Roberto Cacciaglia

Anti-alcohol and anxiolytic properties of a new chemical entity, GET73

N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide (GET73) is a newly synthesized compound structurally related to the clinically used, alcohol-substituting agent, gamma-hydroxybutyric acid (GHB). The present study was designed to assess whether GET73 may share with GHB the capacity to reduce alcohol intake in rats. Additionally, the effect of treatment with GET73 on anxiety-related behaviors and cognitive tasks in rats was investigated. A series of in vitro binding assays investigated the capacity of GET73 to bind to the GHB binding site and multiple other receptors. GET73 (10−9–10−3u2009M) failed to inhibit [3H]GHB binding at both high- and low-affinity GHB recognition sites in rat cortical membranes. GET73 displayed minimal, if any, binding at dopamine, serotonin, GABA, and glutamate receptors in membranes from different rat brain areas. Acute treatment with low-to-moderate, non-sedative doses of GET73 (5–50u2009mg/kg, i.g. or i.p.) (a) reduced alcohol intake and suppressed “alcohol deprivation effect” (a model of alcohol relapse) in selectively bred, Sardinian alcohol-preferring (sP) rats, (b) exerted anxiolytic effects in Sprague-Dawley (SD) and sP rats exposed to the Elevated Plus Maze test, and (c) tended to induce promnestic effects in SD rats exposed to a modified water version of the Hebb–Williams maze test. Although the mechanism of GET73 action is currently unknown, the results of the present study suggest that GET73 has a multifaceted pharmacological profile, including the capacity to reduce alcohol drinking and anxiety-related behaviors in rats.

GET73 increases rat extracellular hippocampal CA1 GABA levels through a possible involvement of local mGlu5 receptor

N‐[(4‐trifluoromethyl) benzyl] 4‐methoxybutyramide (GET73) is a newly synthesized compound displaying anti‐alcohol and anxiolytic properties. In light of the importance of the hippocampal CA1 subregion in alcohol addiction and anxiety‐like behaviors—this in vivo microdialysis study characterized the effect of GET73 on extracellular GABA levels in the hippocampal CA1 region of the freely moving rat—including a possible role for mGlu5 receptor in mediating this effect. Both intraperitoneal administration (2–10 mg/kg) and local intra‐hippocampal CA1 perfusion with GET73 (50–1000 nM) were associated with a transient, step‐wise increase in dialysate hippocampal CA1 GABA levels. The GET73 (10 mg/kg)‐induced increase in GABA levels was not affected by intra‐CA1 perfusion with either the GABA reuptake inhibitor SKF89976A (0.5 mM) or by local GABAA (bicuculline; 1μM) and GABAB (CGP35348; 500 μM) receptor antagonists. On the contrary, the GET73‐induced increase in GABA levels was partially counteracted by the intra‐CA1 perfusion with the mGlu5 receptor negative allosteric modulator MPEP (300 µM). Interestingly, GET73 at the lowest (2 mg/kg) dose tested, by itself ineffective, fully counteracted the increase in GABA levels induced by the mGlu5 receptor agonist CHPG (1000 µM). Taken together, these findings suggest that the GET73‐induced increase in hippocampal CA1 GABA levels operates independently of local GABA reuptake and/or GABAA or GABAB receptors. Furthermore, the present data lead to hypothesize a possible interaction between GET73 and mGluR5‐mediated regulation of hippocampal CA1 GABA transmission, an effect which may be relevant to the ability of GET73 to reduce alcohol intake in an alcohol‐preferring rat strain. Synapse 67:678–691, 2013.

