N-methyl-D-aspartate (NMDA) receptor, glycine site
The primary target of HA-966 is the glycine modulatory site (also known as the strychnine-insensitive glycine site) on the NMDA receptor complex, where it allosterically modulates NMDA receptor function. The two isomers exhibit significantly different activities at this target: (R)-(+)-HA-966 is a selective ligand at this site with an IC₅₀ of 12.5 μM for inhibiting [³H]glycine binding, whereas (S)-(-)-HA-966 is much less active at this site with an IC₅₀ of 339 μM. Additionally, (S)-(-)-HA-966 may exert its sedative effects through other mechanisms, potentially involving GABAB receptor-related pathways.

In vitro studies show that (R)-(+)-HA-966 inhibits strychnine-insensitive [³H]glycine binding to rat cerebral cortex synaptic membranes with an IC₅₀ of 12.5 μM, and inhibits glycine-potentiated NMDA responses in cultured cerebral cortex slices with an IC₅₀ of 13 μM. In electrophysiological experiments, HA-966 produces a selective block of NMDA responses in rat cortical slice preparations, with maximal antagonism achieved at 250 μM; this antagonism is reversed by glycine (1 mM) or D-serine (100 μM). In guinea pig ileum preparations, (R)-(+)-HA-966 inhibits glutamate-evoked contractions with an IC₅₀ of 150 μM.

(R)-(+)-HA-966 ((+)-HA-966; IV; 10 mg/kg) considerably reduces the effects of systemic NMDA (125, 250, 500, and 1000 mg/kg; iv) on dose-dependent pressor and related tachycardia responses[3]. (+)-HA-966 (30, 100 mg/kg; IP) does not affect dopamine synthesis in the striatum of male BKTO, but it does block amphetamine-induced augmentation of dopamine synthesis in the nucleus accumbens in a dose-dependent manner. Mice (20–30g) showed no effect from increases. [1]

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1. We evaluated the ability of the functional antagonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex, (+)-(1-Hydroxy-3-aminopyrrolodine-2-one) ((+)-HA966), to modulate the antinociceptive action of systemic morphine in a rat model of neuropathic pain produced by chronic constriction injury to the sciatic nerve. Mechanical (vocalization threshold to hindpaw pressure) and thermal (struggle latency to hindpaw immersion into a water bath) stimuli were used. 2. In the mechanical test, morphine (0.05, 0.1 and 0.3 mg kg(-1), i.v.) alone produced dose-dependent effects in both neuropathic and uninjured rats. Likewise, morphine (0.1, 0.3 and 1 mg kg(-1), i.v.) dose-dependently increased struggle latencies of the nerve-injured hindpaw in the hot noxious (46 degrees C) test but was ineffective in the non-noxious warm (44 degrees C) and cold (10 degrees C) test. 3. Pretreatment with (+)-HA966 (2.5 mg kg(-1), s.c.) dose-dependently enhanced the effect of morphine in the mechanical test with the relative potency being nerve-injured hindpaw > contralateral hindpaw > uninjured rat. 4. Likewise, (+)-HA966 dose-dependently enhanced the effect of morphine against a hot (46 degrees C) stimulus and produced, in combination with morphine, a dose-dependent effect against a warm (44 degrees C) stimulus. In the cold (10 degrees C) test, (+)-HA966 reversed the ineffectiveness of the highest dose of morphine. 5. Naloxone blocked the effect of the combination of (+)-HA966 with morphine in all tests. The drug combination produced no motor deficits in animals using the rotarod test. 6. These findings suggest that combined administration of antagonists, acting at the glycine site of the NMDA receptor complex and morphine may be a promising approach in the treatment of neuropathic and acute pain[3].

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(R)-(+)-HA-966 exhibits multiple in vivo activities in animal models. For anticonvulsant effects, it has an ED₅₀ of 4.7 mg/kg (i.p.) against audiogenic seizures in DBA/2 mice and 893 mg/kg (i.v.) against NMDLA-induced seizures. In a dystonia model, 30-60 mg/kg (i.p.) potently reduces the severity of dystonic attacks in genetically dystonic hamsters. For analgesia, it abolishes the late phase of formalin-induced pain in mice with an ID₅₀ of 1.6 mg/kg (s.c.). In psychiatric disease models, it antagonizes phencyclidine-induced hyperactivity but does not affect phencyclidine-induced disruption of prepulse inhibition. Additionally, (R)-(+)-HA-966 selectively blocks amphetamine-enhanced dopamine synthesis in the nucleus accumbens without affecting the striatum. In contrast, (S)-(-)-HA-966 prevents stress- and conditioned fear-induced increases in mesocorticolimbic dopamine metabolism and cocaine sensitization.

