Nipradilol Displays a Unique Pharmacological Profile of Affinities for the Different α1-Adrenoceptor Subtypes

Nipradilol

Displays for

a Unique

the

Pharmacological

Different ƒ¿1-Adrenoceptor

Junji Kinami, Hiroshi Tsuchihashi, Department

of Pharmacology,

major by

selectivity

metabolite

of

radioligand

in

the

pKi

values

for

between

the

inhibition values

of

values

the

-blocking

Keywords:

brain

was

these

possess

of

compounds sites

are

low

activity

affinity

of

these

for related

pigs.

rat

was

in

different for

heart

brain. and

sites

aorta.

There

and

the

mediated and

the

and

-affinity

vasoconstriction

to ƒ¿1 -adrenoceptors;

of

in

sites

isomers brain

and and

characteristics

the ƒ¿1High

the ƒ¿1LOW -affinity

Niigata 950-21,

3) The

of

pKi

denitronipradilol,

by ƒ¿1-

There

were

pA2

values

the

order

good

were

for

the

results

the

contractile the

indicate

2) Nipradilol

nipradilol

sites of

correlations

between

These

in

compounds

correlation

adrenoceptors;

group

a examined

for ƒ¿1High-affinity but

no

values.

were

these

values

the

was

pA2

nitroxy

Japan

spleen

SS ≈ RS•âdenitronipradilol,

the

rat

four heart,

order

Nagatomo

6, 1992

its in

The

RR •†

response

to

nipradilol,

> nipradilol •†

compounds

Kamishin'ei-cho,

November

subtypes

guinea

Affinities

pKi that:

and

is important

for

its its ƒ¿1

.

[3H]-Prazosin, Optical

sites

5-13-2

Pharmacological

and

SR

phenylephrine-induced

1) ƒ¿1High -Affinity isomers

and

rats

Keiko Sasaki and Takafumi

Accepted

effects

[3H]-prazosin.

from

the ƒ¿1LOW -affinity

pKi of

antagonistic

with

aortae

spleen

25, 1992

on ƒ¿1-adrenoceptor

assay

in isolated heart,

of

nipradilol,

binding

determined

Keiko Maruyama,

May

of

Subtypes

Niigata College of Pharmacy, Received

ABSTRACT-The

Profile

Nipradilol, ƒ¿1-Adrenoceptor

subtype, ƒ¿1-

and ƒÀ-•@Blocking

activity,

isomer

Our recent studies on the binding

characteristics

of

[3H]-prazosin for the a,-adrenoceptor subtypes revealed that there are two binding sites having different affinities, high and low-affinity sites, for prazosin in the rat ven tricular muscle, brain and spleen membranes; and these sites are designated as a,High and a1L0 -affinity sites (1, 2). We also found that rat ventricular muscle and brain pos sess both a,High and a1L0 affinity sites, whereas rat spleen had only a,High-affinity sites (1, 2). Most a,-adrenergic an tagonists had different affinities for each site in these tissues, suggesting that there may exist five different a, adrenoceptors (a,High-sites in the ventricular muscle, brain and spleen and a1LoW-sitesin the ventricular muscle and brain). Recently, a, •~-blockers possessing antagonistic po tency for both a, and j3-adrenoceptors have been shown to be useful for the clinical treatment of patients with hypertension and angina. Nipradilol, one of these a, ~ blockers, possesses two asymmetric carbon atoms in its chemical structure and is a mixture of four isomers, as shown in Fig. 1 (3, 4). We previously reported the binding characteristics of nipradilol for (3,-, (32-adrenergic and 5HT,B-serotonergic binding sites by using [125I]-iodo

cyanopindolol (ICYP) and [3H]-CGP 12177, respectively; and the results showed that nipradilol had a higher affin ity for (3-adrenoceptors (with no selectivity for f3, and (32 adrenoceptors) than for 5HT,B-receptors (5). The rank orders of potency of the isomers for 8-adrenoceptors and 5HT,B-receptors were SR > nipradilol > SS > RR > RS and SS > SR >_nipradilol > RS > RR, respectively (5).

Fig.

1.

Chemical

structure

of

nipradilol

and

its isomers.

