[3H]Ethylketocyclazocine Receptor Binding and Ketocyclazocine Analgesia (Morphine/Kappa Receptor) GAVRIL W

Proc. Nati. Acad. Sci. USA Vol. 77, No. 6, pp. 3691-3694, June 1980 Medical Sciences

Multiple opiate receptors: [3H]Ethylketocyclazocine receptor binding and ketocyclazocine analgesia (morphine/kappa receptor) GAVRIL W. PASTERNAK George C. Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, and Departments of Neurology and Pharmacology, Cornell University Medical College, New York, New York 10021 Communicated by Lewis Thomas, March 10, 1980

ABSTRACT The receptor binding of the kappa agonist sites with the pharmacologically defined kappa and mu re- L3llethylketocyclazocine to brain homogenates in vitro and ceptors has proven difficult. To investigate possible relationships etocyclazocine (kappa) analgesia in vivo has been investigated and the mu and corpared to morphine, a mu agonist. Saturation analysis between high- and low-affinity binding proposed of [3Hjetylketocyclazocine binding in both mice and rats and kappa receptors, I have compared the receptor binding of yielded biphasic Scatchard plots similar to those of opiate mu [3H]ethylketocyclazocine, a proposed kappa agonist, to that of agonists, antagonists, enkephalins, and endorphins. Treatment [3H]morphine, a proposed mu agonist, and the effects of nal- of brain membranes with monovalent and divalent cations, oxazone treatment on ketocyclazocine (kappa) analgesia to chelating agents, protein-modifying reagents, and enzymes morphine (mu) analgesia. affected [3H]ethylketocyclazocine binding in a manner similar to that of [3H]morphine. Naloxazone, a long-acting antagonist that selectively abolished high-affinity [fJmorphine, H]- MATERIALS AND METHODS dihydromorphine, [3H]naloxone, and [3H-DAla2,Met Jen- kephalinamide binding in vivo, also selectively blocked high- [3H]Ethylketocyclazocine, [3H]morphine, [3H-DAla2,Met5]- affinity [3H]ethylketocyclazocine binding. Evaluation of anal- enkephalinamide and Formula 963 scintillation fluor were gesia with writhing and tail-flick assays in animals whose obtained from New England Nuclear. Sprague-Dawley rats high-affinity binding sites were blocked by naloxazone dem- (180-250 g) and ICR/CD1 mice (25-85 g) were obtained from onstrated a 6-to 7-fold increase in median effective dose (EDso) Charles River. values of ketocyclazocine. This decrease in analgesic potency Binding studies on both mice and rats were performed as was comparable to morphine's decreased potency in similarly treated mice. These biochemical and pharmacological results described (11). Because binding is linear with preincubated suggest that the analgesic properties of both kappa and mu tissue up to 30 mg wet weight tissue per ml (unpublished ob- agonists may be mediated through the same subpopulation of servation), 2-ml aliquots containing 20 mg wet weight tissue receptors, the high-affinity binding sites. per ml were used routinely. Enzyme or reagent treatment consisted of an additional incubation at 250C for 30 or 20 min, Extensive in vwo pharmacological studies of opiate drugs have respectively, with the stated amount of enzyme or reagent, suggested three classes of opioids with their own distinguishable centrifugation, and resuspension for the binding assay (11, 12, receptors: mu (morphine), kappa (ketocyclazocine), and sigma 14). Naloxazone (10 mg/ml of H20 dissolved with glacial acetic (SKF-10,047) (1). Receptor heterogeneity has also been implied acid) at 200 mg/kg was injected subcutaneously in the back of from biochemical investigations (2). Although early opiate the neck. Control animals received naloxone-HCI (200 mg/kg) binding studies were consistent with a homogeneous population in an identical manner (15, 16). of binding sites (3-6), more recent studies using 3H-labeled li- Tail-flick analgesia was measured as described (17). Only gands with higher specific activities clearly demonstrated animals whose latency after the administration of drug was at nonlinear Scatchard plots for agonists and antagonists (2) and least twice the latency prior to the administration of drug were enkephalin (7), consistent with subpopulations of high- and considered analgesic. Writhing tests were performed with low-affinity sites. Although nonlinear Scatchard plots can result benzoquinone as described (18). Median effective dose (EDo) from other factors, such as negative cooperativity, the concept values for both analgesic assays were determined by least- of two sites is partially supported by both biochemical and squares fit to log-probit analysis. pharmacological differences in high-affinity and low-affinity binding. Treatments that distinguish between agonist and an- RESULTS tagonist binding, including mono- and divalent cations (8-10), protein-modifying reagents (11-13), and enzymes (14), act Scatchard Analysis of [3HJEthylketocyclazocine Binding. primarily on high-affinity binding (2, 10, 11, 14). Selective Scatchard plots of [3H]ethylketocyclazocine binding were blockade of high-affinity binding in vivo by naloxazone, a similar in both rat and mouse brain homogenates. Mouse brain long-acting antagonist, decreased morphine's analgesic potency curves could be resolved into two linear components with Kd over 1 -fold with little effect on lethality, suggesting different values of 0.2 nM and 4.3 nM for high- and low-affinity binding, receptor mechanisms (refs. 15 and 16; A.-Z. Zhang and G. W. respectively (Fig. 1). Sodium chloride (100 mM) decreased the Pasternak, unpublished data). number of high-affinity binding sites in a series of four separate Correlating the biochemically defined high- and low-affinity Scatchard studies by 45-60%. Low-affinity binding, which was lowered by only 5-18%, was far less affected. Studies with rat The publication costs of this article were defrayed in part by page brain homogenates also showed nonlinear Scatchard plots with charge payment. This article must therefore be hereby marked "ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviations: ED50& median effective dose; ICso, concentration giving this fact. 50% inhibition. 3691 3692 Medical Sciences: Pasternak Proc. Natl. Acad. Sci. USA 77 (1980)

