EXHIBIT F 

Case: 1:12-cv-05026 Document #: 112-6 Filed: 02/01/13 Page 1 of 10 PageID #:1426
ABBREVIATiONS: CGP2O71 2-A, (±H2-hydroxy-5-[2(2ydroxy-3-(4-((1-methyl-4-tnfluoromethyl)1H-imidazole-2-yl)phenoxy)propyl)amino)ethoxyJbenzamide monomethane sulfonate; [3H]CGP-1 21 77, (±[3H]43-teiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride; lCl1 1 8551 ,ehro-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol.

843

0026-895X/88/060843.09$02.OO/O Copyright © by The American Society for Pharmacology and Experimental Therapeutics

All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 34:843-851 The Receptor Binding Profile of the New Antihypertensive Agent

Nebivolol and Its Stereoisomers Compared With Various f3-.

Adrenergic Blockers

PETRUS J. PAUWELS, WALTER GOMMEREN, GUY VAN LOMMEN, PAUL A. J. JANSSEN, JOSEE E. LEYSEN

Department of Biochemical Pharmacology (P.J.P., W.G., P.A.J.J., J.E.L.) and of Organic Synthesis (G.V.L., P.A.J.J.), Janssen Research Foundation, B-2340 Beerse, Belgium

Received June 2, 1988; Accepted August 23, 1988

SUMMARY

Nebivolol [the (S,R,R,R)- + (R,S,S,S)-racemic mixture], the 10

stereoisomers, and known fl-adrenergic blockers were investigated in vitro for binding to and f32-adrenergic receptor sites

and various neurotransmitter, peptide, and ion channel binding

sites and for inhibition of neurotransmitter uptake. Selective

labeling of /3 and fl2-adrenergic receptor sites in rabbit and rat

lung, respectively, was obtained with [3H]CGP-1 21 77 and [3H]

dihydroalprenolol in the presence of an appropriate concentration

of the selective 32-adrenergic blocker lCl 1 1 8-551 or the selective

f31-adrenergic blocker CGP 2071 2-A. Nebivolol revealed high

affinity and selectivityfor f31-adrenergic receptor sites in the rabbit

lung membrane preparation (K value = 0.9 n and 2/f.1 ratio = 50). The drug dissociated slowly from these receptor sites. The

activity resided in the (S,R,R,R)-enantiomer (R 67 1 38); the

(R,S,S,S)-enantiomer (A 67 1 45) revealed 175 times lower

adrenergic binding affinity. Within the series of stereoisomers,

nebivolol and R 67 1 38 showed the best combination of high

affinity and selectivity. Among the reference compounds, only

CGP 2071 2-A shared these properties. Nebivolol bound to S1A

binding sites with a K, value of 20 n. The stereospecific requirements for interaction with these sites were different from those

for the f31-adrenergic receptor site. S1A binding site affinity was

also observed with the potent but nonselective /3-adrenergic

blockers carvedilol, pindolol, and propranolol. In the various other

investigated radioligand binding and neurotransmitter uptake

assays, nebivolol and its stereolsomers showed activity only at

micromolar concentrations or were inactive. Clinical studies have

shown an interesting hemodynamic profile of nebivolol, offsetting

the negative effects on left ventricular performance generally

observed with classical f3-adrenergic blockers. Several hypotheses regarding the mechanism of action of nebivolol are

summarized.

Nebivolol (R 65 824) (nebivolol-hydrochloride is R 67 555),

3,4-dihydro-2H-1-benzopyran-2-methanol], is a pseudosymmetrical molecule with four asymmetric carbon atoms (Fig. 1).

Ten stereoisomers, comprising four enantiomeric pairs and two

mesoforms, were synthesized and isolated.’ Nebivolol, the racemic mixture of the (S,R,R,R)- and (R,S,S,S)-enantiomers, is

being investigated as a new antihypertensive agent. In clinical

studies with hypertensive patients, nebivolol was found to

reduce heart rate and blood pressure but it also improved left

ventricular function (1-3). In animal pharmacological studies,

immediate reduction in blood pressure was observed with nebivolol, after its administration to conscious spontaneously

hypertensive rats. No such effect was observed with known fi- Part of this work was supported by a grant from the Instituut veer Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (Brussels, Belgium).

I Van Lommen et aL, manuscript in preparation.

adrenergic blockers such as atenolol, propranolol, or pindolol.

A further unusual feature observed at low doses of nebivolol

was its apparent lack of negative cardiac inotropic effect in

anesthetized dogs, in comparison with propranolol. Nebivolol

reduced systemic vascular resistance and increased cardiac

output and stroke volume. At equivalent doses, propranolol

reduced cardiac output and stroke volume. Pharmacological

investigations using isolated tissues have revealed a potent

antagonism by nebivolol of isoprenaline-induced effects mediated by 3,-adrenergic receptors in the guinea pig atrium.

However, the compound was 300-fold less active in antagonizing /32-adrenergic receptor-mediated effects in the guinea pig

trachea. A selective action of nebivolol at f3, -adrenergic receptors in vivo is apparent from the greater inhibition of isoprenaline-induced changes of left ventricular contractility mediated

by cardiac f3,-adrenergic receptors, as compared with the reduction in diastolic blood pressure (vascular f32-athenergic receptors) in dogs (3).

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0,

0

844 Pauwels et a!.

F

Fig. 1. Structure ofnebivolol with indication

(a) of the four asymmetric carbon atoms.

In this study, the receptor binding and neurotransmitter

uptake inhibition properties of nebivolol were investigated.

Specific radioligand binding models have been developed for

selective labeling of fi, - and fl2-adrenergic receptor sites. This

was achieved using (i) selective tissues, i.e., rabbit and rat lung

for ,- and f.2-athenergic receptor sites, respectively, (ii) the

selective 3,-adrenergic receptor blocker CGP 20712-A (4) and

fl2-athenergic receptor blocker ICI 118-551 (5, 6), and (iii) [3H]

CGP-12177 and [‘H}dihydroalprenolol as radioligands. The

stereoselectivity of the fl-adrenergic receptor interaction with

the nebivolol stereoisomers was investigated. The dissociation

rate of unlabeled drugs from the fi, - and fl2-adrenergic receptors

was measured by modification of a previously described tissueadsorbed-to-filter method (7, 8). The interaction of nebivolol

stereoisomers with various neurotransmitter receptors, ion

channels, and peptide binding sites was investigated. The potency of these compounds to inhibit monoamine uptake in rat

brain synaptosomes was tested. The fl-adrenergic selectivity

and profile of nebivolol and its stereoisomers were compared

with those of known fl-adrenergic blockers. The biochemical

profile of nebivolol is discussed in light of the reported pharmacological properties and findings in clinical studies.