In this study, we examined the selectivity of nipradilol, its isomers and its major metabolite denitronipradilol (6) for a,-adrenoceptor subtypes and compared them with those of other a, • n-blockers, labetalol, amosulalol and arotinolol. MATERIALS

AND METHODS

Materials [3H]-Prazosin (76.6 Ci/mmole) was purchased from New England Nuclear/Dupont, Ltd., Boston, MA, U.S.A. Nipradilol, 3,4-dihydro-8-(2-hydroxy-3-isopropyl amino)propoxy-3-nitroxy-2H-1-benzopyran, its isomers (Fig. 1) and denitronipradilol Kowa Co., Ltd.

were kindly donated

by

Animals Male Wistar rats weighing 200-350 g were used. Preparation of membrane-enriched fractions The membrane-enriched fractions from rat heart, brain and spleen were prepared as described previously (1, 2). Protein was determined by the method of Lowry et al. (7). Binding assay Displacement analysis for a,-adrenoceptor subtypes was performed in duplicate with [3H]-prazosin as described previously (1, 2). In brief, the membrane sus pension (0.1 mg of heart and brain and 0.25 mg of spleen membrane proteins) was incubated for 45 min at 231C in a total volume of 0.5 ml containing 60 mM Tris-HC1 (pH 7.4) with an appropriate concentration of [3H]-prazosin

Table

1.

pK; values

of nipradilol

and

its optical

in the presence or absence of unlabelled ligand. The con centrations of [3H]-prazosin were 0.04, 0.1 and 0.2 nM for assessing a,High-affinity sites in the brain, heart and spleen membrane, respectively. The affinity of the a,LoW affinity sites for unlabelled ligand were determined with 0.5 nM [3H]-prazosin in the presence of 0.1 pM phenoxy benzamine in the brain membrane, or with 0.6 nM [3H] prazosin in the presence of 1 pM phenoxybenzamine in the heart membrane. Phenoxybenzamine inhibited the a,High-affinity sites completely (1, 2). After the incuba tion period of 45 min, the medium was immediately filtered through a GF/C glass fiber filter and washed with the incubation buffer according to previously described methods (8). The radioactivity on the filter was counted by a Packard 2200 Tri-Carb Scintillation Analyzer. The specific binding was determined by subtracting the non specific binding in the presence of 10 pM of phentolamine from the total binding. Kinetic analysis All kinetic analyses 9801 computer system sion program (9-12). ing only one or two

were performed on an NEC PC with an iterative non-linear regres The data were fitted to models hav receptor binding sites (9 -12). To

quantitate the displacement characteristics, the slope fac tor (nH) for the displacement curves was determined as described previously (9-12). Most K; values of various ligands are expressed as pK; ( log K;) in this report. Pharmacological observations The contractile tension of the rat and guinea pig aorta was determined as described previously (13, 14). Briefly, aortae were cut into rings and freed of excess tissues.

isomers

for

the

a,-adrenoceptor

subtypes

Values in parentheses are the numbers of experiments. Data are the mean values ±S.E. The slope factors (nH) of the displacement curves of all ligands used in the present study were equal to one. *Some of these data were reported previously (11).

