumulation of lipid peroxi-dation products and neutrophils in mucosal tissues weresignificantly inhibited by TMZ treatment also (Tetik et al.1999).

Retinal injury induced by ischemia and reperfusionwas nearly completely reversed by TMZ pretreatment(Mohand-Said et al. 2002; Ozden et al. 2001). Oral TMZtreatment in guinea pigs improved electroretinograms af-ter ischemic and exitotoxic insult (Payet et al. 2004). Thiseffect was mediated by a reduction in glutamate accumu-lation through the inhibition of glial transporter (Payetet al. 2004).

Effects on Reactive Oxygen Species

Antioxidant properties of TMZ are well documented andsome of these effects were mentioned in the previoussections. Oral TMZ treatment reduced lipid peroxidationof red cell membranes (Maridonneau-Parini and Harpey1985). Aggregation, serotonin release, and MDA pro-duction via cyclooxygenase and thromboxane A2 syn-thetase were investigated in rabbit platelets. TMZ attenu-ated the collagen-induced and ADP-induced aggregation.TMZ also decreased the MDA production induced by col-lagen and Ca-ionophore, A-23187, but did not decreasethe production induced by AA. Furthermore, TMZ dose-dependently decreased the MDA production induced byexogenous phospholipase A2 (Shirahase et al. 1988). Areduction in monocrotaline-induced cardiac mitochon-drial dysfunction was reported with TMZ treatment inrats in addition to a lower O. production and MDA con-tent (Guarnieri and Muscari 1988). Repeated administra-tion of TMZ improved the cardiac mitochondrial function,particularly in state 3 of respiration. In addition, the treat-ment with TMZ reduced, in the heart muscle, both theproduction of mitochondrial O. and the content of tissueMDA. TMZ added alone did not significantly change ei-ther the cardiac mitochondrial activity or the mitochon-drial O. production in comparison to control rats. A freeradical scavenger effect was observed when O. was gen-erated by active human neutrophils with high concen-trations of TMZ (Guarnieri and Muscari 1989). On the

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other hand, TMZ did not change either the levels of tis-sue MDA or lipofuscin in the aerobically perfused heart orthe rate of mitochondrial O. generation while it reducedthe O. formation and MDA content in control hearts. Af-ter ischemia and reperfusion, the drug reproduced theseeffects in the hypertrophied hearts and reduced the levelof tissue MDA in control hearts (Guarnieri and Muscari1993).

Pretreatment with TMZ and addition of the drug to thecardioplegic solution reduced oxygen free radical damageas shown by a reduced release of MDA. Moreover, pre-treatment with TMZ enabled patients to undergo surgerywith better left ventricular function (Fabiani et al. 1993).

Cochleovestibular dysfunctions induced by phenazinemethosulfate (PMS) administration were prevented byTMZ. The authors suggested that PMS stimulated oxy-gen radical generation, and TMZ—with its antioxidantproperties—reversed this process (Aubert et al. 1989).

Direct and indirect measurement of free radical forma-tion in isolated rat hearts showed that ischemia-inducedincrease in free radicals was lower when the hearts weretreated with TMZ before the onset of ischemia (Maupoilet al. 1990).

In a rabbit model of ischemia-reperfusion, TMZ treat-ment significantly improved cardiac function and reducedapoptosis, with a concomitant increase in SOD activityand decrease in MDA levels (Ruixing et al. 2007).

Chronic cor pulmonale (CCP) was suggested to in-volve oxidative stress in its pathogenesis. CCP patientswith higher MDA and lower antioxidant enzyme (cata-lase, erythrocyte and plasma glutathione peroxidase) ac-tivities were randomly selected to receive routine treat-ment or routine treatment plus TMZ. After 3 monthsof therapy, addition of TMZ lowered MDA levels andincreased antioxidant enzyme activities (Bayram et al.2005). Whether TMZ treatment improved the clinical pic-ture in CCP patients was not reported (Bayram et al.2005).

