AND DRUG ACTION Differential response of P-adrenergic receptor-mediated heart rate and aortic blodd flow acceleration timolol The apparent affinity of P-adrenergic receptors for timolol in the sinus node and ventricular myocardium was compared in 11normal male subjects. Sinus nodal function was assessed by heart rate, and left ventricular systolic function was assessed by Doppler-derived aortic blood flow acceleration. The dose of isoproterenol required for a heart rate or acceleration increase of 35% (I35)was determined before and 2 hours after an oral 10 mg dose of timolol. The apparent affinity constant ( 4 ) for timolol binding to the receptor was calculated from the serum timolol concentration and the ratio of the 135after tim~loYI,~ before timolol. The for sinus node and ventricular myocardium were not significantly different from one another. The k, for timolol binding to the sinus node (1.14 x 10" mol/L), however, was significantly greater (p < 0.05) than 4 for ventricular myocardium (7.85 x lo5 mom). These findings suggest that P-adrenergic receptors in the sinus node may have an overall greater affinity for P-blocking agents than do receptors in the ventricular myocardium. (CLIN PHARMACOL THER1992;51:296-301.)
G. Dennis Clifton, PharmD, Mary H. H. Chandler, PharmD, Andrew T. Pennell, PharmD, Randall A. McCoy, PharrnD, and Michael R. Harrison, MD Lexington, Ky. The clinical efficacy of P-adrenergic receptor antagonists are primarily attributable to the ability of these agents to inhibit the positive chronotropic and inotropic effects of sympathetic stimulation. Despite the high affinity of P-blocking agents for receptors in both conducting tissue and myocardium, evidence has accumulated that the sinus node and ventricular myocardium differ in their sensitivity to P-adrenergic receptor i~ that in humans b10ckade.l.~In 1978 ~ o n e l l observed
From the Division of Pharmacy Practice and Science, College of Pharmacy, and the Division of Cardiology, College of Medicine, University of Kentucky. Supported in part by the American College of Clinical PharmacyGlaxo Pharmacotherapy Research Award. Received for publication Feb. 25, 1991; accepted Oct. 8, 1991. Reprint requests: G. Dennis Clifton, PharmD, University of Kentucky Medical Center, 800 Rose St., Room C-117, Lexington, KY 40536-0084. 1311134212
the nonselective P-adrenergic receptor antagonist mepindolol inhibited the positive chronotropic effect of isoproterenol much more potently than its positive inotropic effect. Recent studies from our laboratory further support the theory of differential sensitivity. We found propranolol to be significantly more potent in reducing exercise-induced increases in heart rate compared with left ventricular systolic function.' The increased sensitivity of the sinus node to P-adrenergic receptor antagonism compared with the ventricular myocardium suggests that there may be an alteration in the competitive relationship between agonist and antagonist for the receptor sites in those respective tissues. The purpose of this study was to test the hypothesis that the affinity of (3-adrenergic receptors for timolol in the sinus node is greater than the affinity of the receptors in the ventricular myocardium. Affinity of the P-adrenergic receptors for timolol was determined by use of a modification of the standard isoproterenol sensitivity test.6 In this study,
VOLUME 5 1 NUMBER 3
sinus nodal function was assessed by heart rate, and left ventricular systolic function was assessed by Doppler-derived aortic blood flow acceleration. Measurement of ascending aortic blood flow by Doppler echocardiography provides a unique method for pharmacodynamic assessment of cardiovascular response to drugs. Numerous studies in subjects with and without cardiovascular disease have shown that aortic blood flow acceleration is an accurate descriptor of global left ventricular systolic f ~ n c t i o n . ~Further-~ more, an excellent correlation between P-blocker serum concentrations and reduction in aortic blood flow acceleration has been demonstrated in humans.,
METHODS This was an open-label, nonblinded study involving 14 healthy volunteers from 22 to 31 years of age (mean age, 25.2 +- 3.2 years). Subjects were not taking long-term medications and were instructed to not take any prescription medications within the previous week. They also were instructed to not ingest any over-the-counter medication, caffeine, or alcohol for 48 hours before the study and on study days. On the night before the study each subject was admitted to the research unit. On the next morning, after a light, caffeine-free breakfast, each subject had an intravenous catheter inserted in a forearm vein. The catheter was kept patent with a slow infusion of 0.9% sodium chloride solution. The study was performed with the subject in the supine position with continuous ECG and automated blood pressure monitoring. The test for sinus nodal P-adrenergic receptor sensitivity was performed in a fashion similar to that previously except that the dose of isoproterenol to increase the resting heart rate and aortic blood flow acceleration by 35% of baseline (I,,) (rather than an increase in heart rate of 25 beatslmin) was determined. Single intravenous bolus injections of isoproterenol were administered in doubling doses starting with 0.1 pg (i.e., 0.1 pg, 0.2 pg, 0.4 pg, and so forth) and continued until an increase in heart rate of 40% to 50% of baseline heart rate was attained or heart rate exceeded 140 beatslmin. Heart rate was continuously monitored and displayed on an oscilloscope. ECG tracings were recorded starting 1 minute before isoproterenol administration and continued until the peak in heart rate was clearly over. The peak increase in heart rate was determined from the ECG by measuring the three shortest RR intervals before and after each injection. Left ventricular P-adrenergic sensitivity was determined by use of Doppler-derived measurements of aortic blood flow acceleration. Dop-