Materials and Methods

Tissue preparation. Lungs from male New Zealand rabbits (-2

kg) and female rats (-150 g) were dissected and transferred in 0.9%

NaCl. Tissue was homogenized in 10 volumes (volumes per wet weight

tissue, v/w) of buffer (0.25 M sucrose, 0.15 M NaClO4 . H20, 5 mM

EDTA, and 25 mM imidazol, pH 7.4) with a Polytron mixer (3 x 10

sec, 1500 rpm). The homogenate was centrifuged at 830 x g for 10 mm

to precipitate cell nuclei and debris. The pellet was rehomogenized and

similarly centrifuged. The supernatants were combined, and filtered

over cheesecloth, and further diluted up to 40 volumes per wet weight

with 50 mM Tris.HC1, pH 7.7. This suspension was centrifuged at

23,600 x g for 20 mm, to precipitate the cell membranes. The pellet

was washed twice by suspension in Tris . HC1 buffer and centrifuged.

The final pellet was homogenized with a Duall homogenizer in 10 volumes of 50 mM Tris . HC1, pH 8. During the entire preparation

procedure the tissue suspension was kept at 0-4”. The membrane

preparation was distributed into aliquots and stored at -70’ . For binding assays, the membrane preparation was diluted to 100 volumes

(v/w) with 50 mM Tris . HC1, pH 8. Binding assays to fi,- and $2-adrenergic receptor sites. Incubation mixtures were composed of 2 ml of tissue preparation, 0.1 ml of

[H]CGP-12177 or [H]dihydroalprenolol, with or without drug for

binding site occlusion, and 0.1 ml of solvent (10% ethanol), drug for

inhibition, or drug for determination of nonspecific binding. Samples

were mixed and incubated for 15 mm at 37” . The reactions were stopped

by adding 5 ml of ice-cold Tris . HCI buffer, pH 8.0, and rapid filtration

over Whatman GF/B glass fiber filters under vacuum. The filters were

rapidly rinsed twice with 5 ml of ice-cold Tris . HC1 buffer, pH 8.0.

Filters were placed in scintillation vials and radioactivity was extracted

by vigorous shaking in 8 ml of Instagel II (Packard, Warrenville). The

radioactivity was counted in a Packard Tri-carb 4530 liquid scintillation counter. To measure binding to $,-adrenergic binding sites in rabbit lung

membranes, 10 nM IC! 118-551 was added with [3HJCGP-12177 or [3HJ

dihydroalprenolol for occlusion of fl)-adrenergic binding sites. Nonspecific binding was measured in the additional presence of 1 cM CGP

20712-A. To measure binding to 9)-adrenergic binding sites in rat lung

membranes, 300 nM CGP 20712-A was added with [‘H]CGP-12177 or

[3H]dihydroalprenolol for occlusion of9,-adrenergic binding sites. Nonspecific binding was defined in the additional presence of 1 zM IC! 118-551. To measure potencies of drugs for inhibition of binding, 1 nM [3H]

CGP-12177 or [3H]dihydroalprenolol was used. The drugs were added

to the incubation mixtures in at least six concentrations, spanning 4

orders of magnitude. The specific [3H]CGP-12177 or [3Hjdihydroalprenolol binding in the presence of drug was calculated as the percentage of total [3H]CGP-12177 or [3Hjdihydroalprenolol binding and

plotted versus the log of the drug concentration. IC values (concentration inhibiting 50% of specific [3H]ligand binding) were derived

graphically. K, values were calculated according to the Cheng-Prusoff

equation: K, = ICse/(1 + C/Kd) with C being the concentration and Kd

the equilibrium dissociation constant of the [3Hjligand (9).

For saturation binding curves, [3H]CGP-12177 or [3Hjdihydroalprenolol was used at concentrations between 0.05 and 1 nM. K,, and

Bmas values were derived from Scatchard plote. Linear regression lines

were calculated by the method of least squares.

Measurement of ,- and 2-adrenergic receptor dissociation

rates. The in vitro dissociation rates of the unlabeled drugs from the

i3,- and fl2-adrenergic receptor sites were measured using a tissue- absorbed-to-filter method as previously described (7, 8), with modifi- cations. A tissue membrane preparation (see above), saturated with

drug during preincubation with a concentration of 10 x IC value, was

adsorbed to Whatman GF/B glass fiber filters positioned on the filtration apparatus. The drug-loaded tissues, adsorbed to the filters, were

rinsed with warm buffer for various time periods. At the end of the

rinsing period, the tissue, adsorbed to the filter, was incubated with a

sample of [3H]CGP-12177 to quantify free receptors. Calculation of the

half-time of dissociation of the unlabeled drug was as previously de- scribed (7).

Radioligand receptor binding and neurotransmitter uptake

assays. Radioligand binding assays were performed using rat or guinea

pig brain membrane preparations (10). For neurotransmitter uptake, a

crude synaptosomal fraction from rat brain regions was used (10). The

assay conditions for serotonin S2, serotonin SIA, dopamine D2, dopamine D,, a,-adrenergic, a2-adrenergic, histamine H,, cholinergic-muscarinic, t opiate, benzodiazepine, dihydropyridine, biogenic amine, and

metabolite release, substance P and neurotensin receptor binding, and

serotonin, noradrenaline, dopamine and -y-aminobutyric acid uptake

were as previously described.2 Binding to the veratridine site of the

2 Leysen, J. E., W. Gommeren, A. Eens, D. de Chaffoy de Courcelles, J. C.

Stoof, and P. A. J. Janssen. Biochemical profile of risperidone, a new antipsychotic. J. Pharrnacol. Exp. Ther. 247:661-670 (1988).

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RABBIT LUNG RAT LUNG

I 00

 80

 60

- 40

a0

0 20

I

20712A

TP2O;; ‘

Receptor Binding Profile of Nebivolol 845

Na channel was measured with tetraphenylphosphonium ions as previously described (11).

Materials. (-)-[3H]CGP-12177 (34 Ci/mmol) was from Amersham

and 1[propyl-1,2,3-[3Hjdihydroalprenolol-HC1 (48 Ci/mmol) was obtamed from New England Nuclear (Dreieich, Germany). Nebivolol and

stereoisomers were original substances from Janssen Pharmaceutica

(Beerse, Belgium). Other drugs were generously supplied by the companies of origin.

Results

Development of receptor binding models for selective

labeling of th- and $2-adrenergic receptors. Inhibition of

[3H]CGP-12177 binding by the selective fi,- and $2-adrenergic

blockers CGP 20712-A and ICI 118-551, respectively, was

measured in rabbit and rat lung membrane preparations; inhibition curves are shown in Fig. 2. In rabbit lung, CGP 20712-A

showed a monophasic inhibition curve and inhibited 80% of

total [3H]CGP-12177 binding. The inhibition curve of ICI 188-

551 was biphasic; it was noted that less than 15% oftotal bound

[3H]CGP-12177 was inhibited in the nanomolar range. In contrast, ICI 118-551 inhibited, at nanomolar concentrations, 80%

of total [3HJCGP12177 binding in the rat lung membrane

preparation. In this preparation, CGP 20712-A showed a biphasic inhibition curve; only 25% of the total [3H]CGP-12177

binding was inhibited in the nanomolar range. These findings

indicated that rabbit and rat lung membrane preparations were

enriched in $,- and $2-athenergic receptor sites, respectively.