Side Effects

Drug-induced parkinsonism and tremors were detectedin patients treated with TMZ (Martı Masso et al. 2005).It’s important to note that some of these patients were si-multaneously receiving other drugs potentially capable ofinducing parkinsonism. Treatment with TMZ worsenedpreviously diagnosed Parkinson’s disease and gait disor-ders in some patients (Martı Masso et al. 2005). Other mi-nor adverse effects (episodes of headache) were reported:most of which were not considered to be related to TMZ(Barre et al. 2003)

Pharmacokinetics

Pharmacokinetic profile of TMZ was studied and re-viewed before (McClellan and Plosker 1999). The devel-opment of a modified-release (MR) formulation aimed tomaintain the therapeutic plasma concentration with lessfluctuation, a higher Cmin compared to immediate releasefrom (20 mg × 3) and only twice-daily application (35 mg× 2) (Barre et al. 2003; Genissel et al. 2004; Sellier andBroustet 2003). T1/2 was reported ∼8 h in young volun-teers (25 ± 8) and ∼12 h in elderly (72 ± 4) (Barre et al.2003). Cmax was 91.2 μg/L and AUC0−12 was 720 μg/h/Lin young volunteers (Barre et al. 2003). These parameterswere significantly higher in elderly (115μg/L and 1104μg/h/L, respectively) (Barre et al. 2003).

Analysis

The development of new methods capable of determin-ing drug concentration with related validation parame-ters in pharmaceutical dosage forms and biological sam-ples is important. The increasing interest in TMZ led usto review the methods reported for its determination inraw material, pharmaceutical dosage forms, and biologi-cal fluids in addition to the pharmacological effects.

Spectrophotometric Methods

A thorough literature search has revealed that a largenumber of spectrophotometric (ultraviolet or visible)methods were reported for TMZ. It has been analyzed bythe direct UV spectrophotometric method in bulk and intablet dosage form (Krishnamoorthy and Ganesh 2001).In this method, TMZ showed maximum absorbanceat 270 nm and linearity range was obtained between400 and 700 μg/mL. This direct UV spectrophotomet-ric method was validated and successfully applied to thedetermination of TMZ in pharmaceutical dosage forms(Krishnamoorthy and Ganesh 2001)

Murthy et al. (2002a,b) described two simple and sensi-tive visible spectrophotometric methods for the determi-nation of TMZ in raw material and dosage forms. The firstspectrophotometric method was based on oxidative cou-pling between 3-methyl-2-benzothiazolinone hydrazinehydrochloride and TMZ in the presence of ceric am-monium sulphate. The colored sample was measured at520 nm. The second method was based on the oxida-tion/reduction reaction between Folinciocalteu reagentand TMZ. The colored sample was measured at 745 nm.Both methods have been optimized and necessary vali-dation parameters were calculated (Murthy et al. 2002a).Same authors (Murthy et al. 2002b) later developed twoother UV and visible spectrophotometric methods for the

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determination of TMZ in pure or in dosage forms. In thesestudies, o-phenanthroline or potassium ferricyanide wasused in the presence of ferric chloride for obtaining thecolored product. The colored samples were measured at510 nm or 760 nm depending on the agent (Murthy et al.2002b).

Some laboratories developed methods for the deter-mination of TMZ by complexing it with other reagents(Issa et al. 2002a). TMZ was determined with two com-plexation techniques in pharmaceutical dosage forms.The first method was based on the occurring charge-transfer complex with iodine as σ -acceptor in dry 1,2-dichloroethane. The absorbance of the yellow-coloredcomplex was measured at 364 nm. The second methodwas based on the reaction between TMZ and bromcresolgreen in dry 1,2 dichloroethane. The absorbance of thiscomplex was measured at 408 nm (Issa et al. 2002a).Both methods were successfully applied to the determi-nation of TMZ in dosage forms (Issa et al. 2002a). Thesame authors (Issa et al. 2002b) later described threespectrophotometric methods for the determination ofTMZ. The first method involved the use of 2,3-dichloro-5,6-dicyano-p-benzoquinone, the second was based onthe use of 7,7,8,8-tetracyano-quinodimethane, and thethird method was based on the use of p-chloranil asπ-acceptors to give highly colored complex. The col-ored samples were measured in the visible region. Af-ter optimization and validation (Issa et al. 2002b), allthree methods were successfully applied for the deter-mination of TMZ in tablet dosage form. The associationconstants were also calculated and reported (Issa et al.2002b).

