Selective Attenuation by N-0861 (N6-Endonorboran-2-yl-9-Methyladenine) of Cardiac A1 Adenosine Receptor–Mediated Effects in Humans
Background To determine the adenosine receptor subtype selectivity of the novel antagonist N-0861, the A1 and A2 receptor–mediated cardiac effects of adenosine were investigated in 13 patients during continuous intravenous infusion and boluses of adenosine before and after intravenous infusion of N-0861.
Methods and Results Measurements of the atria-to-His (A-H) interval, chest pain severity, and coronary blood flow velocity were made before and after low-dose (69 μg · kg−1 · min−1) intravenous infusion and bolus (2.5 mg) adenosine. Two doses of N-0861 were infused intravenously, and the adenosine protocol was repeated. N-0861 0.25 mg/kg abolished the negative dromotropic effect (A-H interval prolongation) and chest discomfort experienced during infusion of adenosine and attenuated discomfort observed during the boluses of adenosine; however, the increase in coronary blood flow velocity was not significantly affected.
Conclusions These actions of N-0861 support the concept that the negative dromotropic effect and anginalike pain caused by adenosine are A1 adenosine receptor–mediated, whereas the increase in coronary blood flow velocity is due to activation of A2 adenosine receptors. N-0861 appears to be an effective and selective A1 adenosine receptor antagonist in humans.
Adenosine is a naturally occurring nucleoside formed by the degradation of adenine nucleotides (eg, ATP) and S-adenosylhomocysteine.1 The cardiac effects of adenosine are mediated through at least two specific cell-surface adenosine receptor subtypes, the A1 and A2 receptors.2 A1 adenosine receptors present on cardiomyocytes mediate depression of sinoatrial and AV nodal function, whereas A2 adenosine receptors on the vascular endothelial and smooth muscle cells mediate coronary vasodilation.2 Just as the A1 receptor–mediated effects of adenosine have been used for the acute treatment of supraventricular arrhythmias, the A2 receptor–mediated effects have now been exploited clinically as a pharmacological “stress” during radionuclide cardiac scintigraphy (eg, 201Tl) for detection of ischemic heart disease.3 4 Increasing myocardial adenosine concentration, either directly through continuous intravenous infusion of adenosine or indirectly through the administration of dipyridamole (a drug known to inhibit the cellular uptake of adenosine), will cause a mismatch in coronary blood flow that can be detected by radionuclide imaging techniques. However, this latter use of adenosine is not without undesirable side effects due to activation of cardiac and peripheral A1 receptors. Adenosine given intravenously as a bolus or a continuous infusion causes chest pain in more than 50% of patients and causes noticeable electrophysiological effects in 10% to 15%.5 6 When patients experience these unwanted A1 receptor–mediated effects, the testing protocol may be disrupted, leading to premature termination. Therefore, it has been proposed that adenosine combined with a drug that abolishes or attenuates its negative chronotropic, dromotropic, and algogenic effects may prove to be safer and cause less discomfort than adenosine alone in pharmacological stress testing.7 8
The methylxanthines theophylline and caffeine are reversible and competitive antagonists at the adenosine receptors but have narrow selectivity for the A1 versus the A2 adenosine receptor subtype.9 Recently, a novel nonxanthine adenosine receptor antagonist, N-0861, has been reported to be highly selective for the A1 adenosine receptor subtype in laboratory animal models.7 8 Results of radioligand binding assays for adenosine receptors in brain tissues indicate that N-0861 is ≈610-fold more selective for the A1 than A2 adenosine receptors.10 Pharmacological characterization of N-0861 in guinea pig isolated hearts and anesthetized open-chest swine hearts demonstrated that N-0861 is a reversible and selective antagonist of adenosine at the A1 receptor subtype.7 8 Preliminary data in humans reveal that N-0861 is an effective antagonist in preventing first- and second-degree AV block (an A1 receptor–mediated effect) after intravenous boluses of adenosine.11 Toxicology studies have shown N-0861 to have no mutagenic activity and shown the no-effect acute dose in rodents to be 100 mg·kg−1·d−1, and subchronic studies in rats and dogs have identified no-effect intravenous doses of 50 and 30 mg·kg−1·d−1, respectively.12 In humans, doses of N-0861 up to 0.5 mg/kg have been administered intravenously, limited only by pain at the injection site, which was related to the acidic vehicle.11 In the present study, we sought to demonstrate that N-0861 would effectively antagonize the cardiac A1 receptor– but not the A2 receptor–mediated effects of adenosine in humans.
