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BEHAVIORAL PHARMACOLOGY

Characterization of a Novel Bivalent Morphinan Possessing Agonist and µ Agonist/Antagonist Properties

Department of Pharmacology and Physiology, University of Rochester School of Medicine and Dentistry, Rochester, New York (J.L.M., J.M.B.); and Alcohol and Drug Abuse Research Center, McLean Hospital, Harvard Medical School, Belmont, Massachusetts (X.P., W.X., A.Z., S.S.N., J.L.N.)

Received March 1, 2005; accepted July 28, 2005.

	Abstract

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Previous research has shown that compounds with mixed and µ activity may have utility for the treatment of cocaine abuse and dependence. The present study characterizes the pharmacological profile of a bivalent morphinan that was shown to be a opioid receptor agonist and a µ opioid receptor agonist/antagonist. MCL-145 [bis(N-cyclobutylmethylmorphinan) fumarate] is related to the morphinan cyclorphan and its N-cyclobutylmethyl derivative MCL-101 [3-hydroxy-N-cyclobutylmethyl morphinan S-(+)-mandelate]. MCL-145 consists of two morphinans connected by a spacer at the 3-hydroxy position. This compound had K_i values of 0.078 and 0.20 nM for the and µ opioid receptors, respectively, using radioligand binding assays as shown by Neumeyer et al. in 2003. In the guanosine 5'-O -(3-[³⁵ S]thiotriphosphate) binding assay, MCL-145 produced an E_max value of 80% for the opioid receptor and 42% for the µ opioid receptor. The EC₅₀ values obtained for this compound were 4.3 and 3.1 nM for the and µ opioid receptors, respectively. In vivo MCL-145 produced a full dose-response curve in the 55°C warm water tail-flick test and was equipotent to morphine. The agonist properties of MCL-145 were antagonized by the µ-selective antagonist -funaltrexamine and the -selective antagonist nor-binaltorphimine. MCL-145 also acted as a µ antagonist, as measured by the inhibition of morphine-induced antinociception.

Early research in both nonhuman primates and rats showed that agonists could functionally antagonize many cocaine-induced behaviors, including hyperactivity (Ukai et al., 1994; Crawford et al., 1995), place preference (Suzuki et al., 1992; Crawford et al., 1995; Shippenberg et al., 1996), self-administration (Glick et al., 1995; Negus et al., 1997; Mello and Negus, 1998), and sensitization to hyperactivity and stereotypies (Shippenberg et al., 1996). Administration of agonists also attenuated the reinstatement of extinguished drug-taking behavior, in an animal model of relapse (Schenk et al., 1999, 2000). Although highly selective agonists did have utility for attenuating many cocaine-induced behaviors, these selective agonists produced many severe undesirable side effects. The highly efficacious and selective agonists enadoline, U50,488, and spiradoline produced salivation, emesis, and sedation in nonhuman primates (Negus et al., 1997; Mello and Negus 1998).

Bowen et al. (2003) recently reported on a series of compounds with mixed and µ activity. Included in the report are the morphinans MCL-101 and cyclorphan and the benzomorphan Mr2034. These compounds decreased cocaine self-administration in rhesus monkeys and produced fewer side effects than the -selective agonist enadoline (Bowen et al., 2003). It was also noted that compounds with mixed agonist and µ agonist activity produced a more sustained decrease in cocaine self-administration than compounds with agonist and µ antagonist properties (Bowen et al., 2003).

Our current data focused on a new compound, MCL-145, which was a mixed agonist and µ agonist/antagonist. MCL-145 is a novel bivalent ligand related to the morphinan (-)-cyclorphan and its N-cyclobutylmethyl derivative MCL-101. This compound had high affinity for both the and µ opioid receptors. The present study characterizes the pharmacological properties of MCL-145 in the [³⁵S]GTPS binding assay and in mouse antinociceptive tests.

