Life Sciences, Vol. 33, pp. 985-991 Pergamon Pres Printed in the U.S.A.

THE BINDING TO RAT BRAIN HOMOGENATES OF Mr2034, A UNIVERSAL OPIATE

Nicole Johnson and Gavri l W. Pasternak*

The George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettering Cancer Center, and Departments of Neurology and Pharmacology

Cornell University Medical College New York, NY 10021

(Received in final form June 16, 1983)

Summar~

Mr2034 has been proposed as a kappa opiate. While Mr2034 inhibited the binding of the kappa opiate JH-ethylketocyclazocine better than unlabeled ethylketocyclazocine, i t also displaced the binding of ~H-dihydromorphine and ~H-SKF 100~7 more ~otently thap morphine and SKF 10047, respectively. ~H-D-ala -D- leu°-~nkepha~in was displaced equally well by Mr2~34 and D-alaL-D-leu~-enkephalin. Saturat ion studies of JH-Mr2034 binding demonstrated cu rv i l i nea r Scatchard plots which could be dissected into two components by computer: KDI 0.06 nM, Bmax I 2.49 fmoles/mg t issue; and KD2 2.4 nM, Bmax 2 6.57 fmoles/mg t issue. A port ion of the higher a f f i n i t y (K~ 0.06 nM) component was inh ib i ted bY3naloxonazine treatment in v i t ro (50 nM), suggesting that H-Mr203~ bound with very high a f f i n i t y to mu 1 s i tes . Displacement of H-M~2034 binding by opioids was mul t i - phasic, again implying that °H-Mr2034 was binding to more than one c~ass,of s i te . In view of i t~ s imi lar potency in i nh i b i t i ng mu (~H-dihydromorphine), kapp~ (JH-ethylketo~ycla-zocine), sigma (JH-SKF 10047) and delta (H-D-alaL-D- leu°-enkephal in) opioids Mr2034 might be considered a universal opiate.

Recently both in vivo (i-5) and binding experiments (6-11) have suggested multiple classes of opiate and opioid peptide receptors. Thus, understanding the pharmacological actions of a drug requires an intimate knowledge of its aff ini t ies for the various types of binding sites. A number of new and novel opiates have been introduced in the past few years. One compound, Mr2034 [(-)-N-(2-tetrahydrofurfuryl)-normetazocine], has been proposed as a kappa ligand (12). Although several investigators have reported that i t also binds to mu sites, its ful l receptor selectivity has not been ful ly documented. We now present evidence suggesting that Mr2034, in addition to binding with very high aff in i ty to kappa sites, also bound exceedingly well to a number of other postulated subclasses, including mu 1, mu 2, kappa, sigma and delta.

*Address all correspondence to Dr. G.W. Pasternak, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021.

0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

986 Opioid Receptor Binding of Mr2034 Vol. 33, No. i0, 1983

Methods

Unlabeled Mr2034, Mr2035, Mr2266, Mr2267 and radiolabeled 3H-Mr2034 (17 Ci/mmole) were obtained from Boehringer-lngelheim (West Germany). Other opiates were from NIDA. Peptides were purchased from Be~j~e * ) i Jo l la , CA), and al l other 3H-labeled compounds and Formula 973 from New England Nuclear (Boston, MA). Male Sprague-Dawley rats (180-220 g; CrI:CD(SD)BR) were pur- chased from Charles River Breeding Laboratories (Wilmington. MA). Binding assays were performed as previously described (13). Since 3H-Mr2034 binding was l inear with t issue concentration up to 20 mg wet weight t issue/ml, two ml al iquots (20 mg/ml) were then incubated with the stated H- ! igand (I riM) and the appropriate concentration of unlabeled compound. Specif ic binding was defined as that binding displaced by levallorphan (I uM). T r ip l i ca te samples were used in each assay for both tota l and displaced binding. All aH-Mr2034 binding displaced by unlabele~ Mr2034 was also displaced by this concentration of levallorphan. ~inding of JH-Mr2034 was l inear with t issue and equ i l ib - rium was reached in less than 60 minutes at 20°C. Therefore, we rout ine ly incubated al l samples at 25°C for 60 minutes, f i l t e r e d them over Whatman Glass Fiber GF/B f i l t e r s and washed twice with cold buffer. Naloxonazine treatments were performed as described in the l i t e ra tu re (14): t issue was incubated with naloxonazine (50 riM) and washed twice before assaying. Each consisted of an incubation at 37°C for 10 minutes followed by centr i fugat ion (20 min at 49000 xg). All binding experiments were repl icated at least three times. Analysis of saturat ion studies was performed by nonlinear regression analysis and displayed graphical ly as a Scatchard plot .

