Xanthine Derivatives in the Heart: Blessed Or Cursed?

Current Medicinal Chemistry, 2011, 18, 3695-3706 3695 Xanthine Derivatives in the Heart: Blessed or Cursed?

A. J. Szentmiklósi*,#,1, Á. Cseppent#,1, R. Gesztelyi2, J. Zsuga3, Á. Körtvély4 , G. Harmati4 and P.P. Nánási4

1Department of Pharmacology and Pharmacotherapy, University of Debrecen, Medical and Health Science Center, Debrecen, H-4012, Hungary 2Department of Pharmacodynamics, University of Debrecen, Medical and Health Science Center, Debrecen, H-4012, Hungary 3Department of Neurology, University of Debrecen, Medical and Health Science Center, Debrecen, H-4032, Hungary 4Department of Physiology, University of Debrecen, Medical and Health Science Center, Debrecen, H-4012, Hungary Abstract: Methylxanthines, such as theophylline, have been used to treat cardiorespiratory disorders, whereas caffeine is the most widely consumed psychoactive agent in various soft drinks. Because of the worldwide use of these drugs and the recently synthesized xanthine derivatives, an intensive research on the cardiac actions of these substances is under progress. This review focuses on the molecular mechanisms involved in the actions of xanthine derivatives with special reference to their adenosine receptor antagonistic properties. The main basic and human studies on the action of xanthines on impulse initiation and conduction, as well as the electrophysiological and mechanical activity of the working myocardium will be overviewed. The potential beneficial and harmful actions of the methylxanthines will be discussed in light of the recent experimental and clinical findings. The pharmacological features and clinical observations with adenosine receptor subtype-specific xanthine antagonists are also the subject of this paper. Based on the adenosine receptor-antagonistic activity of these compounds, it can be raised that xanthine derivatives might inhibit the cardioprotective action of endogenous adenosine

on various subtypes (A1, A2A, A2B and A3) of adenosine receptors. Adenosine is an important endogenous substance with crucial role in the regulation of cardiac function under physiological and pathological conditions (preconditioning, postconditioning, ischemia/reperfusion injury). Recent clinical studies show that acute administration of caffeine or theophylline can inhibit various types of preconditioning in human subjects. There are no human studies, however, for the cardiovascular actions of long-term administration of these drugs. Upregulation of adenosine receptors and increased effectiveness of adenosine receptor-related cardiovascular functions have been observed after long-lasting treatment with methylxanthines. In addition, there are data indicating that blood adenosine level increases after long-term caffeine administration. Since the salutary actions (and also the adverse reactions) of a number of xanthine derivatives are repeatedly shown, the main goal is the development of novel structures that mimic the actions of the conventional methylxanthines as lead compounds, but their adenosine receptor subtype-specificity is higher, their water solubility is optimal, and the unwanted reactions are minimized. Keywords: Methylxanthines, xanthine derivatives, adenosine, adenosine receptors, subtype-selectivity, risk/benefit ratio.

1. INTRODUCTION phosphodiesterase enzyme and induce mobilization of calcium from the sarcoplasmic reticulum. It is now becoming clearer that the The commonly used methylxanthines theophylline, caffeine and mainstream of the site of actions of methylxanthines are the adeno- theobromine are naturally occurring compounds in various plants. sine receptors, at least, in the therapeutically relevant concentra- These substances are components of most widely consumed bever- tions of 20-50 M [4, 5]. Accordingly, in this paper we discuss the ages, coffee, tea and cocoa. Theophylline and its salts (aminophyl- cardiac actions of methylxanthines when applied in both therapeutic line, choline theophyllinate) or derivatives (enprophylline, doxofyl- and toxic concentrations. We emphasize the cardiac electrophysi- line, bamiphylline) - depicted in Fig. (1) - are used in the therapy of ological effects of xanthines with special reference to antagonistic bronchial asthma and chronic obstructive pulmonary disease actions on adenosine receptors. (COPD), whereas pentoxyphylline in the treatment of peripheral vascular diseases and in the management of cerebrovascular insuf- 2. MECHANISM OF ACTION OF METHYLXANTHINES ficiency, as well as in diabetic neuropathy [1]. Occasionally theo- AND OTHER XANTHINE DERIVATIVES: BASIC STUDIES phylline is applied to prevent sleep apnea in adults. Caffeine is an analeptic and central nervous system stimulant drug being generally 2.1. Basic Studies on Acute Actions of Methylxanthines and used for enhancement of arousal and vigilance, suppression of fa- other Xanthine Derivatives tigue, as well as to shorten reaction time of the motor system. In addition, caffeine has long been used as an adjuvant in combina- Caffeine in millimolar concentrations has been shown to induce tions to potentiate the pharmacological actions of various analgesics various electrophysiological effects on pacemaker fibers and work- such as acetaminophen and acetylsalicylic acid [2, 3]. ing myocardium. These effects display wide interspecies depend- Methylxanthines, like theophylline or caffeine, are also widely ence. Thus, in rabbit sinoatrial nodal cells, caffeine (1 to10 mM) used in cardiology, since these drugs are appropriate medicaments induced a concentration-dependent, atropine-resistant decrease in for treatment of various bradyarrhythmias and disturbances of sinoatrial pacemaker activity. Caffeine increased the slow inward current, reduced the delayed rectifying outward current, and ele- atrioventricular conduction. In some selected cases, theophylline 2+ may be used as classical “ino-dilator” and diuretic [4]. vated the cytoplasmic Ca concentration. Some of the preparations displayed arrhythmias resulting from the caffeine-induced calcium The mechanism of cellular action of methylxanthines is quite overload. The observed sinus bradycardia can also be related to this complex and their action depends on the concentration applied. latter mechanism [6]. In canine cardiac Purkinje fibers, caffeine (0.5 According to the most conventional views, methylxanthines inhibit to 3 mM) induced an oscillatory potential, which could be modulated by altering intracellular calcium concentration. This oscillatory potential explained the arrhythmias observed in Purkinje fibers *Address correspondence to this author at the Department of Pharmacology and Phar- [ ] macotherapy, University of Debrecen, Medical and Health Science Center, H-4012 7 . In human atrial fibers theophylline (0.1 to 1 mM) was shown to Debrecen, Hungary; Tel: +36 52 411717 ext. 55018; Fax: +36 52 427899; E-mail: increase the rate of phase 4 depolarization and the amplitude of the [email protected] oscillatory potential during diastole and induced the development of #These authors contributed equally to this work. spontaneous slow action potentials. These findings represent the

