ELSEVIER International Journal of Cardiology 56 (1996) 17-27

cardiology

Pharmacological preconditioning of ischemic heart disease by low-dose dipyridamole

Franc0 Laghi Pasini” , Francesca Guideri, Daniel Ferber, Giuseppe Galgano, Andrea Bianchi, Silvia Isidori, Laura De Giorgi, Silvia Petri, Pier Leopold0 Capecchi,

Tullio Di Perri Institute of Clinical Medicine, University of Siena School of Medicine, Policlinico ‘Le Scotte’, I-53100 Siena, Italv

Received 24 January 1996; revised 14 May 1996; accepted 14 May 1996

Abstract

Fourteen patients affected with coronary artery disease underwent two consecutive dipyridamole echocardiographic stress tests, in basal conditions and after repeated low doses of intravenous dipyridamole, following the observation that pulse increases in adenosine plasma levels due to repeated intravenous administration of dipyridamole mimic the mechanism of ischemic preconditioning. Echocardiographic, electrocardiographic, haemodynamic parameters, and adenosine plasma levels were measured. After the second test, six patients were completely negative, and in those eight still positive the onset of dyssynergy was delayed.

Keywords: Preconditioning; Dipyridamole; Adenosine; Echocardiography

1. Introduction

The phenomenon of ischaemic pre-conditioning implies an endogenous protective mechanism by which brief periods of ischaemia protect the tissues against the adverse effects of a subsequent prolonged ischaemia. First described by Murry and colleagues [l], preconditioning is currently the subject of inten- sive investigation in various experimental and clini- cal settings. This phenomenon seems to be mediated, at least in part, by endogenous adenosine [2]. Adenosine receptor blockers blunt preconditioning’s protection, and pretreatment with intracoronary

*Corresponding author, Tel: +0577 585743/585741; fax: +0577 44114.

adenosine mimics preconditioning [2,3]. The protec- tive effect of endogenous and exogenous adenosine may be attributed mainly to Al receptors on myocytes without the involvement of A2 vascular receptors [2,4,5], but also to the newly discovered A3 receptor [6].

It has been recently demonstrated that the in- travenous infusion of dipyridamole, a nucleoside transport inhibitor able to increase plasmatic and interstitial levels of endogenous adenosine [7], may mimic ischaemic cardioprotection in that systolic and diastolic alterations of perfused stunned rabbit hearts are attenuated, or it may potentiate the myocardial infarct size-limiting effect of concomitant ischaemic preconditioning in rabbit [8,9].

So far, evidence of ischaemic preconditioning in

0167-5273/96/$15.00 0 1996 Elsevier Science Ireland Ltd. All rights reserved PII SO167-5273(96)02712-X

18 F.L. Pasini et cd. I lmernationcrl Journal of Cardiology S6 (1996) 17- 27

humans derives only from catheterization laborator- ies [lO,l I], although some clinical clue is arising from the prospective evaluation of the relation between antecedent angina and prognosis after acute myocardial infarction [ 121.

To test whether a cardioprotective effect of dipyridamole could be demonstrated in a clinical setting, we evaluated the onset, the extent and the time course of ischaemia during echocardiography- stress test with high dose dipyridamole in patients with chronic coronary artery disease, in basal con- dition and after repeated low doses of intravenous dipyridamole. This treatment was employed with the assumption that it may mimic the pulse release of endogenous adenosine during ischaemic precondi- tioning, by eliciting a short lasting increase of adenosine in plasma and in myocardial interstitial fluid.

2. Materials and methods

2. I. Dose-finding of dipyridamole

The aim of the first part of the study was to find a dose of intravenous dipyridamole and a time interval of administration able to induce pulse increases in adenosine levels to mimic the supposed effects of brief, repeated ischaemia. Two main conditions might be observed: first of all, after each intravenous administration of the drug, adenosine levels should increase transiently, reaching rapidly a maximal peak, then returning to basal values before the following intravenous dipyridamole infusion. Sec- ondly, the plasma adenosine level attained should be ‘subcritical’, that is its increase should not be sufficient, in terms of maximal peak and time duration, to trigger a possible steal-phenomenon at coronary arteries level. Finally, it should be assumed that the pulse increase in plasma levels of adenosine parallels pulse increase in interstitial adenosine that is supposed to be directly involved in cardioprotec- tion [8,13].

