Acute and chronic effects of the M1/M4-preferring muscarinic agonist xanomeline on cocaine vs. food choice in rats.

RATIONALE: We previously showed that the M1/M4-preferring muscarinic agonist xanomeline can acutely attenuate or eliminate cocaine self-administration in mice. OBJECTIVE: Medications used to treat addictions will arguably be administered in (sub)chronic or repeated regimens. Tests of acute effects often fail to predict chronic effects, highlighting the need for chronic testing of candidate medications. METHODS: Rats were trained to lever press under a concurrent FR5 FR5 schedule of intravenous cocaine and food reinforcement. Once baseline behavior stabilized, the effects of 7 days once-daily injections of xanomeline were evaluated. RESULTS: Xanomeline pretreatment dose-dependently (1.8-10 mg/kg/day) shifted the dose-effect curve for cocaine rightward (up to 5.6-fold increase in A 50), with reallocation of behavior to the food-reinforced lever. There was no indication of tolerance, rather effects grew over days. The suppression of cocaine choice appeared surmountable at high cocaine doses, and xanomeline treatment did not significantly decrease total-session cocaine or food intake. CONCLUSIONS: In terms of xanomeline's potential for promoting abstinence from cocaine in humans, the findings were mixed. Xanomeline did produce reallocation of behavior from cocaine to food with a robust increase in food reinforcers earned at some cocaine/xanomeline dose combinations. However, effects appeared surmountable, and food-maintained behavior was also decreased at some xanomeline/cocaine dose combinations, suggesting clinical usefulness may be limited. These data nevertheless support the notion that chronic muscarinic receptor stimulation can reduce cocaine self-administration. Future studies should show whether ligands with higher selectivity for M1 or M1/M4 subtypes would be less limited by undesired effects and can achieve higher efficacy.

Contribution of both M1 and M4 receptors to muscarinic agonist-mediated attenuation of the cocaine discriminative stimulus in mice.

RATIONALE: We previously showed that muscarinic agonists with M(1) and/or M(4) receptor affinities attenuated cocaine discrimination and self-administration in wild-type mice but not in M(1)/M(4) double-knockout mice. OBJECTIVE: This study aims to elucidate the respective contributions of M(1) and M(4) receptors to this effect. METHODS: Knockout mice lacking either the M(1) subtype (M (1) (-/-) ) or the M(4) subtype (M (4) (-/-) ) and wild-type mice were trained to discriminate 10 mg/kg cocaine from saline. Muscarinic ligands were tested for modulation of cocaine discrimination: xanomeline (M(1)/M(4)-preferring agonist), VU0357017 (M(1)-selective partial agonist), 77-LH-28-1 (M(1) agonist), and BQCA (M(1)-selective positive allosteric modulator). RESULTS: Xanomeline produced rightward shifts in the cocaine dose-effect curve in all three genotypes, but most robustly in wild-type mice. VU0357017 produced rightward shifts in the cocaine dose-effect curve in wild-type and M (4) (-/-) mice, but not in M (1) (-/-) mice. Response rates were suppressed by xanomeline in wild-type and M (1) (-/-) but not in M (4) (-/-) mice and were unaltered by VU0357017. 77-LH-28-1 and BQCA also showed evidence of attenuating cocaine's discriminative stimulus, but at doses that suppressed responding or had other undesirable effects. Intriguingly, both VU0357017 and 77-LH-28-1 exhibited U-shaped dose-effect functions in attenuating cocaine discrimination. None of the drugs substituted for the cocaine stimulus. CONCLUSIONS: Attenuation of the cocaine stimulus by VU0357017 depended upon M(1) receptors, and full effects of xanomeline depended upon both M(1) and M(4) receptors. Therefore M(1)-selective agonists and mixed M(1)/M(4) agonists may be promising leads for developing medications that block cocaine's effects.

Effect of linker substitution on the binding of butorphan univalent and bivalent ligands to opioid receptors.

A series of bivalent hydroxy ether butorphan ligands were prepared and their binding affinities at the opioid receptors determined. Addition of a hydroxy group to a hydrocarbon chain can potentiate binding affinity up to 27- and 86-fold at the mu and kappa opioid receptors, respectively. Two bivalent ligands with sub-nanomolar binding affinity at the mu and kappa opioid receptors were discovered.