	Materials and Methods

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In Vitro Studies
[³⁵S]GTPS Binding Studies to Measure Opioid Receptor Coupling to G Proteins. Membranes from Chinese hamster ovary cells stably expressing either the human (L. Toll, Stanford Research Institute, Palo Alto, CA) or µ (G. Uhl, National Institute on Drug Abuse, Baltimore, MD) opioid receptor were used in the experiments. Native CHO membranes were not transfected with any of the human opioid receptors, but membrane preparations were prepared in the same manner. Cells were scraped from tissue culture plates and were centrifuged at 200g for 10 min at 4°C. The cells were resuspended in phosphate-buffered saline, pH 7.4, containing 0.04% EDTA. After centrifugation at 200g for 10 min at 4°C, the pellet was resuspended in membrane buffer, which consisted of 50 mM Tris-HCl, 3 mM MgCl₂, and 1 mM EGTA, pH 7.4. The membranes were homogenized with a Dounce homogenizer, followed by centrifugation at 39,000g for 20 min at 4°C. The membrane pellet was resuspended in membrane buffer, and the centrifugation step was repeated. The membranes were resuspended in assay buffer, which consisted of 50 mM Tris-HCl, 3 mM MgCl₂, 100 mM NaCl, and 0.2 mM EGTA, pH 7.4.

Either the hKOR-CHO (15 µg of protein/tube), hMOR-CHO (10 µg of protein/tube), or native CHO (15 µg of protein/tube) membranes were incubated with 12 different concentrations of the agonist in assay buffer for 60 min at 30°C in a final volume of 0.5 ml. The reaction mixture contained 3 µM GDP and 0.080 nM [³⁵S]GTPS. Basal activity was determined in the presence of 3 µM GDP and in the absence of agonist, and nonspecific binding was determined in the presence of 10 µM unlabeled GTPS. After the 60-min incubation, the membranes were filtered onto 32 glass fiber filters (What-man Schleicher and Schuell, Keene, NH) by vacuum filtration, followed by three washes with 3 ml of ice-cold 50 mM Tris-HCl, pH 7.5. Samples were counted in 2 ml of Ecoscint A scintillation fluid (National Diagnostics, Atlanta, GA). Data are presented as the percentage of agonist stimulation of [³⁵S]GTPS binding over the basal activity, defined as [(specific binding/basal binding) x 100] - 100. All experiments were repeated three times and performed in triplicate.

In Vivo Studies
Animals. Male ICR mice (20–30 g) (Harlan, Indianapolis, IN) were housed in groups of five with food and water available ad libitum before any procedures. Animals were maintained on a 12-h light/dark cycle in a temperature-controlled animal colony. Studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted by the National Institutes of Health.

Chemicals. MCL-145 and MCL-101 were synthesized as described previously (Neumeyer et al., 2000, 2003). MCL-145 and MCL-101 were initially solubilized in dimethyl sulfoxide, and all subsequent dilutions were performed in distilled water. Morphine sulfate was purchased from Mallinckrodt (St. Louis, MO). ICI 174,864, -FNA, and nor-BNI were purchased from Sigma-Aldrich (St. Louis, MO).

Injections. Intracerebroventricular (i.c.v.) injections were performed as described previously (Porreca et al., 1984). Briefly, mice were lightly anesthetized with ether, and an incision was made in the scalp. An injection was made with a 10-µl Hamilton syringe at a point 2 mm caudal and 2 mm lateral from bregma. Compounds were injected at a depth of 3 mm in a volume of 5 µl.

Antinociceptive Testing. Antinociception was assessed using the 55°C warm-water tail-flick test or the acetic acid writhing test. For the tail-flick test, the latency to the first sign of a rapid tail-flick was taken as the behavioral endpoint (Jannsen et al., 1963). Each mouse was first tested for baseline latency by immersing its tail in the water and recording the time to response. Mice not responding within 5 s were excluded from further testing. Mice were then administered the test compound and tested for antinociception 20 min after the injection. A maximum score was assigned (100%) to animals not responding within 15 s to avoid tissue damage. Antinociception was calculated by the following formula: % antinociception = 100 x (test latency - control latency)/(15 - control latency). For the acetic acid writhing test, mice were injected i.p. with 0.6% acetic acid in a volume of 10 ml/kg body weight. They were then placed in a clear Plexiglas observation jar and the number of abdominal writhes recorded for 15 min (Mogil et al., 1999). Percentage of antinociception was calculated using the formula % antinociception = 100 - [(no. writhes individual mouse/mean no. writhes control group) x 100].