In subsequent experiments, the minor population of and $, -

adrenergic receptor sites in rabbit and rat lung was occluded

by addition of 10 nM ICI 118-551 and 300 nM CGP 20712-A to

rabbit and rat lung membrane preparations, respectively. Figs.

3 and 4 show the saturation binding curves of [‘H]CGP-12177

in rabbit and rat lung membrane preparations under such

conditions. Scatchard analysis revealed a single population of

binding sites in each of the tissues, representing , -adrenergic

receptor sites in the rabbit lung and 92-athenergic receptors

sites in the rat lung. Similar findings were obtained with [‘H]

dihydroalprenolol. Kd and Bmax values for [‘HJCGP-12177 and

[:;H]dihythoalprenolol binding are summarized in Table 1. [‘H]

0 , , , ,-, 0

10 9 5 7 6 5 10 9 8 7 6 5 DRUG CONCENTRATION (-log Ml Fig. 2. Inhibition of total [3H]CGP-1 21 77 binding to rabbit and rat lung

membrane preparations by COP 20712-A and ICI 1 18-551 . Binding was

performed with 1 ni [3H]CGP-1 21 77 as described in Materials and

Methods. In rabbit lung, total and nonspecific binding (in the presence of

1 sM CGP 2071 2-A) represent 16,690 ± 2,950 dpm and 3,71 3 ± 623

dpm, respectively. Rat lung, total and nonspecific binding (in the presence

of 1 MM ICI 1 18-551) represent 17,51 1 ± 2,091 dpm and 2,547 ± 387

dpm, respectively. [3H]CGP-121 77 binding is expressed as percentage

of total binding in the absence of unlabeled drugs. f3, $1-adrenergic

receptor site; $2, fl-adrenergic receptor site. Curves were constructed

using mean values ± standard error of three separate experiments

performed in duplicate.

CGP-12177 bound with high affinity to /3,- and $2-adrenergic

receptor sites, the affinity for the f.,-athenergic receptor being

slightly higher. [‘H]Dihydroalprenolol bound with higher affinity to $2 than fi, -adrenergic receptor sites. Its $2-adrenergic

receptor affinity was similar to that of [‘H]CGP-12177. The

density of 3,-adrenergic receptor sites in the rabbit lung membrane preparation was equal to the density of f32-adrenergic

receptor sites in the rat lung membrane preparation. The

receptor densities obtained with [‘Hjdihydroalprenolol binding

were in the same range.

Interaction of nebivolol, its stereoisomers, and various

fl-adrenergic blockers with fi,- and $2-adrenergic receptor sites. Fig. 5 shows the inhibition curves of nebivolol, its denantiomer R 67 138 (S,R,R,R), and its 1-enantiomer R 67 145

(R,S,S,S) on [3H]CGP42177 binding to /3,- and fl2-adrenergic

receptor sites in rabbit and rat lung membrane preparations,

respectively. Nebivolol and R 67 138 were as potent as CGP

20712-A in the inhibition of [3H]CGP12177 binding to rabbit

lung membrane preparation. R 67 145 was 100 times less active

than R 67 138. In contrast, [3H]CGP-12177 binding to rat lung

membrane preparation was only weakly inhibited by nebivolol

and its two enantiomers. Nebivolol and R 67 138 were 100

times less potent than IC! 118-551 whereas R 67 145 was still

10 times less active. The eight remaining stereoisomers of

nebivolol were similarly investigated; the binding affinities for 11- and fl2-adrenergic receptor sites measured with [‘H]CGP- 12177 and [3H]dihydroalprenolol are summarized in Table 2.

Nebivolol and R 67 138 showed the highest affinity for fi,-

adrenergic receptors and they revealed a l/fl2 receptor selectivity of 40- to 50-fold. The most pronounced fl,-adrenergic

selectivity (70-100-fold) was found with R 74 718 (R,R,R,R),

but its fl,-adrenergic affinity was 12-fold less than that of R 67

138.

The $-adrenergic receptor binding affinity and selectivity of

various known -athenergic blockers is shown in Table 3.

Carvedilol, pindolol, and propranolol potently bound to 3, - and

f32-adrenergic receptor sites and lacked selectivity. Levantolol

and labetolol were less potent and also nonselective. CGP

20712-A was potent and highly selective for /3,-adrenergic receptor sites whereas IC! 118-551 was a selective compound for

132-adrenergic receptor sites. Atenolol showed low affinity for

, $,- and fl2-adrenergic receptor sites and only moderate selectivity.

The dissociation rates of the compounds from the , - and $2 adrenergic receptor sites were measured using the tissue-adI’? sorbed-to-filter technique. The half-times of dissociation are

presented in Table 4. Labetolol, pindolol, propranolol, and

levantolol dissociated within a few minutes from both the $,-

and the fl2-athenergic receptor sites. By contrast, nebivolol,

R 67 138 (S,R,R,R), IC! 118-551, and carvedilol dissociated

slowly from the f,- and fl2-adrenergic receptor site.

Interaction of nebivolol, its stereoisomers, and various

19-adrenergic blockers with neurotransmitter receptors,

ion channels, and peptide binding sites, and neurotransmitter uptake. The binding affinity in vitro of the nebivolol

stereoisomers was measured in radioligand binding assays for

neurotransmitter receptor sites, ion channels and peptide binding sites. The binding affinities of the stereoisomers expressed

as -log IC.,, values and K, values are shown in Table 5. The

potencies of the drugs (-log IC50) to inhibit the uptake of

serotonin, norepinephrine, dopamine, and y-aminobutyric acid

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0.6-

0.5

0. L.

0.3#{149}

0.2

0.1-

E

thI 0E

0.0

0.0 0.5

[3H]CGP-12177 CnM)

0.00 0.02

Specific

0.04 0.06

[H]CGP -12177

Rat Lung

0.08

binding (pmol/mt)

LILI)

U-

(I,

Fig. 4. Saturation binding curve (inset) and Scatchard plot of [3H}CGP1 21 77 binding to 92-adrenergic receptor sites in rat lung membrane

preparation. Binding was carried out as described in the legend to

Fig. 3 except that 300 n CGP 2071 2-A was used instead of 10 n lCI

1 18-551 , to block fl1-adrenergic receptor sites. 0, Total binding; U,

specific binding. Derived Kd and B5 values are presented in Table 1.

Q RABBIT LUNG RAT LUNG

100

 80

 60

:- 0

a. 0C-, 20

I

TOG

80

60’ 40

20

0.02 0.OL Specific [3H] CGP- 12177 binding (pmol/mI)

846 Pauwels et a!.

Rabbit Lung

TABLE 1

K,, and B,..1 values of [3HJCGP-12271 and [3H]dihydroalprenolol

binding to $i and 2-adrenergic receptor sites in rabbit and rat lung

membrane preparation.

K,, and B values are the means ± standard error of values obtained in four

separate experiments.