Modulation of prepulse inhibition through both M(1) and M (4) muscarinic receptors in mice.

RATIONALE: Muscarinic cholinergic M(1) and M(4) receptors may participate in schizophrenia's etiology and have been proposed as targets for antipsychotic medications. OBJECTIVE: Here, we investigated the involvement of these receptors in behavioral measures pertinent to schizophrenia using knockout mice lacking M(1) receptors (M(1)-/-), M(4) receptors (M(4)-/-), or both (M(1)-/-M(4)-/-). METHODS: We measured prepulse inhibition (PPI) of startle without drugs and after treatment with scopolamine (0.32-1.8 mg/kg), xanomeline (3.2 mg/kg), oxotremorine (0.032-0.1 mg/kg), clozapine (1.0-5.6 mg/kg), or haloperidol (0.32-3.2 mg/kg). RESULTS: In female (but not male) mice, combined deletion of both M(1) and M(4) receptors decreased PPI relative to wild-type mice, while knockout of either receptor alone had no significant effect. Scopolamine disrupted PPI in wild-type and M(4)-/- mice, but not in female M(1)-/-M(4)-/- or female M(1)-/- mice. When administered before scopolamine, xanomeline restored PPI in wild-type mice and M(1)-/- mice, but not in M(4)-/- mice. In contrast, pretreatment with oxotremorine increased PPI regardless of genotype. Effects of clozapine and haloperidol on PPI were not hindered by either mutation. CONCLUSIONS: Deletion of both M(1) and M(4) receptors can disrupt PPI, suggesting that (at least partially redundant) M(1) and M(4) receptor-dependent functions are involved in sensorimotor gating mechanisms. PPI-disrupting effects of muscarinic antagonists appeared dependent upon M(1) receptor blockade. Our data also suggest that xanomeline exerts antipsychotic-like effects mainly through M(4) receptor stimulation, while stimulation of non-M(1)/M(4) subtypes may also have antipsychotic potential. Finally, our results do not support a role of M(1)/M(4) receptors in mediating antipsychotic-like effects of clozapine.

Attenuation of cocaine's reinforcing and discriminative stimulus effects via muscarinic M1 acetylcholine receptor stimulation.

Muscarinic cholinergic receptors modulate dopaminergic function in brain pathways thought to mediate cocaine's abuse-related effects. Here, we sought to confirm and extend in the mouse species findings that nonselective muscarinic receptor antagonists can enhance cocaine's discriminative stimulus. More importantly, we tested the hypothesis that muscarinic receptor agonists with varied receptor subtype selectivity can blunt cocaine's discriminative stimulus and reinforcing effects; we hypothesized a critical role for the M(1) and/or M(4) receptor subtypes in this modulation. Mice were trained to discriminate cocaine from saline, or to self-administer intravenous cocaine chronically. The nonselective muscarinic antagonists scopolamine and methylscopolamine, the nonselective muscarinic agonists oxotremorine and pilocarpine, the M(1)/M(4)-preferring agonist xanomeline, the putative M(1)-selective agonist (4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride (McN-A-343), and the novel M(1)-selective agonist 1-(1-2-methylbenzyl)-1,4-bipiperidin-4-yl)-1H benzo[d]imidazol-2(3H)-one (TBPB) were tested as substitution and/or pretreatment to cocaine. Both muscarinic antagonists partially substituted for cocaine and enhanced its discriminative stimulus. Conversely, muscarinic agonists blunted cocaine discrimination and abolished cocaine self-administration with varying effects on food-maintained behavior. Specifically, increasing selectivity for the M(1) subtype (oxotremorine < xanomeline < TBPB) conferred lesser nonspecific rate-suppressing effects, with no rate suppression for TBPB. In mutant mice lacking M(1) and M(4) receptors, xanomeline failed to diminish cocaine discrimination while rate-decreasing effects were intact. Our data suggest that central M(1) receptor activation attenuates cocaine's abuse-related effects, whereas non-M(1)/M(4) receptors probably contribute to undesirable effects of muscarinic stimulation. These data provide the first demonstration of anticocaine effects of systemically applied, M(1) receptor agonists and suggest the possibility of a new approach to pharmacotherapy for cocaine addiction.

