Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

Cellular and behavioral interactions of gabapentin with alcohol dependence.

Gabapentin is a structural analog of GABA that has anticonvulsant properties. Despite the therapeutic efficacy of gabapentin, its molecular and cellular mechanisms of action are unclear. The GABAergic system in the central nucleus of the amygdala (CeA) plays an important role in regulating voluntary ethanol intake. Here, we investigated the effect of gabapentin on GABAergic transmission in CeA slices, on ethanol intake, and on an anxiety measure using animal models of ethanol dependence. Gabapentin increased the amplitudes of evoked GABA receptor-mediated IPSCs (GABA-IPSCs) in CeA neurons from nondependent rats, but decreased their amplitudes in CeA of ethanol-dependent rats. Gabapentin effects were blocked in the presence of a specific GABA(B) receptor antagonist. The sensitivity of the GABA-IPSCs to a GABA(B) receptor antagonist and an agonist was decreased after chronic ethanol, suggesting that ethanol-induced neuroadaptations of GABA(B) receptors associated with ethanol dependence may account for the differential effects of gabapentin after chronic ethanol. Systemic gabapentin reduced ethanol intake in dependent, but not in nondependent, rats and reversed the anxiogenic-like effects of ethanol abstinence using an acute dependence model. Gabapentin infused directly into the CeA also blocked dependence-induced elevation in operant ethanol responding. Collectively, these findings show that gabapentin reverses behavioral measures of ethanol dependence and, in turn, dependence reverses the effects of gabapentin on CeA neurons, and suggest that gabapentin represents a potential medication for treatment of alcoholism.

Dose- and time-dependent expression of anxiety-like behavior in the elevated plus-maze during withdrawal from acute and repeated intermittent ethanol intoxication in rats.

BACKGROUND: Withdrawal from acute bolus intraperitoneal (IP) injection of high doses of ethanol elicits anxiety-like behavior (e.g. Doremus et al., 2003; Gauvin et al., 1989, 1992) and conditioned place aversion (Morse et al., 2000). More recently we demonstrated that withdrawal from a single moderate dose of ethanol (2.0 g/kg) is accompanied by elevations in brain reward thresholds, and that repeated intermittent treatment with this dose results in a significant potentiation of reward deficit (Schulteis and Liu, 2006). METHODS: In the current study, the time- and dose-dependent emergence of anxiety-like behavior was measured in the elevated plus-maze at various times (3 to 24 hours) after acute or 3 daily IP injections of ethanol (1.0, 2.0, or 3.0 g/kg). Rats receiving daily handling for 2 days, and a single anxiety opportunity to explore the maze on a third day were divided into 1 of several treatment protocols: (1) NAIVE conditions: vehicle IP on all 3 days; (2) ACUTE conditions: vehicle on the first 2 days, ethanol on the third day; or (3) REPEAT conditions: ethanol on all 3 days. RESULTS: ACUTE ethanol elicited reduced exploration of the open arms of the elevated plus-maze in a dose- and time-dependent fashion: 1.0 g/kg failed to elicit any significant effects, whereas 2.0 and 3.0 g/kg ethanol elicited a significant anxiety-like response at 6 hours and 9 to 12 hours postinjection, respectively. REPEAT treatment was still without effect at any time point tested following 1.0 g/kg ethanol, but extended the time course of anxiety-like behavior after treatment with either 2.0 or 3.0 g/kg doses. REPEAT treatment with 2.0 and 3.0 g/kg ethanol also produced significant hypoactivity in the maze at some time points postinjection. CONCLUSIONS: Withdrawal from a single exposure to ethanol produces transient but significant anxiety-like behavior, and repeated intermittent bouts of intoxication result in a significant extension of the duration of effect. The rapid emergence and progression of negative emotional signs of withdrawal may be a significant factor in determining susceptibility to transition from casual drinking to loss of control and escalating patterns of consumption that result in alcoholism.

Conditioning processes contribute to severity of naloxone-precipitated withdrawal from acute opioid dependence.