The New Compound GET73, N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide, Regulates Hippocampal Aminoacidergic Transmission Possibly Via an Allosteric Modulation of mGlu5 Receptor. Behavioural Evidence of its “Anti-Alcohol” and Anxiolytic Properties

The present article attempts to provide, on the basis of data emerging from studies carried out in our laboratories, a summary of the chemical and pharmacological properties of the new compound N-[(4-trifluoromethyl)benzyl]4- methoxybutyramide (GET73). Particular emphasis is given to findings obtained in vivo and in vitro suggesting that an allosteric modulation of metabotropic glutamate receptor 5 (mGlu5 receptor) by GET73 may represent the mechanism underlying the effects of the compound produced on rat hippocampal glutamate and GABA transmission. Furthermore, behavioural findings demonstrating how this new compound reduces alcohol intake, displays anxiolytic properties, and influences spatial memory in rats are also summarized. Since mGlu5 receptors play an important role in regulating several central actions of drugs of abuse, and the hippocampus is a crucial brain area involved in addiction, anxiety, and spatial memory, a possible link between mGlu5 receptor allosteric modulation and the profiles of action of GET73 is proposed, although to date no studies have yet explored GET73 binding at the mGlu5 receptor orthosteric and/or allosteric sites. Following a brief overview of glutamatergic neurotransmission, mGlu receptor structures and activation mechanisms, the general properties of mGlu5 receptor and its allosteric modulators are described in the first part of the review.

A Phase I randomized clinical trial testing the safety, tolerability and preliminary pharmacokinetics of the mGluR5 negative allosteric modulator GET 73 following single and repeated doses in healthy volunteers

&NA; Preclinical work suggests that the metabotropic glutamate receptor subtype 5 (mGlu5) may represent a novel target to treat neuropsychiatric disorders, including alcohol use disorder and obesity. The goal of this first‐in‐man study was to evaluate the safety, tolerability and pharmacokinetics (PK) of GET 73 (PubChem SID: 329974174), a novel mGluR5 negative allosteric modulator. This was a double‐blind, placebo‐controlled, ascending dose, Phase I study conducted in healthy male volunteers in two experiments. GET 73 was administered as single ascending doses (N = 48; Experiment 1; 10, 30, 100, 300, 450, 600‐mg) or multiple ascending doses (N = 32; Experiment 2; 100, 300, 450, 450‐mg twice a day). Primary endpoints were the incidence of adverse events (AEs) among drug conditions and drug tolerability. The secondary endpoints were the PK parameters of GET 73 and its metabolite MET 2. Single GET 73 doses of up to 600‐mg and repeated ascending doses of up to 450‐mg twice/day were safe and well‐tolerated. There were no serious or severe AEs. All AEs were mild or moderate in severity. Total GET 73 exposure increased with each increased GET 73 dose. A dose‐related increase in mean maximum plasma drug concentration was observed after repeated dosing. Maximum plasma drug concentrations occurred between 0.5 and 2.05 h after administration in all groups for both single and repeated doses. This first‐in‐human study indicates that GET 73, as single or multiple ascending doses, is safe and well‐tolerated when administered to healthy male volunteers. Graphical abstract Figure. No caption available.

GET73 Prevents Ethanol-Induced Neurotoxicity in Primary Cultures of Rat Hippocampal Neurons

AIMSnN-[(4-trifluoromethyl) benzyl] 4-methoxybutyramide (GET73) may be considered a promising therapeutic agent for the treatment of alcohol use disorders. The compound displayed anti-alcohol and anxiolytic properties in rat. In the present study, an in vitro experimental model of chronic ethanol treatment was used to investigate the ability of the compound to counteract the ethanol-induced neurotoxicity.nnnMETHODSnPrimary cultures of rat hippocampal neurons were exposed to ethanol (75 mM; 4 days) and the neuroprotective effects of GET73 were assessed by evaluating cell viability, cell morphology, glutamate levels and reactive oxygen species production.nnnRESULTSnThe exposure to ethanol induced a reduction of cell viability, an alteration of cytoskeleton, a decrease in extracellular glutamate levels and an increase of reactive oxygen species production. The addiction of GET73 (1 and 10 µM) 1 h before and during chronic ethanol exposure prevented all the above ethanol-induced effects. Based on the proposed GET73 mechanism of action, the effects of mGlu5 receptor negative allosteric modulator, 2-methyl-6-(phenylethynyl)-pyridine (MPEP), on ethanol-induced reduction of cell viability were also assessed. The results indicated that the addiction of MPEP (100 µM) 1 h before and during chronic ethanol exposure prevented the ethanol-induced cell viability reduction.nnnCONCLUSIONnThe present findings provide the first evidence that GET73 shows a neuroprotective role against ethanol-induced neurotoxicity in primary cultures of rat hippocampal neurons. Together with previous findings, these results suggest that GET73 possesses multifaceted properties thus lending further support to the significance of developing GET73 as a therapeutic tool for use in the treatment of alcohol use disorders.