Patients recruited for participation in this study were individuals scheduled to have cardiac catheterization for evaluation of chest pain who were determined during coronary angiography to have at least one normal or minimally diseased coronary artery. All patients gave informed consent for participation in this research protocol before the diagnostic cardiac catheterization. Medications and foods containing caffeine and theophylline were stopped 48 hours before the procedure. Blood samples were obtained before the study was begun to document the abstinence from caffeine and theophylline as well as to ensure that the complete blood count, renal function tests, electrolytes, and liver function tests were within the ranges of normal. The research protocol used was reviewed and approved by the University of Florida Institutional Review Board and Gainesville Veterans Affairs Hospital research review subcommittee.
After completion of diagnostic catheterization and angiography, a 6F quadripolar catheter was advanced through a 7F introducer placed in the femoral vein and positioned across the tricuspid valve near the region of the His bundle. This catheter was then manipulated until a clearly identifiable His bundle deflection was recorded. The His bundle electrogram was recorded continuously throughout the study. An 8F guiding catheter was advanced into the ostium of the angiographically normal coronary artery. Through this catheter, a 3F coronary velocity Doppler catheter (NuMed) was advanced into the coronary artery and manipulated until an adequate blood flow velocity signal was obtained. The position of the Doppler catheter was kept constant throughout the study. Once the instrumentation was completed, baseline measurements of the A-H interval, the H-V interval, sinus cycle length, systemic arterial blood pressure, and mean CBFV were made. After the baseline measurements were obtained, adenosine (Adenocard, Fugisawa) was initially infused at a rate of 60 μg·kg−1·min−1 and then increased stepwise until an ≈1.5- to 2-fold increase in the mean CBFV was observed. After each rate change of the adenosine infusion, a 4-minute period was allowed, to achieve a steady-state effect. Measurements of the above electrophysiological/hemodynamic parameters were then recorded on a chart recorder at a paper speed of 100 mm/s. The patients were then questioned about the occurrence of symptoms such as chest pain or shortness of breath. If symptoms were present, the patients were asked to grade their discomfort from 1 to 10 on a modified Borg scale, in which 1 represents very mild pain and 10 represents the worst pain ever experienced. Additional adenosine was given as a bolus injection (range, 1.5 to 4.5 mg) to produce prolongation of the A-H interval and maximize coronary vasodilation, and the electrophysiological and hemodynamic measurements and symptoms experienced at peak effect were recorded. N-0861 was then administered intravenously at 0.10 mg/kg over a 5-minute period, and measurements of the A-H and H-V intervals, sinus cycle length, mean CBFV, and arterial pressure were made. The continuous infusion and bolus injection of adenosine were then repeated at the same dosages administered previously, and measurements of the electrophysiological and hemodynamic parameters were repeated. Thereafter, a second dose of N-0861 was given at a dose of 0.15 mg/kg over a 5-minute period, and the above protocol was repeated. Because of the long half-life of N-0861 in humans,11 a second dose of N-0861 was administered to yield a cumulative dose of 0.25 mg/kg.
Twenty-four hours after the completion of the study, the patients underwent a detailed physical examination and laboratory analysis. The values of hemoglobin, hematocrit, white blood cell count, platelet count, electrolytes (Na+, K+, Cl−, CO2−), renal function tests (BUN, creatinine), liver function tests (bilirubin, AST, ALT, LDH), and urinalysis (pH, specific gravity, glucose, bilirubin, sediment) were compared with precatheterization values.