The reaction of piperazine derivatives with iron (III)ions to produce yellow orange complexes was exhaus-tively utilized by Abou-Attia et al. (2003) to developmethods for TMZ determination. The complexation be-havior, stability constants, and optimum reaction condi-tion were investigated in this reported study (Abou-Attiaet al. 2003).

Three different spectrophotometric methods have beendescribed for the determination of TMZ in the presenceof its acid-induced degradation products (Bebawy et al.2004). The first method was based on measurement offirst derivative absorbance value at 282 nm. The sec-ond was based on first derivative of the ratio spectraat the same wavelength. The third method was relatedto the separation of TMZ from its acid-induced degrada-tion products followed by densitometric measurement ofthe spots at 215 nm. All proposed methods were suc-cessfully applied for the determination of TMZ in rawmaterial, laboratory-made mixtures, and pharmaceuticaldosage forms. The validations of the proposed methodswere also done (Bebawy et al. 2004).

Darwish developed four simple and sensitive ki-netic spectrophotometric techniques for the determina-tion of TMZ (Darwish 2005). These techniques werebased on oxidation with alkaline KMnO4, couplingwith 4-chloro-7-nitrobenzofuron, condensation with1,2-naphthoquinone-4-sulphonic acid sodium salt, andcharge-transfer complexation with p-chloranil. The ab-sorbance of the final colored product was then measuredat different wavelengths (Darwish 2005). The analyti-cal performance of these methods were calculated andreported. These methods were successfully applied forthe determination of TMZ in commercial tablet dosageforms.

Chromatographic Methods

Analytical laboratories face the continual challengeof balancing investment in new technology—to highthroughput and performance—with the need to run ex-isting methods and tests to support current production(Adamovics 1997; Ahuja and Scypinski 2001; Christian2004; Ohannesian and Streeter 2002; Synder et al. 1997).High-performance liquid chromatography (HPLC) is oneof these key analytical separation technologies.

HPLC Methods

The applicability of HPLC to the pharmaceutical indus-try and biological samples made it the mainstay of phar-maceutical research development and independent re-searcher laboratories in the 1970s (Adamovics 1997;Ahuja and Scypinski 2001; Christian 2004; Ohannesianand Streeter 2002; Synder et al. 1997). In the pharmaceu-tical industry, HPLC is employed throughout the wholedrug analysis process, including drug discovery screen-ing, raw material analysis, stability studies, impurity test-ing, pharmacokinetic studies, and final product testing.Several HPLC methods using different detectors for de-termination of TMZ were reported. The impressively in-creasing use of HPLC in drug analysis included TMZ also.Generally, reversed phase HPLC (RP-HPLC) was used inthese studies. Several HPLC methods with UV detectorare now available for the determination of TMZ.

Thoppil and Amin described the stability for RP-HPLC method for the determination of TMZ in tabletsand controlled release pellets. Water:acetonitrile: triethy-lamine mixture (90:10:0.1; v/v/v) adjusted to pH 3.3 witho-phosphoric acid was used as the mobile phase. C18 col-umn, 1.0 mL/min flow rate, and 270 nm wavelengthwere used (Thoppil and Amin 2001). The RSD% val-ues for concentrations for within-day and between-dayassays were reported for showing the sensitivity of theproposed method (Thoppil and Amin 2001). Another

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method utilizing reversed-phase liquid chromatography(RP-LC) and UV detection reported the determinationof TMZ in human plasma using liquid–liquid extractionfor sample cleanup procedure with 10 ng/mL sensitiv-ity (Jeoung et al. 2005). TMZ was extracted from plasmasamples with saturated K2CO3 solution into an ethylac-etate phase. This method was also applied to real sampleand for pharmacokinetic studies (Jeoung et al. 2005).

