Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1629-1632, March 1990 Pharmacology Dextrorphan binds to opioid receptors in guinea-pig brain membranes and is an antagonist at opioid receptors in myenteric plexus (binding/guinea-pig ileum/levorphanol/stereoselectivity) AVRAM GOLDSTEIN AND ASHA NAIDU Department of Pharmacology, Stanford University, Stanford, CA 94305 Contributed by Avram Goldstein, December 11, 1989

ABSTRACT Dextrorphan (+)-tartrate, purified by re- LaRoche); [D-Ala,MePhe4,Gly-ol5]enkephalin (DAGO; Pe- peated crystallization to remove all traces of the enantiomer ninsula Laboratories) (2); trans-3,4-dichloro-N-methyl- levorphanol, binds to IL, 6, and K sites on guinea-pig brain N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide methane- membranes with lower affinities (by a factor of400-3200) than sulfonate (U50,488; Upjohn) (3); (-)-naloxone hydrochloride levorphanol. In the guinea-pig ileum myenteric plexus longi- (NAL; Endo Laboratories, New York); normorphine hydro- tudinal muscle preparation (GPI), dextrorphan, at 100-200 chloride (NOR) and (+)-naloxone hydrochloride (National ,uM, inhibits the electrically stimulated twitch, but this action Institute on Drug Abuse); N-methyl-D-aspartic acid (NMDA), is not blocked or reversed by naloxone; both (+)- and (-)- aminophosphonovalerate, norepinephrine (levarterenol), at- naloxone produce similar non-opioid twitch inhibition at com- ropine, and eserine (physostigmine) (Sigma). parable concentrations. At 10-20 jaM, dextrorphan' blocks and Preparation ofDEXp. [3H]LEV (Ro 1-5431/701, Hoffmann- reverses the twitch inhibition due to ,u and KWagonists, but the LaRoche; 17.4 Ci/mmol; 1 Ci = 37 GBq) was purified' by blockade can be overcome only partially by increasing the preparative reversed-phase HPLC (Waters, C18 AgBondaPak, agonist concentration. We conclude that dextrorphan is an 3.9 x 300 mm, isocratic 75% CH3CN in 0.01 M sodium opioid ligand with low affinity and with antagonist effect on tartrate, 1.5 ml/min, 0.6-ml fractions). To 1 g of DEX (+)- opioid receptors in the GPI. tartrate was added 106 cpm of the purified [3H]LEV. Just enough boiling water was added to bring the material into The opioid receptors are highly stereoselective. Levorphanol solution, and repeated recrystallizations were carried out in (LEV, 3-hydroxy-N-methylmorphinan) is a high-affinity ,- this manner by overnight storage at 4°C. As LEV (+)-tartrate selective agonist, whereas its enantiomer dextrorphan (DEX) is somewhat less soluble in water than DEX'(+)-tartrate, it apparently has about three orders ofmagnitude lower affinity was possible to eliminate most of the tracer LEV and with it, than LEV at opioid binding sites (1). However, when a chiral presumably, any contaminant LEV initially present. After compound is obtained by resolution of a racemic mixture, each recrystallization step the redissolved DEX was mea- chemical standards of purity could not exclude the presence sured by absorbance at 279 nm, and the [3H]LEV content'was of 1 part per 1000 of an undesired enantiomer. We therefore assessed by radioactivity measurement in a scintillation considered the possibility that LEV, contaminating the DEX counter. supplied by the manufacturer, might be responsible for the Binding Assays. These were conducted as described (1) apparent low-affinity binding of DEX and thus that the with guinea-pig brain membranes, in TB and KHB, using receptors do not recognize DEX at all. Accordingly, ultra- radioligands and competing ligands in assays highly selective pure DEX (DEXp) was prepared as described under Mate- for u, 8, and K opioid binding sites. In an experiment with rials and Methods. This compound proved to be indistin- mouse brain membranes, [3H]LEV was used to label primar- guishable from the commercial starting material (DEXC) in its ily ,u sites. Ki values were computed from competition curves binding to guinea-pig brain membranes in standard ,u, 5, and to obtain ligand selectivity profiles, as described (1). K assays in two