O H N O HN CH O 3 H3C N H N O N N H3C N N O N N O N N CH3 CH3 CH3 Theophylline Caffeine Enprophylline CH3 OH O N O O CH O 3 O O N H C H3C N H3C N 3 N N N N O N N O N N O N CH 3 CH CH3 3 Pentoxyphylline Doxofylline Bamiphylline

Fig. (1). Chemical structures of most frequently used methylxanthine drugs. major electrophysiological basis helping to understand the theo- specific adenosine receptor activation. All subtypes of adenosine phylline-induced atrial ectopic activity [8]. In working myocardium receptors are available in the heart [24]. A1 adenosine receptors are 2+ caffeine stimulated the L-type Ca -current (ICa) [9, 10]. This effect negatively coupled to adenylyl cyclase, and are responsible for the can be ascribed to the ability of caffeine to activate the ryanodine reduction of sinoatrial rate, AV-conduction, atrial contractile force, channels in the sarcoplasmic reticulum. Caffeine is known to in- attenuation of the positive inotropic effect of catecholamines, as crease the calcium-induced calcium release, amplifying thus the well as for the protection against myocardial ischemia/reperfusion slow inward current [11]. In contrast, there are studies indicating an injury. A2A receptors are positively coupled to adenylyl cyclase. inhibitory action of caffeine on ICa [12, 13]. In rat ventricular myo- They are expressed in rat ventricular myocardium and are known to cytes, Varro et al. [14] found that high concentration (20 mM) of counteract some A1 adenosine receptor-mediated anti-adrenergic caffeine reduced the amplitude of the inward rectifier potassium actions [25]. A2B adenosine receptors couple positively to adenylyl and inward calcium currents. Zhang et al. [15] described a caffeine- cyclase, and mitogen-activated protein kinase. These receptors were activated large-conductance plasma membrane cation channel in suggested to modulate cellular hypertrophy [26]. A3 receptors, like cardiac myocytes. It was suggested that this can be an additional A1 adenosine receptors, are negatively linked to adenylyl cyclase. mechanism by which caffeine and theophylline may contribute to Their activation could inhibit the -adrenergic receptor activation- generation of cardiac arrhythmias. induced enhancement of cAMP, as well as provides tolerance [ ] It is important to take into account that these changes occur in a against hypoxic injury 27 . To understand all cardiac actions of concentration range of 1 to 20 mM of methylxanthines. This range methylxanthines, it is necessary to shortly survey the main effects is well above those xanthine concentrations (20-50 M) which can of adenosine on the cardiac pacemakers, conduction systems and be reached during therapeutic interventions or by consuming caf- the working myocardium. feine-containing soft drinks. The cardiac actions of methylxanthines Regarding the action of adenosine on impulse initiation and could not be explained by their phosphodiesterase inhibiting action conduction, the first described action of adenosine was slowing of or by mobilization of intracellular calcium, since these effects of the heart rate [28]. In spontaneously beating isolated right atrial methylxanthines are very slight at concentrations below 100 M. myocardium, His bundles, and Purkinje fibers of guinea pigs, On the other hand, methylxanthines have been shown to antagonize adenosine showed a characteristic suppressive effect on the sponta- adenosine receptors effectively within their therapeutic concentra- neous firing rate in these specific pacemaker cells [29-35]. A1 tion range [16]. Therefore, it is conceivable that cardiac activity of adenosine receptor activation enhanced the inward rectifier potas- methylxanthines can be related to their antagonistic actions directed sium current (IK-Ado), and consequently hyperpolarized the cell to endogenous adenosinergic signalization [17]. membrane resulting in reduction of the slope of phase 4 depolarization [36-38]. Adenosine has also been reported to inhibit the pace- Adenosine is an endogenous, crucial signaling purine nucleo- [ ] side that plays prominent role in regulation of cardiovascular func- maker current (If) in sinoatrial and AV nodal cells 39-41 . In human, Honey and his colleagues were the first to describe in 1930 tion, having also an important role in pathogenesis of various car- [ ] diovascular diseases. Adenosine is much implicated in the control 42 that adenosine induced sinus bradycardia. Endogenously re- of local blood flow of various organs [18], regulation of cardiac leased adenosine might have a role in hypoxia- or ischemia-induced pacemaker activity, AV conduction, myocardial contractility, and bradycardia, since the adenosine receptor antagonist aminopylline cardioprotection [19-23]. Adenosine acts through G protein-coupled antagonized the hypoxia-induced suppression of sinoatrial pace- receptors on the target cells. Until recently, four subtypes of adeno- maker activity in spontaneously beating guinea pig right atrial sine, namely A , A , A and A , have been identified structurally, preparations. As for humans, Watt has proposed that adenosine 1 2A 2B 3 might be involved in bradyarrhythmias related to sick sinus syn- functionally and pharmacologically. These adenosine receptors [ ] were characterized on the basis of their specific pharmacological drome 43, 44 . profile (rank order of potency of agonists and antagonists), G pro- Drury and Szent-Györgyi were the first to identify adenosine- tein coupling, and signaling mechanism involved in the subtype- induced heart block [28]. Later, Stafford observed that adenosine Xanthine Derivatives in the Heart Current Medicinal Chemistry, 2011 Vol. 18, No. 24 3697 caused a concentration-dependent atrioventricular conduction block adenosine, like hypoxia, exerted a concentration-dependent prolon- in guinea pig hearts [45]. Adenosine is also capable to induce an gation of AV conduction in perfused guinea pig heart. Actions on impaired or blocked of atrioventricular nodal conduction in humans conduction delay induced by both hypoxia and adenosine could be [42, 46]. This is generally a short-lived, not too serious, adverse antagonized by aminophylline, an adenosine receptor antagonist. reaction, but prolonged complete heart block requiring intubation Belardinelli et al. [72] finally suggested that endogenously released and temporary pacing has also been reported [47]. A1 adenosine adenosine might play an important role in AV conduction distur- receptors are abundantly expressed in the AV junction. Adenosine bances also in case of acute hypoxia. Similar conclusions have been acts on the proximal part of atrioventricular junction (atrio-nodal drawn in other studies [73, 74]. region and nodal region), whereas no effect was observed on the [ ] [ ] In our previously reported experiments 75 , sinoatrial pace- nodal-His bundle 48 . In the negative dromotropic effect of adeno- maker activity of spontaneously beating guinea pig left atria was sine, activation of the inward rectifier potassium current and the recorded using extracellular electrodes. During moderate hypoxia, concomitant hyperpolarization of the cell membrane may have a the sinoatrial firing rate gradually decreased. This bradycardia was crucial role. In addition, inhibition of L-type calcium channels has [ ] effectively antagonized by aminophylline, while enhanced in the also been a suggested as underlying mechanism 39 . presence of the adenosine deaminase inhibitor coformycin and the Adenosine is thought to be exerted a strong depressant effect on membrane purine transport uptake blocker dipyridamole, indicating atrial myocardial contractility. The negative inotropic effect of the crucial role of the accumulated adenosine in the sinus bradycar- adenosine has been described in electrically driven rat [49], guinea dia occurring under hypoxic conditions. [ ] pig, and human atrial preparations 50-53 . The negative inotropic A similar experiment is presented in Fig. (2), using another xan- effect in atrial myocardium is related to the marked shortening of thine-type adenosine receptor antagonist, 8-p-(sulphophenyl)theo- action potentials [22, 49, 51, 54, 55]. Based on microelectrode re- 42 phylline. This xanthine prevented the moderate hypoxia-induced cordings and K flux measurements, Jochem and Nawrath were the decrease in sinoatrial pacemaker activity practically completely. As first to report that adenosine activated a potassium conductance [ ] this compound acts on only at extracellular sites, therefore an addi- 55 . Belardinelli and Isenberg, using voltage-clamp technique in tional inhibitory action of enzyme phosphodiesterase can be ex- single atrial myocytes, showed that the adenosine-activated conduc- cluded. tance was an inwardly rectifying potassium current [36]. It was also revealed that the adenosine- and acetylcholine-activated conduc- tances are identical, and these channels are associated with the re- 120 ceptors via a pertussis toxin-sensitive G-protein. Schrader et al. *** ** reported that adenosine reduced the rate of rise and amplitude of the ** slow action potential in guinea pig atrial preparations [56]. Now it is 100 clear that the mechanism of the depressant effects of adenosine on the atrial myocardium is related to activation of the inwardly- rectifying potassium current (IK-Ado) and a blockade of the slow inward current mediated by L-type Ca2+ channels [57]. 