Agonist Effects of MCL-145. To further determine the in vivo opioid receptor profile of MCL-145, mice were pretreated with a µ-(-FNA, 20 nmol i.c.v., -24 h), -(ICI-174,864, 4 nmol i.c.v., -20 min), or (nor-BNI, 3 nmol i.c.v., -24 h)-selective antagonist. Control mice received a vehicle injection (5 µl of distilled water, i.c.v., -24 h or -20 min). Then, mice received MCL-145 (5 nmol i.c.v.). Antinociception was assessed 20 min after agonist injection.

Antagonist Effects of MCL-145. Mice were treated concomitantly with morphine sulfate (3 nmol i.c.v.) and varying doses of MCL-145 i.c.v. Control mice received a vehicle injection (5 µl of distilled water i.c.v., -20 min). Antinociception was assessed 20 min after agonist injection.

Statistical Analysis. IC₅₀ values were calculated by least-squares fit to a logarithm-probit analysis. All dose-response lines were analyzed using the regression methods described by Tallarida and Murray (1986). Regression lines, ED₅₀ (dose producing 50% antinociception) values, and 95% CL were determined (Tallarida and Murray, 1986). All data points shown are the mean of seven to 10 mice, with standard error of the mean represented by error bars. Statistical analysis of the [³⁵S]GTPS binding data and the antinociceptive data used the Student's t test.

	Results

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In Vitro Studies
Affinity, Selectivity, and Efficacy of MCL-145. The binding of the novel bivalent morphinan MCL-145 (Fig. 1) to all the opioid receptors was measured. MCL-145 had a K_i value of less than 0.08 nM for inhibiting the binding of [³H]U69,593 to the receptor in CHO membranes stably transfected with the human opioid receptor. MCL-145 had a 2- and 120-fold lower affinity for the µ and receptors, respectively (Table 1) (Neumeyer et al., 2003).

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical structures of the bivalent ligand MCL-145 and the monomeric parent compound MCL-101.

View this table: [in this window] [in a new window]   TABLE 1 Ki inhibition values of µ-, -, and -opioid binding to CHO membranes by MCL compounds Membranes were incubated with varying concentrations of the compounds in the presence of either 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole, or 1 nM [3H]U69,593 to measure binding to µ, , and sites, respectively. After equilibrium binding was reached, membranes were filtered onto glass fiber filters. Data are expressed as the mean Ki value ± S.E.M. for three experiments performed in triplicate (table from Neumeyer et al., 2003).

To characterize the efficacy of MCL-145, the [³⁵S]GTPS binding assay was used. CHO membranes stably transfected with the human µ and opioid receptors were used for this assay. The native CHO membranes were not transfected with any of the human opioid receptors. MCL-145 produced an E_max value, percentage of maximal stimulation, of 80 ± 2% for the opioid receptor (Fig. 2) and 42 ± 14% for the µ opioid receptor (Fig. 2). The EC₅₀ values obtained for this compound were 4.3 ± 0.5 and 3.1 ± 0.3 nM for the and µ opioid receptors, respectively. The well characterized agonist U69,593 produced an E_max value of 77 ± 11% with an EC₅₀ value of 36 ± 5 nM. The µ-selective agonist [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin produced an E_max value of 116 ± 4% with an EC₅₀ value of 55 ± 7 nM.

View larger version (22K): [in this window] [in a new window]   Fig. 2. [35S]GTPS binding data for MCL-145. (), titration of MCL-145 in the [35S]GTPS binding assay using hKOR-CHO membranes. , titration of MCL-145 in the [35S]GTPS binding assay using hMOR-CHO membranes. , titration of MCL-145 in the [35S]GTPS binding assay using native CHO membranes.