Rabb lung /, Rat lung 02 K5 B K5 B

flu (mo//mg oftissue flu tmoI/mg of tissue

I3HICGP-12177 0.14 ± 0.01 9.3 ± 0.7 0.24 ± 0.04 10.7 ± 0.7

(3HjDihydroalprenolol 0.89 ± 0.23 5.1 ± 1.3 0.21 ± 0.03 7.7 ± 0.4

Fig. 3. Saturation binding curve (inset) and Scatchard

plot of [3HJCGP-1 21 77 binding to fl,-adrenergic receptor sites in rabbit lung membrane preparation. Binding

was carried out in the presence of 1 0 nM ICI 118-551

to block fl2-adrenergic receptor sites. Nonspecific

binding was defined in the presence of 1 M CGP

2071 2-A (0, total binding; #{149}, Specific binding). Curves

were constructed using mean values of binding data

from four separate experiments. SB, specific [3H]CGP1 2177 binding, total bound [3H]CGP-1 21 77 in the

presence of 10 nM ICI 118-551 minus nonspecifically

bound. F, free [3H]CGP-1 21 77 concentration, added

concentration of [3H]CGP-1 21 77 minus the total concentration bound. K,, value was given by the reciprocal

value of the slope of the lines. B,,,a value was given

by the intersection point with the abscissa (in pmol/

ml). Lines were calculated using the method of least

squares. Values are presented in Table 1.

l0      010  DRUG CONCENTRATION(-logM)

Fig. 5. Inhibition of [3H]CGP-1 21 77 binding to  and f2-adrenergic

receptor sites in rabbit and rat lung membrane preparation, respectively,

by nebivolol (0), R 67 138 (#{149}),R 67 1 45 (x), CGP 2071 2-A (U), and ICI

1 18-551 (0). Rabbit lung binding was in the presence of 10 nM CI 118-

551 ; total binding, 13874 ± 1 327 dpm; and nonspecific binding, 2475 ± 1 03 dpm. Rat lung binding was in the presence of 300 n CGP

20712-A; total binding, 13384 ± 2199 dpm; and nonspecific binding, 1 841 ± 228 dpm. [3H]CGP-1 21 77 binding is expressed as percentage

of total binding in the presence of 10 n ICI 1 18-551 and 300 n CGP

2071 2-A for rabbit and rat lung, respectively. Curves were constructed

using mean ± standard error values of three separate experiments in

duplicate.

in crude synaptosomal preparations are shown in Table 6.

Several of the nebivolol stereoisomers bound to SIA binding

sites labeled with [3H]8hythoxy2(din..propylammno)tetralin

nebivolol, R 65 825, R 67 138, R 65 260, R 74 716, R 74 829,

and R 67 142 showed K, values between 20 and 40 nM. In the

various other investigated radioligand binding and neurotransmitter uptake assays, nebivolol and its stereoisomers showed

only activity at micromolar concentrations or were inactive.

In order to better visualize the profile of the nebivolol stereoisomers, pie charts have been constructed for nebivolol, R 67

138, and R 67 145 using the reciprocal of the K, values for

receptor binding and IC50 values for inhibition of monoamine

uptake (Fig. 6). The pie chart shows the relative contribution

of each activity in the sum of activities of the drug presented

in Tables 2, 5, and 6. For nebivolol, $,-adrenergic receptor

binding accounts for 93%, f32-adrenergic receptor binding for

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Receptor Binding Profile of Nebivolol 847

TABLE 2

Binding affinity of nebivolol stereoisomers for  and fi2-adrenergic receptor sites measured with two ligands

a, -log 1Gw (M), mean value ± standard deviations. Numbers in parentheses, number of experiments. b, K, values (nM). Binding was performed as described in Materials

and Methods in the presence of 300 n CI 1 1 8-551 with rabbit lung membrane preparation and 1 0 n CGP 2071 7-A with rat lung membrane preparation to measure

111- and 02-adrenergic receptor sites, respectively.

lHlCGP-12177 binding (1 nM) [HlDihydroalrenod binding (1 nM) A-number,

Configuration Rabbe lung 0, Rat lung 02 Rabbit lung 0 Rat lung 02 Nebivolol a. 8.13±0.05(3) 6.6(2) 8.72±0.09(4) 6.57±0.05 (4)

S,R,R,R + R,S,S,S b. 0.88 48 55 0.91 44 48

R65825 a. 7.53±0.05(3) 6.15±0.07(3) 7.92±0.09(4) 6.13±0.11 (3)

S,R,S,S ± R,S,R,R b. 3.5 144 41 5.7 281 49

R 67 138 a. 8.17 ± 0.06 (3) 6.76 ± 0.05 (3) 8.95 ± 0.1 (4) 6.96 ± 0.15 (3)

S,R,R,R b. 0.8 34 42 0.54 19 35

R 67 145 a. 5.93 ± 0.1 1 (3) 5.66 ± 0.05 (2) 6.53 ± 0.05 (3) 5.66 ± 0.1 1 (3)

R,S,S,S b. 140 423 3 138 367 2.6

R65260 a. 7.65±0.07(2) 6.70±0.14(2) 8.2(2) 6.40±0.14(2)

S,R,R,S b. 2.7 39 15 3 68 23

R74716 a. 6.15±0.07(2) 5.65±0.2(2) 6.8(2) 5.69±0.07(2)

R,S,S,R b. 84 433 5.15 75 375 5

R74829 a. 6.5(2) 6.20±0.14(2) 7.05±0.07(2) 6.15±0.2(2)

S,R,S,R b. 38 122 3.2 43 125 2.9 R74714 a. 6.60±0.14(2) 5.95±0.07(2) 7.25±0.07(2) 6.0(2)

S,R,S,S b. 30 217 7.2 27 166 6.1

R67142 a. 7.5(2) 6.10±0.14(2) 7.90±0.14(2) 6.0(2)

R,S,R,R b. 3.8 153 40 6 166 28 R 74 721 a. 5.95 ± 0.07 (2) 5.15 ± 0.07 (2) 6.40 ± 0.07 (2) 5.30 ± 0.14 (2)

R,R,S,S b. 133 1370 10 193 857 4.4

R 74 723 a. 5.1 (2) 5.30 ± 0.14 (2) 5.40 ± 0.14 (2) 5.19 ± 0.07 (2)

S,S,S,S b. 945 971 1 1935 1187 0.6

R74718 a. 7.05±0.07(2) 5.2(2) 7.65±0.07(2) 5.35±0.07 (2)

R,R,R,R b. 11 1222 111 11 744 68

TABLE 3

Binding affinity of various fl-adrenergic blockers for  and 2-adrenergic receptor sites measured with two ligands

a, -log lC (M), mean value ± standard deviation. Numbers in parentheses, number of experiments. b. K value (nM). Binding was performed as described in the legend

to Table 2.

[HJC GP-12177 binding (1 flM) l3HlDihy droalprenold tnding (1 nM)

. Rabbd lung 13, Rat lung 132 Ratio P2/P’

. Rabbd lung 0 Rat lung $2 Rate P2 CGP20712-A

Atenolol a. b. a. b.

