Neuroscience Letters, 20 (1980) 347-350 347

© Elsevier/North-Holland Scientific Publishers Ltd.

SELECTIVITY OF A SERIES OF CLONIDINE-LIKE DRUGS FOR ~l A N D ~2 ADRENOCEPTORS IN RAT BRAIN

R.J. SUMMERS, B. JARROTT and W.J. LOUIS

Clinical Pharmacology and Therapeutics Unit, Department of Medicine, University of Melbourne, A ustin Hospital, Heidelberg, Victoria 3084 (Australia)

(Received July 21st, 1980; Accepted August 15th, 1980)

The relative selectivity of a series of imidazoline derivatives and substituted guanidines structurally related to clonidine for ~ and ~2 adrenoceptors has been examined in binding studies using membranes prepared from rat cerebral cortex and the radioligands [3H]prazosin and [3H]clonidine. There was a 150- fold difference between the most and least selective clonidine-like drugs in their relative affinity for c~ and t~2 adrenoceptors.

A number of imidazoline derivatives and substituted guanidines structurally related to clonidine have been shown to be potent displacers of [3H]clonidine binding to ~2 adrenoceptors in membranes prepared from rat cerebral cortex [8, 9, 11, 13]. Clonidine is also known to act on postjunctional cq receptors, since its pressor effects in the pithed rat are antagonized by prazosin [12]. This agonist action of clonidine at c~l adrenoceptors would tend to antagonize its central antihypertensive action which appears to be mediated by aa adrenoceptors [10]. Although other factors such as bioavailability and metabolism are important in determining the antihypertensive effects of imidazolines and substituted guanidines, agonist compounds with a greater selectivity for a2 adrenoceptors might be expected to have a wider effective dose range than clonidine. In addition such compounds may prove superior to clonidine as ligands for binding studies to o~2 adrenoceptors. Prazosin (2(4-(2-furoyl)piperazine-l-yl)-4-amino-6,7-dimethoxyquinazoline) is a potent antihypertensive drug known to selectively block al adrenoceptors [1] and which selectively labels this ~ adrenoceptor subgroup in membranes from rat brain [5]. This study utilizes the selectivity of [3H]clonidine for o~: adrenoceptors and [3H]prazosin for o~1 adrenoceptors to determine the relative selectivity of a series of clonidine-like drugs for adrenoceptors in rat brain. The results illustrate that guanfacine and CP 14,304 have greater selectivity for ~2 adrenoceptors than clonidine.

Rat cerebral cortex was dissected [7] and homogenized in Tris • HCI buffer pH 7.6 at 4°C [13]. One ml of homogenate (containing 20 mg wet wt. tissue or 1.6-1.8 mg protein) was added to 1 ml Tris buffer (pH 7.6 at 25°C) and incubated for 30 min at 25°C with [3H]prazosin (0.2-0.5 nM final concentration; 33 Ci/mmol) and

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displacing agents . Non-speci f ic b inding was de te rmined in ident ical tubes con ta in ing

in add i t ion 10 / ,M indo ramin . F i l t ra t ion , washing and count ing o f samples were as

previously desc r ibed [1 l] . [3H]Prazosin was k ind ly d o n a t e d by Pf izer U.K. Ltd . and

was pur i f ied at least weekly by thin layer c h r o m a t o g r a p h y on silica gel plates using

e t hy l ace t a t e /d i e thy l amine ( 1 9 : l ) as the runn ing solvent . The c lonidine- l ike drugs

were o b t a i n e d as fol lows: guanabenz and i n d o r a m i n (Wyeth) ; c lonidine , ST600,

ST91 (Boehringer) ; Baya 6781 (Bayer); naphazo l ine and phe n to l a mine (Ciba) ; CP

14, 304-18 (Pf izer U.K. Ltd.) ; lofexid ine (Mer re l -Na t iona l ) ; t i amenid ine (Hoechs t

AG) ; guanfac ine and 44549 (Sandoz) .

Specif ic [3H]prazosin b inding to m e m b r a n e s p repared f rom rat cerebra l cor tex

was r ap id and sa turable . Specific b ind ing represented 93.6 +_ 0.6070 (n = I l) o f total

b inding . These results conf i rm the repor t o f Greengrass and Bremner [5]. Sca tchard

analysis o f d a t a revealed a single popu la t i on o f b inding sites with a Bmax o f 9.4

p m o l / g and a KD o f 0.113 nM (n = 3). Hil l analysis o f the da t a gave coeff ic ients

near uni ty indica t ing a lack of coopera t iv i ty in b inding .