TMZ was determined in its tablet dosage form usingRP-LC and the measurement was carried out at 210nm wavelength with UV detector (Altiokka et al. 2006).Sensitivity was reported as 1.2 × 10−7 M. The authorsapplied their method for the direct analysis of TMZ intablet dosage forms (Altiokka et al. 2006). LC with elec-trochemical detector was used for the trace determina-tion of TMZ in human blood plasma. After extraction andevaporation steps, TMZ was detected at +1.1 V using 0.01M KH2PO4:0.05 M NaH2PO4:methanol (75:5:22; pH 3.2)mobile phase. The LOD value was obtained as 55 pg/mLin plasma. All necessary validation parameters were cal-culated and reported (Bari et al. 1999).

Courte and Bromet (1981) described the fluorescencedetection for the determination of TMZ in plasma atthe ng/mL level. The standard deviations of within-dayand between-day assays, calibration values, and limit ofsensitivity were reported in nanomolar concentrations.The sensitivity of the method was allowed for phar-macokinetic study in human after the extraction pro-cess (Courte and Bomet 1981). In the recently publishedwork, Khedr et al. (2007) described a LC with fluores-cence detection method for the sensitive determinationof TMZ in spiked human plasma after precolumn deriva-tization with 9-fluorenylmethyl chloroformate. The pro-posed method comprises the precolumn derivatizationwith 9-fluorenylmethyl chloroformate and injection ontoa reversed-phase column. TMZ was detected by a fluo-rescence detector using 265 nm excitation and 311 nmemission wavelengths. The sensitivity was reported as1.5 ng/mL. All necessary validation parameters weregiven (Khedr et al. 2007).

Several LC with mass spectrometric detection are avail-able for the determination of TMZ in biological samplesand bioequivalence study (de Jager et al. 2001; Dinget al. 2007; Jiao et al. 2007a, 2007b; Medvedovici et al.2005; Wang et al. 2007). These assays have adequatesensitivity within a relatively short analysis time. Wanget al. reported liquid–liquid extraction followed by LC-electrospray ionization mass spectrometry (LC-ESI-MS)to investigate the pharmacokinetic profiles and bioequiv-alence of TMZ (Wang et al. 2007). The mobile phaseconsisted of a mixture of methanol:formic acid (0.05%)(80:20; v/v) and the flow rate was 1.0 mL/min. Themethod was validated in the concentration range of 0.4–

120.0 ng/mL. The LOQ value was 0.4 ng/mL in a 0.5mL plasma sample (Wang et al. 2007). de Jager et al.(2001) proposed a rapid, selective, and sensitive LC-MS method for the determination of TMZ. The methodwas applied to the pharmacokinetic study of TMZ tabletdosage form. The liquid–liquid extraction procedure wasused for plasma samples. The linearity range was ob-tained as 1.51–383.00 ng/mL and the retention time was∼1.75 min (de Jager et al. 2001).

Medvedovici et al. (2005) developed and validated an-other method for the analysis of TMZ in plasma sam-ples using LC-APCI-MS/MS. The suggested sample prepa-ration procedure was simple, robust, and included onlyprotein precipitation with trifluoro acetic acid (TFA).Reversed-phase conditions with tandem mass spectro-metric detection were utilized. Ionization was realizedwith an atmospheric pressure chemical ionization inter-face. The suggested and validated method was used toassess the bioequivalence of two marketed immediate-release and MR dosage forms (Medvedovici et al. 2005).