The dose-finding study was performed in healthy volunteers belonging to the staff of the Institution in which the study was planned. According to the available data concerning the pharmacokinetics of the drug (half-life after intravenous administration

11.622.2 h) [14], the dose response curve of adeno- sine increase following drug administration and the half-life of plasma adenosine [ 15,161, dipyridamole was administered at the dose of 1 mg at 6 min intervals (Study A, a=3), 2 mg at 15 min intervals (Study B, n=3), 2 mg at 30 min intervals (Study C. n=5) (Fig. 1). Each dose was repeated five times, in accordance with several studies showing that re- peated brief periods of ischaemia induced precondi- tioning of the heart in animal models in vivo [ 17- 20]. Adenosine plasma levels were measured by high performance liquid chromatography technique as reported in details elsewhere [ 15,161. Blood samples for adenosine determinations were withdrawn at the following times: Study A 0 (baseline), 1 (end of the bolus), 3, 6 min (baseline of the following bolus) for each single 1 mg drug intravenous administration up to 40 min; Study B 0 (baseline), 1, 5: 10, 15 min (baseline of the following bolus) for each 2 mg drug intravenous administration up to 75 min; Study C 0 (baseline), 5, 10, 20, 30 min (baseline of the following bolus) up to 150 min.

2.2. Echocardiography stress test

The reproducibility and intrasubject variability of the technique in our ‘laboratory was measured. In details, two relevant parameters to the response after dipyridamole, such as left ventricular end-diastolic and end-systolic volumes, were calculated two times in 40 subjects, by one observer who was blinded to the results of the previous analysis. Statistical corre- lation between the results of the two tests was performed by linear regression analysis. Left ven- tricular end-diastolic volumes in the first and in the second test were 176.55k37.07 ml and 170.75k37.99 ml (mean t standard deviation), respectively (r=0.97; P<O.OOl). Left ventricular end-systolic volumes in the first and in the second test were 74.45k21.88 ml and 69.15k23.64 ml, (mean If: standard deviation), respectively (r=0.96; P<O.OOl). Standard deviation of the mean of in- dividual differences was calculated for left ventricu- lar end-diastolic volume and left ventricular end- diastolic volume, as described elsewhere [21,22], being 5.07 and 4.70 for left ventricular end-diastolic volume and left ventricular end-systolic volume, respectively.

F.L. Pasini et al. I International Journal of Cardiology 56 (1996) 17-27 19

nmol/l AD0 wo-

SW-

400-

A

800-

ZOO-

study A

s.s.ll. = 15.9; n = 3.

600-

100 I 1 0 20 40 60 a0 loo l20 l40 loo

lnh

-l#xaatam* +tm#xaUuL* -tm#1atisnmlm lnrrvsla ltlumh lnltrvsla

DIPYWIMMW

Fig. 1, Dose-finding of dipyridamole. Effect of repeated low-dose intravenous dipyridamole administrations on plasma levels of adenosine. Data are expressed as standard error of the means (S.E.M.) in the one-way analysis of variance (ANOVA). Study A: S.E.M.= 15.9; II =3. Study B: S.E.M.=15.5; n=3. Study C: S.E.M.=8.24; n=5.

Fourteen patients (Table l), referring to our Institution with a history of effort chest pain and angiographically assessed coronary artery disease, were enrolled in the study. They all gave an in- formed consent to participate in the study according to the principles of the Declaration of Helsinki. Eight had a history of previous myocardial infarction. No

patients were receiving antianginal treatment at the time of the study since at least 48 h. All patients were asymptomatic at the time of dipyridamole echocardiography. All the 14 patients who entered the study had a positive dipyridamole echocardiog- raphy test result.