Al l the c lonidine- l ike drugs were capable , but to a vary ing degree, o f d isplac ing

[3H]prazosin b ind ing . Values for ICs0 were ob ta ined by p rob i t analysis f rom

exper iments at 3 d i f ferent drug concen t ra t ions and the Ki value ob ta ined using the

Cheng and P r u s o f f equa t ion [2]. These values are shown in Table I together with

values ob ta ined for [3H]clonidine, which has been used to label c~2 ad renocep to r s .

High rat ios indica te high selectivity for c,2 ad renocep to r s . Guanfac ine and CP

14,304 d i sp layed by far the highest selectivity in this g roup with ra t ios o f 3220 and

TABLE 1

DISPLACEMENT OF [3H]PRAZOS|N AND [3H]CLONID1NE BINDING FROM MEMBRANES PREPARED FROM RAT CEREBRAL CORTEX BY CLONIDINE-LIKE DRUGS

Inhibition constant (Ki) values have been calculated from the IC50 for inhibition of binding using the Cheng and Prusoff [2] equation ICso = Ki (1 + D/KI~). Values given are means +_ S.E.M. for 4-7 experiments conducted in duplicate.

Drug K, [3H]Prazosin Ki [3H]Clonidine a K, [3H]Prazosin

K, [3H]Clonidine

Guanfacine 6117 _+ 1310 CP 14,304-18 2102 + 173 Yiamenidine 2092 +_ 442 ST 600 2592 +_ 636 Clonidine 593 + 102 Baya 6781 324 + 88 Guanabenz 212 ± 27 ST91 1174 _+ 403 Lofexidine 181 + 49 Naphazoline 124 +_ 16 44-549 40 +_ 2

a From ref. 11.

1.9 ~- 0.3 3220 0.8 ± 0.01 2627 2.3 + 0.5 910 4.6 ± 0.9 564 2.2 +_ 0.2 269 1.2 ± 0.4 269 0.9 + 0.2 236

12.4 +_ 4.2 95 2.3 + 0.2 79 2.0 _+_ 0.8 62 1.8 +~ 0.3 22

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2630, followed by tiamenidine, ST600, clonidine, Baya 6781, guanabenz and ST91 with ratios of 910, 564, 269, 236 and 95, respectively. Lofexidine, naphazoline and 44549 were the least selective with ratios of 79, 62 and 22, respectively. The ratios obtained here for clonidine and naphazoline are very comparable to values recently reported [8]. These results demonstrate that there is a 150-fold difference between the most and least selective clonidine-like drugs in their relative affinity for o~1 and a2 adrenoceptors.

Of the drugs tested guanfacine and CP 14,304 displayed the highest selectivity for c~2 adrenoceptors. This is supported by studies in the peripheral nervous system which provide evidence that guanfacine is more selective than clonidine for prejunctional a2 adrenoceptors [4]. Labelled guanfacine is available at a specific activity of 24.2 Ci/mmol and it will be of interest to compare the sites labelled by this ligand and [3H]clonidine. The degree of selectivity for c~l and o~2 adrenoceptors could be of value in the treatment of hypertension since it is known for clonidine that the correlation between plasma concentration and hypotensive effect is seen only at low dose levels - at higher dose levels the hypotensive effect is considerably smaller than expected, probably reflecting the increasing importance of the peripheral pressor component [3].

Drugs which are less selective than clonidine, such as lofexidine and, particularly, 44549, may be expected to have an even narrower therapeutic range, whereas those such as guanfacine and CP 14,304, which have poor displacing activity against prazosin binding to c~l receptors, would be expected to have a wider therapeutic range. It may also prove possible to extend the therapeutic range of these drugs by blocking their effects on peripheral ~ adrenoceptors using prazosin.

In conclusion, the present study shows that clonidine-like drugs vary in their selectivity for o~1 and a2 adrenoceptors identified by [3H]prazosin and [3H]clonidine binding, respectively. The most selective drugs were CP 14,304-18 and guanfacine and the least selective naphazoline, lofexidine and 44549. These differences could be important in the development of new selective ligands for 0~2 adrenoceptors and play a part in the determination of the effective therapeutic range of these drugs.

The authors thank the National Health and Medical Research Council of Australia for support and Mrs. Jeanette Brewer for technical assistance.

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