Jiao et al. (2007b) described a convenient method forthe identification and quantification of TMZ in humanplasma using methanol as the protein precipitating agentfollowed by LC-MS. The described method did not re-quire any extraction or evaporation steps. Detection wasperformed on a single quadrupole mass spectrometerwith selected ion monitoring mode via electrospray ion-ization source (Jiao et al. 2007b). Ding et al. (2007) de-veloped a sensitive and selective LC-ESI-MS method forthe determination of TMZ in human plasma. The methodwas applied to a pharmacokinetic study of TMZ in healthyChinese volunteers. Following liquid–liquid extractionwith a mixture of cyclohexane-diethyl ether (1:1; v/v),LC separation was achieved on a phenomenex Luna C18(250 mm × 4.60 mm, 5 μm) column with a mobile phaseof 10 mM ammonium acetate buffer solution containing0.1% acetic acid-methanol (45:55; v/v) (Ding et al. 2007).Recently, Jiao et al. (2007a) developed a LC/ESI-MSmethod for the determination of TMZ in human plasmaand applied their method to a bioequivalence study onChinese volunteers. The chromatographic separationwas performed using an Xterra MS C18 (150 mm ×4.60 mm, 5 μm) column with a mobile phase consist-ing of methanol and water (40:60; v/v) adjusted to pH2.0. The single quadrupole mass spectrometer was oper-ated by selected ion monitoring mode. The LOQ valuewas obtained as 2.5 ng/mL. The method was fully vali-dated and the proposed method was successfully appliedto the bioequivalence study for its two kinds of tablets.The necessary pharmacokinetic values were determinedand reported in detail in this study. This study also indi-cated that these two products were bioequivalent in theChinese population (Jiao et al. 2007a).

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High-Performance Thin-Layer Chromatographic(HPTLC) Methods

Only one HPTLC method was found for the anal-ysis of TMZ in the literature (Thoppil et al. 2001).TMZ is separated from related impurities on pre-coated silica gel aluminium plate (60 F-254) usingn-buthanol:water:methanol:ammonia (20%)(14:0.2:0.2:2; v/v/v/v) mixture as mobile phase. Thespots were detected using densitometric scanner at 254nm. This method was developed for the determinationof the purity and the separation of TMZ with its relatedimpurities. The validated HPTLC method is precise,specific, accurate, and stable (Thoppil et al. 2001).

Gas Chromatographic (GC) Methods

Fay et al. (1989) described a gas chromatography-massspectrometric (GC-MS) method for the determination ofTMZ in biological fluids. The method involved a sim-ple extraction step for TMZ from human blood, plasma,and urine samples with dichloromethane after alkaliniza-tion and analysis by GC on fused-silica column of OV-1701 with helium as carrier gas at a 2 mL/min flow rate.The linear responses were obtained within 1–200 ng/mLrange for blood and plasma and within 0.5–10.0 μg/mLfor urine. Detection limit was as low as 1 ng/mL (Fay et al.1989). Barre et al. (2003) used heptafluorobutyric anhy-dride as a derivatizating agent after alkalinization of bi-ological sample. After derivatization, methylene chloridewas used for the extraction procedure. The evaporatedresidue was dissolved in ethylacetate and injected intothe gas chromatograph. Plasma and urine TMZ amountswere assayed using a nitrogen-phosphorus selective de-tector (NPD). The LOQ values were found as 10 μg/L and0.25 mg/L in plasma and urine samples, respectively. Theproposed method was successfully applied to obtain phar-macokinetic profile of MR formulation of TMZ in the el-derly and renal failure patients (Barre et al. 2003).

Electroanalytical Methods

The application of electroanalytical methods to the analy-sis of pharmaceuticals and biological samples has rapidlyexpanded since the recent developments of new, rapid,and sensitive voltammetric methods and new electrodematerials (Hart 1990; Ozkan 2007; Ozkan et al. 2003;Smyth and Vos 1992; Uslu and Ozkan 2007a; Wang2000). In electroanalytical methods, the redox reactionsoccurring at the electrodes are heterogeneous and takeplace in the interfacial region between the solution andthe electrode surface (Hart 1990; Ozkan 2007; Ozkanet al. 2003; Smyth and Vos 1992; Uslu and Ozkan 2007;