80 Adenosine exerts quite different actions on contractility in atrial and ventricular myocardium. Under baseline conditions, adenosine had no negative inotropic action in ventricles - it displayed rather a 60 untreated moderate positive inotropic effect [58-64]. Importantly, adenosine had no effect on action potential duration or baseline L-type cal- [ ] cium current 65 . In contrast, in ventricular myocardium when of pre-hypoxia control % 40 adenylate cyclase was previously activated using various com- 8-SPT pounds, including isoprenaline, histamine and dopamine, adenosine displayed a negative inotropic action [58, 63, 64, 66-68]. Under these conditions adenosine or adenosine analogues attenuated all 20 catecholamine-stimulated outward and inward currents. With the 0 10 20 30 40 50 60 70 80 90 exception of rat and ferret, no adenosine-activated potassium current can be demonstrated in ventricular myocardium [39]. In addi- minutes during hypoxia tion, the antiadrenergic action of adenosine is due to an inhibition of adenylate cyclase activity with the concomitant reduction of Fig. (2). Action of 50 M 8-p-(sulphophenyl)theophylline (8-SPT) on the cAMP content [10, 28, 29, 45]. On the other hand, adenosine has moderate hypoxia-induced decrease in sinoatrial pacemaker activity of been shown to exert significant anti-adrenergic effect on myocar- spontaneously beating guinea pig right atria. Asterisks indicate significant dial preparations [64, 66]. Accordingly, there is a concept that differences between the 8-SPT treated (n=6) and the untreated control (n=9) adenosine acts as a negative feedback modulator of -adrenoceptor- groups. mediated responses in the heart [64, 66]. Although ventricular myocardium contains primarily A1 adenosine receptors, there are studies In electrically stimulated guinea pig atria, under normoxic con- showing that A2A and A3 adenosine receptors are also expressed in [ ] ditions, theophylline induced a significant increase of contractile ventricular myocytes 24 . force within a concentration range of 0.1-1 mM, which is higher Accordingly, methylxanthines are capable to antagonize the ac- than usual adenosine receptor-antagonist concentrations [76]. Ac- tions of both exogenous and endogenous adenosine. The competi- cording to Collis et al. [77], the positive inotropic actions of theo- tive antagonism between methylxanthines and exogenous adenosine phylline and enprophylline were not due to adenosine receptor in various cardiac functions has long been established [69-71]. blockade, but rather to inhibition of phosphodiesterase. According [ ] A great mass of experimental evidence is available for estab- to experiments of Bellemann and Scholz 78 , the positive inotropic lishing the ability of methylxanthines to antagonize the effects of actions of high concentration (0.6 mM) of theophylline can be asso- endogenous adenosine accumulated in certain conditions, like hy- ciated with an increase of sarcolemmal calcium influx and an action poxia, ischemia, or increased metabolic activity. Regarding atriov- on intracellular calcium binding or storage sites. In electrically entricular conduction, Belardinelli et al. [72] demonstrated that driven left atrial preparations of guinea pigs, adenosine reduced the contractile force and shortened the duration of intracellularly re- 3698 Current Medicinal Chemistry, 2011 Vol. 18, No. 24 Szentmiklósi et al. corded action potentials. The similarity between the adenosine- and In our experiments, the actions of long-term oral caffeine treat- the hypoxia-induced alterations of myocardial electrical and me- ment were studied on the electrical and mechanical activity of A1 chanical activities is striking, since both the adenosine- and hy- adenosine receptor selective N6-cyclopentyladenosine (CPA) on poxia-induced changes could readily be prevented by aminophyl- guinea pig atrial myocardium, as well as on the antagonistic action line. Extreme changes in action potential morphology due to long- of methylxanthines on the CPA-induced decrease of mechanical lasting hypoxia could also be reversed by aminophylline. On the activity in atrial myocardium. Slow action potentials were gener- basis of these results it is assumed that the increased tissue level of ated in potassium-depolarized (25 mM) guinea pig atrial muscles endogenous adenosine in acute myocardial hypoxia might contrib- treated by 1 M isoproterenol. CPA decreased the rate of rise and ute to functional impairment of atrial myocardium [21]. In nor- the duration of slow action potentials (Fig. 3, Table 1) in a concen- moxic conditions, methylxanthines, in therapeutically applied con- tration-dependent manner. A very strong sensitization in the CPA- centrations, do not alter or minimally affect the functional activity induced electrophysiological changes was observed after a 2 week of myocardium and pacemaker tissues. It can be supposed that caffeine treatment. stimulatory actions of methylxanthines applied in therapeutic con- On the basis of these experiments it can be suggested that after centrations occur only under pathological conditions (e.g. hypoxia, long-lasting caffeine treatment a strong sensitization develops in the ischemia), when endogenous tissue adenosine levels are substan- CPA-induced depression of slow Ca2+ influx and outward K+ out- tially elevated. flow. It is thought that under these conditions Vmax represents the slow inward Ca2+ current, whereas duration of slow action potential 2.2. Basic Studies on Chronic Actions of Xanthine Derivatives is related to the outward potassium current [56]. In these experi- In contrast to the extensive studies performed on the regulation ments, guinea pigs were fed with caffeine for 10 weeks, their aver- of CNS adenosine receptors, there is only limited information age serum caffeine concentration was 42.3 ± 3.6 M. In electrically available on the effect of chronic methylxanthine treatment on the driven left atrial preparations, sensitization to CPA-induced de- cardiovascular action of adenosine. In the study by Wu et al. [79] crease of mechanical activity developed. Its maximum was observed at week 2 of caffeine treatment, then the potency ratio mod- the action of a 2-week theophylline treatment on myocardial A1 adenosine receptors was investigated. Cardiac adenosine receptors erately diminished (Table 1). From week 2, the Emax values were were upregulated by chronic exposure to theophylline and this also elevated in atrial muscles of caffeine-treated groups. It is note- upregulation was accompanied by an increased sensitivity of ven- worthy, that at week 10 of caffeine treatment a moderate tolerance tricular myocytes to the electrophysiological and biochemical ef- developed in the adenosine-antagonistic action of various methylx- fects of adenosine receptor agonists. Similarly, chronic (2 weeks) anthines (e.g. theophylline, caffeine, and DPCPX), i.e. their anti- theophylline treatment resulted in upregulation of atrial and ven- adenosine pA2 values were reduced. From these studies, an upregulation of A1 adenosine receptors due to chronic caffeine treatment, tricular A1 adenosine receptors in rats [80]. It must be noted, however, that only the indirect, antiadrenergic, negative inotropic and resulting in alterations in electrical and mechanical parameters of chronotropic effects of adenosine receptor agonist on atrial muscle atrial myocardium, can be concluded. were sensitized, but the direct cardioinhibitory actions were not altered. Chronic caffeine consumption (3 weeks) potentiated the A2 3. HUMAN STUDIES adenosine receptor-related hypotensive action of adenosine in rats [81]. According to Varani et al. [82], repeated caffeine administra- There are a number of early reports, indicating that the resting tion upregulated human platelet adenosine A receptors, which is sinus frequency or the increase during exercise fails to alter signifi- 2A cantly after therapeutic doses of caffeine [85]. Interestingly, in vari- accompanied with an increased antiaggregatory action of the A2A adenosine receptor agonist 2-hexenyl-5’-N-ethylcarboxamido- ous pathological states or in healthy volunteers at various sub- adenosine. Similar results were obtained also in rat platelets [83]. maximal and maximal exercise intensities, when tissue concentra- Interestingly, in an experiment with repeated (23 days) adminis- tion of endogenous adenosine elevates, methylxanthines exert no- ticeable effects on the sinoatrial pacemaker. It is suggested by Watt tration of a selective A1 adenosine receptor antagonist KW-3902 (8- [ ] (noradamantan-3-yl)-1,3-dipropylxanthine) Kusaka and Karasawa 43 that sick sinus syndrome is an adenosine-mediated disease. In [84] failed to observe any alteration in the A adenosine-receptor- this syndrome, oral theophylline proved to be efficacious in increas- 1 ing sinus rate at rest and during exercise, and also to reduce cardiac related bradycardic action of 5’-N-ethylcarboxamidoadenosine [ ] (NECA). pauses 86, 87 . In a randomized, controlled trial (The THEOPACE SOLVENT-TREATED CAFFEINE-TREATED