7.86±0.05(3)

1.6

5.65±0.07(2)

396

5.2

1222

4.8(2)

7493

763

19

8.35(2)

2.1

6.25±0.07(2)

266

5.3(2)

835

4.75±0.07(2)

2960

397

11

Levantolol a.

b.

6.90±0.07(2)

15

6.60±0.14(2)

49 3.2

7.5(2)

15

6.50±0.14(2)

53 3.5

Labetolol a. b.6.7(2)

24

7.0(2)

19 0.79

7.40±0.14(2)

19

6.79±0.07 (2)

30 1.58

Carvedilol a. b.8.65 ± 0.07 (2)

0.24

9.0 (2)

0.19 0.79

9.02 ± 0.09 (4)

0.43

8.81 ± 0.02 (3)

0.25 0.58

Pindolol a. b.8.13 ± 0.1 1 (3)

1.4

8.30 ± 0.1 (3)

1.0 0.7

8.66 ± 0.1 1 (3)

1.0

8.33 ± 0.15 (3)

0.8 0.8

Propranolol a. b.7.83 ± 0.05 (3)

2.8

8.5 (2)

0.62 0.22

8.6 (2)

1 .2

8.75 (2)

0.29 0.24

ICI 1 18-551 a.

b.

6.60 ± 0.14 (2)

49

8.60 ± 0.07 (2)

0.49 0.01

7.12 ± 0.09 (4)

36

8.43 ± 0.10 (3)

0.62 0.02

1.7%, and SIA binding site binding for 4.1%. The contribution

of the other activities listed in Tables 5 and 6 is negligible. The

relative contribution of each activity of R 67 138 (S,R,R,R) was

similar. However, the chart of R 67 145, the (R,S,S,S)-enantiomer, which only weakly bound to f,-adrenergic receptors, is

completely different. For this compound, binding to SIA sites

accounts for 33%, fl,-adrenergic receptor binding for 22%, and

/32-adrenergic receptor binding, the veratridine site of the Na

channel, and the uptake of serotonine and dopamine between

7 and 10%.

The profile of various f3-adrenergic blockers is shown in

Tables 7 and 8. It reveals that carvedilol, pindolol, and propranolol also potently bind to SIA binding sites with K, values

of 3, 15, and 84 nM, respectively. In addition, carvedilol and

labetolol inhibited [:IHIWB 401 binding to a,-adrenergic receptor sites with a K, value of 3 and 42 nM, respectively.

Discussion

Specificity of the ,- and -adrenergic receptor binding model. In agreement with previous reports (see Ref. 12)

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848 Pauwels et a!.

TABLE 4

Half-time of dissociation of nebivolol stereoisomers and

fl-adrenergic blockers from $- and fl2-adrenergic receptor sites Values are mean ± standard deviation. Numbers in parentheses, number of

experiments. T, Rabbit lung fi, Rat lung (32 mm Nebivolol

R 67 138

R 67 145

lClll8-551

Carvedilol

CGP 20712-A

Levantolol

Atenolol

Propranolol

Pindolol

Labetolol

81 ± 19 (1 0)

109 ± 35 (8)

38 ± 8 (5)

54±3 (4)

47 ± 18 (9)

21 ± 6 (6) 1 1 ± 6 (5) 1 0 ± 3 (4)

8 ± 1 (6)

7 ± 1 (3)

6 ± 2 (4)

32 ± 5 (11)

37 ± 7 (4)

7 ± 2 (3)

47±22(6)

61 ± 28 (3)

7 ± 3 (2)

1 3 ± 5 (3)

7 ± 1 (2)

6 ± 2 (2)

we found that rabbit lung was mainly enriched in fi, -adrenergic

receptor sites and rat lung in f.2-athenergic receptor sites. We

obtained selective labeling of ,- and fl2-adrenergic receptor

sites in these tissues, respectively, with the nonselective radioligands [‘HJCGP-12177 and [3H]dihydroalprenolol (Table 1),

in the presence of an appropriate concentration of the selective

$2-adrenergic blocker ICI 118-551 or the selective /31-adrenergic

blocker CGP 20712-A. Using these conditions, Scatchard analysis ofthe binding data ofthe radioligands indicated the presence

of only one binding site in the tissues, respectively (Figs. 3 and

4). The use of selective - and 9,-adrenergic blockers for

occlusion of the receptor sites, respectively, was recently also

applied by Nanoff et al. (13) for selective labeling of 3,- and f32-

adrenergic receptor sites in rat cardiac microsomes.

Selectivity of nebivolol for $- and fi2-adrenergic receptor sites compared with its stereoisomers and other

fi-adrenergic blockers. Nebivolol was a potent blocker of f,-

adrenergic receptor sites in rabbit lung. It showed a subnanomolar K, value of 0.9 nM, measured with E:IH]CGp42177 as

well as [:IH]dihythoalprenolol Its d-enantiomer R 67 138 was

equipotent whereas its 1-enantiomer R 67 145 was 175-fold less

potent. The least active stereoisomer, R 74 723 (S,S,S,S),

showed a K, value of 945 nM. CGP 20712-A, pindolol, propranolol, and carvedilol showed f3,-adrenergic affinity in the same

range as nebivolol. In contrast, levantolol, labetolol, and atenolol were 17-, 27-, and 450-fold less active than nebivolol.

In addition, nebivolol showed a pronounced /3,-athenergic

selectivity inasmuch as it was 50 times less potent at 32- adrenergic receptor sites in the rat lung membrane preparation.

In the series of stereoisomers, the R 67 138 (S,R,R,R)-enantiomer showed the optimal combination of high potency and

selectivity for f3,-adrenergic receptors. Nebivolol, which is a

racemic mixture of the (S,R,R,R) and (R,S,S,S)-enantiomers,

shared these properties. The inactive (R,S,S,S)-enantiomer

apparently had little effect on the activity. The presence of this

compound can only reduce the concentration of the active

enantiomer by half. Using [‘H]dihydroalprenolol, the K, value

of nebivolol was indeed two times higher than that of R 67 138.