CBFV data were reported as the percent increase above baseline13 and were used as a surrogate measure of coronary blood flow both at baseline and during drug administration. This assumption could be made because the CBFV is linearly related to coronary blood flow at a given constant arterial diameter.14 15 16 Nonstenosed coronary arteries were used in our investigation so that any increase in CBFV could be used as an index of the increased flow. The duration of the increase in the A-H interval and mean CBFV caused by the adenosine boluses was determined as the period of time in which the A-H interval and mean CBFV exceeded a value 10% above baseline. Fig 1⇓ illustrates changes in the A-H interval and mean CBFV measured every 3 seconds from an individual patient after a bolus injection of adenosine (2.4 mg). Asterisks in the figure denote the peak effect of the A-H interval prolongation and mean CBFV increase. The doubled arrows represent the measured duration of these two responses.
Values are expressed as mean±SD. Statistical analysis was based on Student’s t test (two-sample) for paired data and two-way ANOVA for multiple comparisons among control and interventions. Differences between the group means (control versus intervention) were considered significant at P<.05.
Thirteen patients (8 men and 5 women) 45 to 66 years old (mean, 56 years) were recruited for participation in this study. Pretest caffeine levels were essentially undetectable (0.6±0.5 μg/mL). The entire study protocol was successfully completed in all 13 patients, and no adverse events or complications were experienced by any patient during administration of adenosine or of N-0861, alone or combined with adenosine.
CBFV measurements were made in the left anterior descending coronary artery in 9 patients, in the left circumflex in 3, and in the right coronary artery in 1. The baseline electrophysiological and hemodynamic measurements are summarized in Table 1⇓.
Effect of Adenosine Alone
The intravenous infusion of adenosine (mean dose, 69±12 μg·kg−1·min−1) produced a 61% increase in the mean CBFV (9±5 to 14±8 cm/s). At this rate of adenosine infusion, no significant effect was noted on the A-H interval, systolic or diastolic arterial blood pressure, or sinus cycle length compared with baseline (Table 1⇑). When additional adenosine (2.4±1.2 mg) was administered as a rapid intravenous bolus, a significant prolongation of the A-H interval (94±16 to 177±66 ms) was obtained, and a further increase in the mean CBFV (14±8 to 23±13 cm/s) was noted compared with the infusion of adenosine alone (Table 1⇑). Likewise, the systolic and diastolic arterial blood pressures were significantly (P<.05) reduced after the bolus injection of adenosine compared with baseline (138±24 versus 110±27 and 77±13 versus 60±14 mm Hg, respectively). Although the sinus cycle length was prolonged by 18% after the bolus injection of adenosine, this increase in sinus cycle length did not reach statistical significance (Table 1⇑).
Effect of N-0861 Alone
At the 0.10-mg/kg and cumulative 0.25-mg/kg doses, N-0861 alone had little effect on the A-H interval (90±14 versus 91±14 versus 92±14 ms), sinus cycle length (900±234 versus 923±265 versus 986±299 ms), or mean CBFV (9±5 versus 10±6 versus 10±5 cm/s) compared with baseline. Conversely, N-0861 alone (0.10 and 0.25 mg/kg) caused a small but significant rise in the systolic arterial blood pressure compared with baseline (138±24 versus 145±25 versus 154±24 mm Hg). The diastolic blood pressure also exhibited an upward trend. No patient experienced any adverse symptoms related to the N-0861 infusion.
Reversal by N-0861 of the Cardiac Effects of Adenosine
N-0861 at the 0.10-mg/kg dose partially antagonized the A1 receptor–mediated effects of adenosine. During both continuous infusion and bolus administration of adenosine, N-0861 at the cumulative dose of 0.25 mg/kg completely abolished the prolongation of the A-H interval and increase in sinus cycle length caused by adenosine. In contrast, N-0861 did not antagonize the A2 adenosine receptor–mediated effect but instead caused a small increase in mean CBFV (23±13 to 30±13 cm/s) and a significant (P<.05) increase in the systolic and diastolic arterial blood pressures (110±27 to 128±34 and 60±14 to 73±17 mm Hg, respectively). The electrophysiological and CBFV data that were recorded after the rapid adenosine bolus before and after N-0861 are summarized in Fig 2⇓.