Two-dimensional echocardiographic dipyridamole

20 F.L. Pusini et al. I International Journal of Cnrdiolog~ .56 (1996) 17-27

Table I Clinical variables of the study patients

n=14

Age (years) (meankstandard deviation) Male/female Previous acute myocardial infarction Site of acute myocardial infarction

6.527 IO/4 8

Anterior 2 Inferior 6

Diabetes 2 Beta-blockers 0 Nitrates 0 Calcium antgonists 0

stress tests were performed as described by Picano and coworkers [23,24]: 0.56 mg/kg over 4 min, no dose for 4 min and then, if the test remained negative, 0.28 mg/kg over 2 min; the maximal cumulative dose was therefore 0.84 mg/kg over 10 min. Patients were instructed to fast for at least 3 h before the test and to avoid tea, coffee and cola- drinks, the xanthine content of which can limit the dipyridamole action, for the preceding 24 h. Two dimensional echocardiograms were continuously monitored and recorded during and up to 10 min after dipyridamole test. Commercially available im- aging system ATL Ultramark 9 Color Doppler Ultrasound System with a 2.5 MHz transducer was used. The test terminated if new wall motion dyssynergy was detected by two-dimensional ech- ocardiography or if symptoms were judged to be unacceptable by the physician performing the test. Aminophylline was administered (240 mg intraven- ous as a bolus) as soon as dyssynergy was demon- strated. In the baseline studies, as well as during stress testing, all standard echocardiographic views were obtained when possible; during the test new areas of abnormal wall motion were identified on multiple views by rapidly moving the ultrasound transducer through various positions. Positivity of the test was based on the detection of a transient dyssynergy of contraction, which was absent, or of a lesser degree in the baseline examination. Correla- tion between left ventricular segments and coronary distribution was established according to Feigen- baum [25]. A Wall Motion Score Index was derived for rest and peak dipyridamole echocadiograms in each patient; the left ventricle was divided in 14

segments according to the American Society of Echocardiography Committee of Standards 1261. Each segment was graded as normal, normal motion at rest, with normal or increased wall motion (hy- perkinesia) after dipyridamole (score = 1); hypo- kinetic, marked reduction of endocardial motion (score=2); akinetic, virtual absence of inward mo- tion (score = 3) and dyskinetic or aneurismatic, paradoxical wall motion away from the centre of the left ventricle in systole (score=4). The Wall Motion Score Index was derived by the summation of individual segment scores divided by the number of interpreted segments. Inadequately visualized seg- ments were not scored, any region that was akinetic or dyskinetic at rest was not considered for analysis.

The time to onset (time to positivity, in min) and the dose of dipyridamole (tolerated dose, in mg/kg) required to obtain positivity were registered, together with the duration of wall motion abnormalities (duration of dyssynergy).

In the case of a negative test the time of onset was arbitrarily assumed to be 10 min (total time of the test) and the dose of dipyridamole the total adminis- tered dose (0.84 mg/kg) for statistical analysis.

Echocardiograms were blindly evaluated by two experienced observers: if there was any disagreement over the interpretation, a third observer reviewed the study and subsequent majority judgement was con- sidered binding.

For each patient two stress tests were performed with a time lag of 24 h. The first test was performed in basal conditions on day 1. On day 2 the patients underwent five repeated, bolus administrations of 2 mg dipyridamole at 30 min intervals, according to the findings from the dose-response study. One h after the last administration, a second echocardiog- raphy dipyridamole stress test was performed. The second test was always performed within 24 h from the first one.

Haemodynamic parameters (systolic and diastolic blood pressure, heart rate; rate-pressure product) were monitorized throughout the tests.

