Design, Synthesis and Biological Evaluation of Aminoalkylindole Derivatives as Cannabinoid Receptor Ligands with Potential for Treatment of Alcohol Abuse

Attenuation of increased endocannabinoid signaling with a CB1R neutral antagonist might offer a new therapeutic direction for treatment of alcohol abuse. We have recently reported that a mono-hydroxylated metabolite of the synthetic aminoalkylindole cannabinoid JHW-073 (3) exhibits neutral antagonist activity at CB1Rs and thus may serve as a promising lead for the development of novel alcohol abuse therapies. In the current study, we show that systematic modification of an aminoalkylindole scaffold identified two new compounds with dual CB1R antagonist/CB2R agonist activity. Similar to the CB1R antagonist/inverse agonist rimonabant, analogues 27 and 30 decrease oral alcohol self-administration, without affecting total fluid intake and block the development of alcohol-conditioned place preference. Collectively, these initial findings suggest that design and systematic modification of aminoalkylindoles such as 3 may lead to development of novel cannabinoid ligands with dual CB1R antagonist/CB2R agonist activity with potential for use as treatments of alcohol abuse

Repeated administration of phytocannabinoid Δ9-THC or synthetic cannabinoids JWH-018 and JWH-073 induces tolerance to hypothermia but not locomotor suppression in mice, and reduces CB1 receptor expression and function in a brain region-specific manner

These studies probed the relationship between intrinsic efficacy and tolerance / cross-tolerance between Δ9-THC and synthetic cannabinoid drugs of abuse (SCBs) by examining in vivo effects and cellular changes concomitant with their repeated administration in mice. Dose-effect relationships for hypothermic effects were determined in order to confirm that SCBs JWH-018 and JWH-073 are higher efficacy agonists than Δ9-THC in mice. Separate groups of mice were treated with saline, sub-maximal hypothermic doses of JWH-018 or JWH-073 (3.0 mg/kg or 10.0 mg/kg, respectively) or a maximally hypothermic dose of 30.0 mg/kg Δ9-THC once per day for 5 consecutive days while core temperature and locomotor activity were monitored via biotelemetry. Repeated administration of all drugs resulted in tolerance to hypothermic effects, but not locomotor effects, and this tolerance was still evident 14 days after the last drug administration. Further studies treated mice with 30.0 mg/kg Δ9-THC once per day for 4 days, then tested with SCBs on day 5. Mice with a Δ9-THC history were cross-tolerant to both SCBs, and this cross-tolerance also persisted 14 days after testing. Select brain regions from chronically treated mice were examined for changes in CB1 receptor expression and function. Expression and function of hypothalamic CB1Rs were reduced in mice receiving chronic drugs, but cortical CB1R expression and function were not altered. Collectively, these data demonstrate that repeated Δ9-THC, JWH-018 and JWH-073 can induce long-lasting tolerance to some in vivo effects, which is likely mediated by region-specific downregulation and desensitization of CB1Rs

Atypical Pharmacodynamic Properties and Metabolic Profile of the Abused Synthetic Cannabinoid AB-PINACA: Potential Contribution to Pronounced Adverse Effects Relative to Δ9-THC

Recreational use of marijuana is associated with few adverse effects, but abuse of synthetic cannabinoids (SCBs) can result in anxiety, psychosis, chest pain, seizures and death. To potentially explain higher toxicity associated with SCB use, we hypothesized that AB-PINACA, a common second generation SCB, exhibits atypical pharmacodynamic properties at CB1 cannabinoid receptors (CB1Rs) and/or a distinct metabolic profile when compared to Δ9-tetrahydrocannabinol (Δ9-THC), the principal psychoactive cannabinoid present in marijuana. Liquid chromatography tandem mass spectrometry (LC/MS) identified AB-PINACA and monohydroxy metabolite(s) as primary phase I metabolites (4OH-AB-PINACA and/or 5OH-AB-PINACA) in human urine and serum obtained from forensic samples. In vitro experiments demonstrated that when compared to Δ9-THC, AB-PINACA exhibits similar affinity for CB1Rs, but greater efficacy for G-protein activation and higher potency for adenylyl cyclase inhibition. Chronic treatment with AB-PINACA also results in greater desensitization of CB1Rs (e.g., tolerance) than Δ9-THC. Importantly, monohydroxy metabolites of AB-PINACA retain affinity and full agonist activity at CB1Rs. Incubation of 4OH-AB-PINACA and 5OH-AB-PINACA with human liver microsomes (HLMs) results in limited glucuronide formation when compared to that of JWH-018-M2, a major monohydroxylated metabolite of the first generation SCB JWH-018. Finally, AB-PINACA and 4OH-AB-PINACA are active in vivo, producing CB1R-mediated hypothermia in mice. Taken collectively, the atypical pharmacodynamic properties of AB-PINACA at CB1Rs relative to Δ9-THC (e.g., higher potency/efficacy and greater production of desensitization), coupled with an unusual metabolic profile (e.g., production of metabolically stable active phase I metabolites) may contribute to the pronounced adverse effects observed with abuse of this SCB compared to marijuana

Tamoxifen Isomers and Metabolites Exhibit Distinct Affinity and Activity at Cannabinoid Receptors: Potential Scaffold for Drug Development.