Results

F i rs t we performed saturat ion studies with 3H-Mr2034 to establish i ts a f f i n i t y for the opioid binding s i tes. Scatchard plots of the resul ts (Fig. 1) c lear ly showed a curv i l inear plot consistent with more than one type of binding s i te . Computer analysis of the saturation data indicated a high a f f i n i t y (K~ 0.06 nM; Bmax 2.49 fmoles/mg t issue) and a lower a f f i n i t y component w~th greater capacity (K D 2.38 nM; Bmax 5.57 fmoles/mg t issue) . Previous work with other °H-ligands demonstrated that they bound with highest a f f i n i t y to Mu I s i tes. We therefore examined the ef fects of Mu I blockade in v i t ro with naloxonazine on ~H-Mr2034 binding. As noted in the control t issue, the binding was consistent with both high (KD 0.04 riM; Bmax 0.73 fmoles/mg t issue) and lower a f f i n i t y (K~ 2.45 nM; Bmax 9.3 fmoles/mg t issue) components. As seen with the other H-l igands, naloxonazine treatment decreased the higher a f f i n i t y component 7~ , whereas no decrease in the number of lower a f f i n i t y si tes was found. The a f f i n i t y constants were unchanged by the treatment. 3 However, one s ign i f i cant di f ference between the actions of naloxonazine on H-Mr2034 ~inding and i ts actions on other H- opioids was noted. Unlike the other ~H-ligands, a s ign i f icant port ion of the high a f f i n i t y binding remained fol lowing the naloxonazine treatment. Although not conclusive, the presence of residual high a f f i n i t y binding, which was seen in al l three separate experiments, raised the poss ib i l i t y that the high a f f i n i t y binding component seen in control t issue consisted of more than one class of s i te . I t should also be noted that the Mu I binding of other 3H-labeled compounds usually was approximately 0.8-1.5 fmoles/mg t issue (7,8). This corresponded well ~i th the decrease of 1.76 fmoles/mg t issue seen with naloxonazine treatment of ~H-Mr2034.

We examined the a b i l i t y of a number of opioids to displace 3H-Mr2034 binding (Table 1). Unlabeled Mr2034 was far more potent than any of the other ~ompound~ teste~ (p<O.O01). Most dramatic was the very low potency of

H-D-ala~-D-leuJ-enkephalin whose IC50 was far above 100 nM.

Vol. 33, No. i0, 1983 Opioid Receptor Binding of Mr2034 987

While the other opiates tested were al] less potent than unlabeled Mr2034, they did not di f fer that much between themselves. Oftentimes the use of a single ICso value has been misleading since the actual displacements were multiphaslc, suggesting that the radiolabeled compound§ were binding to more than one class of site. Multiphasic displacements of °H-Mr2034 were also seen (Fig. 2). In all cases, the displacement curves of unlabeled Mr2034 were far to the lef t of other opioids, confirming the information in Table I . More interesting, a portion of Mr2034 binding was displaced by low concentrations of the other opioid while the remainder was less sensitive. This was part icularly evident with the displacement by D-ala2-D-leu5-enkephalin. While a portion of binding was inhibited by concentrations below 0.5 nM, the remain~oer was v i r t u a l l y unaffected by concentrations up to 15 nM. Although th is i n i t i a l displacement was small, i t was consistent ly foun~ with a l l experiments. Although not shown in the f igure, a l l speci f ic JH-Mr2034 binding could be displaced by the opioids i f s u f f i c i e n t l y high concentrations were used.

1 5 o | ; I I

• C o n t r o l o N a l o x o n a z i n e

- 1 0 %

5 o l

o •

m

0 2 , 5 5 7 , 5 1 0

3H-Mr 2034 Binding (fmoles/mg tissue)

Figure 1: Scatchard plot of 3H-Mr2034 saturat ion iN control and naloxonazine- treated t issue.

Rat brain homogenates were prepared and binding performed with 3H-Mr2034 (0.1 to 5.1 nM). Only speci f ic binding is presented and the values represent the means of t r i p l i c a t e determinations From a typical experiment. The experiment was repl icated 3 times with s imi lar resu l ts . Computer analysis of the saturation curves demonstrated two components in the control t issue (K D 0,06 nM with a Bmax of 2.49 fmoles/mg t issue; and K 0 ?.38 nM with a Bmax of 6.57 fmoles/mg t issue) as well as in the na#oxonazine-treated t issue (K D 0.04 nM with a Bmax of 0.73 fmoles/mg t issue; and K D 2.45 nM with a Bmax of 9.3 fmoles/mg t issue).