MCL-101 (Fig. 1) is the monomeric parent compound of MCL-145 and produced similar maximal stimulation of [³⁵S]GTPS binding. Interestingly, MCL-101 showed no antagonism, even at higher concentrations. MCL-101 produced E_max values of 64 ± 13 and 102 ± 8% for the µ and receptors (Fig. 3). The EC₅₀ value for the stimulation of [³⁵S]GTPS binding mediated by the and µ receptor was 10 ± 2.7 and 31 ± 8.2 nM, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 3. [35S]GTPS binding data for MCL-101. , titration of MCL-101 in the [35S]GTPS binding assay to hKOR-CHO membranes. , titration of MCL-101 in the [35S]GTPS binding assay to hMOR-CHO membranes.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Dose-response curves for i.c.v. MCL-145, morphine, and MCL-101 in the 55°C warm-water tail-flick test. Antinociception was assessed 20 min after agonist injection.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Time-response curves for i.c.v. () MCL-145 and () MCL-101. Antinociception was tested in the 55°C tail-flick test 20 min after agonist injection.

View larger version (12K): [in this window] [in a new window]   Fig. 6. Agonist properties of MCL-145 at the µ, , and receptors. Antinociceptive responses in the 55°C tail-flick test in mice pretreated with i.c.v. µ-, -, or -selective opioid antagonists. Pretreatment time for nor-BNI and -FNA was -24 h. ICI 174,864 pretreatment time was -20 min before agonist injection. Antinociceptive testing was done 20 min after MCL-145 injection. **, P < 0.01 in comparison with MCL-145 alone.

Antagonist Properties of MCL-145. To assess the antagonist activity of MCL-145 at the µ receptor, morphine and MCL-145 were administered concomitantly, and antinociception was assessed 20 min after the injection. Figure 7 shows that a low dose, 0.05 nmol, of MCL-145 antagonized morphine-induced antinociception. These data demonstrated that MCL-145 acted as a µ antagonist at low doses. In vivo, MCL-101 demonstrated no antagonist activity when administered with morphine (Neumeyer et al., 2000). Likewise, MCL-101 did not exhibit antagonism in the [³⁵S]GTPS binding assay.

View larger version (12K): [in this window] [in a new window]   Fig. 7. MCL-145 antagonism of morphine-induced antinociception. Mice were injected concomitantly with varying doses of morphine i.c.v. and 0.05 nmol of MCL-145. Antinociception was assessed 20 min after agonist injection in the 55°C tail-flick test. *, P < 0.05 and **, P < 0.01 in comparison with morphine dose alone.

View larger version (12K): [in this window] [in a new window]   Fig. 8. MCL-145 did not antagonize U50,488 antinociception in vivo. Mice were injected i.c.v. with 30 nmol of U50,488 and 0.05 nmol of MCL-145. Antinociception was assessed 20 min after the injection in the acetic acid writhing assay. Pretreatment (-24 h) with the -selective antagonist nor-BNI resulted in significant antagonism of U50,488 antinociception. **, P < 0.01 compared with U50,488 alone.

	Discussion

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Several studies using nonhuman primates have demonstrated the efficacy of using a agonist and a µ antagonist to attenuate many cocaine-induced behaviors. The advantage of using a compound with activity at both receptors is that a reduction in side effects is seen, compared with a highly selective agonist (Negus et al., 1997; Mello and Negus, 1998; Bowen et al., 2003). Of particular interest was the previous data obtained with MCL-101. The study demonstrated that MCL-101 dose dependently produced a sustained decrease in cocaine self-administration with minimal side effects (Bowen et al., 2003). The promising results obtained in vivo with MCL-101 prompted continuing development of novel ligands which would bind the µ and receptors (Neumeyer et al., 2003).

MCL-145 is related to the morphinan cyclorphan and its N-cyclobutylmethyl derivative MCL-101. The novel bivalent ligand MCL-145 was synthesized by coupling two identical pharmacophores of MCL-101 with a connecting spacer at the 3-hydroxy position (Neumeyer et al., 2003). Previous data obtained both in vitro and in vivo with MCL-101 demonstrated that this compound had high affinity at both µ and receptors (Neumeyer et al., 2000). One question of interest in synthesizing MCL-145 was whether the bivalent ligand would have altered pharmacology or advantages compared with the monovalent parent compound.