Fig 3⇓ summarizes the effects of N-0861 on the duration of the responses (A1 and A2) to bolus administration of adenosine. N-0861 at the cumulative dose of 0.25 mg/kg significantly reduced the duration of A1 adenosine receptor–mediated prolongation of the A-H interval compared with baseline. That is, N-0861 essentially abolished the adenosine-induced A-H interval prolongation by reducing the duration of this effect from 9±4 to 0.5±1.4 seconds, whereas it significantly prolonged the duration of the increase in the mean CBFV from 22±12 to 35±15 seconds.
Combined Effect of Adenosine and N-0861 on Cardiac Rate-Pressure Product
The baseline rate-pressure product averaged 9409±2891 bpm×mm Hg. During the infusion of adenosine (69±12 μg·kg−1·min−1), the rate-pressure product increased nonsignificantly to 10 075±2979 bpm×mm Hg. However, when N-0861 was added to the adenosine infusion, the rate-pressure product rose significantly, to 10 223±2748 bpm×mm Hg (P<.05).
Effect of N-0861 on Algogenic Effects of Adenosine
Nine of the 13 patients reported some degree of chest discomfort, which could be attributed to adenosine during the continuous infusion (mean, 4 on Borg scale) and/or bolus (mean, 7 on Borg scale) administration of adenosine. No ST-segment changes were noted to suggest the presence of myocardial ischemia. N-0861 prevented the sensation of chest discomfort in all patients during the infusion of adenosine and attenuated it after the bolus injection of adenosine (mean, 5 on Borg scale) (Table 2⇓). This attenuation of adenosine-induced chest pain by N-0861 mirrored the antagonism of the A-H interval prolongation. In none of the patients was there a relationship between the severity of chest discomfort and the increase in the mean CBFV.
Effect of N-0861 on Laboratory Indexes
N-0861 had no effect on the total blood count, electrolyte profile, renal function tests, liver function tests, or urinalysis from samples collected 24 hours after administration of this antagonist compared with precatheterization (before the administration of N-0861) laboratory values. Measurements of the total urinary output before and after administration of N-0861 were not performed because of the confounding influences of the clinical catheterization procedure and the need for posttest hydration.
The major finding of this study was that N-0861 is a competitive and selective antagonist of the A1 adenosine receptor in humans. N-0861 reversed the negative dromotropic effects of adenosine, which is A1 receptor–mediated, but did not attenuate the coronary dilation, which is mediated by the A2 adenosine receptor. In the presence of N-0861, the increase in CBFV observed during administration of adenosine was somewhat higher than in its absence (Table 1⇑). Furthermore, in the majority of patients, adenosine induced a dose-dependent chest discomfort. N-0861, a selective and specific A1 adenosine receptor antagonist,7 8 10 significantly attenuated the sensation of adenosine-induced chest pain in a competitive manner (Table 2⇑). This provides the strongest evidence thus far that chest discomfort associated with adenosine administration is mediated via the A1 receptor. Also, in the presence of N-0861, the rate-pressure product was slightly increased during the adenosine infusion compared with that of adenosine alone. Thus, adenosine, when administered with N-0861, behaves pharmacologically as a short-acting, selective A2 receptor agonist.
Adenosine alone prolonged the A-H interval (A1 receptor–mediated effect) and increased the mean CBFV (A2 receptor–mediated effect). The dose required to induce the A1 receptor–mediated effect was much higher than that required to cause the A2 receptor–mediated effect. This finding is in full agreement with data obtained in isolated guinea pig hearts.7 In this latter study, a 100-fold greater concentration of adenosine was required to induce an A1 as opposed to an A2 receptor–mediated effect. Also consistent with the results of animal studies in anesthetized open-chest swine hearts,8 a higher dose of adenosine was necessary to cause peripheral vasodilation (fall in systemic blood pressure) as opposed to coronary vasodilation (increase in CBFV).