Table 1. Effect of ions, chelators, and protein-modifying reagents on [3H]ethylketocyclazocine and [3H]morphine binding in rat brain [3H]Ethylketo- cyclazocine [3H]Morphine binding binding Change, Change, Treatment cpm % cpm % Monovalent cations* None 2120± 27 - 1460± 115 NaCl 5mM 1610± 102 -23 956 87 -34 15 mM 1270 + 57 -40 773 61 -53 50 mM 1130 + 93 -47 470 57 -68 100 mM 883 ± 72 -58 392 30 -73 LiCl 25mM 1860 ± 45 -12 1110 ± 132 -24 50mM 1460+ 75 -31 777 ± 100 -47 100 mM 1150 + 24 -46 669 ± 77 -54 KCl 25mM 2020 + 42 -5 1570 + 77 +8 50 mM 1850 + 48 -13 1300 ± 89 -11 50 100 150 200 250 100 mM 1700 ± 26 -20 1030 ± 36 -30 [3H ]Ethylketocyclazocine binding, fmol/20 mg tissue Divalent cations and chelatorst NaCl 100 mM 735 22 - 417 + 60 FIG. 1. Scatchard analysis of [3H]ethylketocyclazocine binding. + 1 mM MnCl2 1060 ± 32 +45 810 + 96 +94 Mouse brains were prepared and assayed with [3H]ethylketocycla- + 1 mM CaCl2 730 44 -1 615 + 90 +47 zocine (0.042-3.6 nM) at 25°C in the presence (0) and absence (0) + 1 mMEGTA 712 40 -3 555 + 72 +33 of NaCl (100 mM). Only specific binding is reported (fmol/20 mg of + 1 mMEDTA 488 22 -34 198 ± 19 -53 tissue). The experiment has been repeated four times. + 1 mM EDTA + 1 mMMnCl2 905 19 +23 837 + 78 +101 similar Kd values (0.23 nM and 5.5 nM for and low-af- Protein-modifying reagentst high- Nothing 1780± 61 - 1360 ± 70 finity binding, respectively), similar numbers of high- and Jodoacetamide low-affinity binding sites per mg of tissue, and a 60% decrease 1 mM 1590 58 -11 1290 + 38 -6 in high-affinity binding sites by sodium ions (100 mM). 5 mM 1110 21 -37 713 ± 56 -48 Biochemical Characterization of [3H]Ethylketocyclazo- 10 mM 522 19 -71 232 + 74 -83 cine and 13H]Morphine Binding in Vitro. Cations and che- N-Ethylmaleimide lators have proven useful in evaluating the agonist-antagonist 1,uM 2010± 134 +13 1310+ 9 -4 character of opiates (8-10). Several mono- and divalent cations 10,uM 1700 60 -5 969 + 17 -29 and chelators produce similar agonist-like effects on both 100,uM 612± 38 -66 318+ 65 -77 [3H]ethylketocyclazocine and [3H]morphine binding (Table * Rat brains were prepared and assayed with [3H]ethylketocyclazocine 1). Sodium's inhibition of [3H]morphine binding (concentration (1.6 nM) or [3H]morphine (1.2 nM) and the designated cations. giving 50% inhibition, IC50, 20 mM) and [3H]ethylketocyclo- Values represent specific binding (cpm I SEM). All values were zocine binding (IC50, 50 mM) was similar to results with [3H1- obtained from the same experiment using the same homogenate. dihydromorphine (10). The slightly decreased sensitivity of The experiment has been repeated three times. t Rat brains were prepared and assays were performed with divalent [3H]ethylketocyclazocine to sodium corresponds to its lower cations and chelators with [3H]ethylketocyclazocine (3.4 nM) and "sodium ratio" of 6.4 (19). Lithium ions lowered the binding [3H]morphine (0.94 nM) in the presence and absence of NaCl (100 of both 3H-labeled ligands slightly less than sodium, whereas mM). Values represent specific binding (cpm + SEM). All values potassium ions produced effects only at high concentrations. were obtained from the same homogenate in the same experiment. As previously described for a variety of 3H-labeled ligands The experiment has been repeated three times. EGTA, ethylene (10), manganese chloride markedly increased the binding of glycol bis(3-aminoethyl ether)-N,N,N',N'-tetraacetate. Rat brains were prepared, treated with the appropriate reagent for both 3H-labeled agonists in the presence of sodium, whereas 20 min at 25°C, and assayed with [3H]ethylketocyclazocine (1.8 nM) calcium chloride was much less effective. EDTA treatment and [3H]morphine (1.2 nM). Values represent specific binding (cpm potentiated the sodium's inhibition on both 3H-labeled ligands, ± SEM). All values reported were obtained from the same homog- whereas EGTA, a chelator relatively specific for calcium, had enate in the same experiment. The experiment has been repeated little effect. The binding of both 3H-labeled ligands was in- three times. hibited in a dose-dependent manner by iodoacetamide and N-ethylmaleimide. The IC50 values for iodoacetamide and Influence of Naloxazone on [3H]Ethylketocyclazocine N-ethylmaleimide inhibition of [3H]ethylketocyclazocine Binding and Ketocyclazocine Analgesia. Naloxazone is a binding (5.5 mM and 43 ,M, respectively) and for [3H]mor- narcotic antagonist which selectively blocks high-affinity opiate phine binding (4.4 mM and 28 ,uM, respectively) are quite agonist, antagonist, and enkephalin binding sites in vivo for similar to results with [3H]dihydromorphine (11). several days (15, 16). To investigate high-affinity [3H]ethyl- Trypsin and chymotrypsin treatment of tissue homogenate ketocyclazocine receptor binding, I performed saturation lowered both [3H]ethylketocyclazocine and [3H]morphine analysis in mice treated with naloxone and naloxazone (Fig. 3). binding (Fig. 2). Plotted semilogarithmically (Fig. 2), both Whereas naloxone treatment was without effect after 24 hr, 3H-labeled ligands demonstrate biphasic curves for trypsin and naloxazone treatment abolished high-affinity [3H]ethylketo- chymotrypsin. The breaks in the curves for both 3H-labeled cyclazocine binding. ligands occur at the same concentrations of enzymes as [3H]- To correlate high- and low-affinity kappa binding sites and naloxone (6). analgesia, I studied ketocyclazocine analgesia in mice treated Medical Sciences: Pasternak Proc. Natl. Acad. Sci. USA 77 (1980) 3693