40 40

30 30

20 control 20 control CPA 30 nM 10 10 CPA 1 nM CPA 100 nM 0 0

-10 -10 CPA 10 nM

-20 CPA 300 nM -20 CPA 30 nM -30 -30 Membrane potential (mV) Membrane potential (mV)

-40 -40

-50 -50 0 50 100 150 200 0 50 100 150 200 Time (ms) Time (ms)

Fig. (3). CPA-induced, concentration-dependent depression of maximum velocity of depolarization (Vmax) and shortening of the duration of slow action potentials generated in guinea pig atrial trabeculae depolarized by 25 mM potassium and treated with 1 M isoproterenol. The preparations were stimulated at a frequency of 0.5 Hz, transmembrane potentials were recorded using conventional sharp microelectrodes. Xanthine Derivatives in the Heart Current Medicinal Chemistry, 2011 Vol. 18, No. 24 3699

Table 1. Effects of Long-Term Caffeine Treatment on the CPA-Induced Alterations in the Mechanical and Electrical Activity of Guinea Pig Atrial Myocardium

Parameter Solvent-treated Caffeine-treated

Week 1

pD2 7.73 ± 0.10 8.35 ± 0.06*

Emax 89.5 ± 3.5 92.8 ± 2.5 n 5 6 Concentration ratio 4.17 Week 2

pD2 7.54 ± 0.24 8.43 ± 0.04**

Emax 83.5 ± 3.2 93.0 ± 3.2* n 6 6 Concentration ratio 37.8 ± 4.8 7.76

Vmax (% reduction) 31.0 ± 5.5 95.7 ± 1.8**

APD75 (% shortening) 5 69.7 ± 9.6* n 4 Week 6

pD2 7.78 ± 0.10 8.58 ± 0.08*

Emax 90.0 ± 1.6 95.2 ± 1.8 n 5 6 Concentration ratio 6.31 Week 10

pD2 7.86 ± 0.08 8.55 ± 0.07*

Emax 85.8 ± 2.4 97.3 ± 1.1* n 5 4 Concentration ratio 4.90

Data are mean values ± S.E.M, EC50 = the concentration producing half-maximum response (its negative based logarithm: pD2), Emax = maximum effect expressed in % of pre-CPA contractile force, n = number of experiments, Vmax = rate of rise of depolarization, APD75 = duration of action potential at 75% of repolarization. Changes in Vmax and APD75 were measured at 10 nM CPA concentration. Concentration ratios denote the ratios of EC50 values of CPA concentration-response curves (solvent-treated versus caffeine-treated). Aster- isks indicate significant differences between the caffeine-treated and the solvent-treated control groups.

Study) it was, however, shown that oral theophylline reduced only endogenous adenosine in development of atrial fibrillation and the the minor symptomes of sick sinus syndrome, while was not effec- possibility of an adenosine antagonist-based therapy. As theophyl- tive in patients suffering from severe forms of the disorder [88]. line is not a specific adenosine receptor antagonist, it is hopeful that Human electrophysiological studies are also available to demon- A1 adenosine receptor selective antagonists (e.g. synthetic methylx- strate that oral theophylline suppressed the symptoms in sick sinus anthines) can be more efficient drugs to terminate this type of atrial syndrome [89, 90]. Benditt et al. [90] reported that theophylline fibrillation [74]. It is noteworthy that after caffeine consumption treatment was useful in young patients with recurrent symptomatic different changes in sinus rhythm were observed in healthy humans bradyarrhythmias. Furthermore, it was suggested that theophylline exposed to various submaximal and maximal exercise intensities. might be a reasonable alternative strategy in the treatment of Thus, some investigators reported that caffeine increases the heart chronic symptomatic bradycardia in the elderly [91, 92]. rate under these circumstances [100-103]. Recently, McClaran and [ ] Sinus node dysfunction, primarily sinus bradycardia, is fre- Wetter 104 reported that in non-habitual caffeine users, low doses quently associated with heart transplantation [93]. Bertolet and his of caffeine decreased the heart rate during submaximal exercise, but coworkers [94] observed that use of theophylline for posttransplan- not at near maximal and maximal exercise. It is tempting to suppose tation bradyarrhythmias was efficient in increasing the heart rate that because of the imbalance between oxygen requirement and and reduced the need for implantation of a permanent pacemaker. consumption in maximal physical exercise, endogenous adenosine Reversibility of prolonged chronotropic dysfunction by theophyl- release can substantially be increased. In this case caffeine might line and aminophylline following orthotopic cardiac transplantation counterbalance the endogenous adenosine-related suppression of was also reported by a number of investigators [95-97]. Oral or sinoatrial pacemaker activity. intravenous theophylline proved to be effective in not only sinus There are conflicting reports available on the possible arrhyth- node disturbances, but also in abnormal impulse conduction. Thus, mogenic action of caffeine or theophylline. According to a meta- Alboni et al. [97] described that oral theophylline represents a valid analysis, moderate consumption of caffeine does not increase the therapy in most patients with atrial fibrillation associated with a frequency or severity of cardiac arrhythmias in healthy persons, in slow ventricular response. Intravenous theophylline proved to be a patients with ischemic heart disease, or in ones having pre-existing useful method in the management of bradycardia secondary to AV serious ventricular ectopy. There is an increase, however, in the block and offers a pharmacological alternative to patients where frequency of ventricular extrasystoles in heavy coffee consumers other pharmacological strategies or cardiac pacemaker implantation drinking 9 or more cups daily [105]. It was recently found that may be contraindicated [98]. An interesting observation was re- moderate tea intake can be associated with a lower prevalence, ported by Bertolet et al. [99], who demonstrated the effectiveness of while higher coffee consumption with a slightly higher prevalence theophylline in atrial fibrillation due to adenosine accumulation in of ventricular arrhythmias in patients hospitalized with acute myo- acute myocardial infarction. After