Tamoxifen (Tam) is a selective estrogen receptor (ER) modulator (SERM) that is an essential drug to treat ER-positive breast cancer. Aside from known actions at ERs, recent studies have suggested that some SERMs like Tam also exhibit novel activity at cannabinoid subtype 1 and 2 receptors (CB1R and CB2Rs). Interestingly, cis- (E-Tam) and trans- (Z-Tam) isomers of Tam exhibit over a 100-fold difference in affinity for ERs. Therefore, the current study assessed individual isomers of Tam and subsequent cytochrome P450 metabolic products, 4-hydroxytamoxifen (4OHT) and 4-hydroxy-N-desmethyl tamoxifen (End) for affinity and activity at CBRs. Results showed that Z-4OHT, but not Z-Tam or Z-End, exhibits higher affinity for both CB1 and CB2Rs relative to the E-isomer. Furthermore, Z- and E-isomers of Tam and 4OHT show slightly higher affinity for CB2Rs, while both End isomers are relatively CB1R-selective. When functional activity was assessed by G-protein activation and regulation of the downstream effector adenylyl cyclase, all isomers examined act as full CB1 and CB2R inverse agonists. Interestingly, Z-Tam appears to be more efficacious than the full inverse agonist AM630 at CB2Rs, while both Z-Tam and Z-End exhibit characteristics of insurmountable antagonism at CB1 and CB2Rs, respectively. Collectively, these results suggest that the SERMs Tam, 4OHT and End elicit ER-independent actions via CBRs in an isomer-specific manner. As such, this novel structural scaffold might be used to develop therapeutically useful drugs for treatment of a variety of diseases mediated via CBRs

SERM isomers reduce basal G-protein activity via CB1 and CB2Rs.

<p>The ability of SERMs to modulate basal G-protein activity via [A] CB1R, [B] CB2R and [C] MORs was evaluated by examining [³⁵S]-GTPγS binding in the presence or absence of a receptor-saturating concentration (10 μM) of all compounds. G-protein modulation by full agonists CP-55,940 (10 μM) and DAMGO (10 μM) was examined to serve as positive controls for activation of [A-B] CBRs and [C] MORs, respectively. G-protein modulation by the inverse agonists AM-281 and AM-630 was examined to serve as positive controls for regulation of [A] CB1 and [B] CB2R signaling. The mean ± SEM of [³⁵S]GTPγS binding is presented as percent of G-protein activity in the presence of vehicle. ^a,b[³⁵S]GTPγS binding produced by individual SERMs acting at hCB1 [A], hCB2 [B] or hMOR [C] receptors designated by different letters above bars, is significantly different (P<0.05, one-way ANOVA; Tukey <i>Post-hoc</i> test). *,**Bar graphs comparing E and Z isomers of individual SERMs that are designated by asterisks, are significantly different from activity at respective receptors (P<0.05, 0.01; student’s t-test).</p

SERM isomers modulate forskolin-stimulated AC activation via G_i/o proteins and CB2Rs.

<p>[A-C] Modulation of forskolin-stimulated cAMP production by SERMs (10 μM) in intact CHO-hCB2 and CHO-hMOR cells was evaluated. Drugs were examined in CHO-hCB2 cells (+/- 100 ng PTX pretreatment) and in CHO-hMOR cells not expressing CBRs. Intracellular cAMP values (mean ± SEM) are presented as percent response compared to levels observed in the presence of vehicle. Statistics revealed that no drug altered basal cAMP levels in CHO-hCB2 cells treated with PTX (P<0.01; student’s <i>t</i>-test) or in CHO-hMOR cells (P<0.01; one-sample <i>t</i>-test).</p

Modulation of adenylyl cyclase activity by SERM isomers in CHO-hCB2 cells.

<p>Modulation of adenylyl cyclase activity by SERM isomers in CHO-hCB2 cells.</p

Competition binding of SERM isomers employing CHO-hCB1 and CHO-hCB2 membranes.

<p>Competition binding of SERM isomers employing CHO-hCB1 and CHO-hCB2 membranes.</p

SERM isomers exhibit mid-nanomolar to low-micromolar affinities for CB1 and CB2Rs.

<p>A measure of affinity (K_i) of E and Z isomers of Tam, 4OHT, and End for respective CB1 and CB2Rs was obtained by conducting competition binding studies, employing 0.2 nM [³H]-CP-55,940 and increasing concentrations of test compounds. K_i values (mean ± SEM) were derived from non-linear regression analysis of the curves shown in [A-C]. Individual K_i values and statistical analysis of pK_i values are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.t001" target="_blank">Table 1</a>. Filled squares and circles represent binding of respective E and Z isomers to CB1Rs, open squares and circles represent binding of respective E and Z isomers to CB2Rs.</p

Antagonism of CP-55,940 inhibition of forskolin-stimulated AC activity by SERM isomers in intact CHO-hCB1 cells.

<p>CHO-hCB1 cells were pre-incubated for 30 min with receptor saturating concentrations of individual SERMs and were subsequently co-incubated for 7 min with increasing concentrations of CP-55,940. Measurements of CP-55,940 effects alone on potency (IC₅₀) and efficacy (E_MAX) of intracellular cAMP were obtained and were compared to the shifts in IC₅₀ and E_MAX values observed in [A-D]. All IC₅₀, EC₅₀, and K_B values (mean ± SEM) were derived from non-linear regression analysis of the curves shown in [A-D] and are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167240#pone.0167240.t003" target="_blank">Table 3</a> with statistical analysis. Open squares represent the concentration-effect curve for CP-55,940 alone, while filled symbols represent the action of CP-55,940 in the presence of the SERM indicated [A-C] or the selective CB1R inverse agonist AM-281 [D].</p