N-0861 did not antagonize the A2 receptor–mediated increase in CBFV. In fact, N-0861 tended not only to increase the mean CBFV but also to prolong the duration of this increase. Again, this mirrors the 20% increase in coronary blood flow and 15% prolongation of the duration of coronary blood flow increase reported in the anesthetized open-chest swine hearts.8 A possible explanation for this latter observation is that N-0861, by maintaining or increasing the rate-pressure product, increased the myocardial oxygen demand, which in turn leads to a further increase in coronary vasodilation and thereby flow. Although plausible, this explanation is less likely to apply when the mean CBFV was further increased by the intravenous boluses of adenosine. A more likely explanation, based on the facts that adenosine depresses atrial contractility and also attenuates the positive inotropic effects of catecholamines on the ventricular myocardium, is that N-0861 antagonizes these effects of adenosine and thereby increases or maintains atrial and ventricular contractility. As a consequence, myocardial oxygen demand and overall cardiac performance will increase, which in turn will cause an increase in the coronary blood flow and systemic blood pressure. This hypothesis is supported by the observation that N-0861 prevents the reduction in left ventricular pressure induced by adenosine in the anesthetized open-chest swine heart.8 N-0861 did cause a dose-related rise in the systolic arterial blood pressure that contributed to the measured increase in the rate-pressure product. Similar observations were also made in the anesthetized pig.8
Adenosine is known to induce a dose-dependent activation of visceral nociceptors.17 When administered to patients with coronary artery disease, it is known to provoke chest pain that is similar in quality and location to their typical anginal pain.18 This pain can be worsened after administration of the adenosine uptake blocker dipyridamole and lessened with the nonselective adenosine receptor antagonist theophylline.17 Some investigators have hypothesized that the A1 receptor subtype is the mediator of the adenosine-induced pain. However, our study is the first to use a proven selective A1 receptor antagonist to antagonize adenosine-mediated chest pain. N-0861 caused a dose-dependent, selective antagonism of the adenosine-induced chest pain. From this observation, we conclude that the A1 receptor subtype mediates the electrophysiological effects of adenosine as well as nociception. Consistent with our interpretation that the chest pain caused by adenosine is A1 and not A2 receptor mediated, in none of the patients was there a relationship between the severity of chest discomfort and the increase in mean CBFV. However, chest pain was reported in patients despite complete antagonism of the adenosine-induced A-H interval prolongation by N-0861. Hence, it appears that less A1 receptor activation is needed to cause pain than to produce measurable A-H interval prolongation. Alternatively, the A1 receptor may not be the only adenosine receptor subtype that mediates algogenic effects of the nucleoside.
In summary, N-0861 in humans is a safest-use, selective, and competitive A1 adenosine receptor antagonist. On the basis of its well-documented effects in laboratory animals and the present study results, we propose that the electrophysiological and nociceptive effects of adenosine in humans are, in large part, mediated by the A1 receptor. As previously proposed,19 the results reported in this study reveal that in humans, adenosine combined with N-0861 acts as a short-acting A2 receptor agonist. The combination of adenosine and N-0861 should be a better pharmacological stressor than adenosine alone because of the increase in the rate-pressure product and coronary blood flow. With the unwanted A1 receptor–mediated effects (AV block and chest pain) effectively antagonized by N-0861, this combination should also be safer and more comfortable for the patient than adenosine alone. Moreover, N-0861 alone may prove useful in situations of increased endogenous adenosine, such as in myocardial infarction, after cardiopulmonary bypass, in cardiac arrest, and in cardiac transplant rejection, which may cause (1) bradyarrhythmias,20 21 22 23 24 25 (2) an increase in the defibrillatory threshold,26 and (3) an attenuation of the cardiac effects of catecholamines (A1 adenosine receptor–mediated effects).
Selected Abbreviations and Acronyms
|bpm||=||beats per minute|
|CBFV||=||coronary blood flow velocity|
Discovery Therapeutics, Inc (Richmond, Va) generously supplied N-0861 as well as the funding for other supplies, which made this project possible.
- Received September 20, 1995.
- Revision received October 27, 1995.
- Accepted November 9, 1995.
- Copyright © 1996 by American Heart Association
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