Dataset for Phase I randomized clinical trial for safety and tolerability of GET 73 in single and repeated ascending doses including preliminary pharmacokinetic parameters

The data in this article outline the methods used for the administration of GET 73 in the first time-in-human manuscript entitled “Phase I randomized clinical trial for the safety, tolerability and preliminary pharmacokinetics of the mGluR5 negative allosteric modulator GET 73 following single and repeated doses in healthy male volunteers” (Haass-Koffler et al., 2017) [1]. Data sets are provided in two different manners. The first series of tables provided includes procedural information about the experiments conducted. The next series of tables provided includes Pharmacokinetic (PK) parameters for GET 73 and its main metabolite MET 2. This set of data is comprised by two experiments: Experiment 1 references a single ascending dose administration of GET 73 and Experiment 2 references a repeated ascending dose administration of GET 73.

Administration of the metabotropic glutamate receptor subtype 5 allosteric modulator GET 73 with alcohol: A translational study in rats and humans:

Preclinical work suggests that GET 73 (N-[4-(trifluoromethyl)benzyl]-4-methoxybutyramide), a novel metabotropic glutamate receptor subtype 5 negative allosteric modulator, may represent a novel pharmacological treatment for alcohol use disorder. Two independent experiments evaluated the effect of acutely administered GET 73 (0, 30, and 100 mg/kg, intragastrically) on alcohol-induced hypolocomotion (n=72) and sedation/hypnosis (n=36) in rats. In healthy male volunteers (n=14), an open-label, randomised, crossover study was conducted to compare adverse events and pharmacokinetic parameters, in two experiments in which 300 mg GET 73 was administered, with and without alcohol, once and thrice. In rats, when administered with alcohol-vehicle, 100 mg/kg, but not 30 mg/kg, GET 73 reduced spontaneous locomotor activity. When administered with alcohol, no dose of GET 73 altered either alcohol-induced hypolocomotion or sedation/hypnosis. In humans, both single and thrice 300 mg GET 73 administration were well tolerated, in the presence and absence of alcohol, with no differences in adverse events. There were no significant differences in relative bioavailability between administering 300 mg GET 73 in the presence or absence of alcohol.

In Vitro Functional Characterization of GET73 as Possible Negative Allosteric Modulator of Metabotropic Glutamate Receptor 5

The present study was aimed to further characterize the pharmacological profile of N-[4-(trifluoromethyl) benzyl]-4-methoxybutyramide (GET73), a putative negative allosteric modulator (NAM) of metabotropic glutamate subtype 5 receptor (mGluR5) under development as a novel medication for the treatment of alcohol dependence. This aim has been accomplished by means of a series of in vitro functional assays. These assays include the measure of several down-stream signaling [intracellular Ca++ levels, inositol phosphate (IP) formation and CREB phosphorylation (pCREB)] which are generally affected by mGluR5 ligands. In particular, GET73 (0.1 nM–10 μM) was explored for its ability to displace the concentration-response curve of some mGluR5 agonists/probes (glutamate, L-quisqualate, CHPG) in different native preparations. GET73 produced a rightward shift of concentration-response curves of glutamate- and CHPG-induced intracellular Ca++ levels in primary cultures of rat cortical astrocytes. The compound also induced a rightward shift of concentration response curve of glutamate- and L-quisqualate-induced increase in IP turnover in rat hippocampus slices, along with a reduction of CHPG (10 mM)-induced increase in IP formation. Moreover, GET73 produced a rightward shift of concentration-response curve of glutamate-, CHPG- and L-quisqualate-induced pCREB levels in rat cerebral cortex neurons. Although the engagement of other targets cannot be definitively ruled out, these data support the view that GET73 acts as an mGluR5 NAM and support the significance of further investigating the possible mechanism of action of the compound.