NMDA Receptor; Endogenous Metabolite; NINJ1

Transmitters mediating 'fast' synaptic processes in the vertebrate central nervous system are commonly placed in two separate categories that are believed to exhibit no interaction at the receptor level. The 'inhibitory transmitters' (such as Glycine and GABA) are considered to act only on receptors mediating a chloride conductance increase, whereas 'excitatory transmitters' (such as L-glutamate) are considered to activate receptors mediating a cationic conductance increase. The best known excitatory receptor is that specifically activated by N-methyl-D-aspartate (NMDA) which has recently been characterized at the single channel level. The response activated by NMDA agonists is unique in that it exhibits a voltage-dependent Mg block. We report here that this response exhibits another remarkable property: it is dramatically potentiated by glycine. This potentiation is not mediated by the inhibitory strychnine-sensitive glycine receptor, and is detected at a glycine concentration as low as 10 nM. The potentiation can be observed in outside-out patches as an increase in the frequency of opening of the channels activated by NMDA agonists. Thus, in addition to its role as an inhibitory transmitter, glycine may facilitate excitatory transmission in the brain through an allosteric activation of the NMDA receptor. [1]

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Neo-angiogenesis is important for tumor growth. Glycine is a non-toxic amino acid with suspected anti-angiogenic effects. This study was designed to evaluate anti-angiogenic effects of glycine in colorectal cancer. Glycine was added to cultures of human and rat colorectal cancer cells (CRC), and endothelial cells (HUVEC). Glycine's direct impact was monitored using MTT assays. Angiogenesis in HUVEC was monitored using 3D sprouting and migration assays. VEGF and CRC-conditioned media were used to stimulate angiogenesis. The glycine receptor (GlyR) was detected using Western blotting and inhibited using strychnine. The WAG-Rij/CC-531 model of metastatic CRC was used to evaluate glycine's impact in vivo. Tumor growth and vessel density were monitored in rats fed with or without 5 % glycine for 14 days. VEGF and conditioned media significantly increased proliferation, migration, and capillary formation to up to 267 %. Glycine completely neutralized this effect and strychnine completely blunted glycine's effect. GlyR was detected in HUVEC. Tumor volume, weight, and vessel density decreased by 35 % (p = 0.02), 34 % (p = 0.03), and 55 % (p = 0.04) in glycine-fed animals. Glycine inhibits angiogenic signaling of endothelial cells and tumor growth. Glycine would be a promising additive to standard and targeted cancer therapies. [3]

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First recognized more than 30 years ago, Glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1 and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death [4].

Frontocortical NMDA receptors are pivotal in regulating cognition and mood, are hypofunctional in schizophrenia, and may contribute to autistic spectrum disorders. Despite extensive interest in agents potentiating activity at the co-agonist glycine modulatory site, few comparative functional studies exist. This study systematically compared the actions of the Glycine reuptake inhibitors, sarcosine (40-200 mg/kg) and ORG24598 (0.63-5 mg/kg), the agonists, glycine (40-800 mg/kg), and D-serine (10-160 mg/kg) and the partial agonists, S18841 (2.5 mg/kg s.c.) and D-cycloserine (2.5-40 mg/kg) that all dose-dependently prevented scopolamine disruption of social recognition in adult rats. Over similar dose ranges, they also prevented a delay-induced impairment of novel object recognition (NOR). Glycine reuptake inhibitors specifically elevated glycine but not D-serine levels in rat prefrontal cortical (PFC) microdialysates, while glycine and D-serine markedly increased levels of glycine and D-serine, respectively. D-Cycloserine slightly elevated D-serine levels. Conversely, S18841 exerted no influence on glycine, D-serine, other amino acids, monamines, or acetylcholine. Reversal of NOR deficits by systemic S18841 was prevented by the NMDA receptor antagonist, CPP (20 mg/kg), and the Glycine modulatory site antagonist, L701,324 (10 mg/kg). S18841 blocked deficits in NOR following microinjection into the PFC (2.5-10 μg/side) but not the striatum. Finally, in rats socially isolated from weaning (a neurodevelopmental model of schizophrenia), S18841 (2.5 and 10 mg/kg s.c.) reversed impairment of NOR and contextual fear-motivated learning without altering isolation-induced hyperactivity. In conclusion, despite contrasting neurochemical profiles, partial glycine site agonists and glycine reuptake inhibitors exhibit comparable pro-cognitive effects in rats of potential relevance to treatment of schizophrenia and other brain disorders where cognitive performance is impaired. [2]