Dzfferenti p-blocker binding 297 pler measurements were initiated when heart rate began to increase and continued until Doppler values returned to baseline. Doppler recordings were made from the suprasternal notch position by use of a nonimaging transducer angulated to record the maximal flow signal in the ascending aorta as defined by the audio output and a light emitting diode. The instrument used in this study (Exerdop, A.H. Robins Co., Inc., Richmond, Va.) operates at 3 MHz and measures modal velocity from the received Dopplershifted flow velocity signal with a sampling rate of 5 msec. The highest velocity for each systole is reported as the peak modal velocity for that beat. Peak acceleration is derived internally at a high sample rate of 200 Hz with a two-point interpolated slope algorithm. The accuracy of this instrument has been established previously in animal studiesg and confirmed in clinical investigation~.'~ The peak increase in acceleration was determined by taking the mean of the highest 10 consecutive Doppler measurements before and after each bolus injection. Blood pressure was monitored by use of an automated blood pressure monitor (Paramed model 9350, Paramed Technology Inc., Mountain View, Calif.) just before each injection and every 2 minutes after infusion for 6 minutes. After baseline heart rate and acceleration were reestablished (+ 10% of original baseline), the next dose was administered. Subjects were asked to not talk during the study period, and environmental stimulation was kept to a minimum. After completion of the first isoproterenol doseresponse curve, 10 mg timolol was administered orally. After 2 hours a blood sample for determination of timolol concentration was collected. After the first blood sample, a second dose-response curve to isoproterenol was obtained and the I,, for heart rate and aortic blood flow acceleration determined in a manner identical to that described previously. A second blood sample for timolol was collected at the end of the study. The mean of the two plasma sample timolol concentrations for each subject was used for determination of the affinity rate constants. This study was approved by the University of Kentucky Medical Institutional Review Board (Lexington, Ky.) and was conducted in the University of Kentucky General Clinical Research Center (MOlRR02602). Timolol assay. Plasma timolol concentrations were quantitated on the basis of modifications of a previously described HPLC method.', In brief, heparinized plasma (2 ml) was shaken gently with NaOH (4 mol/L, 0.1 ml), internal standard (phenacetin, 0.25 pg), and methyl tert-butylether (5 ml) for 10 minutes.
CLIN PHARMACOL THER MARCH 1992
Fig. 1. Dose of isoproterenol necessary to increase acceleration (ACC) and heart rate (HR) by 35% above baseline (I,,). Bars depict the mean I,, for each parameter.
After centrifugation, the upper layer was transferred and evaporated to dryness. The residue was reconstituted in mobile phase (waterlacetonitrile, 80 :20, containing 1% triethylamine and adjusted to pH 3 with orthophosphoric acid) and an aliquot injected into the chromatograph. The stationary phase consisted of a C,, HPLC column (15 cm X 4.6 mm internal diameter; 5 pm particle size; Supelcosil LC-18, Supelco, Inc., Bellefonte, Pa.). The mobile phase flow rate was 1 mllmin and ultraviolet detection was performed at a wavelength of 295 nm. The interday coefficients of variation (n = 4) were 13.5% and 5.6% at concentrations of 20 nglml and 100 nglml, respectively. The intraday coefficients of variability (n = 4) were 7.3% and 3.4% at concentrations of 5 ng/ml and 100 nglml, respectively. The standard curves were linear over a 0 to 100 nglml timolol plasma concentration range (3> 0.99). All assays of study samples were completed in a single day. Timolol for standard curves was supplied by Merck Sharp and Dohme Research Laboratories (West Point, Pa.). Pharmacodynamic analysis. A log dose-response curve for heart rate and acceleration was determined, and the dose required to increase the resting heart rate and acceleration by 35%, representing isoproterenol sensitivity, was estimated by interpolation. The dose ratio (DR) of isoproterenol for heart rate and peak acceleration were calculated for each subject as the I,, after timolol administration divided by the I,, before timolol administration. According to the classic recep-
tor theory of drug antagonism, the following relationship should exist between DR and drug concentration (P): (DR - 1)
= k,P
in which k, is the apparent association (affinity) constant for the binding of timolol to its receptor. From the DR and P, the k, for timolol binding to sinus node (heart rate) and ventricular myocardium (peak aortic acceleration) were calculated. Statistical analysis. Significance was determined by the paired t test and by the Mann Whitney U test (for the isoproterenol response). Differences were considered significant at p < 0.05.