A standard 12-lead electrocardiogram (ECG) using a Cardioline Delta 300 ECG was recorded and ST changes (ST elevation or depression of more than 1 mm measured 0.08 s after the J point) and the QT interval corrected for the heart rate according to Bazet [27] at baseline and at ischaemia or at the end

F.L. Pasini et al. I International Journal of Cardiology 56 (1996) 17-27 21

of dipyridamole test were evaluated [28]. The onset of angina was registered.

Adenosine plasma levels were measured at the following times during the stress test (either on day 1 and on day 2): 0 (baseline), 4, 8 and 10 min. During the pharmacological conditioning with repeated bolus administrations of dipyridamole, adenosine plasma levels were measured at the times reported above, i.e. 0 (baseline), 10, 20, 30 min after each single administration (time 30 min after each bolus administration corresponds to the time 0 of the following bolus). Adenosine assay was performed in high performance liquid chromatography on heparin- ized blood withdrawals treated with 50 PM dipyridamole (Boehringer Ingelheim, Germany), and 10 PM EHNA (Wellcome Italia, Pomezia, Italy); the mobile phase was composed of 35 ,uM phosphate buffer, acetonitrile, and methanol (95:2.5:2.5) (flux rate 1 ml/min, wavelength 254 nm).

Echocardiography and ECG were registered also during the pharmacological preconditioning period, in order to rule out the emergency of ischaemia.

2.3. Statistical analysis

Data were expressed as meankstandard deviation. Time to positivity, duration of dyssynergy, and tolerated dose were analyzed by means of paired Student’s t-test; heart rate systolic blood pressure, diastolic blood pressure, rate-pressure product, and QTc were analyzed by the Student-Neuman-Keul’s test for multiple measurements. Since values of Wall Motion Score Index are non-parametric data obtained before and after dipyridamole treatment in the same subjects, variations in this index were evaluated by the Wilcoxon test. Probability (P) value <0.05 was considered to be statistically significant.

3. Results

3. I. Dose-finding of dipyridamole

Pulse administration of 1 mg dipyridamole at 6 min intervals produced a progressive increase in adenosine plasma levels without any recovery to baseline (Fig. 1A). By employing 2 mg dipyridamole at 15 min intervals, a pulse increase of adenosine

plasma levels was obtained, but also baseline adeno- sine concentration progressively increased as a con- sequence of the still too short intervals (Fig. 1B). Prolongation of the intervals up to 30 min attained pulse increases in adenosine plasma levels with a almost complete recovery to baseline at the time of the following administration of 2 mg dipyridamole (Fig. 1C).

3.2. Echocardiographic jindings during dipyridamole test

Each dipyridamole stress test required approxi- mately 30 min. Side-effects due to dipyridamole were always minor and well tolerated by all the patients.

Baseline left ventricular dyssynergy of contraction was found in 10 patients; in fact, eight patients had a history of previous myocardial infarction, and in all of them dyssynergy was present in the region involved by the necrosis.

On day 1, during the first dipyridamole stress test, all the patients showed transient regional asynergy (those four patients with a normal baseline left ventricular contraction developed hypokinesia, four patients developed akinesia in the segments previ- ously hypokinetic and hypokinesia in previously normal regions, and in six patients hypokinesia evolved in akinesia). The involved myocardial walls were the interventricular septum in ten patients, the lateral wall in four and the inferior wall in two.

Mean Wall Motion Score Index raised from sig- nificantly from baseline to the onset of dyskinesia (Table 2).

On day 2, rhythmic intravenous administration of dipyridamole was performed in all the patients; during this time, none of the patients presented chest pain, nor anyone of them developed electrocar- diographic or echocardiographic signs of ischaemia.

After the second stress test, transient dyssynergy occurred only in eight patients; the six remainders showed the same contraction pattern as in the baseline examination, and thus they were considered negative for ischaemia.