application of theophylline, a cardial infarction [106]. According to the Danish Diet, Cancer, and rapid (< 5 min) and long-term conversion of atrial fibrillation to Health Study [107], in which 47 949 subjects were participated, sinus rhythm was observed. These findings highlighted the role of consumption of caffeine was not associated with an elevated risk of 3700 Current Medicinal Chemistry, 2011 Vol. 18, No. 24 Szentmiklósi et al. atrial fibrillation or flutter. Similarly, at modest dose of caffeine, no postglomerular and preglomerular vessels. Adenosine constricts the action on the incidence of ventricular arrhythmia could be observed preglomerular vessels by activation of A1 adenosine receptors, in patients with ventricular tachycardia or fibrillation [108, 109]. whereas exerts a vasodilator action on the postglomerular arterioles [ ] Regarding the effects of methylxanthines on the working myo- by stimulation of A2 receptors 120 . Activation of A1 receptors [ ] decreases renal perfusion pressure, glomerular filtration, and tubu- cardium, the picture is quite complex. Conrad and Prosnitz 110 [ ] studied the cardiovascular actions of aminophylline-infusion in lar sodium reabsorption 121 . It is supposed that adenosine may play a role in the kidney-mediated fluid retention in heart failure healthy male volunteers and found that aminophylline exerted a [ ] mild positive inotropic effect, but this action could be prevented by 122 . In this case, use of selective A1 adenosine receptor antago- pretreatment with the -adrenergic receptor antagonist metoprolol nists might provide therapeutic benefits in the setting of congestive or propranolol. The hemodynamic action of intravenously applied heart failure. At present, one of the primary approaches of treatment aminophylline was also studied in healthy human subjects and no of heart failure is diuretic therapy. On the basis of clinical experi- significant improvement was found on cardiac performance during ences, however, it became clear that the generally applied diuretics [ ] (e.g. furosemide) may reduce glomerular filtration rate in patients tread-mill exercise after using of the compound 111 . These find- [ ] ings are in good agreement with the further study of Conradson suffering from heart failure 120, 123 . In contrast, selective block- using theophylline and enprophylline [112]. According to the hemo- ade of A1 adenosine receptors by CVT-124 was shown to reduce [ ] afferent arteriole pressure, increase the urinary flow and sodium dynamic studies of Parker et al. 113 , aminophylline was effective [ ] to increase cardiac output in patients suffering from heart failure, excretion 124, 125 . It is logical to assume that treatment with se- but no change was observed in patients with cor pulmonale [114] or lective A1 adenosine antagonists can be more effective than the chronic obstructive pulmonary disease [115]. conventional diuretic therapy. Theophylline and aminophylline were extensively used over the The chemical structures of some novel A1 adenosine receptor last 70 years to increase inotropy in patients suffering from heart selective compounds are presented in Fig. (4). The newest drug in failure [113, 116], and to treat patients with bronchial asthma [117]. this category is rolofylline (KW-3902). In a randomized, placebo- However, because of the narrow therapeutic ratio and due to its controlled, dose-finding study (PROTECT Pilot Study), it was ob- cardiac, gastroenteric and CNS side effects, theophylline has lost served that A1 adenosine receptor blockade with rolofylline could popularity in the last decades. According to current AHA and ESC prevent renal impairment in patients with acute heart failure and positively affected the acute symptoms as well as the 60 days out- guidelines, the two primary pharmacological approaches in symp- [ ] tomatic heart failure are ACE inhibitors/ARBs and diuretics. Theo- come 126 . The large clinical trial, PROTECT, began in May 2007 phylline exerts a slight positive inotropic action combined with a and was completed in January 2009. 2033 patients from 173 sites of good vasodilator activity, therefore, it can be an ideal drug for com- 19 countries were recruited in this study. The results were disap- bined inotropic and vasodilator treatment of heart failure. In addi- pointing as in the larger trial the benefits observed in the PROTECT tion, theophylline and its derivatives have also moderate diuretic Pilot Study could not be replicated. Rolofylline showed no differ- activity based on adenosine receptor antagonism [118], decreasing ence from placebo regarding the primary end-points in patients with thus the circulating blood volume and preload of the heart. Endoge- acute heart failure. In the study, there was a trend toward a lower nous adenosine was also shown to play an important role in patho- incidence of serious adverse cardiac side reactions, but the inci- genesis of heart failure. Plasma adenosine levels are increased in dence of neurological events (seizures and strokes) were higher. As a consequence, the drug has been dropped from development [127- patients with heart failure in parallel with the severity of the disease ] [ ] 129 . The clinical trial with another A1 adenosine receptor-selective 119 . In the nephron, A1 and A2 adenosine receptors are widely [ ] distributed and they have significant functional role. Adenosine can blocker, naxifylline was also discontinued 130 . At present, it is not influence the glomerular filtration rate, renal hemodynamics, and clear whether the basic principle is a pitfall or there might be prob- the tubuloglomerular feedback. This purine nucleoside reduces lems with the physicochemical properties of the investigated drugs? glomerular filtration by inducing selective vasoactive actions on the The resolution of this issue should be very important for the future