RESULTS After the administration of timolol, a 35% increase in both heart rate and acceleration was achieved in 11 of the 14 subjects. The findings reported represent data from those 11 subjects. Zsoproterenol sensitivity. The I,, for isoproterenol was greater for heart rate (0.612 5 .576 pg) compared with acceleration (0.373 2 0.154 pg), suggesting a lower sensitivity to isoproterenol of the sinus node compared with the ventricular myocardium (Fig. 1). However, the difference was not statistically significant. The I,, for both heart rate and acceleration were significantly increased after timolol administration compared with heart rate and acceleration before timolol administration. ( p < 0.01). The DR for heart
VOLUME S 1 NUMBER 3
Dzferential p-blocker binding. 299
Fig. 2. The apparent k, for timolol binding to the ventricular myocardium (LV) and sinus node (SN). Bars depict the mean k, for each parameter. * p < 0.05.
rate (1 12.4 -+ 57.8) was significantly greater (p < 0.05) than the DR for acceleration (77.1 + 46.5). Timolol administration did not have a statistically significant effect on the blood pressure response to isoproterenol. The reductions in diastolic blood pressure at the I,, for heart rate and acceleration before timolol (-5.3 + 9.0 and -5.6 -+ 6.4 mm Hg) were not significantly different from those observed after timolol (-1.1 k 8.7 and 0.01 k 4.7 mm Hg). Timolol concentrations. Two hours after oral timolol administration the mean plasma timolol concentration was 34.2 + 11.6 nglml (range, 14.7 to 49.3 nglml). These concentrations were not significantly different (p = 0.65) from the end-of-study plasma timolol levels of 32.1 k 15.1 nglml (range, 11.8 to 63.7 nglml) . The mean plasma timolol concentration for the study period was 33.1 -+ 12.5 nglml. Apparent receptor sensitivity for timolol. From the DR and P data the k, values were calculated that represented apparent P-receptor sensitivity to timolol in the sinus node and ventricular myocardium, respectively (Fig. 2). Values of k, for heart rate (1.14 X lo6 k 4.88 x lo5 mol1L) were significantly greater (p < 0.05) than k, values for acceleration (7.85 x 10' -+ 4.00 X lo5 molIL), suggesting that the P-adrenergic receptors in the sinus node have greater affinity for timolol than P-receptors in the ventricular myocardium.
DISCUSSION The purpose of this study was to determine whether the apparent in vivo k, for P-receptor binding of timolol is different for sinus nodal tissue compared with ventricular myocardium. The use of heart rate to measure response to sympathetic stimulation or blockade, as performed in this study, has been shown to be a reliable indicator of sinus nodal P-adrenergic receptor The use of ascending aortic blood flow acceleration measured by Doppler to assess the contractile response of the left ventricular myocardium to sympathetic stimulation and blockade represents a unique and novel approach for assessment of ventricular myocardial P-adrenergic receptor sensitivity. Previous work by us and others has shown the usefulness and accuracy of the Doppler technique in estimation of acute changes in myocardial systolic function induced by pharmacologic or physiologic interventions.9.12,14-17 Timolol was chosen as the probe P-adrenergic blocker in this study because of its pharmacologic and pharmacokinetic properties. Timolol is minimally (10%) bound to plasma proteins; it is available as the pure active levo-isomer; and there is no evidence of an active metabolite.18 These properties remove or minimize some confounding variables that may otherwise have to be considered with other P-adrenergic blocking agents. Previous investigators have shown an asynchrony of
CLIN PHARMACOL THER MARCH 1992
the inotropic and chronotropic responses after the administration of oral or intravenous propranolol to normal subjects and patients with ischemic heart disStudies have shown that the negative chronotropic response to administration of propranolol both precedes and outlasts the negative inotropic re~ ~ o n s e . 'In . ~ humans, .~ we recently showed that the concentration-effect relationship for S-(-)-propran0101 and its negative inotropic effect differ from its negative chronotropic effect. Propranolol is significantly more potent (lower ECSo) in reducing exerciseinduced increases in heart rate compared with aortic blood flow acceleration. The current study supports these findings, inasmuch as the k, for timolol binding to the sinus node (as determined by heart rate) was significantly greater than the k, for binding to the ventricular myocardium (as determined by aortic blood flow acceleration). Both pharmacokinetic and pharrnacodynamic factors may contribute to differences in tissue response to timolol. The design of this study precludes pharmacokinetic variability as a factor, unless distribution of the drug to separate cardiac tissues differ. Drugmediated response in tissues depends on two tissue factors and two factors related to drug-receptor interaction.19 The tissue factors include the density of drug receptors and the efficiency of the functions that convert receptor stimulus into tissue response. The drugreceptor related parameters include the k, and the intrinsic efficacy of the drug for the receptor. In the present study, both receptor interaction and tissue factors may contribute to the variable drug responsiveness of the sinus node and myocardium. The method used in this study for determining receptor affinity for the antagonist in different tissues assumes a receptor population that is homogeneous with respect to density and subtype. If there is a greater relative density of one subtype of P-adrenergic receptor in sinus nodal versus myocardial tissue, this may influence considerably the observed k, values. It is, in fact, well accepted that both PI- and P2-adrenergic receptors coexist in the human heart. In the atria approximately 33% to 50% of the total P-adrenergic receptor population is of the p2 subtype.20By contrast, in the ventricles the amount of P2-adrenergic receptors comprise 20% to 25% of the p~pulation.~'Previous studies have shown that isoproterenol induced increases in chronotropy and inotropy are mediated through both (3,- and P2-adrenergic receptors.20 It is plausible that the difference in observed k, values for sinus node and ventricular myocardium are caused by differences in the distribution of P-adrenergic subtypes in those re-