RATIONALE: Single injections with morphine can induce a state of acute opioid dependence in humans and animals, typically measured as precipitated withdrawal when an antagonist such as naloxone is administered 4-24 h after morphine. Repeated treatment with morphine results in further increases in naloxone potency, and prior work has shown that this progressive shift in naloxone potency requires repeated naloxone experience under some but not all experimental conditions. OBJECTIVE: The current study sought to further characterize the experimental conditions that support naloxone experience-dependent and experience-independent potentiation of precipitated suppression of operant responding in morphine pretreated rats, and to assess more directly whether conditioning mechanisms may contribute to the former process. METHODS: Rats trained on an FR15 schedule for food received a total of five vehicle or morphine injections (5.6 mg/kg SC) at 4, 8, or 22 h prior to an operant session in which a cumulative dose-effect function for naloxone-induced suppression of responding was determined. Separate groups of animals at each interval between morphine and naloxone received cumulative naloxone dosing after all morphine pretreatments (NAL ALL DAYS) or after just the first and last morphine pretreatment (NAL FIRST/LAST). Additional groups of rats at the 4 h MOR-NAL interval received most of their naloxone cumulative dose-effect experience in either the home cage or in the operant context with levers retracted. RESULTS: Vehicle-pretreated (Morphine-Naive) rats showed little change in the naloxone dose-effect function even after five cumulative dose-effect determinations. With a single morphine pretreatment, naloxone potency was increased at 4 or 8 h post-morphine, but not at 22 h. With repeated morphine treatment, all MOR-NAL intervals resulted in significant shifts in naloxone potency across treatment days even when naloxone was administered only after the first and last morphine pretreatment. However, much greater shifts in naloxone potency were observed at 4-h and 8-h intervals when naloxone was administered on all treatment days. At the 22 h MOR-NAL interval, there was no further potentiation in naloxone potency with additional naloxone experience provided on the intermediate days. Finally, when the repeated naloxone experience occurred in the home cage at the 4-h interval, naloxone potency was identical to that seen after limited naloxone experience (NAL FIRST/LAST), and significantly less than naloxone potency in groups receiving repeated naloxone experience in the operant context. CONCLUSIONS: The results suggest that conditioned withdrawal mechanisms may play a significant role in the initial development of opioid dependence.

Repeated experience with naloxone facilitates acute morphine withdrawal: potential role for conditioning processes in acute opioid dependence.

Single injections with morphine can induce a state of acute opioid dependence in humans and animals, typically measured as precipitated withdrawal when an antagonist such as naloxone is administered 4-24 h after morphine. Repeated treatment with morphine at 24-h intervals can result in a progressive shift in potency of naloxone to produce such acute withdrawal signs, including suppression of operant responding for food reward. The current study characterized fully both morphine and naloxone dose-effect functions in an effort to establish the relative contributions of repeated morphine vs. repeated naloxone (Nal) experience to these potency shifts. Rats trained on an FR15 schedule for food received four vehicle or morphine injections (0.56-5.6 mg/kg sc), spaced 24 h apart. Four hours after each morphine pretreatment (Repeat Nal), or 4 h after the fourth and final morphine pretreatment only (Single Nal), a cumulative dose-effect function for naloxone-induced suppression of responding was determined. Vehicle-pretreated (Morphine Naive) rats showed little change in the naloxone dose effect function even after four cumulative dose-effect determinations. By contrast, a progressive increase in naloxone potency was observed following successive pretreatments with morphine under Repeat Nal conditions, and the magnitude of naloxone potency shift was morphine dose dependent. At a morphine dose of 5.6 mg/kg, repeated naloxone experience in the presence of morphine was not an absolute requirement to produce an increase in naloxone potency across days, but repeated naloxone could potentiate the magnitude of the observed shift, indicating both experience-independent and experience-dependent processes at work. At lower doses of morphine (1.0 and 3.3 mg/kg) no shift in naloxone potency was observed across days of morphine treatment in the absence of repeated naloxone experience (Single Nal conditions), indicating an increasing contribution of naloxone experience-dependent processes as dose of morphine was decreased. It is argued that these experience-dependent processes in the progressive shift of naloxone potency observed in the current study may reflect an important role of conditioning in the early development of opioid dependence.