The affinity of HA-966 for the NMDA receptor glycine site is assessed using radioligand binding assays. Rat cerebral cortex synaptic membranes are prepared and incubated with 0.5-2 nM [³H]glycine (or the selective ligand [³H]MDL 105,519) and various concentrations of HA-966 (0.1-1000 μM) in 50 mM Tris-HCl buffer (pH 7.4) at 4°C for 30-60 minutes. Non-specific binding is defined using 1 mM glycine or 10 mM D-serine. The reaction is terminated by rapid vacuum filtration through Whatman GF/B glass fiber filters, followed by three washes with ice-cold buffer. After drying the filters, retained radioactivity is measured using a liquid scintillation counter to calculate specific binding inhibition percentages, and IC₅₀ and Ki values are obtained by fitting competition binding curves using non-linear regression.

Primary cortical neurons from neonatal rats are cultured at densities of 1-2×10⁵ cells/well in 24-well plates in Neurobasal medium with B27 supplement for 10-14 days. For whole-cell patch-clamp recordings, the external solution is Mg²⁺-free, and NMDA (10-100 μM) plus glycine (1-10 μM) are applied via a fast perfusion system. Various concentrations of HA-966 (1-1000 μM) are pre-applied for 1-2 minutes to assess inhibition of NMDA-evoked currents. Alternatively, for cAMP detection assays, HEK293 cells expressing recombinant NMDA receptors are seeded into 96-well plates, treated with various concentrations of HA-966 for 15-30 minutes, lysed, and intracellular cAMP accumulation is measured by ELISA.

Animal/Disease Models: SD (SD (Sprague-Dawley)) rats (11 to 12 weeks old) [3]
Doses: 10 mg/kg
Route of Administration: IV
Experimental Results: Dramatically attenuated systemic NMDA-induced dose-dependent pressor and associated tachycardia responses.

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1. The effects of the glycine/NMDA receptor antagonist, (+)-HA-966 on the neurochemical and behavioural responses to amphetamine have been determined in the mouse and rat. 2. In vehicle-treated control mice, (+)-HA-966 (30-100 mg kg-1) did not affect dopamine synthesis in either the nucleus accumbens or striatum and was without marked effect on spontaneous locomotor activity. 3. In the mouse, (+)-HA-966 (30 and 100 mg kg-1) dose-dependently blocked the enhancement of dopamine synthesis induced in the nucleus accumbens by amphetamine, but was without effect on the increase in dopamine synthesis in the striatum. 4. Intracerebroventricular administration of the glycine/NMDA receptor antagonist, 5,7-dichlorokynurenic acid, in the mouse (10 micrograms) also significantly attenuated amphetamine-enhanced DOPA accumulation in the nucleus accumbens, but not in the striatum. 5. The decrease of dopamine synthesis in striatum and nucleus accumbens induced by the dopamine receptor agonist, apomorphine, was unaffected by (+)-HA-966 (100 mg kg-1). 6. (+)-HA-966 (30 mg kg-1) failed to attenuate the hyperactivity induced by the systemic administration of amphetamine in the mouse, but totally prevented the hyperlocomotion following infusion of amphetamine into the rat nucleus accumbens. In contrast, stereotyped behaviour induced by infusion of amphetamine into the rat striatum was not altered following pretreatment with (+)-HA-966 (30 mg kg-1). 7. The results are consistent with a selective facilitatory role of glycine/NMDA receptors on mesolimbic dopaminergic neurones.[1]

Early studies indicate that following intravenous administration, the effects of HA-966 exhibit an appreciable delay (several minutes), and the effects are significantly reduced in hepatectomized mice, suggesting that the compound may require hepatic metabolism to an active metabolite to fully exert its pharmacological effects. Parameters such as oral bioavailability and detailed plasma half-life have not been systematically reported. The compound is commonly administered via intraperitoneal, subcutaneous, or intravenous routes, with distinct pharmacodynamic effects typically observed within 15-60 minutes post-administration in animal studies.