spective tissues. Further study with selective agonists and antagonists will be needed to determine if heterogeneity in receptor subtypes are responsible for the difference in receptor affinity for P-adrenergic blocking agents observed in this study. The methods used to assess left ventricular function and P-adrenergic receptor sensitivity and affinity may also be responsible for the differences seen. Although peak aortic blood flow acceleration is extremely sensitive to alterations in the inotropic state of the myocard i ~ m , it~cannot , ~ ~be ~regarded ~ ~ as a true index of myocardial contractility because it may be influenced by changes in loading conditions23724and heart In human subjects paced at rest, Doppler measurements of acceleration have been shown to vary inversely with changes in heart rate.25 This is most likely caused by a reduction in venous return and reduced filling pressures. Elevations in heart rate induced by isoproterenol are less likely to influence acceleration because, as with exercise, venous return is unchanged or increased.26 Reductions in afterload induced by isoproterenol may also increase aortic blood flow acceleration independent from its effects on myocardial contractility. Despite this potential, two factors suggest that our conclusions are valid. First, the fact that timolol did not significantly alter the effect of isoproterenol on diastolic blood pressure suggests that the changes in afterload were similar before and after P-blockade. Second, if timolol did block the afterload reducing effect of isoproterenol, a greater dose ratio for acceleration compared to heart rate would be expected (because the positive inotropic effect of isoproterenol would be overestimated in the pre-timolol phase). Just the opposite was actually observed. Further study with afterload independent indexes of inotropism may be needed to confirm these findings.
References Bloomfield DA, Sowton E. Rate-independent effects of propranolol: the differentiation between chronotropic, inotropic and peripheral vascular responses. Circ Res 1967;21(suppl):243-7. Hunt D, Sloman G, Clark RM, et al. Effect of betaadrenergic blockade on the systolic time intervals. Am J Med Sci 1970;259:97-113. Bonelli J. Demonstration of two different types of betareceptors in man: selective blockade of the positively inotropic and the positively chronotropic effect of isoproterenol. Int J Clin Pharmacol 1978;16:313-9. Boudolas H, Beaver BM, Kates RE, et al. Pharmacodynamics of inotropic and chronotropic responses to oral therapy with propranolol. Chest 1978;73:146-53. Clifton GD, Pennell AT, Harrison MR. Pharmacody-
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6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
namics of propranolol on left ventricular function: assessment by Doppler echocardiography. CLINPHARMACOL THER1990;48:431-8. Cleaveland CR, Rangno RE, Shand DG. A standardized isoproterenol sensitivity test. The effects of sinus arrhythmia, atropine and propranolol. Arch Intern Med 1972;130:47-52. Bennett ED, Barclay SA, Davis AL, Mannering D, Mehta N. Ascending aortic blood velocity and acceleration using Doppler ultrasound in the assessment of left ventricular function. Cardiovasc Res 1984;18:632-38. Stein PD, Sabbah HN, Albert DE, Snyder JE. Continuous-wave Doppler for the noninvasive evaluation of aortic blood velocity and rate of change of velocity: evaluation in dogs. Med Instrum 1987;21: 177-82. Mehta N, Bennett D, Mannering D, Dawkins K, Ward DE. Usefulness of noninvasive Doppler measurement of ascending aortic blood velocity and acceleration in detecting impairment of the left ventricular functional response to exercise three weeks after acute myocardial infarction. Am J Cardiol 1986;57:1052-8. McDevitt DG, Frisk-Holmberg M, Hollifield JW, Shand DG. Plasma binding and the affinity of propran0101 for a beta receptor in man. CLINPHARMACOL THER 1976;20:152-7. Klein C, Gerber JG, Gal J, Nies AS. Beta-adrenergic receptors in the elderly are not less sensitive to timolol. THER1986;40:161-4. CLINPHARMACOL Harrison MR, Smith MD, Friedman BJ, DeMaria AN. Uses and limitations of exercise Doppler echocardiography in the diagnosis of ischemic heart disease. J Am Coll Cardiol 1987;10:809-17. Lennard MS, Parkin S. Timolol determination in plasma and urine by high performance liquid chromatography with ultraviolet detection. J Chromatogr 1985;338:249-52. Bryg RJ, Labovitz AJ, Mehdirad AA, Williams GA, Chaitman BR. Effect of coronary artery disease on Doppler-derived parameters of aortic flow during upright exercise. Am J Cardiol 1986;58:14-9. Mehdirad AA, Williams GA, Labovitz AJ, Bryg RJ, Chaitman BR. Evaluation of left ventricular function during upright exercise: correlation of exercise Doppler
Dzferential P-blocker binding. 30 1
16.