These rings, about 2 mm in width, were mounted in 12-m1 organ baths. The Krebs-Henseleit solution had the follow ing composition: 118 mM NaCI, 4.7 mM KCI, 2.5 mM CaC12, 25 mM NaHCO3, 1.2 mM MgSO4i 1.2 mM KH2PO4, and 11 mM glucose. The temperature of the so lution was maintained at 37 ± 1 V and aerated with a mix ture of 95% 02 and 5% CO2. The contractile tension of these preparations was recorded on a potentiometric recorder (Hitachi APD-74) with a strain gage transducer and a carrier amplifier (Nihon Kohden RP-3 or San'ei Instrument Co., Ltd. 6M52). The aorta was always stretched to 0.5 -1 g to obtain the optimum response. Concentration-response curves were determined for phenylephrine before and after addition of each com pound, and their pA2 values were calculated by the previ ously described equation (15). RESULTS Table 1 summarizes the pKi values of nipradilol, its isomers and denitronipradilol for a1-adrenoceptor sub types in [3H]-prazosin binding to brain, heart and spleen membranes. Both alHigh and a1Low-affinity sites were found in the brain and heart membranes, while only alHigh-affinity sites existed in the spleen membranes (1, 2). These five a1-adrenoceptors had different affinities for various antagonists (1, 2). The binding affinity of labetalol for these subtypes were distinguishable among the compounds used in the present study: brain alHigh> heart alHigh = spleen alHigh > brain a1Low> heart a1Low• The pKi values of the other a1 •n-blockers, arotinolol and amosulalol, for alHigh-affinity sites were higher than those for a1Low-affinity sites. On the other hand, the pKi values of nipradilol for various subtypes were similar except for heart alHigh > heart a1Lo,. The pKi values of nipradilol and related compounds for the alHigh-affinity site were sig nificantly lower than those of prazosin, amosulalol, labetalol and arotinolol. The order of potency of nipradilol, its isomers and denitronipradilol for alHigh affinity sites was SR > nipradilol RR > SS = RS > denitronipradilol in the heart, brain and spleen. Thus, the orders of potency were similar for the alHigh-affinity sites in these three tissues. On the other hand, the orders of potency for the a1Lowaffinity sites was not consistent in the heart and brain. The rank order of the pKi values of denitronipradilol was heart a1Low> heart alHigh > spleen alHigh brain alHigh ? brain a1Low.The rank order of the pKi values of the optical isomers of nipradilol for these subtypes was different for each compound: SR, brain a1Low brain alHigh >_ heart alHigh = spleen alHigh > heart alHigh SS, heart a1Low> brain a1Low? heart alHigh brain alHigh ? spleen alHigh; RR, brain a1Low> heart alHigh heart a1Low? brain alHigh > spleen alHigh;RS, brain a1Low

Table

2.

adrenoceptors

Values means

pA2

values

of

in rat and

in parentheses ± S.E.

nipradilol guinea

and pig

are the numbers

its

optical

isomers

for

a,

aorta

of experiments.

Data

are the

heart a,LoW, heart alHigh brain alHigh? spleen alHigh• Table 2 also summarizes the pA2 values of the contrac tile tension of rat and guinea pig aorta. a1-Adrenoceptor subtypes in the rat and guinea pig aorta were alHigh and a1Low-affinity sites, respectively, as determined by the pA2 values of prazosin (10.80 in rat aorta and 8.30 in guinea pig aorta) (16). All compounds antagonized the phenylephrine-induced contractile response in a competi tive manner when analyzed by Schild plots (data are not shown). The pA2 value of nipradilol in the rat aorta was lower than the value of amosulalol and labetalol, and it was higher than that of arotinolol. On the other hand, the pA2 values of SS, RS and denitronipradilol were lower than the value of arotinolol. The order of pA2 values of these compounds was SR= nipradilol= RR > SS RS denitronipradilol in the rat aorta. The pA2 of nipradilol in the guinea pig aorta was lower than that of amosulalol and was higher than that of arotinolol. On the other hand, the pA2 values of RS and denitronipradilol were lower than the value of arotinolol. The order of pA2 values of these compounds was SR >_nipradilol RR >_ SS denitronipradilol > RS in guinea pig aorta. The pA2 values of these compounds in the guinea pig aorta were similar to those in the rat aorta except for those of denitronipradilol. Figure 2 shows the relationship between the pKi values of these compounds for al High-affinitysites in the three tis sues and the pA2 values for the antagonistic potency in the contractile response to phenylephrine of rat and guinea pig aorta. These results suggested a good correla tion between the pKi values of the alHigh-affinity sites and the pA2 values in rat aorta (r = 0.90 0.94, P < 0.001) . The pA2 values in the guinea pig aorta showed less correlation (r=0.74-0.79, P<0.01) with the pKi values than those obtained in the rat aorta. There was no correlation be tween the pKi values of the a1Low-affinitysites and the pA2 values in both rat and guinea pig aorta (heart vs. rat

Fig. spleen

2.