Univalent and bivalent ligands of butorphan: characteristics of the linking chain determine the affinity and potency of such opioid ligands.

Bivalent morphinan compounds containing ester linkers were synthesized and their binding affinities at the mu, delta, and kappa opioid receptors determined. Addition of methyl groups adjacent to the hydrolytically labile ester linkage increased stability while only partially affecting binding affinity. The resulting bivalent ligands with optimized spacer length and structure show potent binding profiles with the most potent compound (4b), having K(i) values of 0.47 nM for both the mu and kappa opioid receptors, and 4a, having K(i) values of 0.95 and 0.62 nM for the mu and kappa receptors, respectively. Both 4a and 4b were partial agonists at the kappa and micro receptors in the [(35)S]GTPgammaS binding assay.

Synthesis and pharmacological evaluation of hydrophobic esters and ethers of butorphanol at opioid receptors.

We synthesized several hydrophobic esters and ethers of butorphanol and assessed their affinities at opioid receptors in CHO cell membranes. Tested compounds displayed moderate to high affinities to the mu and kappa receptors. The findings accord with previous evidence of a lipophilic binding pocket in the opioid receptors that can be accessed to afford good binding affinity without the need for a phenolic hydrogen-bond donor group. The most potent (K(i)=61 pM at mu and 48 pM at kappa) novel agent was (-)-N-cyclobutylmethylmorphinan-3-yl-14-ol phenoxyacetate (4d).

In vivo characterization of (-)(-)MCL-144 and (+)(-)MCL-193: isomeric, bivalent ligands with mu/kappa agonist properties.

Once opioid receptor dimers were postulated, a goal has been to synthesize and screen novel opioids, with the hope of furthering our knowledge of the structure-activity relationship of opioid ligands with the opioid receptors. The aim of the current study was to address whether two isomeric bivalent ligands would have pharmacological differences after central administration, in vivo. The two compounds, (-) bis(N-cyclobutylmethyl-morphinan-3-yl) sebacoylate dihydrochloride (MCL-144) and 1-((+)N-cyclobutylmethylmorphinan-3-yl)-10-((-) N-cyclobutylmethylmorphinan-3-yl)sebacolyate (MCL-193) are each linked by a 10-carbon chain ester. The active (-) enantiomer for both ligands is 3-hydroxy-N-cyclobutylmethyl morphinan ((-)MCL-101), a N-cyclobutylmethyl analogue of cyclorphan (J Med Chem 43:114-122, 2000). MCL-144 contains two active levo rotatory (-)(-) pharmacophores, while MCL-193 contains one active (-) and one inactive (+) pharmacophore of MCL-101. In vitro analysis demonstrated that all three compounds, (-)(-)MCL-144, (+)(-)MCL-193 and (-)MCL-101 were kappa agonists and mu partial agonists. (-)(-)MCL-144 and (-)MCL-101 had much higher affinity for both the mu and kappa opioid receptors compared to (+)(-)MCL-193. In vivo, (-)(-)MCL-144 and (+)(-)MCL-193 produced full dose-response curves, in the 55 degrees C tail-flick test, with each compound having an ED(50) value of 3.0 nmol after intracerebroventricular (i.c.v.) administration. The analgesic properties of both compounds were antagonized by the mu-selective antagonist, beta-funaltrexamine and the kappa-selective antagonist nor-binaltorphimine. Concomitant, i.c.v., administration of either (-)(-)MCL-144 or (+)(-)MCL-193 with morphine, did not significantly antagonize morphine-induced antinociception at any dose tested. In antinociceptive tests, (-)(-)MCL-144 and (+)(-)MCL-193 had the same pharmacological properties, demonstrating that having two active pharmacophores separated by a 10-carbon spacer group did not increase the antinociceptive efficacy of the compound. Additionally, it was also of interest to compare (-)(-)MCL-145 and (-)(-)MCL-144, as the only difference between these bivalent ligands is the spacer region connecting the two pharmacophores, yet (-)(-)MCL-145 produced an ED(50) value 10-fold lower than (-)(-)MCL-144 (ED(50) values = 0.3 nmol and 3.0 nmol, respectively).