Regarding behavioral toxicity, (R)-(+)-HA-966 at effective anticonvulsant doses (e.g., 30-100 mg/kg i.p. in mice) does not cause significant sedation or ataxia, with normal performance in rotarod tests. In contrast, (S)-(-)-HA-966 produces sedative effects at lower doses (3-5 mg/kg i.p.), and high doses (5 mg/kg i.p.) suppress weight gain. The sedative/ataxic effects of racemic HA-966 are primarily attributable to the (S)-(-)-isomer. No lethal toxicity data for HA-966 within experimental dose ranges have been reported in the available literature. This compound is intended for scientific research use only and is not for human therapeutic applications.

[1]. R-(+)-HA-966, a glycine/NMDA receptor antagonist, selectively blocks the activation of the mesolimbic dopamine system by amphetamine. Br J Pharmacol. 1991 Aug;103(4):2037-44.

[2]. Discriminative stimulus effects of R-(+)-3-amino-1-hydroxypyrrolid-2-one, [(+)-HA-966] a partial agonist of the strychnine-insensitive modulatory site of the N-methyl-D-aspartate receptor. J Pharmacol Exp Ther. 1995 Dec;275(3):1267-73.

[3]. The antinociceptive effect of combined systemic administration of morphine and the glycine/NMDA receptor antagonist, (+)-HA966 in a rat model of peripheral neuropathy. Br J Pharmacol. 1998 Dec;125(8):1641-50.

The glycine site on the N-methyl-D-aspartate (NMDA) receptor complex, which is insensitive to strychnine, is a target for the development of various therapeutic drugs, including anxiolytics, antidepressants, antiepileptics, anti-ischemic drugs, and cognitive enhancers. This study investigated the discriminative stimulative effect of the ineffective glycine site partial agonist (+)-HA-966 [R-(+)-3-amino-1-hydroxypyrrolidone-2-one]. Male Swiss-Webster mice were trained in a T-maze to distinguish between (+)-HA-966 (170 mg/kg, intraperitoneal injection) and saline, with behavior controlled by food. Other glycine partial agonists, such as 1-amino-1-cyclopropanecarboxylic acid and D-cycloserine, although known to differ from (+)-HA-966 in other pharmacological effects, were able to completely substitute for the discriminative stimulative effect of (+)-HA-966. The glycine site antagonist 7-chlorokynurenic acid cannot replace (+)-HA-966. Similarly, other functional NMDA antagonists acting on non-glycine sites of the NMDA receptor cannot replace (+)-HA-966: neither high-affinity ion channel blockers (dizocide) nor low-affinity ion channel blockers (ibergerine), the competitive antagonist NPC 17742 [2R,4R,5S-2-amino-4,5-(1,2-cyclohexyl)-7-phosphonohepanoic acid], nor the polyamine antagonist isifenprodil can replace (+)-HA-966. Although the complete agonist glycine does not act as a substitute, this compound completely blocks the discriminative stimulatory effect of (+)-HA-966. In another group of trained mice to distinguish between 0.17 mg/kg dezocephalpine and saline, (+)-HA-966 produced at most 50% of the dezocephalpine-related responses. These data suggest that the discriminative stimuli of (+)-HA-966 are based on its partial agonist effect at the strychnine-insensitive glycine site. [2]

C4H9CLN2O2

152.58

152.0353

C, 31.49; H, 5.95; Cl, 23.23; N, 18.36; O, 20.97

42585-88-6

42585-88-6 (HCl); 75195-65-2 (hydrate); 111821-58-0 (S-isomer)； 123931-04-4 (R-isomer)

121225697

Typically exists as solids at room temperature

0.375

3

3

9

115

0

C1CN(C(=O)C1N)O.Cl

HA-966 hydrochloride; S50J1C89KU; 42585-88-6; UNII-S50J1C89KU; 2-Pyrrolidinone, 3-amino-1-hydroxy-, monohydrochloride;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)