buffer systems. In guinea-pig ileum myenteric Guinea-Pig Ileum Assay. The electrically stimulated GPI, plexus longitudinal muscle preparation (GPI), DEXC and mounted in KRB, was used as described (4, 5). Concentra- DEXp, at 100-200 ,M, were equipotent in inhibiting the tions of substances added to the bath were incremented twitch by a non-opioid mechanism. At 10-20,uM they were geometrically, usually by doublings, and IC50 or IC30 values equipotent in blocking or reversing twitch inhibition due to (concentrations of agonists causing 50% or 30%6 inhibition of opioid agonists.' twitch amplitude) were obtained by interpolation on semi- logarithmic plots. MATERIALS AND METHODS RESULTS Drugs and Reagents. Reagents were Baker analytical grade or equivalent. Tris-HCI buffer (TB, mM concentrations): 50, The amount oftracer LEV added initially to DEXC, at a molar pH 7.4. Krebs-Hepes buffer (KHB): NaCI 118, KCl 4.8, ratio of 1:54 million (Table 1, cycle 0), was far too little to CaC12 2.5, MgCl2 1.2, Hepes (N-2-hydroxyethyl piperazine- contribute any activity of its own in binding assays or the N'-2-ethanesulfonic acid, Research Organics) 25,' pH GPI. After the fourth recrystallization, the added LEV (and adjusted to 7.4 with NaOH. Krebs-Ringer buffer (KRB): therefore any LEV present initially as a contaminant of same as KHB with KH2PO4 1.2 and NaHCO3 25 instead of Hepes, and, in addition, glucose 11, choline chloride 0.02; Abbreviations: LEV, levorphanol; DEXC, commercial dextrorphan; mepyramine maleate (125 nM) were also present. KRB was DEXp, purified dextrorphan; GPI, guinea-pig ileum myenteric plexus longitudinal muscle preparation; DAGO, [D-Ala2,MePhe4,Gly- bubbled constantly with 5% CO2 in 02 at 37°C to maintain pH o15]enkephalin; NAL, (-)-naloxone hydrochloride; NOR, normor- 7.4. phine hydrochloride; NMDA, acid; BREM, Drugs and suppliers are as follows: dextrorphan (+)-tartrate bremazocine; DPDPE, [D-penicillamine,N-meth~yl-D-asparticD-penicillamineSjenkepha- (DEX) and levorphanol (+)-tartrate (LEV) (Hoffmann- lin. 1629 Downloaded by guest on September 23, 2021 1630 Pharmacology: Goldstein and Naidu Proc. Natl. Acad Sci. USA 87 (1990) Table 1. Removal of added [3H]LEV (17.4 Ci/mmol) from DEX LEV by repeated recrystallization of the (+)-tartrate salt A ------I ------K. 8 Purification Cycle LEV, pmol DEX, mmol LEV/DEX factor 0 43.2 2.35 1.84 x 10-8 1.0 DEXc 8 . 1 5.63 1.12 5.03 x 10-9 3.7 2 0.850 0.692 1.23 x 10-9 15. 3 0.0958 0.315 3.04 x 10-10 61. L K 8 DEX 4 0.0157 0.191 8.22 x 10-11 220. p g---L-.-. -K-.------8 Tracer LEV was added to DEXC tartrate and repeated recrystal- lizations were carried out. Residual [3H]LEV and DEX in each supernatant were determined by radioactivity measurement and l absorbance at 279 nm, respectively. The final product was called -10 -9 -8 -7 DEXp. DEXC) had been reduced, relative to DEX, by a factor of 220 -7 -6 -5 -4 (Table 1, cycle 4). We then compared DEXp (the material from the fourth LOG Ki with DEXC in the binding assay and the bioassay. Fig. cycle) FIG. 2. Ligand selectivity profiles of LEV, DEXC, and DEXp. 1 shows that the crude and purified ligands competed iden- Data are log(K,) values, computed from IC50 in standard binding tically for LEV binding sites in mouse brain membranes. Fig. assays with guinea-pig brain membranes, as described (1). Radioli- 2 presents the ligand selectivity profiles (1) of LEV, DEXc, gands were [3H]DAGO (for IL), [3H]DPDPE (for 8), and [3H]BREM and DEXp in guinea-pig brain membranes using standard , displaced by U50,488 (for K) in a paired-tube procedure (1). Note 8, and K binding assays. Again, the affinities of DEXC and different abscissa scales for LEV (upper) and DEX (lower). Each DEXP were the same. Thus, it is evident from both figures assay was carried out in both TB (solid lines) and KHB (broken sites could not lines), in at least three independent experiments. As documented that the affinities of DEXC for opioid binding elsewhere (1), SEM for each log (Kj) was -0.1 log unit, too small to be due to