14 ~Y4

0~ 3

N la- 2 3ISisA

0 g 50

I I 25 50 75 100 125 0 1 2 3 [3H]Ethylketocyclazocine binding, Trypsin, ,ug/ml fmol/20 mg tissue

if II FIG. 3. Effects of in vivo naloxazone and naloxone treatment on B 100 [3H]ethylketocyclazocine binding. Mice were treated with either naloxazone (0) or naloxone (0) (200 mg/kg); 18 hr later brain homogenates were prepared and assayed with [3Hlethylketocyclazocine (0.16-18 nM). Binding is reported as fmol/20 mg of tissue and rep- 50 resents specific binding only. The experiment has been repeated three times. 100

with [3H]ethylketocyclazocine are also consistent with high- 0 and low-affinity binding components with affinities similar to 50 the other [3H]opiates. Because sodium ions selectively decrease \ agonist binding, the inhibition of [3H]ethylketocyclazocine 50 binding was anticipated. Sodium's decrease of [3H]ethylketocyclazocine binding parallels that of [3H]morphine and [3H]- dihydromorphine (2, 8). Sodium acts primarily on high-affinity 25- binding, as determined by Scatchard analysis for the mu agonist [3H]dihydromorphine, the antagonist [3H]naloxone, and [3H]enkephalin. Likewise, high-affinity [3H]ethylketocyclazocine binding is particularly sensitive to sodium. 0 10 20 30 40 50 Treatment of brain homogenates with divalent cations and Chymotrypsin, pg/ml chelators, protein-modifying reagents, and even enzymes has FIG 2. Effects of trypsin and chymotrypsin. (G A) Trypsin. Rat similar effects on both [3H]ethylketocyclazocine and [3H]- brain homogenate was prepared, incubated with the designated morphine binding. The ability of these treatments to differ- trypsin concentration, and assayed with both [3H]et'hylketocyclazo- entiate between agonist and antagonist binding at the con- cine (0, 1.3 nM) and [3H]morphine (0, 1.2 nM). (I rithmic plot. Specific control binding is 1260 cpm fo[ns3H]ethylketo- centrations examined (10, 14) helps explain the slight differ- cyclazocine and 940 cpm for [3H]morphine. The expe,riment has been repeated three times. (B) Chymotrypsin. Rat brain Ihomogenate was Table 2. Ketocyclazocine analgesia in naloxazone- and naloxone- prepared, incubated with the designated chymotrypsiin concentration, and assayed with [3H]ethylketocyclazocine (0, 1.8 nM) and [3H]- treated mice: Writhing assay morphine (0, 1.5 nM). (Inset) Semilogarithmic plot. Specific control Keto- Naloxone group Naloxazone group binding is 1370 cpm for [3H]ethylketocyclazocine arnd 1130 cpm for cyclazocine, Inhibition, Inhibition, [3H]morphine. The experiment has been repeated three times. mg/kg Writhes % Writhes N