988 Opioid Receptor Binding of Mr2034 Vol. 33, No. 10, 1983

I nh i b i t i on of aH-Mr2034 Binding

Compound (n determinations)

Mr2034 (9) Morphine (3) Ethylketocyclazocine (3) SKF 10,047 (3) )Fala ? -D-leub-enkephalin (3)

IC50 of 3H-Mr2034 binding (nM)

4.8 + 1.4 15.3 ¥ 1.5 21.7 ~ 0.9 34.7 ¥ 7.7

> 100-

Rat brain homogenates were prepared and the i nh i b i t i on of 3H-Mr2034 binding (I nM) determined using seven concentrations of unlabeled compounds (0.05-100 nM). Excluding D-alaL-D-leu b enkephalin, the potencies of the remaining opiates d i f fered s i g n i f i c a n t l y (p < 0.001) as determined in by analysis of variance (F3,14 = 20.36

5000

4000 ~ i [ A ,---. e

E 3000 Q_ ~1:~ 2000 Mr2034 ~ .c: 1000

CI~ 5000

4000

3000 2000

1000

0 # 0

i

B : D-ala2-D-leu5-Enkephalin

Mr2034

I I I I

' ' c'lli ' ' ° Ethylketocyclazocine -L r [ ~ 4 7

I I ~ , L L / / I I

1 I0 I00 0 1 I0 IO0

[Displacer] nM

Figure 2: I nh ib i t i on of 3H-Mr2034 by Mr2034 and other opioids.

Rat brain homogenates were prepared and binding performed with 3H-Mr2034 ( i nM). Only spec i f ic binding is presented and the values represent the means ~ SEM of t r i p l i c a t e samples from a typ ica l experiment. The experiments were repl icated three times with s imi lar resu l ts . Each displacement was performed with both the opioid compound and Mr2034 in the same experiment as a contro l . Displacers included: Mr2034 (closed c i r c l e s ) , morphine (open c i r c l es ) , ethylketoc~clazoc~ne (open squares), SKF 10,047 (closed t r iang les ) and O-ala~-D-leu~-enkephalin (open t r iang les ) .

Vol. 33, No. i0, 1983 Opioid Receptor Binding of Mr2034 989

The greater potency of unlabeled Mr2034 re la t i ve to the others could be explained by i ts binding to a s i te which did not bind the other compounds, or merely to a far greater potency. To examine this question, we compared the a b i l i t y of unlabeled Mr2034 to i nh ib i t the binding of a number of JH-labeled opioids with the i r prototypic ligands (Table 2). In al l cases, unlabeled Mr2034 was equipotent or more potent than the prototypic compound for each 3H-labeled opiate l igand. The fu l l displacement turves ~Fig 3) again demonstrated mult i-phasic displacements for D-ala--D-leu -enkephalin. In te res t ing ly , unlabeled Mr2034 was equipotent to unlabeled D-ala2-D-leu 5- ~nkephalin. The absence of multiphasic displacements with the other H-compounds might be explained by the high potency of Mr2034. Perhaps lower

concentrations might have been needed to define the i n i t i a l displacements.

I t has been reported that Mr2034 was an agonist (12). We therefore determined i ts sodium sh i f t (Table 3). The addit ion of sodium chlor ide (I00 mM) to the assay shi f ted the displacement curve 6 .4- fo ld . By contrast, the antagonist analog of Mr2034, Mr2266, which was equipotent to Mr2034 in the displacement of 3H-naloxone showed l i t t l e sh i f t in these assays.

Table 2: Inh ib i t i on of~H-Opioid Binding

IC50 (nM)

3H-dihydro- 3H-ethylketo- Compound (n determinations) morphine cyclazocine

Mr2034 (5) Morphine (4) Ethylketocyclazocine (4) SKF 10,047 (3) D-ala2-D-leu3-enkephalin (4)

0 . 9 8 + 0 . 1 7 1 . 3 + 0 . 4 3.9 ¥ 1 . 4

- 5 . 9 + 3 . 0

3H-SKF i0,047

1.4 + 0.6

3.9 + 1.7

3H-D-al a2-D_ leu 5-

enkephal i n

2 . 4 + 0 . 5

Rat brain homogenates were prepared and the inh ib i t ion of 3H-ligand binding (al l at 1 nM) determined using seven concentrations of unlabeled compounds. The resul ts are the means ± s.e.m, of n separate experiments.

Table 3: Sodium shi f ts

_ IC50 (nM)

Compound Without NaCl With NaCl Ratio

Mr2034 0.52 + 0.07 3.33 + 1.15 6.4 (p<O.05) Mr2266 0.80 ¥ 0.171 1.21 ¥ 0.33 1.5 N.S.

Rat brain homogenates were prepared and assays performed with 3H-naloxone (I nM) and seven concentrations of Mr2034 and Mr2266 in the presence and absence of NaCI (i00 mM). Values are the means ± s.e.m, of three separate experiments. Signif icance levels for the IC50 sh i f t s with sodium were determined by Students t - t e s t .