17.
18.
19.
20.
21. 22.
23.
24.
25.
26.
with post-exercise two-dimensional echocardiographic results. Circulation 1987;75:413-9. Clifton GD, Harrison MR, DeMaria AN. Influence of beta-adrenergic blockade upon hernodynamic response to exercise assessed by Doppler echocardiography. Am Heart J 1990;120:579-85. Harrison MR, Clifton GD, DeMaria AN. Hemodynamic effects of calcium channel and beta-receptor antagonists: evaluation by Doppler echocardiography. Am Heart J 1991;121:126-33. Gengo FM, Green JA. Beta-blockers. In: Evens WE, Schentag JJ, Jusko WJ, eds. Applied pharmacokinetics: principles of therapeutic drug monitoring. Spokane, Washington: Applied Therapeutics, 1986:735-8 1. Kenakin TP. Drug Antagonism. In: Kenakin TP, ed. Pharmacologic analysis of drug-receptor interaction. New York: Raven Press. 1987:205-44. Motomura S, Zerkowski HR, Daul A, Brodde OE. On the physiologic role of beta-2 adrenoceptors in the human heart: in vitro and in vivo studies. Am Heart J 1990;119:608-19. Brodde OE. Cardiac beta-adrenergic receptors. IS1 Atlas Sci: Pharmacol 1987;l:107-12. Noble MIM, Trenchard D, Guz A. Left ventricular ejection in conscious dogs. I. Measurement and significance of the maximum acceleration of blood from the left ventricle. Circ Res 1966;19: 139-47. Wallmeyer R, Wann SL, Sagar KB, et al. The influence of preload and heart rate on Doppler echocardiographic indexes of left ventricular performance: comparison with invasive indexes in an experimental preparation: Circulation 1986;74:181-6. Bedotto JB, Eichhorn EJ, Grayburn PA. Effects of left ventricular preload and afterload on ascending blood flow velocity and acceleration in coronary artery disease. Am J Cardiol 1989;64:856-9. Harrison MR, Clifton GD, Sublett KL, DeMaria AN. Effect of heart rate on Doppler indexes of systolic function in humans. J Am Coll Cardiol 1989;14:929-35. Weiner N. Norepinephrine, epinephrine, and the sympathomimetic arnines. In: Gilman AG, Goodman LS, Gilman A, eds. Goodman and Gilman's The pharmacologic basis of therapeutics. 6th ed. New York: MacMillan, 1975:138-75.
G. Dennis Clifton, PharmD, Mary H. H. Chandler, PharmD, Andrew T. Pennell, PharmD, Randall A. McCoy, PharrnD, and Michael R. Harrison, MD Lexington, Ky. The clinical efficacy of P-adrenergic receptor antagonists are primarily attributable to the ability of these agents to inhibit the positive chronotropic and inotropic effects of sympathetic stimulation. Despite the high affinity of P-blocking agents for receptors in both conducting tissue and myocardium, evidence has accumulated that the sinus node and ventricular myocardium differ in their sensitivity to P-adrenergic receptor i~ that in humans b10ckade.l.~In 1978 ~ o n e l l observed
From the Division of Pharmacy Practice and Science, College of Pharmacy, and the Division of Cardiology, College of Medicine, University of Kentucky. Supported in part by the American College of Clinical PharmacyGlaxo Pharmacotherapy Research Award. Received for publication Feb. 25, 1991; accepted Oct. 8, 1991. Reprint requests: G. Dennis Clifton, PharmD, University of Kentucky Medical Center, 800 Rose St., Room C-117, Lexington, KY 40536-0084. 1311134212
the nonselective P-adrenergic receptor antagonist mepindolol inhibited the positive chronotropic effect of isoproterenol much more potently than its positive inotropic effect. Recent studies from our laboratory further support the theory of differential sensitivity. We found propranolol to be significantly more potent in reducing exercise-induced increases in heart rate compared with left ventricular systolic function.' The increased sensitivity of the sinus node to P-adrenergic receptor antagonism compared with the ventricular myocardium suggests that there may be an alteration in the competitive relationship between agonist and antagonist for the receptor sites in those respective tissues. The purpose of this study was to test the hypothesis that the affinity of (3-adrenergic receptors for timolol in the sinus node is greater than the affinity of the receptors in the ventricular myocardium. Affinity of the P-adrenergic receptors for timolol was determined by use of a modification of the standard isoproterenol sensitivity test.6 In this study,
VOLUME 5 1 NUMBER 3
sinus nodal function was assessed by heart rate, and left ventricular systolic function was assessed by Doppler-derived aortic blood flow acceleration. Measurement of ascending aortic blood flow by Doppler echocardiography provides a unique method for pharmacodynamic assessment of cardiovascular response to drugs. Numerous studies in subjects with and without cardiovascular disease have shown that aortic blood flow acceleration is an accurate descriptor of global left ventricular systolic f ~ n c t i o n . ~Further-~ more, an excellent correlation between P-blocker serum concentrations and reduction in aortic blood flow acceleration has been demonstrated in humans.,