This difference in K, value was not observed with [‘H]CGP12177, probably because the potency difference was within the

experimental variation. Within the presently investigated

series of /3-adrenergic blockers, only two compounds showed

combined high affinity and selectivity for ,-athenergic receptors; these were nebivolol and CGP 20712-A. The latter had a

2-fold lower affinity but was still 10 times more selective than

nebivolol. Atenolol, generally referred to as a selective ,- adrenergic blocker, was 300 to 400 times less potent and 2 to 4

times less selective than nebivolol. Striking differences were

TABLE 5

Receptor binding profile of nebivolol stereoisomers

a, -log lCw (N), mean value ± standard deviation. Numbers in parentheses, number of experiments. b, K values (nM). Experimental details are described in Materials and Methods. Tested up to a concentration of 10’ M, the nebivolol stereolsomers showed no interaction with dopamine D receptors ([3H]Sch 23390, rat stnatum),

cholinergic muscarinic receptors ([3Hjdexetimide, rat striatum), benzodiazepine receptors ([3Hjflunitrazepam, rat forebrain), u-opiate receptors ([3H)sufentanit, rat forebrain),

substance P binding sites ([3H]substance P, rat stnatum), or neurotensin binding sites ([3Hlneurotensin, guinea pig forebrain). In addition, nebivolol showed a -log lC value of 6.3 (one experiment) for the biogenic amine and metabolite release site ([3Hlketanserin, rat striatum). Inhibition of 3H-Iigarld binding

Adrenergic-a, Adrenergic-a2 Serotonin S,5 Serotonin S Histamine H, Dopamine 02 Veratndlnesite of Nebivolol a. 5.50 ± 0.07 (4) <5.0 7.56 ± 0.05 (3) 5.65 ± 0.07 (2) 5.25 ± 0.07 (2) 5.0 5.75 ± 0.07 (2) 5.6

b. 1160 20 700 2400 4000 889 2512 R65825 a. 5.55±0.07(2) <5.0 7.30±0.14(2) 5.60±0.1(2) 5.07±0.17(2) 5.0 5.47±0.24(2) 5.42±0.1(2) b. 1033 38 780 3756 4000 1694 3802 A 67 138 a. 5.55 ± 0.07 (2) 5.00 ± 0.07 (2) 7.50 ± 0.08 (3) 5.70 ± 0.07 (2) 5.27 ± 0.03 (2) 5.34 ± 0.03(2) 5.92 ± 0.03 (2) 5.20 ± 0.07(2) b. 1033 4670 24 623 2300 1806 600 6310 R67145 a. 4.95±0.07(2) <5.0 6.90±0.08(3) 5.20±0.2(2) <5.0 5.37±0.03(3) 5.90±0.14(2) 5.42±0.1(2) b. 41 10 95 2330 1660 629 3802 A 65 260 a. 5.65 ± 0.07 (2) 4.97 ± 0.03 (2) 7.3 (2) 5.37 ± 0.1 (2) 5.00 ± 0.1 (2) 5.25 ± 0.07 (2) 6.5 (2) 5.30 ± 0.14 (2)

b. 820 5000 38 1328 4300 2222 158 5012 R74716 a. 4.92±0.03(2) <5.0 7.27±0.03(2) 5.45±0.14(2) <5.0 <5.0 5.30±0.14(2) 5.5(2) b. 4300 40 1100 2505 3162

R74829 a. 5.32±0.03(2) <5.0 7.35±0.13(3) 5.52±0.17(2) <5.0 <5.0 5.55±0.14(2) 5.30±0.14(2)

b. 1740 34 965 1409 5012 R74714 a. 5.6(2) <5.0 6.62±0.1(2) 5.27±0.1(2) <5.0 5.22±0.17(2) 5.45±0.2(2) 5.37±0.10(2) b. 915 180 1673 2098 1774 4266 A 67 142 a. 5.35 ± 0.07 (2) <5.0 7.42 ± 0.10 (2) 5.9 (2) 5.1 (2) 5.2 (2) 5.45 ± 0.07 (2) 5.37 ± 0.03 (2) b. 1620 28 390 3404 4000 1774 4266 A 74 721 a. 5.47 ± 0.03 (2) 4.92 ± 0.03 (2) 6.32 ± 0.1 (2) 5.71 ± 0.13 (2) 5.00 ± 0.07 (2) 5.3 (2) 5.70 ± 0.07 (2) 5.32 ± 0.17 (2) b. 1220 4400 360 522 4300 1980 997 4786 R74723 a. <5.0 <5.0 6.05±0.07(2) 5.12±0.1(2) <5.0 5.15(2) 6.25±0.07(2) 5.37±0.03(2) b. 670 2323 2798 280 4266 A 74 718 a. 5.72 ± 0.03 (2) <5.0 5.95 ± 0.07 (2) 6.55 ± 0.07 (2) 5.20 ± 0.07 (2) <5.0 5.75 ± 0.13 (2) <5.0

b. 678 840 88 2704 889

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TABLE 6

Neurotransmitter uptake profile of nebivolol stereolsomers

a, -log lC (M), mean value ± standard deviation. Numbers in parentheses, number

of experiments. b, lC (nM). Experimental details are described in Materials and

Methods. Tested up to a concentration of 1 O M. the nebivolol stereoisomers

showed no interaction with [3H]-y-aminobutyric acid uptake in rat cortex. InhibiSon of IHlneurotransmitter uptake

U #{149}0 0 0 #{149} U 0 0 0 #{149} 0 0 % I 32 SIA S2 “2 H, D2 DHP vera 5-HT u NA u GA Neb,volol 92.95 1.70 4.09 0.12 007 0.01 0.03 0.02 0.09 033 0.24 0.14 020 R67138 93.80 2.21 3.13 0.12 007 0.02 0.03 004 013 012 0.11 008 015 R67145 22.07 7.31 32.53 1.33 0.75 0.31 0.31 1.86 491 8 13 9.78 347 724

Fig. 6. Pie charts of the receptor binding and neurotransmitter uptake profile of nebivolol, R 67 138, and

R 67 145. Pie charts were constructed using the

reciprocals of K, values in Table 2 and 5 and lC

values in Table 6. The table shows the per cent

contribution of each activity in the sum of the indicated

activities of a nebivolol-stereoisomer. DHP, dihydropyridine binding site, vera, veratndine binding site of

the Na channel; 5-HT, 5-hydroxytryptamine; NA, nor- adrenaline; DA, dopamine.

Receptor Binding Profile of Nebivolol 849

Serotonin Noradrenaline Dopam,ne

Nebivolol a. 6.47 ± 0.1 (2) 6.25 ± 0.07 (2) 6.40 ± 0.07 (2)

b. 340 565 400

R 65 825 a. b.

6.57 ± 0.1 (2)

269

6.27 ± 0.03 (2)

531

6.65 (2)

223

R67138 a.

b.

6.17±0.03(2)

676

6.00±0.07(2)

1000

6.3(2)

501

R 67 145 a. b.

6.5 (2)

316

6.05 ± 0.07 (2)

891

6.37 ± 0.1 (2)

427

R 65 260 a.

b.

6.02 ± 0.03(2)

944

6.11 ± 0.17(3)

805

6.35 ± 0.2(2)

473

R74716 a.

b.

6.15±0.07(2)

712

5.8(2)

1584

6.15±0.2(2)

750

R 74 829 a.

b.

6.15 ± 0.14 (2)

727

6.20 ± 0.14 (2)

647

6.62 ± 0.1 (2)

240

R74714 a.

b.

6.70±0.2(2) 211 6.22±0.17(2)

620

6.4(2)

398

A 67 142 a.

b.

6.25 ± 0.2 (2)

562

5.97 ± 0.17 (2)

1103

6.27 ± 0.1 (2)

536

R74721 a.

b.

6.47±0.03(2)

334

6.47±0.03(2)

334

6.37 ±0.3(2)

508

A 74 723 a.

b.