O O O H H H C H H3C N H3C N 3 N O N N N

N O N N O N N O N

CH3 CH3 CH3

DPCPX (CPX) Rolofylline (KW3902, NAX) Naxifylline (BG-9719, CVT-124) O O OH O H H H3C N N N H3C N

N O N N O N

CH 3 OH Toponafylline (BG-9928) PSB-36

Fig. (4). Chemical structures of DPCPX and new A1 adenosine receptor selective antagonists. Xanthine Derivatives in the Heart Current Medicinal Chemistry, 2011 Vol. 18, No. 24 3701 trends of drug development. Currently there is only one A1 recep- fer et al. [154], as well as Claeys and his associates [155] observed tor-selective xanthine derivative, toponafylline under clinical trial that blockade of adenosine receptors with aminophylline limited for treatment of heart failure [130]. ischemic preconditiong in human beings. Tomai and his coworkers [150] described that adenosine receptor blockade by bamiphylline 4. CURRENT PROBLEMS AND FUTURE PERSPECTIVES inhibited the ischemic preconditioning during coronary angioplasty. The same group [156] has also demonstrated that bamiphylline Independently of the above mentioned facts, it appears that improved exercise-induced myocardial ischemia, increased the theophylline receives a renewed attention as according to a number ischemic threshold, prolonged exercise duration, delayed the onset of studies. This drug - applied in relatively low doses - was able to of anginal pain, and reduced the ST segment depression - possibly exert significant anti-inflammatory action in patients with bronchial via selective blockade of A1 adenosine receptors. asthma and chronic obstructive pulmonary disease [117, 131-135]. On the basis of this recognition, a new era of use of methylxanthi- 4.2. Possible Implications for Postconditioning nes may start, the significance of which could affect not only the treatment conceptions of cardiorespiratory diseases, but also the Myocardial ischemia is always followed by reperfusion associ- synthesis of new and more effective methylxanthines. When con- ated myocardial necrosis and apoptosis. This reperfusion injury can sidering the ongoing and future use of methylxanthines, we should be diminished by postconditioning, which was first described by take into account a number of issues. According to the title of this Zhao et al. [157]. Postconditioning includes a brief ischemic epi- work, we should wage the benefits and risks of xanthine treatment. sode immediately at the onset of reperfusion after a prolonged ischemic episode. This intervention was shown to reduce the size of The main facts are as follows. (1) caffeine is the most widely the infarct area [157-159]. Although, the literature data are conflict- consumed and relatively safe psychoactive agent all over the world. ing, it seems likely that A2A and A2B adenosine receptors may play (2) Although treatment guidelines have regulated theophylline to critical role in postconditioning [160-162], however, involvement of third-line treatment of bronchial asthma and chronic obstructive A and A adenosine receptors have also been emerged [163]. pulmonary disease [136, 137], the rate of theophylline users among 1 3 [ ] asthmatic and COPD patients is relatively high 138 . (3) The anti- 4.3. Possible Implications for Ischemia/Reperfusion Injury inflammatory action of theophylline, observed in low dose, will likely facilitate the worldwide use of theophylline again [117, 135]. Adenosine is known to exert a protective effect against (4) There is an intense, lengthy interdisciplinary endeavor for de- ischemic injury. In the adenosinergic cardioprotection, practically, veloping new, more selective and more effective xanthine deriva- all subtypes of adenosine receptors contribute. A1 adenosine recep- tives. Although, according to large studies the moderate consump- tor-induced cardioprotection operates when the receptors are acti- tion of caffeine-containing beverages does not associate with an vated prior to and during ischemia. During reperfusion, A1 adeno- increased cardiovascular mortality and morbidity, there are some sine receptor activation is not effective [163-167]. Although, in concerns that the use of methylxanthines should be taken into ac- human, the expression of A3 adenosine receptors is relatively poor, count in point of view of possible cardiovascular consequences. there are pharmacological proofs to support the protective role of Thus, it was emerged that methylxanthines are able to eliminate the A receptor activation both in the pre- and post-ischemic period, as [ ] 3 preconditioning actions of endogenous adenosine 139 . As adeno- well as during ischemia [160, 168-170]. In contrast to A1 and A3 sine receptors are highly implicated in ischemia/reperfusion proc- adenosine receptors, A2A and A2B adenosine receptors do not play a ess, the same suggestion might also be conceivable for treatment significant role in the pre-ischemic and ischemic phase, but are with various xanthines having adenosine receptor-antagonist prop- highly effective during reperfusion [163, 164, 170-173]. Several erties. As for the possible implication of theophylline and xanthine experimental studies provide evidence that blockade of any of the structures, we will focus exclusively on four interesting aspects of known adenosine receptor subtypes could somehow diminish the cardiac activity: possible implications for (1) preconditioning, (2) cardioprotective action of adenosine [163, 174]. postconditioning, (3) ischemia/reperfusion injuries, and (4) epigenetic processes in the heart. 4.4. Possible Implications for Epigenetic Mechanisms

4.1. Possible Implications for Preconditioning The term “epigenetics” describes all meiotically and mitotically heritable alterations in gene expression that are not coded in the It is widely believed that adenosine is a highly effective en- DNA sequence itself [175]. Epigenetic mechanisms involves DNA dogenous cardioprotective agent, which plays important role in methylation, RNA-associated silencing and histone modification. ischemic preconditioning. It has been observed that a short period Histone modifications as epigenetic modifiers are post-translational of ischemia increases the tolerance against a subsequent sustained modifications of histones, including acetylation and methylation of ischemia [140, 141]. This might be the basis of the experience that conserved lysine residues. Histone deacetylases (HDACs) regulate the infarct size is smaller in patients who had several anginal at- cellular signaling and gene expression. Until recently, distinct genes tacks before acute myocardial infarction [140]. Preconditioning may encoding 18 mammalian HDACs (grouped into Class I, II and III) occur in two phases: an early phase, called also early precondition- have been identified [176]. Histone deacetylase 2 (HDAC2) is a ing, and a delayed phase often mentioned as a second window of critical factor for embryonic development and cytokine signaling protection. The delayed phase of ischemic preconditioning is sup- relevant for immune responses. Pathologic activity of this enzyme posed to implicate synthesis of cytoprotective proteins [142]. There can lead to various cardiac disturbances such as myocardial hyper- [ ] is good