Wang 2000). They are very rapid methods that havebeen applied successfully to trace measurements of im-portant pharmaceuticals thanks to the high sensitivityand selectivity that they provide (Hart 1990; Ozkan 2007;Ozkan et al. 2003; Smyth and Vos 1992; Uslu and Ozkan2007; Wang 2000). An adsorptive stripping square wavevoltammetric method was described for the determina-tion of TMZ in tablets and spiked urine samples based onmeasuring the single and sharp peak in acetate buffer atpH 5.0 (Ghoneim et al. 2002). Adsorptive oxidation peakoccurred at 0.75 V (vs. Ag/AgCl). The oxidation peak waslinear between 5 × 10−8 and 5 × 10−6 M, with a detec-tion limit of 2 × 10−8 M, in raw material. The detectionlimit was also reported as 1.7 μg/mL for urine. In urinesamples, linear response was between 17 and 85 μg/mL.Cyclic voltammetric studies were also performed for theinvestigation of the process. The solution conditions, suchas pH, nature of the buffer, and instrumental parameters,were optimized for the determination of TMZ in tabletsand urine samples (Ghoneim et al. 2002).

Flow Injection Analysis Methods

A flow injection method using ion-selective electrode wasreported for the determination of TMZ (Issa et al. 2005).The useful pH ranges were 1.5–3.8 and 4.5–7.5 and theresponse was linear in the concentration range between3.2 × 10−5 and 1 × 10−2 M for TMZ. The selectivityof this proposed ion-selective electrode was investigated.It showed very good selectivity for TMZ with respect toa number of sugars, amino acids, and inorganic cations(Issa et al. 2005). TMZ amount was determined in phar-maceutical dosage forms under batch and flow injectionconditions. In this study, in addition to flow injectionanalysis method, the authors used conductometric titra-tion method and performed potentiometric analysis usinga conventional electrode and graphite-coated wire elec-trode under batch condition for comparison. No statisti-cal difference was found between this method and others(Issa et al. 2005).

Altiokka et al. (2006) used a direct flow injectionmethod with UV detection mode. As the solvent system,methanol:water (10:90; v/v) mixture was used. UV detec-tor was set at 210 nm. The sensitivity of this FIA methodwas 7.04 × 10−7 M. The proposed method was supportedby necessary validation parameters and was comparedwith the simple UV spectrophotomeric method, whichwas also proposed by the same authors (Altiokka et al.2006).

Chemiluminescence Method

TMZ was determined by the chemiluminogenic reac-tion in the presence of potassium permanganate in

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polyphosphoric acid solution as a catalyst (Palilis andCalokerinos 2000). This method was used for the deter-mination, with very low detection limits, of TMZ in bio-logical samples without any pretreatment procedure. Thelinearity was between 5 and 1000 ng/mL. The sensitiv-ity was 5 ng/mL. Recovery results also showed the appli-cability of this method to biological samples (Palilis andCalokerinos 2000).

Conclusion

Conventional treatment of ischemic heart disease in-cludes β-blockers, Ca channel blockers, and nitrates.These agents are expected to restore the imbalance be-tween the requirement and supply of oxygen of themyocardium, and are together called as “hemodynamicagents” as they also alter hemodynamic parameters. Un-fortunately, these “hemodynamic” agents cannot con-trol the symptoms consistently, and many patients needto undergo revascularization procedures despite carefultreatment. “Metabolic modulation” therefore seems to bethe missing component of the optimal treatment strat-egy in angina, and TMZ, by altering the substrate prefer-ence, is a promising agent in this new group of modulatordrugs. As for the analysis of the drug, an overview of thecurrent state-of-art analytical methods for the determina-tion of TMZ has been presented. The literature containsdifferent analytical techniques for the determination ofTMZ. Most of the reported methods are LC, which re-quire elaborate procedures and spectrophotometric tech-niques. Only a limited number of other methods werereported. All of these published methods related with thedetermination of TMZ in raw material, pharmaceuticaldosage forms, and biological samples. Some of them arerelated to the bioequivalency and bioavailability studies.We have covered both the newer and older analyticaltechniques. Of all the techniques reported here, the LCmethods, especially with MS detection, seem to be moresensitive and resulted in a better resolution in biologicalmatrices.

Conflict of Interest

Pubmed and Scopus were used to gather the studies, andthose within the scope of our review and expertise wereanalyzed.

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