1 . 9 + 0 . 4

990 Opioid Receptor Binding of Mr2034 Vol. 33, No. i0, 1983

Discussion

The resul ts indicated the Mr2034 was a unique opiate compound. Previous studies have implied the existence of two cla~ses of mu sites (7). The decrease in the higher a f f i n i w component of H-Mr2034 binding was seen with naloxonazine treatment indicated that ~H-Mr2034 bound with high potency to the mu I s i te . However, a portion of the high a f f i n i t y binding component of 3H-Mr2034 remained fol lowing naloxonazine treatment, suggesting ti lat H- !4r2034 also bound with a similar high a f f i n i t y to another type of s i te . Tile exact determination of the pharmacological spec i f i c i t y of the remaining high a f f i n i t y binding has not been fur ther iden t i f i ed .

The displacement studies c lear ly demonstrated Mr2034's high potency for al l classes of opioid binding s i tes. In al l cases examined, Mr2034 was equal to or more potent than the prototypic compound with which i t was being compared. Thus, MrZ034 was more potent o F mu receptors than morphine and as potent on delta si tes as D-ala2-D-leu5-enKephalin. 3H-Ethylketocycla- zocine has been e f fec t i ve l y used to label kappa si tes (8-10). Therefore, the ab i l i t y of Mr2034 to displace °H-ethylket?cyclazocine binding implied that i t , too, was a potent kappa drug. Mr2034 s similar potency on 3H-SKF 10,047 binding demonstrated that i t also bound to sigma si tes. Previous work has shown that most benzomorphans did not discriminate between kappa and sigma

7500

.~ 6000

ooo

-r 1500

1500

"~ 1200

=.. ~ 9 0 0

~- 600

i n -

' ' A

i i C I

~ ine

Mr2054

0 1 10 100

,.o! ._~E 2500 2000

1500 T c:~ c:~ .S I000

o 500 ,A

1500

I"-.- ~ 1200 ',:a'- E O d ~ 9oo u_~

~ 600

3 0 0

o

[Displacer 1 nM

' ' B '

~i -leu5-Enkephalin

Mr2034 ~ i

' ' D

Mr2034

~' / I 1~0 o 1 lOO

Figure 3: Inh ib i t i on of 3H-opioid binding by Mr2034 and other opioids.

Rat bra~n homogenates were prepared and binding performed with the stated °H-ligands (a l l at I nM). Only speci f ic binding is presented and the values represent the mean + SEM of t r i p l i c a t e samples from a typical experiment. The experiments were repl icated three times with similar resu l ts . Each displacement was performed with both the opioid compound and Mr2034 in the same experiment as a control . Displacers are the same as those in Figure I .

Vol. 33, No. i0, 1983 Opioid Receptor Binding of Mr2034 991

sites, as exidenced by differences in the displacement of 3H-ethylketocycla- zocine and ~H-SKF 10,047 binding (8). However, the prototypic kappa drug ketocyclazocine dXsplaced H-ethylketocyclazoc~ne binding 5-fold more effectively than ~H-SKF 10,047~ implying that H-SKFIO,047 was indeed labeling sites not labeled by H-ethylketocyclazocine (8).

The displacement of 3H-Mr2034 binding by the other opioids i l l us t ra ted an important concept. The resul ts from Table I alone, which demonstrated that none of the classical opioids were as e f fec t i ve as MRS034 i t s e l f on 3H-Mr2034 binding, might be interpreted to mean that °H-Mr2034 was labe l l ing a separate and d is t inc t s i te . However, Mr2~34 was equipotent or more potent than the other opioids on the binding o~ JH-la~eled dihydro- morphine, ethy~ketocyclazocine, SKFIO,047 and D-ala~-D-leu -enkephalin, implying that H-Mr2034 was most l i~e ly labeling mu, kappa, sigma and delta s i tes . While a compound l i ke D-alaL~D-leuJ-enkephalin would be expected to potently displace the binding of H-Mr2034 to delta s i tes, i t would not be expected to displace 3H-Mr2034 binding to mu 2, kappa or sigma binding sites~ Thus, the accurate in terpre ta t ion of the binding of a novel opioid l i ke H-Mr2034 required knowing both the sens i t i v i t y of JH-Mr2034 binding ~ o other opioids as well as the sens i t i v i t y of the binding of the other H-opioids to unlabeled Mr2034.

In conclusion, Mr2034 appeared to show similar a f f i n i t i e s for al l the 3H-ligands tested. In view of this very high potency for al l tested classes of opioid binding s i tes, Mr2034 might be considered a "universal opiate".

Acknowledgements

We thank Drs. Posner, Ahern, Merz, Pollman, Ensiger and Hawks for the i r assistance with these studies. This work was supported by a grant from NIOA (DA00~615). GWP is a rec ip ient of a Teacher-lnvestigator Award from NINCDS (NSO0415).

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