24 hr earlier with either naloxone or naloxazoine. Ketocycla- 0 77 75 zocine's analgesic potency is attenuated markecdly by naloxa- 0.05 55 29 - 0.1 28 64 zone treatment in both the writhing assay )le 2) and tail- (Tal 0.2 10 87 88 0 flick assay (Table 3). 0.4 40 47 DISCUSSION 0.5 - 37 51 The biochemical characteristics of [3H]ethylke 0.8 30 70 ~tocyclazocine mg/kg 0.077 + 0.005 0.47 ± binding are quite similar to the binding of othEer [3H]opiates, ED50, 0.002 especially [3H]morphine. Saturation studies wilth a variety of Groups of mice were treated with naloxone or naloxazone (200 high specific activity [3H]opiates, including dihyidromorphine, mg/kg). Twenty-four hours later each group was subdivided into groups of eight animals and tested for morphine analgesia with the naloxone (6), enkephalin (7), morphine, naltre xone, and di- writhing assay. Writhing was assessed with two 1-min intervals at 15 prenorphine (A.-Z. Zhang and G. W. Pasternak unpublished and 20 min after ketocyclazocine, which corresponded to 5 and 10 min data), yield biphasic Scatchard curves suggestive of high- and after benzoquinone (3 mg/kg intraperitoneally). ED5o values were low-affinity components. The biphasic Scatchard plots obtained determined by least-squares fit to a log-probit analysis. 3694 Medical Sciences: Pasternak Proc. Nati. Acad. Sci. USA 77 (1980) Table 3. Ketocyclazocine analgesia in naloxazone- and naloxone- through the low-affinity binding sites. Likewise, nonanalgetic treated mice: Tail-flick assay differences in pharmacological action between kappa and mu Ketocyclazocine, mg/kg Naloxone group* Naloxazone group* opioids may result from differences in their low-affinity binding interactions. 5 38% (3/8) 10 57% (4/7) I thank Mary Buatti for her excellent technical assistance, Carol 15 75% (6/8) D'Anella for secretarial help, and Drs. David Ahern, Charles Inturrisi, 40 - 29% (2/7) and Jerome Posner for their aid. This work was supported in part by 50 38% (3/8) a Pharmaceutical Manufacturers Association Research Starter Grant, 60 - 63% (5/8) a Lilly Research Grant, New York State Health Research Council ED50, mg/kg 7.4 ± 0.4 55.0 ± 0.4 Award 1609, National Institutes of Health Grant BRSG 5 S07 RR 05534, and National Cancer Institute Core Grant CA-08748. G.W.P. Mice were treated with either naloxone or naloxazone (200 mg/kg) is a recipient of National Institute of Neurological and Communicative and tested for analgesia in the tail-flick assay 24 hr later. To be considered analgetic, the tail-flick latency after ketocyclazocine treatment Disorders and Stroke Teacher-Investigator Award 1 K07NS415-01. was at least twice the baseline value. Baseline latencies ranged from 3 to 5 sec. ED50 values for morphine analgesia were determined by a 1. Martin, W. R., Eades, C. G., Thompson, J. A., Huppler, R. A. & least-squares fit to a log-probit plot. Gilbert, P. E. (1976) J. Pharmacol. Exp. Ther. 197,517-532. * Percentage of mice with analgesia is shown. Values in parentheses 2. Pasternak, G. W. & Snyder, S. H. (1975) Nature (London) 253, are the no. of analgesic mice/no. of mice tested. 