evidence that A1 and A3 adenosine receptors have crucial trophy or heart failure 177 . Thus, inhibitors of HDAC2 enzyme role in adenosine-mediated preconditioning in both experimental are considered as important cardiac drugs, to be applied e.g. for animals [143-149] and human subjects [150, 151]. In preconditiong, prevention of myocardial hypertrophy [176, 178, 179]. It is notice- the role of adenosine is supported by strong pharmacological evi- able that Class IIa HDACs (HDAC 4, 5 and 9) repress myocardial dence, indicating that various xanthine-type adenosine receptor hypertrophy and Class I and Class IIa enzymes have opposing roles blockers, like 8-p-(sulphophenyl)theophylline, 8-(4-carboxyeth- in regulation of cardiac myocyte growth and heart diseases [180]. enylphenyl)-1,3-dipropylxanthine (BW A1433), and 8-cyclopentyl- The roles of various HDACs are quite complex and they might also 1,3-dipropylxanthine (DPCPX) effectively eliminated precondition- be involved in pathogenesis of cardiac arrhythmias and acute coro- ing [147, 152]. Recently, Riksen et al. [139] and Meijer et al. [153] nary syndromes [179]. have shown that acute caffeine administration prevented protection This epigenetic mechanism might be significant in patients suf- in two different human models of ischemic preconditioning. Schae- fering from bronchial asthma and COPD. It was repeatedly de- 3702 Current Medicinal Chemistry, 2011 Vol. 18, No. 24 Szentmiklósi et al. scribed that low concentrations of theophylline activated HDAC trial for treating heart failure (Fig. 4). Xanthine-type A2A antago- and suppressed the activation of inflammatory genes [134, 181]. nists were also under clinical trial in Parkinson disease, e.g. istrade- This action could be observed at low (1 to 10 M) theophylline fylline (KW-6002) and ST-1535. ST-1535 is under phase I trial, concentrations, whereas is lost at higher theophylline levels (100 whereas the study with istradefylline was discontinued [130]. Two M). Theophylline activates different subtypes of HDACs via an xanthine-type A2B adenosine receptor antagonists are under clinical intracellular site of action, but it appears that it has a relatively more development for bronchial asthma (ATL 844 and GS 6201) [130]. [ ] selective action on Class I than on Class II HDACs 182 . To our It is surprising that relatively few subtype-selective xanthine- best knowledge, there are no reports on the action of theophylline type antagonists were chosen for human studies and approximately and other xanthine derivatives on the cardiac HDACs, but it should half of the trials were discontinued because of ineffectiveness or be strictly considered whether these low theophylline levels might adverse reactions. It appears that we should consider primarily the affect also the physiological or pathological cardiac functions? At cardiac actions of caffeine, theophylline and their derivatives. It present, we do not know whether theophylline can be regarded as a would be a useful option to develop compounds, like 8-p-(sulpho- friend or a foe on the heart in this regard. Because the actions of phenyl)theophylline (8-SPT; Fig. 5), 8-(4’-carboxymethyloxy- various HDACs are very complex, the outcome of activation of phenyl)-1,3-dipropylxanthine or 8-(4’-carboxymethyloxyphenyl)- these enzymes can be either beneficial or harmful. Extensive clini- 1,3-dipropylxanthine-2-aminoethylamide as theophylline deriva- cal studies are necessary to reveal the significance of this issue. tives, which are not able or very poorly able to penetrate the blood brain barrier [189]. With the use of these types of compounds, the 5. CONCLUSIONS central nervous system effects, like seizures, could effectively be avoided. The main goal of this paper was to review the current state of the medical use and perspectives of xanthine structures, such as The actual concern is that how xanthine structures could influ- theophylline, caffeine and some of the novel adenosine subtype- ence pre- and postconditioning, as well as ischemic-reperfusion specific xanthine derivatives. It can be stated that the conventional injuries. As highlighted above, there are clinical experiences indi- methylxanthine theophylline is still frequently prescribed world- cating that methylxanthines can inhibit preconditioning and the wide, but the frequency of adverse reactions and the relatively low question emerged, whether these adenosine receptor antagonists can efficacy of this drug have led to reduced use in the pulmonological blunt the cardioprotective activity of endogenous adenosines. First, practice. The recent recognition of the specific anti-inflammatory these clinical observations refer exclusively to acute administration action of low theophylline doses as a HDAC activator will possibly of methylxanthines. Until recently, there are no chronic human significantly accelerate the worldwide application of theophylline. trials. Second, there are experimental findings that adenosine recep- It is tempting to speculate that the anti-inflammatory property of tors are upregulated and the adenosine-induced physiological re- theophylline or theophylline-derivatives could be used in not only sponses are concomitantly increased under chronic exposure to in bronchial asthma and COPD, but also in ischemia/reperfusion methylxanthines [79-83]. In this paper, we clearly demonstrated that injuries, such as heart transplantations or acute myocardial infarc- - at least in guinea pigs - the long-term administration of oral caf- tion. In the reperfusion phase of these cardiovascular events the feine administration may strongly enhance the A1 adenosine recep- crucial role of the inflammatory processes are widely accepted tor-induced electrical and mechanical responses in electrically [183-186]. It has been shown that pretreatment with aminophylline driven left atrial myocardium. These findings strongly suggest that before coronary artery bypass grafting or heart transplantation im- under chronic administration of caffeine or theophylline, adenosine proved the mechanical and biochemical functions of the heart in receptors participating in preconditioning or ischemia-reperfusion human subjects [187, 188]. Regarding caffeine, this drug possibly processes are upregulated. Therefore, it can be predicted that long- remains the most widely consumed psychoactive substance in the lasting exposures to these methylxanthines do not worsen, but future. There are intensive endeavors to synthesize new adenosine rather improve the effectiveness of these endogenous cardioprotec- subtype-selective xanthine derivatives. Two A1 subtype selective, tive mechanisms. In addition, it should be taken into account that xanthine-type adenosine receptor antagonists were under clinical caffeine is capable of inducing a dose-related increase of plasma trials (rolofylline, naxifylline), but these studies were discontinued. adenosine levels [190]. These factors together could likely ensure At present, only toponafylline (BG-9928) is in a phase IIb clinical the integrity of endogenous cardioprotective mechanisms. At any rate, in the light of a number of large prospective studies it can be