trace contamination by LEV. shown effectively here. The ligand selectivity profiles ofLEV and DEX were quite be similar except for the large difference in affinity at each ofthe concentrations (>400 IAM) NAL itself, and also its enantio- three sites. Interestingly, however, the actual K, ratios mer (+)-NAL, inhibited the twitch (not shown). (DEXC/LEV) were significantly different at the three types of In view of the findings ofChoi et al. (6) that dextrorotatory sites-3200 and 2500 at ,A 2500 and 1000 at 8, 790 and 400 at opiates block NMDA receptors, we considered the possibil- K (data for TB and KHB, respectively). These differences ity that the naloxone-resistant agonist effects seen here might provide additional evidence that the binding of DEXC could also be due to NMDA receptor-inhibition. However, neither not be due to LEV contamination. in the unstimulated nor in the stimulated GPI did NMDA In the GPI, DEXC and DEXp were tested on six strips. No itself have any effect (to 400 ,uM), nor did the NMDA inhibition was seen until concentrations of 100-200 FM were antagonist aminophosphonovalerate (to 400 gM) inhibit the reached, and then both inhibited the twitch with essentially electrically stimyjated twitch. We conclude that NMDA the same IC50 (Table 2). Within-strip comparisons with NOR receptors, if present, do not mediate the electrically stimu- were computed in order to normalize for strip-to-strip sen- lated twitch and therefore that twitch inhibition by DEX is sitivity variations. DEXP was used in all subsequent exper- unrelated to NMDA receptors. iments on GPI, unless otherwise specified. As shown above, DEX, in the range 1-10 juM, clearly Typical GPI records are shown in Fig. 3. Record A shows interacted with opioid receptors in the binding assay, but in the well-known rapid onset and reversal (by wash) of twitch the'GPI no agonist effects were observed except at much inhibition by NOR. Record'B displays the slower onset of higher concentrations and'then not by an opioid receptor- LEV inhibition and its' prompt reversal by NAL. Records C mediated action. The possibility'remained that DEX could and D show the twitch inhibition by a very high concentration act as' an antagonist in the GPI. Accordingly, we tested its of DEX and the failure of NAL to block it. At very high ability to reverse (and block) the ,u agonists LEV and DAGO as well as the highly selective K agonist U50,488. The GPI 800 contains no (or very few) 8 receptors. i Fig. 3, record E, shows that LEV caused 60% inhibition, 700 A which was reversed slowly (over 10 min) to 24% inhibition 600 after addition of DEX. Above 64 uM DEX had no further antagonist effect. Thus, at a concentration much lower than n 500 required to inhibit the twitch, DEX could reverse the agonist m0 400 effect of LEV on the GPI. Reversal was only partial, usually about half, and varied from strip to strip. C 300 t Pretreatment with DEX (10-20 ,uM) in the GPI blocked the 200 - twitch inhibition by LEV, DAGO, and U50,488 (Fig. 4)'. The blockade could be overcome only partially by raising the 100- agonist concentration. Inasmuch as parallel shifts of the logarithmic dose-response curves were not observed, the -8 -7 -6 -5 -4 -3 conventional computation of antagonist apparent dissocia- Log conc. DEX (M) tion constant, K& (7), would have no precise meaning. How- ever, as rough measures of antagonist potencies, "empirical FIG. 1. Competition for LEV binding sites on mouse brain DEX. The results membranes. DEXC (circles) and DEXp (triangles), prepared as de- K, values" were computed for (Table 3) scribed, were compared in a competition assay with [3H]LEV (0.28 show characteristic values of KI for NAL, the affinity of nM) in TB. Each determination was in triplicate; SEM values were which is typically an order of magnitude greater at ,u (LEV) too small to depict except for the one instance shown. than at K (U50,488) receptors. Empirical Ke values for DEX Downloaded by guest on September 23, 2021 Pharmacology: Goldstein and Naidu