Relationship and

8, arotinolol;

aorta,

r=0.26;

between

the pA2 values

the

pK; values

in the rat and

guinea

of nipradilol pig aorta.

and

its isomers

1, Nipradilol;

for

ajHigh-affinity

2, SR; 3, SS; 4, RR;

sites

in the

rat heart,

5, RS; 6, amosulalol;

brain

and

7, labetalol;

9, denitronipradilol.

brain vs. rat aorta, r=0.56;

guinea pig aorta, r=0.43).

r=0.28;

heart vs.

brain vs. guinea pig aorta

DISCUSSION Nipradilol is a potent R-adrenergic blocking agent that has direct vasodilating and a,-blocking properties. It pos sesses two asymmetric carbon atoms and is a mixture of four isomers (3, 4). There is one asymmetric carbon atom at the 3 position of the benzopyran ring and another at the 2' position of the aryloxypropanolamine group. Denitronipradilol, which has weak a,-adrenergic activity, is a major metabolite (6). We demonstrated that a, adrenoceptor subtypes exist in various tissues and that most of the a,-adrenergic antagonists had various affin ities for each subtype in these tissues (1, 2). The present study also showed that nipradilol, its isomers and denitronipradilol had different affinities for various a, adrenoceptor subtypes. The rank order of pK; values of

the isomers and denitronipradilol was also different for these subtypes. Denitronipradilol and SS were selective for a,Low-affinity sites in the heart, but RR was selective for a,L0 -affinity sites in the brain. These results suggested that these subtypes have different affinities for various a, adrenoceptor antagonists and that the configuration of the 3 position of the benzopyran ring and the 2' position of the aryloxypropanolamine residue was important for the recognition of the a,-adrenoceptor subtypes. The order of potency of these compounds for the aiHigh affinity sites in the heart, brain and spleen was generally SR > nipradilol >_ RR >_ SS RS > denitronipradilol. Therefore, the results suggest that the R configuration of the nitroxy group of the benzopyran ring and the S configuration of the 2' position at the aryloxypropanola mine residue were important for the a,-antagonistic ac tions of these drugs. We previously reported that nipradilol and its isomers were not selective for (3, and P2 adrenoceptors and that the order of potency of these com pounds for (3-adrenoceptor subtypes and 5HT,B-receptors

was SR > nipradilol > SS > RR > RS and SS > SR >_nipr adilol > RS > RR, respectively (5). Previous reports (14, 17-20) have also shown that p-blockers possessing the S configuration are more potent than those of the R configu ration among aryloxypropanolamines. On the other hand, the R configuration of the nitroxy group of nipradilol played an important role in the a1-blocking ac tivity. These results suggested that these three receptors, a1-, (3 and 5HT1B-receptors, had different characteristics in the recognition sites for the optical isomers of nipradilol. Based on recently available data, an increasing number of complex classification schemes for the a-adrenoceptor subtypes are being published. In blood vessels, there are two distinct subtypes (a1H & a1L) of a1-adrenoceptors that are distinguished by their affinities for prazosin and

correlation and

pig

types,

al.

The

The

in

the

guinea to

may

the

the

proposed a similar classification (a1A & a1B) in rat brain, spleen, heart and other tissues. The a!A-subtype has a high affinity for WB-4101, while the a1B-subtype has a low affinity for this drug and is susceptible to chlorethylcloni dine (CEC); these subtypes have similar affinities for

ed

with

of

all

is

to

that

the

rat

guinea

aorta

the

affinity

vascular

a1H

binding

the

pKi

pA2

and

a1Low

affinity

present are

imply

binding

from

the

sites

a1Low

Recently, ment

arotinolol

and 3

90-, those

(5,

higher

11).

while

cological tor

profile

and

guinea

of

brain

pig

the a1Low

alLow different

for

heart

the

and

treat

diseases.