563-565. 3. Pert, C. B. & Snyder, S. H. (1973) Science 179, 1011-1014. 4. Simon, E. J., Hiller, J. M. & Edelman, I. (1973) Proc. Nati. Acad. ences between the two 3H-labeled ligands. The small amount Sci. USA 70, 1947-1949. of antagonist activity indicated by [3H]ethylketocyclazocine 5. Terenius, L. (1973) Acta Pharmacol. Toxicol. 32,377-384. binding studies is significantly less than ketocyclazocine and 6. Pasternak, G. W. & Snyder, S. H. (1974) Mol. Pharnacol. 10, pentazocine and far less than nalorphine or cyclazocine (19) and 183-193. therefore may not be detectable in current in vvo assays of 7. Lord, J., Waterfield, A., Hughes, J. & Kosterlitz, H. W. (1977) antagonist activity. Nature (London) 267,495-499. Naloxazone blocks high-affinity [3H]ethylketocyclazocine 8. Pert, G. B., Pasternak, G. W. & Snyder, S, H. (1973) Science 182, binding in a manner similar to all other narcotics and enkeph- 1359-1361. alins tested, including [3H]dihydromorphine, [3H]naloxone, 9. Simon, E. J., Hiller, J. M., Groth, J. & Edelman, I. (1975) J. [3H-DAla2,Met5]enkephalinamide (15, 16), [3Hjnaltrexone, Pharmacol. Exp. Ther. 192,531-537. 10. Pasternak, G. W., Snowman, A. & Snyder, S. H. (1975) Mol. [3H]dipremorphine, [3H-Leu5]enkephalin, and [3H-Met5]- Pharmacol. 11,735-744. enkephalin (A.-Z. Zhang and G. W. Pasternak, unpublished 11. Pasternak, G. W., Wilson, H. A. & Snyder, S. H. (1975) Mol. data). Thus, high-affinity [3H]ethylketocyclazocine or kappa Pharmacol. 11,340-351. receptor binding is quite similar to high-affinity mu agonist, 12. Wilson, H. A., Pasternak, G. W. & Snyder, S. H. (1975) Nature antagonist, and enkephalin binding. The marked attenuation (London) 253, 448-450. of ketocyclazocine analgesia with blockade of high-affinity 13. Simon, E. J. & Groth, J. (1975) Proc. Nati. Acad. Sci. USA 72, bonding and its close similarity to results with morphine anal- 2404-2407. gesia imply that these same high-affinity sites may mediate both 14. Pasternak, G. W. & Snyder, S. H. (1975) Mol. Pharmacol. 11, ketocyclazocine and morphine analgesia. Analgesic differences 478-484. between opiate mu and kappa agonists, antagonists, and mixed 15. Pasternak, G. W., Childers, S. R. & Snyder, S. H. (1980) Science arise from differences in their mo- 208,514-516. agonist-antagonists may 16. Pasternak, G. W., Childers, S. R. & Snyder, S. H. (1980) J. lecular interactions with the same high-affinity population of Pharmacol. Exp. Ther., in press. binding sites, as illustrated by their varying sensitivity to sodi- 17. D'Amour, F. E. & Smith, D. L. (1941) J. Pharnacol. Exp. Ther. um. Previous studies demonstrated that in vivo high-affinity 72,74-79. receptor blockade in mice with naloxazone did not change the 18. Pearl, J. & Hoeris, L. (1966) J. Pharmacol. Exp. Ther. 154, median lethal dose (LD50) value for morphine despite the 319-323. 11-fold increase in its analgetic ED50 value. It was suggested 19. Pert, C. B., Snyder, S. H. & May, E. L. (1976) J. Pharmacol. Exp. that those factors responsible for lethality might be mediated Ther. 196,316-322.