In the 14 patients, during the second test Wall Motion Score Index measured at the time of onset of dyssynergy during the first test was not different

22 F.L. Pasini et al. I International humal of Curdiology 56 (1996) 17-27

Table 2 Effect of preconditioning with pulse low-dose dipyridamole intravenous administration on standard dipyridamole test (Date are expressed as mean 2 standard deviation)

Before preconditioning (n = 14): dipyridamole test +

After preconditioning (n = 14): dipyridamole test + (n = 8) dipyridamole test - (n=6)

Basal Peak 1 Basal Time of peak 1 Peak 2

Heart rate (beatslmin) 6527 89t20'."*** 6657 9124""" 93r7h*** Systolic blood pressure (mmHg) 143*17 143?15”.b n.s. 138z9 144t 18” ns. 1452 16b n.s.

Diastolic blood pressure (mmHg) 85215 85-C 14".h n.s. SO?5 8126" n.s. 82?~5~ n.s. Rate pressure product (beats/min/mmHg) 940321619 12850i-3099".b*** 9197k1334 12687+-1630'*** 1274211630h***

QTc 0.42t0.02 0.47+0.02b.c*** 0.42t0.01 0.43+-0.01' n.s. 0.45t0.03h*'k Wall Motion Score Index 1.44kO.43 1.71 iro.44’.“*** 1.4420.43 1.44kO.43’ ns. 1.59-+0.49d*

Time to positivity (min) 4.052.2 x.ot2.0** Duration of dyssynergy (mm) 6.7k2.09 2.01-2.08 Tolerated dose (mg/kg) 0.38ZO.25 0.72t0.15*"

“.hReadings with the same superscript are not significantly different. ‘Readings are significant at P<O.OOl. “Readings are significant at PcO.05.

***P<o.ool **zJ<o.o1 *p<o.o5 Wilcoxon test: Wall Motion Score Index; Student-Neuman-Keul’s test for multiple measurements: QTc, heart rate, systolic and diastolic blood pressure, rate pressure product; Student’s t-test for paired data: tolerated dose, duration of dyssynergy, time to positivity.

from that at baseline; conversely, it was reduced in a significant manner at the end of the test (Table 2).

The time to onset of positivity increased and, as a consequence, the duration of dyssynergy was shor- tened. The dose of dipyridamole required to obtain positivity or tolerated dose, raised significantly (Table 2).

As the differences observed between the two stress tests could be referred to the six patients resulted negative in the second dipyridamole test, a further analysis was performed only for the group of eight patients remaining positive also in the second stress test.

In these patients, the dyssynergy involved the same ventricular regions as in the first test; however, the time to onset of positivity was delayed, the duration of dyssynergy was shortened, and the tolerated dose increased (Table 3). The Wall Motion Score Index, evaluated at time of positivity in the first stress, was not significantly different from baseline, whereas at the end of the test it was significantly prolonged, as it was during the first dipyridamole test (Table 3).

3.3. Electrocardiographic jindings

During the first dipyridamole test, six out of 14 patients with positive echocardiographic results,

showed diagnostic electrocardiographic changes (ST segment depression of more than 1 mm, 0.08 s after the J point); the site of the ST segment depression was not strictly related to the site of dyssynergy.

All the patients resulting positive to dipyridamole administration showed a significant QTc interval prolongation at positivity (Table 2).

Electrocardiographic alterations in the second stress test did not differ from those observed in the first test; but they were delayed in the time, concomi- tantly with echocardiographic changes.

In the second dipyridamole stress test, QTc inter- val measured at the time of onset of dyssynergy during the first test was not significantly different from that at baseline, whereas at the end of the test it was prolonged significantly (Table 2). In those eight patients remaining positive also in the second dipyridamole test, the QTc interval was prolonged at the onset of positivity, but it was not significantly modified at the time of onset of positivity in the first dipyridamole test (Table 3).

3.4. Occurrence of chest pain

During the first test, ten out of 14 patients had chest pain of variable intensity and duration; chest pain relief was always obtained by aminophylline administration.