METHODS This was an open-label, nonblinded study involving 14 healthy volunteers from 22 to 31 years of age (mean age, 25.2 +- 3.2 years). Subjects were not taking long-term medications and were instructed to not take any prescription medications within the previous week. They also were instructed to not ingest any over-the-counter medication, caffeine, or alcohol for 48 hours before the study and on study days. On the night before the study each subject was admitted to the research unit. On the next morning, after a light, caffeine-free breakfast, each subject had an intravenous catheter inserted in a forearm vein. The catheter was kept patent with a slow infusion of 0.9% sodium chloride solution. The study was performed with the subject in the supine position with continuous ECG and automated blood pressure monitoring. The test for sinus nodal P-adrenergic receptor sensitivity was performed in a fashion similar to that previously except that the dose of isoproterenol to increase the resting heart rate and aortic blood flow acceleration by 35% of baseline (I,,) (rather than an increase in heart rate of 25 beatslmin) was determined. Single intravenous bolus injections of isoproterenol were administered in doubling doses starting with 0.1 pg (i.e., 0.1 pg, 0.2 pg, 0.4 pg, and so forth) and continued until an increase in heart rate of 40% to 50% of baseline heart rate was attained or heart rate exceeded 140 beatslmin. Heart rate was continuously monitored and displayed on an oscilloscope. ECG tracings were recorded starting 1 minute before isoproterenol administration and continued until the peak in heart rate was clearly over. The peak increase in heart rate was determined from the ECG by measuring the three shortest RR intervals before and after each injection. Left ventricular P-adrenergic sensitivity was determined by use of Doppler-derived measurements of aortic blood flow acceleration. Dop-
Dzfferenti p-blocker binding 297 pler measurements were initiated when heart rate began to increase and continued until Doppler values returned to baseline. Doppler recordings were made from the suprasternal notch position by use of a nonimaging transducer angulated to record the maximal flow signal in the ascending aorta as defined by the audio output and a light emitting diode. The instrument used in this study (Exerdop, A.H. Robins Co., Inc., Richmond, Va.) operates at 3 MHz and measures modal velocity from the received Dopplershifted flow velocity signal with a sampling rate of 5 msec. The highest velocity for each systole is reported as the peak modal velocity for that beat. Peak acceleration is derived internally at a high sample rate of 200 Hz with a two-point interpolated slope algorithm. The accuracy of this instrument has been established previously in animal studiesg and confirmed in clinical investigation~.'~ The peak increase in acceleration was determined by taking the mean of the highest 10 consecutive Doppler measurements before and after each bolus injection. Blood pressure was monitored by use of an automated blood pressure monitor (Paramed model 9350, Paramed Technology Inc., Mountain View, Calif.) just before each injection and every 2 minutes after infusion for 6 minutes. After baseline heart rate and acceleration were reestablished (+ 10% of original baseline), the next dose was administered. Subjects were asked to not talk during the study period, and environmental stimulation was kept to a minimum. After completion of the first isoproterenol doseresponse curve, 10 mg timolol was administered orally. After 2 hours a blood sample for determination of timolol concentration was collected. After the first blood sample, a second dose-response curve to isoproterenol was obtained and the I,, for heart rate and aortic blood flow acceleration determined in a manner identical to that described previously. A second blood sample for timolol was collected at the end of the study. The mean of the two plasma sample timolol concentrations for each subject was used for determination of the affinity rate constants. This study was approved by the University of Kentucky Medical Institutional Review Board (Lexington, Ky.) and was conducted in the University of Kentucky General Clinical Research Center (MOlRR02602). Timolol assay. Plasma timolol concentrations were quantitated on the basis of modifications of a previously described HPLC method.', In brief, heparinized plasma (2 ml) was shaken gently with NaOH (4 mol/L, 0.1 ml), internal standard (phenacetin, 0.25 pg), and methyl tert-butylether (5 ml) for 10 minutes.