Proc. Natl. Acad. Sci. USA 87 (1990) 1631 Table 2. Twitch inhibition by NOR, DEXC, DEXp, and LEV in the GPI NOR DEXC DEXP LEV n 11 6 5 8 log(IC50) -6.82 ± 0.14 -3.75 ± 0.07 -3.90 ± 0.14 -7.77 ± 0.10 IC50 150 nM 180 ± 31MM 130 49 AM 17 nM log diff. from NOR 0 3.05 ± 0.07 2.93 + 0.07 -0.80 + 0.07 IC50 ratio to NOR 1.0 1100 850 0.16 IC50 ratio to LEV - 6900 5300 1.0 n is number of independent experiments with different strips. log(IC50) values were obtained by interpolation. NOR and DEX data shown here were obtained in the same strips, but log(IC50) of NOR in all experiments (n = 50) was virtually identical, -6.76 ± 0.04 (IC50 = 170 nM). "log diff. from NOR" was computed for each strip to normalize for differences in strip sensitivity; data are means of these individual differences, NOR and LEV concentrations are nM; DEX concentrations are 1AM. were about 1000 times greater, with a similar preference for line concentration established by each stimulus was maxi- Ax receptors over K. The DEX blockade of the u agonist mally effective. DAGO, shown in Fig. 4B, yielded empirical K1 = 3.7 nM, but this experiment was not replicated. DEX, at concentrations that blocked the opioid agonists, 0 --A0 did not block twitch inhibition caused by norepinephrine or 80 atropine, consistent with a specific action on opioid recep- s- tors. The cholinesterase inhibitor eserine (100 nM) reversed a-10 70--0 0 60 DEX 0 \ \0 DEX 1 0 and blocked DAGO and U50,488 (not shown), as first dem- .2 onstrated with morphine as agonist by Kosterlitz and Wa- 50 -0-0 0 0 terfield (8). The blockade was grossly similar to that pro- 40 --0 3. duced by DEX, in that it could not be overcome fully by 30 - agonist. Eserine, at these concentrations, did not by itself enhance the twitch amplitude, suggesting that the acetylcho- 20 - 10- 0- 1 10 100 100DO LEV concentration (nM) A NOR 240 nM Wash

-C B i -CQ) ttttuutittttkwuku-ILLttuuLiuuutltttI I------I -C LEV NAL 1 min (2 29 nM 100nM ai

c . 1111IMillilliffiffil 10 100 1000 DAGO concentration (nM) Wash 200XM In icI)U - 10-- C 50- 0 D 7 0u E- NAL DEX 6a0- DEX 0 0 DEX 20 200 nM 200 pM -C 5 -C 4~0- 0 3 31 0~~~ cqi 21 0- LEV LEV DEX DEX 7.3 nM 58 nM 8.0 aM 64 pM 0 - o 1 10 100 100NO FIG. 3. Typical records from the GPI. Time marker on line B U50,488 concentration (nM) applies to all records. Note mechanical artifact at each wash. Records C and D are from same strip; in record D, NAL pretreatment FIG. 4. Blockade of opioid agonists by DEX. Preincubation (4 was 3 min before adding DEX. Record E shows excerpts from a min) with DEX (A.M concentration indicated on curves) was followed 41-min continuous record (gap durations are indicated) in which LEV by stepwise incrementation ofagonist concentration. Typical results concentrations were doubled in a stepwise manner, followed by are shown for k agonists LEV (A) and DAGO (B) and K agonist stepwise doubling ofDEX; there were no washes in this experiment. U50,488 (C). Downloaded by guest on September 23, 2021 EmE 1632 Pharmacology: Goldstein and Naidu Proc. Natl. Acad. Sci. USA 87 (1990) Table 3. Potencies of NAL and DEX as antagonists to LEV, Was it possible, then, that DEX acted as an antagonist in DAGO, and U50,488 the concentration range (about 1-10 ,uM) corresponding to its Antagonist affinities at , and K binding sites? This proved to be the case. DEX blocked (and reversed) the twitch inhibitions due to the Agonist NAL, nM DEX, AtM ,u-selective agonists LEV and DAGO as well as the K- LEV 4.0, 5.8 3.5 ± 1.3 (6) selective agonist U50,488. Moreover, consistent with its U50,488 17., 25. 