Table 3

F.L. Pasini et al. I International Journal of Cardiology 56 (1996) 17-27 23

Effect of preconditioning with pulse low-dose dipyridamole intravenous administration on standard dipyridamole test (Data are expressed as mean+ standard deviation)

Before preconditioning (n=8): dipyridamole test +

Basal Peak 1

Heart rate (beats/min) 6426 925p*** Systolic blood pressure (mmHg) 143519 1432 13”.h n.s. Diastolic blood pressure (mm/Hg) 82?11 81 z! 10”,b n.s. Rate pressure product (beats/min/mmHg) 91711’1249 13 251+3495”.h***

QTc 0.42?0.02 0.47~?0.02~.‘*** Wall Motion Score Index 1.5520.56 1.81 +0.57b,d**

Time to positivity (min) 4.022.2 Duration of dyssynergy (mm) 6.722.09 Tolerated dose (mg/kg) 0.38kO.25

After preconditioning (n=8): dipyridamole test +

Basal Time of peak 1 Peak 2

63%7 90?4”*** 91 z4”*** 135+6 1422 16” n.s. 145123h n.s. 7826 8123” n.s. 81-C8h n.s.

8443 +548 12 1201 1238a*** 12 290tl 102h*** 0.42?0.02 0.43tO.Ol‘n.s. 0.4720.02”*** 1.5520.56 1.55 i-0.56%~. 1.81 t-0.57h**

8.0t2.0** 2.0-+2.08**

0.7210.15**

“.bReadings with the same superscript are not significantly different. ‘Readings significant at P<O.OOl. dReadings significant at PCO.01 ***P<o.ool; **p<o.o1; *p<o.o5. Wilcoxon test: Wall Motion Score Test; Student-Neuman-Keul’s test for multiple measurements: QTc, heart rate, systolic and diastolic blood pressure, rate pressure product; Student’s r-test for paired data: time to positivity, duration of dyssynergy, tolereted dose,

After the second dipyridamole stress test, the occurrence of chest pain was delayed in six out of the ten patients who experienced symptoms during the first test; the four remainders did not refer chest pain at all.

case symptoms promptly recovered after amino- phylline administration.

3.7. Metabolic findings

3.5. Haemodynamic jindings

Haemodynamic behaviour of study patients during the dipyridamole tests is displayed in Table 2 and Table 3. All the patients had heart rate and rate pressure product significantly but slightly increased.

During the two dipyridamole tests adenosine plasma levels increased (Table 4 and Fig. 2). During rhythmic intravenous administration of dipyridamole (2 mg) adenosine plasma levels increased in a pulsed manner (Fig. 2).

4. Discussion 3.6. Adverse effects to dipyridamole stress test

No patients presented significant arrhythmias or severe hypotension. All patients during the first test and those with a positive response after the second test, received intravenous aminophylline (240 mg) for obvious asynergy or severe chest pain. In every

In the present study, repeated low doses (2 mgX5)

of dipyridamole, administered at 30 min intervals prior to high dose dipyridamole echocardiography stress test, induced pulse increase in plasma adeno- sine and markedly modified the ischaemic response elicited in coronary artery disease patients. Six out of

Table 4 Adenosine plasma levels (nmol) during DET before and after preconditioning with pulse low-dose dipyridamole intravenous administration- (Data are expressed as mean?standard deviation, n= 14)

0.56 mg/kg 0.28 mg/kg

0 4’ 8’ 10’ 15’ 20’ 25’

Before preconditioning 211214 732252 6951r78 796221 650264 486293 244232 After preconditioning 2442 12 652227 637%86 716244 554270 416%50 260’- 26

F.L. Pusini et al. I International Journal of Cur&logy 56 (1996) 17-27

Dipyrldamola ,,$& s

Ill

n Fig. 2. Adenosine plasma level changes during day 1 (baseline) and day 2 (pre-conditioning).