CLIN PHARMACOL THER MARCH 1992
Fig. 1. Dose of isoproterenol necessary to increase acceleration (ACC) and heart rate (HR) by 35% above baseline (I,,). Bars depict the mean I,, for each parameter.
After centrifugation, the upper layer was transferred and evaporated to dryness. The residue was reconstituted in mobile phase (waterlacetonitrile, 80 :20, containing 1% triethylamine and adjusted to pH 3 with orthophosphoric acid) and an aliquot injected into the chromatograph. The stationary phase consisted of a C,, HPLC column (15 cm X 4.6 mm internal diameter; 5 pm particle size; Supelcosil LC-18, Supelco, Inc., Bellefonte, Pa.). The mobile phase flow rate was 1 mllmin and ultraviolet detection was performed at a wavelength of 295 nm. The interday coefficients of variation (n = 4) were 13.5% and 5.6% at concentrations of 20 nglml and 100 nglml, respectively. The intraday coefficients of variability (n = 4) were 7.3% and 3.4% at concentrations of 5 ng/ml and 100 nglml, respectively. The standard curves were linear over a 0 to 100 nglml timolol plasma concentration range (3> 0.99). All assays of study samples were completed in a single day. Timolol for standard curves was supplied by Merck Sharp and Dohme Research Laboratories (West Point, Pa.). Pharmacodynamic analysis. A log dose-response curve for heart rate and acceleration was determined, and the dose required to increase the resting heart rate and acceleration by 35%, representing isoproterenol sensitivity, was estimated by interpolation. The dose ratio (DR) of isoproterenol for heart rate and peak acceleration were calculated for each subject as the I,, after timolol administration divided by the I,, before timolol administration. According to the classic recep-
tor theory of drug antagonism, the following relationship should exist between DR and drug concentration (P): (DR - 1)
= k,P
in which k, is the apparent association (affinity) constant for the binding of timolol to its receptor. From the DR and P, the k, for timolol binding to sinus node (heart rate) and ventricular myocardium (peak aortic acceleration) were calculated. Statistical analysis. Significance was determined by the paired t test and by the Mann Whitney U test (for the isoproterenol response). Differences were considered significant at p < 0.05.
RESULTS After the administration of timolol, a 35% increase in both heart rate and acceleration was achieved in 11 of the 14 subjects. The findings reported represent data from those 11 subjects. Zsoproterenol sensitivity. The I,, for isoproterenol was greater for heart rate (0.612 5 .576 pg) compared with acceleration (0.373 2 0.154 pg), suggesting a lower sensitivity to isoproterenol of the sinus node compared with the ventricular myocardium (Fig. 1). However, the difference was not statistically significant. The I,, for both heart rate and acceleration were significantly increased after timolol administration compared with heart rate and acceleration before timolol administration. ( p < 0.01). The DR for heart
VOLUME S 1 NUMBER 3
Dzferential p-blocker binding. 299
Fig. 2. The apparent k, for timolol binding to the ventricular myocardium (LV) and sinus node (SN). Bars depict the mean k, for each parameter. * p < 0.05.
rate (1 12.4 -+ 57.8) was significantly greater (p < 0.05) than the DR for acceleration (77.1 + 46.5). Timolol administration did not have a statistically significant effect on the blood pressure response to isoproterenol. The reductions in diastolic blood pressure at the I,, for heart rate and acceleration before timolol (-5.3 + 9.0 and -5.6 -+ 6.4 mm Hg) were not significantly different from those observed after timolol (-1.1 k 8.7 and 0.01 k 4.7 mm Hg). Timolol concentrations. Two hours after oral timolol administration the mean plasma timolol concentration was 34.2 + 11.6 nglml (range, 14.7 to 49.3 nglml). These concentrations were not significantly different (p = 0.65) from the end-of-study plasma timolol levels of 32.1 k 15.1 nglml (range, 11.8 to 63.7 nglml) . The mean plasma timolol concentration for the study period was 33.1 -+ 12.5 nglml. Apparent receptor sensitivity for timolol. From the DR and P data the k, values were calculated that represented apparent P-receptor sensitivity to timolol in the sinus node and ventricular myocardium, respectively (Fig. 2). Values of k, for heart rate (1.14 X lo6 k 4.88 x lo5 mol1L) were significantly greater (p < 0.05) than k, values for acceleration (7.85 x 10' -+ 4.00 X lo5 molIL), suggesting that the P-adrenergic receptors in the sinus node have greater affinity for timolol than P-receptors in the ventricular myocardium.