6.25 ± 0.2 (2)

562

5.97 ± 0.1 (2)

1074

6.12 ± 0.03 (2)

758

R74718 a. b.

6.35±0.07(2)

446

6.35±0.07(2)

1189

6.02±0.03(2)

954

observed with the compounds in the half-times of dissociation

from the is,- and f32-adrenergic receptor sites. Nebivolol can be

considered a slowly dissociating drug from the fi,- as well as

the 32-adrenergic receptor sites. A slow drug receptor dissociation may increase the duration of action of the drug, although

pharmacokinetic and metabolic processes may play a major

role (7). The biggest advantage of slow receptor dissociation is

the achievement of a stable receptor blockade, which is relatively insensitive to sudden variations in free concentrations of

endogenous agonist or to fluctuations in the free concentration

of the drug. This was also observed with R 67 138, IC! 118-551,

and carvedilol whereas the other reference compounds dissociated within minutes from the fl-adrenergic receptor sites.

Biochemical profile of nebivolol compared with its

stereoisomers and other $-adrenergic blockers. Besides

high binding affinity for /3,-adrenergic receptor sites, nebivolol

bound to SIA binding sites with a K, value of 20 nM, i.e., about

20-fold higher than its K, value for the /3,-adrenergic receptor

site. Several of the nebivolol stereoisomers bound to the S1A

binding sites with a similar potency; hence, the stereospecific

requirements for interaction with these sites appeared to be

distinct from those for the /3,-adrenergic receptor site (Table 5).

As a result, some of the stereoisomers bound more potently to

the S1A binding sites than to the f,-adrenergic receptor sites.

This was the case for the 1-enantiomer of nebivolol, R 67 145

(R,S,S,S) (see Fig. 6), and for R 74 716 (R,S,S,R), R 74 829

(S,R,S,R) and R 74 723 (S,S,S,S). Carvedilol, pindolol and

propranolol also bound to SIA binding sites, although their f3,-

adrenergic receptor affinity was at least 10-fold higher than

their SIA binding site affinity. At the same time, carvedilol

showed a,-adrenergic receptor affinity; it was 14-fold lower

than its fl,-adrenergic affinity. Labetolol showed a,-adrenergic

affinity in the same range as its fl,-adrenergic affinity. Hence,

it appeared that only CGP 20712-A, IC! 118-551, levantolol,

and atenolol are characterized by fl-adrenergic affinity free

from other tested binding affinities.

Mode of action of nebivolol as antihypertensive agent.

Clinical and in vivo pharmacological studies with nebivolol

revealed an interesting hemodynamic profile, different from

that of classical $-adrenergic blockers (see introduction). Observed reductions in heart rate can probably be attributed to

,-athenergic receptor blockade. However, improved left yentricular function, reduction in systemic vascular resistance, and

related increased cardiac output seen with nebivolol are not

properties of classical fl-adrenergic blockers. Also, the immediate reduction in blood pressure, obtained after administration

of nebivolol to conscious spontaneous hypertensive rats, has

not been observed with known fl-adrenergic blockers. Recent

observations have revealed that the particular hemodynamic

profile is specifically obtained with nebivolol, whereas the fi,-

adrenergic active enantiomer R 67 138 (S,R,R,R) showed the

activities of a typical f-adrenergic blocker. Hence, the properties of nebivolol apparently resulted from the combined activities of the two enantiomers. The presently investigated biochemical properties do not provide a direct clue for the explanation of the beneficial effects.

Drug properties reported to be related to decreased vascular

resistance such as a,-adrenergic or serotonin 52 antagonism, Nebivolol R 67 138 R 67 145

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850 Pauwels eta!.

TABLE 7

Receptor binding profile of various fl-adrenergic blockers

a, -log IC50 (M), mean value ± standard deviation. Numbers in parentheses, number of experiments. b, K, values (nM). Binding was performed as described in the legend

to Table 5. Tested up to a concentration of 1 0 M, the (3-adrenergic blockers showed no interaction with dopamine 0, receptors ([3HJSch 23390, rat stnatum), cholinergic

muscarinic receptors ((3Hjdexetimide, rat stnatum), benzodiazepine receptors (3Hlflunitrazepam, rat forebrain), M-opiate receptors ([3Hjsutentanil, rat forebrain), substance

P binding sites ([3Hlsubstance P. rat stnatum), or neurotensin binding sites ([3Hjneurotensin. guinea pig forebrain). Inhibition of 3H-Iigand binding

Adrenergic-a, Adrenergic-a2 Serotonin S,5 Serotonin S Histamine H, Dopamine D2 ,-OpEate CGP20712-A a. <5.0 <5.0 5.10±0.1 (3) <5.0 <5.0 5.6(2) 5.5(2) <5.0 b. 595 992 854 Atenolol a. <5.0 <5.0 <5.0 <5.0 <5.0 <5.0 <5.0 <5.0 b. Levantolol a. 5.65 ± 0.07 (2) <5.0 6.3 (2) 5.42 ± 0.1 (2) <5.0 <5.0 <5.0 <5.0 b. 820 375 1,167 Labetolol a. 6.95 ± 0.08 (3) <5.0 6.40 ± 0.07 (3) <5.0 <5.0 <5.0 5.05 ± 0.07 (2) <5.0 b. 42 298 2,408

Carveditol a. 8.03±0.15(3) 5.30 ± 0.05 8.35±0.07(2) 6.17±0.03(2) 5.15±0.07(2) 6.26±0.05(3) 5.00±0.1(3) 5.90 ± 0.2 b. 3.4 (3) 3.4 207 3,034 213 2,700 (2)

2,168

Pindolol a. 5.3 (2) <5.0 7.61 ± 0.1 (3) <5.0 <5.0 <5.0 <5.0 <5.0

b. 1,825 15 Propranolol a. <5.0 <5.0 6.95 ± 0.07 (2) 5.1 (2) <5.0 <5.0 <5.0 b. 84 2,466

Id 1 18-551 a. <5.0 <5.0 4.6 (2) 5.25 ± 0.07 (2) <5.0 <5.0 <5.0 <5.0

b. 18,840 1,629

TABLE 8

Neurotransmitter uptake profile of various -adrenergic blockers

a, -log lC (M), mean value ± standard deviation. Numbers in parentheses, number

of experiments. b, lC (nM). Uptake was performed as described in the legend to

Table 6. Tested up to a concentration of 1 0 M, the (3-adrenergic blockers showed

no interaction with l3Hh-aminobutync acid uptake in rat cortex.

lnhitxt,on of HJ-neurotransm,tter uptake Serotonin Noradrenal’ne Dopam,ne

CGP20712-A a. 5.60±0.14(2) 5.35±0.07(2) <5.0

b. 2,577 4,466

Atenolol a. <5.0 <5.0 <5.0

b.