18. ± 2. (6) higher affinity for ;L than for K binding sites, its potency was greater in blocking LEV (and in one experiment, DAGO) Preincubation (4 min) with NAL or DEX, followed by stepwise than in blocking U50,488. doubling ofagonist concentration. & values for NAL and "empirical loga- K1 values" for DEX (see text) were computed from shifts in agonist However, instead of the typical parallel shift of the logarithmic dose-response curves at IC30 according to the equation rithmic dose-response curve seen with reversible competi- K, = C/(DR - 1), where C is antagonist concentration and DR is tive antagonists, the agonist curve was shifted and flattened dose ratio for equal effect in the presence and absence of antagonist in the presence of DEX, with restriction of the agonist effect (7). At antagonist concentrations used here (20-200 nM NAL, 1-40 to about 50% inhibition. jM DEX), all DR values were >1.5. Data are from independent DEX did not block twitch inhibitions due to norepinephrine experiments or are means ± SEM with the number of experiments or atropine, a result consistent with specific interaction at in parentheses. opioid receptors. Furthermore, we demonstrated that NMDA receptors play no role here. Kosterlitz and Waterfield (8) showed long ago that LEV and DEX both inhibit cholinester- DISCUSSION ase and in the GPI block the inhibition due to morphine. We a reported here was to found that the cholinesterase inhibitor eserine produced The chief purpose of the experiments partial blockade of u and K agonists very similar to that by ascertain whether DEX, the (+)-enantiomer of the opioid DEX, in that there seemed to be a noncompetitive component. agonist LEV, could bind to opioid receptors, albeit with low The preponderance of evidence therefore suggests that affinity. Inasmuch as DEX is produced by resolution of the DEX agonism at high concentrations is not an opioid effect racemate, it was possible that the observed low-affinity but that its occupancy of opioid receptors at concentrations binding of DEX was due to trace contamination by LEV, in the range demonstrated to be effective in binding studies undetectable by chemical means. This kind ofartifact may be results in blockade and reversal of opioid agonists. The present whenever the affinity of one synthetic enantiomer is partially noncompetitive character of the blockade could be orders of magnitude lower than that ofthe other. We showed due, at least in part, to cholinesterase inhibition by DEX. any possible that repeated recrystallizations ofDEX reduced We thank Louise I. Lowney for technical assistance and Profes- LEV content by >200-fold without any change in affinities at sors H. W. Kosterlitz, A. Herz, and B. M. Cox for helpful comments ,&, 6, or K opioid binding sites. We concluded, therefore, that on the manuscript. This work was supported by Grant DA-1199 from DEX itself, despite its "wrong" chirality, does indeed bind the National Institute on Drug Abuse. to opioid receptors, with affinities about three orders of 1. Goldstein, A. & Naidu, A. (1989) Mol. Pharmacol. 36, 265-272. magnitude lower than LEV. 2. Gillan, M. G. C. & Kosterlitz, H. W. (1982) Br. J. Pharmacol. DEX has been regarded generally as inert, but a ligand that 77, 461-468. binds to a receptor at the same site as a known agonist, 3. Vonvoigtlander, P. F., Lahti, R. A. & Ludens, J. H. (1983) J. whatever the concentration required, must itself be an ago- Pharmacol. Exp. Ther. 224, 7-12. an Accordingly, we studied the effects of 4. Kosterlitz, H. W. & Waterfield, A. A. (1975) Annu. Rev. Phar- nist or antagonist. macol. Toxicol. 15, 29-48. DEX in the GPI, which contains functional ,u and K receptors. 5. Goldstein, A. & Schulz, R. (1973) Br. J. Pharmacol. 48,655-666. Increasing concentration caused no twitch inhibition until 6. Choi, D. W., Peters, S. & Viseskul, V. (1987) J. Pharmacol. about 50 jLM. Then a classical logarithmic dose-response Exp. Ther. 242, 713-720. curve was obtained, the same for crude and purified DEX, 7. Kosterlitz, H. W. & Watt, A. J. (1968) Br. J. Pharmacol. 33, nor blocked 266-276. but the twitch inhibition was neither reversed by 8. Kosterlitz, H. W. & Waterfield, A. A. (1975) Br. J. Pharmacol. NAL. A similar result was reported by Kachur et al. (9) with 53, 131-138. dextromethorphan. We concluded that this was not an opioid 9. Kachur, J. F., Morgan, D. W. & Gaginella, T. S. (1986) J. agonist effect. Pharmacol. Exp. Ther. 239, 661-667. Downloaded by guest on September 23, 2021