DISCUSSION The purpose of this study was to determine whether the apparent in vivo k, for P-receptor binding of timolol is different for sinus nodal tissue compared with ventricular myocardium. The use of heart rate to measure response to sympathetic stimulation or blockade, as performed in this study, has been shown to be a reliable indicator of sinus nodal P-adrenergic receptor The use of ascending aortic blood flow acceleration measured by Doppler to assess the contractile response of the left ventricular myocardium to sympathetic stimulation and blockade represents a unique and novel approach for assessment of ventricular myocardial P-adrenergic receptor sensitivity. Previous work by us and others has shown the usefulness and accuracy of the Doppler technique in estimation of acute changes in myocardial systolic function induced by pharmacologic or physiologic interventions.9.12,14-17 Timolol was chosen as the probe P-adrenergic blocker in this study because of its pharmacologic and pharmacokinetic properties. Timolol is minimally (10%) bound to plasma proteins; it is available as the pure active levo-isomer; and there is no evidence of an active metabolite.18 These properties remove or minimize some confounding variables that may otherwise have to be considered with other P-adrenergic blocking agents. Previous investigators have shown an asynchrony of
CLIN PHARMACOL THER MARCH 1992
the inotropic and chronotropic responses after the administration of oral or intravenous propranolol to normal subjects and patients with ischemic heart disStudies have shown that the negative chronotropic response to administration of propranolol both precedes and outlasts the negative inotropic re~ ~ o n s e . 'In . ~ humans, .~ we recently showed that the concentration-effect relationship for S-(-)-propran0101 and its negative inotropic effect differ from its negative chronotropic effect. Propranolol is significantly more potent (lower ECSo) in reducing exerciseinduced increases in heart rate compared with aortic blood flow acceleration. The current study supports these findings, inasmuch as the k, for timolol binding to the sinus node (as determined by heart rate) was significantly greater than the k, for binding to the ventricular myocardium (as determined by aortic blood flow acceleration). Both pharmacokinetic and pharrnacodynamic factors may contribute to differences in tissue response to timolol. The design of this study precludes pharmacokinetic variability as a factor, unless distribution of the drug to separate cardiac tissues differ. Drugmediated response in tissues depends on two tissue factors and two factors related to drug-receptor interaction.19 The tissue factors include the density of drug receptors and the efficiency of the functions that convert receptor stimulus into tissue response. The drugreceptor related parameters include the k, and the intrinsic efficacy of the drug for the receptor. In the present study, both receptor interaction and tissue factors may contribute to the variable drug responsiveness of the sinus node and myocardium. The method used in this study for determining receptor affinity for the antagonist in different tissues assumes a receptor population that is homogeneous with respect to density and subtype. If there is a greater relative density of one subtype of P-adrenergic receptor in sinus nodal versus myocardial tissue, this may influence considerably the observed k, values. It is, in fact, well accepted that both PI- and P2-adrenergic receptors coexist in the human heart. In the atria approximately 33% to 50% of the total P-adrenergic receptor population is of the p2 subtype.20By contrast, in the ventricles the amount of P2-adrenergic receptors comprise 20% to 25% of the p~pulation.~'Previous studies have shown that isoproterenol induced increases in chronotropy and inotropy are mediated through both (3,- and P2-adrenergic receptors.20 It is plausible that the difference in observed k, values for sinus node and ventricular myocardium are caused by differences in the distribution of P-adrenergic subtypes in those re-
spective tissues. Further study with selective agonists and antagonists will be needed to determine if heterogeneity in receptor subtypes are responsible for the difference in receptor affinity for P-adrenergic blocking agents observed in this study. The methods used to assess left ventricular function and P-adrenergic receptor sensitivity and affinity may also be responsible for the differences seen. Although peak aortic blood flow acceleration is extremely sensitive to alterations in the inotropic state of the myocard i ~ m , it~cannot , ~ ~be ~regarded ~ ~ as a true index of myocardial contractility because it may be influenced by changes in loading conditions23724and heart In human subjects paced at rest, Doppler measurements of acceleration have been shown to vary inversely with changes in heart rate.25 This is most likely caused by a reduction in venous return and reduced filling pressures. Elevations in heart rate induced by isoproterenol are less likely to influence acceleration because, as with exercise, venous return is unchanged or increased.26 Reductions in afterload induced by isoproterenol may also increase aortic blood flow acceleration independent from its effects on myocardial contractility. Despite this potential, two factors suggest that our conclusions are valid. First, the fact that timolol did not significantly alter the effect of isoproterenol on diastolic blood pressure suggests that the changes in afterload were similar before and after P-blockade. Second, if timolol did block the afterload reducing effect of isoproterenol, a greater dose ratio for acceleration compared to heart rate would be expected (because the positive inotropic effect of isoproterenol would be overestimated in the pre-timolol phase). Just the opposite was actually observed. Further study with afterload independent indexes of inotropism may be needed to confirm these findings.
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