O CH 3 O F N F H3C N H H3C N F CH3 N N N O O N N O N N

CH3 O CH3 CH3

Istradefylline (KW-6002) GS 6201 (CVT-6883)

O H H3C N O N S OH O N N O

CH3

8-p-(sulphophenyl)theophylline

Fig. (5). Chemical structures of istradefylline, GS 6201, and 8-SPT. Xanthine Derivatives in the Heart Current Medicinal Chemistry, 2011 Vol. 18, No. 24 3703 ascertained that chronic, but moderate coffee consumption (which and mechanical activity of the atrial myocardium. Arch. Int. Pharmacodyn. is the main source of caffeine intake) does not increase generally Ther, 1979, 238, 283-295. [22] Szentmiklosi, A.J.; Nemeth, M.; Cseppento, A.; Szegi, J.; Papp, J.G.; the risk of coronary disease and heart failure. There might be, of Szekeres, L. Effects of hypoxia on the guinea-pig myocardium following course, adverse reactions associated mainly to the cardiac and neu- inhibition of adenosine deaminase by coformycin. Arch. Int. Pharmacodyn. rohumoral actions of acute caffeine exposition, but these reactions Ther., 1984, 269, 287-294. might occur in a specific vulnerable population, in elderly and hy- [23] Vinten-Johansen, J.; Thourani, V.H.; Ronson, R.S.; Jordan, J.E.; Zhao, Z.Q.; Nakamura, M.; Velez, D.; Guyton, R.A. Broad-spectrum cardioprotection pertensive patients, or when the possible adverse reactions are ge- with adenosine. Ann. Thorac. Surg., 1999, 68, 1942-1948. netically determined [4, 191, 192]. The extremely high coffee con- [24] Auchampach, J.A.; Bolli, R. Adenosine receptor subtypes in the heart: sumption, however, carries large cardiovascular risk. therapeutic opportunities and challenges. Am. J. Physiol., 1999, 276, H1113- 1116. In conclusion, at present we are in a position to apply rational [25] Norton, G.R.; Woodiwiss, A.J.; McGinn, R.J.; Lorbar, M.; Chung, E.S.; drug design also in the platform of adenosinergic pharmacology as Honeyman, T.W.; Fenton, R.A.; Dobson, J.G., Jr.; Meyer, T.E. 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Received: April 13, 2011 Revised: July 08, 2011 Accepted: July 10, 2011