14 positive patients became negative to the test, since they did not develop any new wall motion abnormality during the second high dose infusion of dipyridamole. In patients in which also the second test remained positive, a significant increase in the dose of dipyridamole necessary to induce ischaemia and a delay in the onset of asynergy were registered. Anyway, the site and the extent of ischaemia were exactly reproducible in both tests.

Very interestingly, QTc interval prolongation, which has been shown to be a sensitive marker of actual myocardial ischaemia [28], was abolished or delayed during the second test in dipyridamole test- and dipyridamole test+ patients, respectively. QTc interval behaved according to other electrocar- diographic, and echocardiographic changes.

The dipyridamole test may offer a rather complex stratification of the ischaemic response: beyond the positivity or negativity, the time of onset of is- chaemia and the amount of dipyridamole able to evoke the wall motion abnormalities may be quan- tified as expression of severity of coronary reserve impairment. Moreover, short-term (1 day) repro- ducibility of dipyridamole echocardiography test has been shown to be very high [24,29]. Picano and coworkers have demonstrated that in a group of 87 patients with rest and/or effort angina, all 60 patients with a positive test had a positive repeat test and the 27 negative were also negative on the second day [29]. The timing of asynergy and the dose of drug necessary to induce ischaemia were also very similar

between the two tests [29]. In our study the two dipyridamole echocardiography tests were performed within 24 h, hence, we can reasonably suggest that the difference in the responses obtained could not be attributed to the intrinsic variability of the method.

The intravenous administration of dipyridamole induces a significant increase in plasma adenosine likely due to a block of adenosine uptake into erythrocytes and endothelium, thereby leading to a potentiation in its dilatatory action on small vessels dependent on the activation of low affinity A2 receptors 130-321. One can assume that dipyridamole-dependent ischaemia is triggered by the vasodilating activity of endogenous adenosine through the activation of low affinity A2 receptor. Accordingly, we have shown that during high dose dipyridamole infusion, a plasma concentration of adenosine in the micromolar range (0.x-1.2 PM) is reached, and that therefore an activation of vascular A2 receptors may occur. Repeated intravenous bolus administrations of low dose dipyridamole (2 mg X5) are able to induce pulse increases in plasma adeno- sine levels in coronary artery disease patients with a positive echocardiography stress test. According to Wang and coworkers [7], Hintze and Vatner [33], and Knabb and coworkers [ 131, we can assume that an increase in interstitial adenosine after dipyridamole administration is also obtained. During the pharmacological preconditioning period, none of the patients developed new echocardiographic or electrocardiographic signs of ischaemia nor they complained for angina1 pain This may suggest that during the dipyridamole bolus infusions, the increase in endogenous adenosine was not sufficient, in terms of maximal amount and time duration, to elicit any haemodynamic response able to trigger coronary steal phenomena and subsequent ischaemia. How- ever, such increases profoundly affected the follow- ing echocardiography stress test, in that a marked reduction of the pharmacological-elicitable ischaemia was observed. We can hypothesize two possible mechanisms underlying such an effect: either the second high dose dipyridamole was no more able to induce a coronary haemodynamic derangement or, although a coronary steal phenomenon still occurred, this was not followed by clinical, echocardiographic and electrocardiographic signs, since ischaemia of cardiomyocytes did not eventually developed. In any

F.L. Pasini et al. I lntemational Journal of Cardiology 56 (1996) 17-27 25

case, it is reasonable to state that the effect of dipyridamole is dependent on the enhancement of adenosine concentration.