The labetalol,

(3-adrenoceptors

10000

affinity

the

were

used

ischemic

were 1200

to

and sites

similar

affinities

had

a1Low for

a

towards

respec

arotinolol

than

has

0.6

3000-fold

subtypes,

nipradilol

selectivity

the and

aorta.

been

labetalol

Thus

alHigh

while

alHigh-affinity had

sites.

values. in

of

and

al-adrenoceptor

nipradilol

alLow-affinity

values

pA2

types

alHigh

heart

Amosulalol, for

correlat pKi

amosulalol,

to

for

selectivity

sites,

the

for

1300

alL

Furthermore,

• p-blockers,

nipradilol,

to

new

have or

a1

than

tively

in

• (3-blockers

of

higher

the

hypertension

affinities

170-,

in

not

the

vasoconstriction,

subtypes

al

of

that

in

a1Low

al-adrenoceptors

be

respectively.

involved

affinity

may

sites,

findings

sites

that

aorta

the

alHigh-affinity

the

with

simi

vascular

the

when

compared

suggest pig

the

that

were that

were

aorta

for

suggest

but from

rat

8.30, study

affinity

binding

compounds the

and

present

higher

for

also

subtypes

10.80

results

hand,

some

were

guinea

Our

other

in

observations

rat

to

of

values

compounds

These

aorta.

the

the a

different

the

values

the

were

for

We

for

hand,

is and

a1L-subtypes

a1Low-subtypes. prazosin

[3H]-prazosin

were

On

and alHigh

affinities and

(alHigh)-subtypes,

sites

(a1Low)-subtypes. sites,

rat for

of

a1H

aorta

WB

a1L-subtype

WB-4101

different

had

than sites

for

the

other

these

for

the

the

and

for

affinity

on

denitronipradilol

pig

to

the

and sub

respectively.

and

that

pig

On

high

26),

of

rat

prazosin

affinity

alHigh

aorta.

the

classification

values

guinea

a1H1gh-affinity

lar

the

(16). that

low The

based

pA2

and

respectively

has

sites

pig

8.45,

has

(25,

suggesting

identical

showed of

2),

of and

aorta

(25).

sites (1,

be

aorta

CEC

affinity

prazosin

values

CEC

affinity

guinea

a1L-adrenoceptor

9.89

rat

to

pig

insensitive a1Low

the

sensitive

and

pA2 were

in

is

a1Low

and

that

a1H

a1L-subtypes

and

for

rat

demonstrated

contained

a1H-subtype

4101

values

the

(25)

aorta

and

pKi

in

respectively.

a1H

guished by their affinities for [3H]-prazosin in the rat brain, spleen, and heart (1, 2). On the other hand, Mor row and Creese (22) reported that rat brain contains two subtypes (ala & alb) with similar affinities for prazosin but different affinities for phentolamine. Han et al. (23, 24)

(r=0.74-0.79, P<0.01) between the pKi values for the alHigh-affinity sites and the pA2 values in guinea pig aorta, as compared to that in the rat aorta. There was no

the

values et

guinea

showed

(26). The a1L-subtype has a low affinity for prazosin in the guinea pig aorta, and the a1N-subtype has a higher affinity for HV-723 and WB-4101 than for prazosin. On the other hand, Han and Minneman (27) classified the subtype in the rat aorta as a1B. Four subtypes (alA, a1B, aic & a1D) have been cloned (28-31), and the a1B, aic and a1D-sub types are susceptible to CEC. These classifications (cloned a1A, a1B, aic & a1Di ala & alb, and a1A & a1B) do not in clude the subtypes with low affinities for prazosin. It was revealed that there was a good correlation be tween the pKi values obtained for the alHigh-affinity sites and the pA2 values in the rat aorta (r=0.90-0.94, P<0.001). There was a significant but lower correlation

pA2

Muramatsu

yohimbine (21). Our results also show that there are two distinct receptor subtypes (alHigh& a1LoW)that are distin

prazosin. The relationship between alHigh and a1LoW-sub types and a1A and a1B-subtypes in the rat brain, spleen and heart has not yet been clarified (1, 2). Muramatsu et al. (25) recently proposed that the al-adrenoceptors in blood vessels can be divided into three subtypes (alH, alL & a1N) by their antagonist affinity and susceptibility to CEC. The alH-subtype has a high affinity for prazosin, susceptible to CEC and includes alB-subtypes in the dog carotid artery and atypical a1A-subtypes in the rat aorta

between

the

affinity

alHigh

unique

the

and

pharma

a1-adrenocep

subtypes.

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