MCL-145 demonstrated interesting pharmacological properties. In the [³⁵S]GTPS binding assay, which is a measure of G protein activation, the compound produced dose-dependent agonist and antagonist stimulation for the µ and receptors. However, high concentrations of MCL-145 also inhibited [³⁵S]GTPS binding below basal in native CHO membranes. This would indicate that much of the apparent antagonism and basal inhibition of [³⁵S]GTPS binding was not receptor mediated and was perhaps a direct effect on the G protein, or may be a result of nonspecific tissue binding. In vivo, MCL-145 acted as a full agonist at the receptor, whereas it had mixed agonist/antagonist activity at the µ receptor. The bivalent structure of MCL-145 significantly changed its pharmacological characteristics in comparison with the monovalent parent compound MCL-101. MCL-101 showed no antagonism in the [³⁵S]GTPS binding assay, but it did produce similar E_max values. In vivo, MCL-101 acted as an agonist at both the µ and receptors, but it showed no antagonist properties (Neumeyer et al., 2000). It seems that the bivalent nature of MCL-145 allows it to interact in a different manner with both the µ and receptors.

There are no data to indicate the exact mechanism of how MCL-145 is interacting with the receptor. It is possible that one pharmacophore of the ligand is binding the receptor and that the spacer and adjacent pharmacophore are left free extracellularly. Alternatively, the compound may be metabolized to monomers, each with the ability to bind receptor. However, it seems unlikely that these monomers are MCL-101, based on the in vitro and in vivo data obtained with MCL-145. If MCL-145 is being metabolized to monomers, it is also possible that one of the monomeric pharmacophores is left with a portion of the spacer region. In this configuration, it is plausible that the activity of the ligand would be different from that of MCL-101. It is also not unreasonable to postulate that MCL-145 is capable of binding and bridging two receptors. There is evidence to suggest that G protein-coupled receptors, and in particular opioid receptors, are capable of both homo- and heterodimerization. In the endogenous state, the µ and receptors may not be complexed, but with the addition of a bivalent ligand, such as MCL-145, they could be capable of forming dimers. The concept of G protein-coupled receptor dimerization is still a relatively new field of study with many interesting implications. The role of opioid receptor dimers is now being considered in processes such as tolerance (He et al., 2002). It is intriguing to consider the possibility that such heterodimers, particularly -µ, could also play a role in addiction.

In summary, MCL-145 is a relatively short-acting, bivalent agonist and µ agonist/antagonist. Future studies will assess the ability of MCL-145 to attenuate morphine tolerance and dependence.

	Footnotes

 
This work was supported by Grants K05-DA00360 and T32 DA07232 (to J.M.B.) and DA014251 (to J.L.N.) from the National Institute on Drug Abuse. Part of this work was presented as a meeting abstract: Mathews JL, Xiong W, Gu X-H, Neumeyer JL, and Bidlack JM (2003) In vivo characterization of MCL-145: a morphinan derivative possessing mixed kappa agonist and mu agonist/antagonist properties, in Abstracts of 34th International Narcotics Research Conference (no. 153), Perpignan, France, July 6–11, 2003; available at http://www.inrcworld.org.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.084343.

ABBREVIATIONS: U50,488, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methane-sulfonate hydrate; MCL-101, 3-hydroxy-N-cyclobutylmethyl morphinan S-(+)-mandelate; Mr2034, (-)-(1R,5R,9R,2'S)-5,9-dimethyl-2'-hydroxy-2-tetrahydrofurfuryl-6,7-benzomorphan-D-tartrate; MCL-145, bis(N-cyclobutylmethylmorphinan) fumarate; GTPS, guanosine-5'-O-(3-thio)triphosphate; CHO, Chinese hamster ovary; hKOR, human opioid receptor; hMOR, human µ opioid receptor; ICI 174,864, N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (where Aib is -aminoisobutyric acid); -FNA, -funaltrexamine; nor-BNI, nor-binaltorphimine; CL, confidence limits; U69,593, (5,7,8)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl) benzeneacetamide.

Address correspondence to: Dr. Jean M. Bidlack, Department of Pharmacology and Physiology, Box 711, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642-8711. E-mail: jean_bidlack{at}urmc.rochester.edu

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