The first hypothesis could be consistent with a reduced vasodilatory effect of the full dose of dipyridamole due to a down regulation of A2 coronary receptors stimulated by pulse increase in endogenous plasma adenosine. This phenomenon, called receptor desensitation, has been observed also for Al and A2 receptors in different systems [5,34,35]. Recently Hussain and Mustafa [36] have developed an in vitro model of coronary artery in tissue culture to study A2 adenosine receptor. In vascular rings treated with 2-chloroadenosine, the relaxation responses to other specific adenosine agonists were attenuated compared to control ar- teries, thus confirming that a desensitation phenom- enon involving A2 receptors may occur [36]. How- ever, no clear evidence so far exists in humans that the increase in endogenous adenosine or the adminis- tration of exogenous adenosine are followed rapidly by a desensitation. During adenosine thallium sci- ntigraphy for the diagnosis of coronary artery dis- ease, a progressive large increase in coronary flow is obtained with intravenous adenosine, titrated from initial dose of 50 pglkglmin to 140 ~gfkglmin, with dose increases every minute, without evidence of rapid desensitation [37]. Therefore, although we cannot completely rule out such a mechanism, it seems unlikely that a significant reduction in the vasodilatory response may occur as a consequence of an A2 receptor desensitation due to low, short lasting increases in endogenous adenosine. On the other hand, a down regulation of the Al receptor on cardiomyocytes should antagonize the possible car- dioprotective activity of pulsed dipyridamole ad- ministrations [5].

The second possible explanation of our findings is that a preconditioning-like phenomenon is triggered by increases of adenosine induced pharmacological- ly. Such pulse increases of adenosine, could mimic the events occurring in the course of subcritical ischaemic episodes unable to elicit either evident contractile deficit or chest pain. This interpretation is in agreement with data shown by Miura and co- workers [8] and Mosca and coworkers [9]. The former in 1992 showed that in rabbits the administra- tion of a single dose of dipyridamole potentiated the

myocardial infarct size-limiting effect of ischaemic preconditioning [8]. Such an enhancement was abolished by Cphenyltheophylline, a non-selective adenosine receptor blocker [8]. However, in this model dipyridamole alone (without ischaemic pre- conditioning) failed to show any activity. The au- thors suggested that a single dose dipyridamole was not able to increase interstitial adenosine concen- tration up to a ‘threshold level’ for triggering the preconditioning mechanism [8]. This could be at- tained with the association of an ischaemic precondi- tioning, as suggested by Miura and coworkers, or by repeated bolus administrations of the drug as in our experimental setting. On the other hand, Mosca and coworkers [9] have shown that perfusion with dipyridamole during 5 min prior to ischaemia protected rabbit hearts from systolic and diastolic alterations of stunning secondary to prolonged is- chaemia. Although in the in vivo setting it has been found that preconditioning did not protect against stunning [38], our findings claim that pulse increases in plasma adenosine do protect myocardium from ischaemia-dependent contractile deficit. Therefore, we can suggest that the effects of dipyridamole administrations which we have observed in coronary artery disease patients are due to an adenosine- mediated, possibly Al-dependent cardioprotective activity of the drug, in a very similar way to that considered for preconditioning. In fact, plasma con- centrations of endogenous adenosine during pulse dosing of dipyridamole (up to about 430 nmol/l) are able to activate high affinity Al receptors on car- diomyocytes [35].

Moreover, short-term (1 day) reproducibility of dipyridamole echocardiography stress test as ob- served in our study as well as in other laboratories, seems to suggest that the so-called ‘second window’ of protection of preconditioning [39] does not occur with this model.

The main limitation of the study concerns the open design without a placebo control, with a relatively small number of patients involved. Moreover it will be necessary to extend the study to other stress tests, mainly exercise, to evaluate the impact of dipyridamole on different mechanisms leading to myocardial ischaemia. The number of dipyridamole doses to be administered and the duration of the protection elicited by the drug have to be deter-

26 F.L. Pasini et al. I lntemational Journal of Cardiology 56 (1996) 17-27

mined, but this point seems to be scarcely relevant in terms of interpretation of the present findings.

In conclusion, we found that dipyridamole, ad- ministered as repeated low-dose intravenous boluses, is able to abolish or to markedly reduce myocardial ischaemia induced by high dose of the drug during echocardiography stress test in coronary artery dis- ease patients. Such an effect seems to be attributable to pulse increases in endogenous plasma adenosine. Therefore, low repeated doses of dipyridamole do mimic the ischaemic preconditioning phenomenon.

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