Levantolol a. 6.63 ± 0.1 (3) 6.55 ± 0.1 4 (2) 5.62 ± 0.03 (2)

b. 236 288 2,374

Labetolol a. 5.75 ± 0.07 (2) 6.0 (2) 5.52 ± 0.1 (2)

b. 1,789 1,000 3,029

Carvedilol a. 6.30 ± 0.16 (4) 5.62 ± 0.1 (2) 6.23 ± 0.2 (3)

b. 528 2,406 627

Pindolol a. 5.0 (2) 5.61 ± 0.07 (3) 5.1 (3)

b. 10,000 2,417 7,943

Propranolol a. 5.95 5.50 ± 0.07 (2) 5.1

b. 1,122 3,160 7,943

ICI 1 18-551 a. 5.72 ± 0.03 (3) 5.48 ± 0.17 (4) 5.35 ± 0.07 (2)

b. 1,880 3,250 4,460

Ca2 entry blockade, or dopamine D, antagonism were not

observed with nebivolol or the stereoisomers. In this study,

nebivolol and both separated enantiomers showed SIA binding

site affinity; however, this affinity was also observed with

carvedilol, pindolol, and propranolol. S,A binding sites have

been proposed to have a role in cardiovascular function. Hypotension and bradycardia have been observed with 8-hydroxy2-di-N-propylamino-tetralin, considered as the prototype of SIA

agonists, in very particular experimental conditions (14, 15).

However, the reported cardiovascular effects ofthis SIA agonists

are unlike the hemodynamic effects of nebivolol. Neither did

carvedilol, pindolol, and propranolol share the hemodynamic

effects of nebivolol. In the absence of a sufficient number of

drugs that specifically act as agonists and antagonists on the

S,A binding sites in the absence of controlled clinical data,

hypotheses on the role of the SIA binding sites must be regarded

with caution. Several hypotheses regarding the mechanism of

action of nebivolol can be proposed. One possibility is an

antagonistic action at presynaptic $-adrenergic receptors involved in the release of adrenaline and noradrenaline from

adrenergic neurones (16). Otherwise, emerging molecular biological data on the amino acid sequences of cloned receptors

have revealed dissimilarities in the sequence of the 3-adrenergic

receptor obtained from various tissues and species (17, 18).

Therefore, it seems not unlikely that different, as yet unidentified, receptor subtypes exist, which may have particular functions. Such possibilities could be explored with nebivolol. Acknowledgments We sincerely thank Suzy De Cauwer for secretarial work. References 1. De Cree, J., H. Geukens, J. Leempoels, and H. Verhaegen. Haemodynamic

effects in man during exercise of a single oral dose of nebivolol (R 67 555):

new beta- 1 -adrenoceptor blocking agent: a comparative study with atenolol,

pindolol, and propranolol. Drug Dev. Res. 8:109-117 (1986).

2. De Cree, J., H. Geukens, C. Cobo, and H. Verhaegen. Subacute hemodynamic

effects of nebivolol in man at rest and during exercise. Angiology 38:440-448 (1987).

3. Van de Water, A., W. Janssens, J. Van Nueten, R. Xhonneux, J. De Cree, H.

Verhaegen, R. S. Reneman, and P. A. J. Janssen. The pharmacological and

hemodynamic profile of nebivolol, a chemically novel, potent and selective

(3,-adrenergic antagonist. J. Cardiovasc. Pharmacol. 1 1: 552-563 (1988).

4. Dooley, D. J., and H. Bittiger. Quantitative assessment of central (3,- and 2- adrenoceptor regulation using CGP 20712-A. J. Pharmacol. Methods 18:131- 136 (1987).

5. O’Donnell, S. R., and J. C. Warst.all. Evidence that ICI 118-551 is a potent,

highly beta,-selective adrenoreceptor antagonist and can be used to characterize beta-adrenoreceptor populations in tissues. Life Sci. 27:671 (1980).

6. Bilski, A., S. Dorries, D. Fitzgerald, R. Jessup, H. Tucker, and J. Wale. IC!

118-551, a potent beta,-adrenncepthr antagonist. Br. J. Pharmo.coL 69:P292 (1980).

7. Leysen, J. E., and W. Gommeren. The dissociation rate of unlabelled dopamine antagonists and agonists from the dopamine-D2 receptor application of

an original filter method. J. Recept. Res. 4:817-845 (1984).

8. Leysen, J. E., and W. Gommeren. Drug-receptor dissociation time, new tool

for drug research: receptor binding affinity and drug-receptor dissociation

profiles of serotonin-52, dopamine-D2, histamine-H, antagonists, and opiates.

DrugDev. Res. 8:119-131 (1986).

9. Cheng, Y.-C., and W. H. Prusoff. Relationship between the inhibition con- sLant (K,) and the concentration of inhibitor which causes 50 per cent

inhibition (I) ofan enzymatic reaction. Biochem. Pharmacol. 22:3099-3108 (1973).

10. Leysen, J. E., P. Van Gompel. W. Gommeren, R. Woestenborghs, and P. A. J.

 by guest on January 25, 2013 molpharm.aspetjournals.org Downloaded from 

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Receptor Binding Profile of Nebivolol 851

Janssen. Down regulation of serotonin-52 receptor sites in rat brain by 15. Di Francesco, G. F., M. A. Petty, and J. R. Fozard. Antihypertensive effects chronic treatment with the serotonin-S2 antagonists: ritanserin and setope- of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in conscious dogs. rone. Psychopharmacology 88:434-444 (1986). Eur. J. PharrnocoL 147:287-290 (1988).

11. Pauwels, P. J., and P. M. Laduron. TPP accumulation in rat brain synap- 16. Misu, Y., and T. Kubo. Presynaptic (3-adrenoceptors. Med. Res. Ret’. 6:197-

tosomes as a probe for Na channels. Eur. J. Pharmacol. 132:289-293 (1986). 225 (1986).

12. Nahorski, S. R. Identification and significance ofbeta-adrenoceptor subtypes, 17. Marx, J. L. Receptors highlighted at NIH Symposium. Science (Wash. D. C.)

in Towards Understanding Receptors, Current Reviews in Biomedicine I 238:615-616 (1987).

(J. W. Lamble, ed). Elsevier/North Holland (1981). 18. Dohiman G. H., M. G. Caron, and R. J. Lefkowitz. A family of receptors

13. Nanoff, C., M. Freissmuth, and W. Sch#{252}tz. The role of a low (3,-adrenoceptor coupled to guanine nucleotide regulatory proteins. Biochemistry 26:2657- selectivity of [3H]CGP-12177 for resolving subtype-selectivity of competitive 2664 (1987).

ligands. Naunyn-Schmiedeberg’s Arch. Pharmacol. 336:519-525 (1987). _______________________________________________________________

14. Fozard, J. R., A. K. Mir, and D. N. Middlemiss. Cardiovascular response to

8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in the rat: site of Send reprint requests to: Dr. P. J. Pauwels, Department of Biochemical

action and pharmacological analysis. J. Cardiovasc. Pharmacol. 9:328-347 Pharmacology, Janssen Research Foundation, B-2340 Beerse, Belgium

(1987).

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