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Based on the results, BUN and serum creatinine levels significantly increased following exposure to lead. Glycine supplementation (500 and 1,000 mg/kg, IP) decreased BUN and creatinine serum levels (P<0.001). Biomarkers of OS were also reduced in renal tissue following glycine therapy in Pb-exposed mice (P<0.001). Histopathological changes were observed in mice treated with lead as tubular dilation, protein cast, vacuolization, and inflammation. In this regard, glycine inhibited histopathological alterations in kidney caused by lead exposure. Conclusion: It was found that Glycine treatment significantly mitigated Pb-induced renal injury most likely through alleviating OS and the associated deleterious outcomes on the kidney tissue. [5]

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Both Glycine and d-serine significantly increased extracellular 5-HT levels for 10 min, whereas dopamine levels remained unchanged. L-serine, in contrast, had no significant effects on 5-HT levels. Conclusions: It is possible that the increase in 5-HT in response to Glycine and d-serine was mediated by N-methyl-D-aspartate receptors. The transient increase in 5-HT in the PFC might be associated with the alleviation of negative symptoms in patients with schizophrenia and the amelioration of sleep quality in patients with insomnia[6].

Cell viability and proliferation [3]
For colorimetric Thiazolyl Blue (MTT)-assays, 4 × 103 cells per well were seeded in 100 μL of Glycine-free medium on a 96-well plate. ECM was used as vehicle and HUVEC were pretreated with either the vehicle or 25 ng/ml of VEGF. After 24 h, glycine (0, 0.01, 0.1, or 1 mM) was added to cell cultures. For each concentration, replicates of four wells were used. After 48 h, 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheniltetrazolium bromide (MTT) reduction activity was measured to assess mitochondrial function as a marker of cell growth and viability. Ten microliters of MTT solution was dissolved in PBS and added to the culture. The cells were incubated at 37 °C for 4 h. The fluid was subsequently discharged and the precipitated formazan crystals were then dissolved using 2-propanol. The optical density value of each well was measured after 10 min at 570 nm using a microplate reader. The measured data were corrected to the blank values without cells. The experiments were repeated three times.

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Endothelial cell migration assay [3]
The experimental design was modified from the Culture-Inserts system. The culture inserts were transferred to a 24-well plate and 2 × 105 HUVEC/ml in ECM (supplemented with or without 25 ng/ml of VEGF) was seeded in each well. After 24 h, the inserts were removed using sterile tweezers and 500 μm cell-free gaps were created. The cells were subsequently washed with PBS to remove detached cells. ECM was used as vehicle and either 1 mM of Glycine or the vehicle was then added to each plate. In an additional control group, strychnine (50 μmol/l) was added to block possible GlyR-mediated effects. Photomicrographs of each assay were taken at four randomly selected sites along the newly-created cell-free gaps at 20× magnification, every hour for 8 h. The photomicrographs were analyzed using Axiovision 4.8 software and the examiner was blinded for the treatment group. The changes in the surface coverage were determined as the initial cell-free gap size minus the final cell-free gap size and the surface coverage rate (SCR) was calculated as a ratio of the migrated distance to time.

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3D-sprouting angiogenesis assay [3]
The impact of Glycine on endothelial cell differentiation and capillary formation was assessed using spheroid sprouting angiogenesis assays. HUVEC were trypsinized and suspended in growth medium containing 20 % Methocel. Using an 8-channel multipipette, 25 μl drops were put on a quadratic petri dish and cultured as hanging drops. After 12 h, a spheroid with approximately 400 cells formed in each of the hanging drops. The collagen matrix was prepared by adding 0.1 % acetic acid until the consistency of the matrix became viscous. A prepared collagen was mixed with Medium199 and neutralized with 0.2 M NaOH. The spheroids were washed with PBS and, after centrifugation (1000 rpm, 5 min, room temperature), approximately fifty spheroids were mixed with 1 ml of the prepared collagen before being added to a 48-well plate and incubated for 30 min to promote polymerization. Supplement-free ECM was added to the collagen beds with or without the addition of either 25 ng/ml of VEGF or the conditioned media which contained 1 mM of glycine or no glycine at all. To block GlyR-mediated effects, strychnine (50 μM) was added to additional cell cultures in combination with a 1 mM glycine containing medium with or without VEGF. After 24 h, the spheroids were fixed using 4 % Para-Formaldehyde (PFA) and the capillary-like structures were examined using phase contrast microscopy. Ten spheroids per group were analyzed and the assay was repeated three times with different HUVEC samples. The average cumulative length of all sprouts per spheroid was calculated. The examiner was blinded for the treatment group.

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Where indicated, mouse cells were treated with 5 mM glycine at the time of cell death induction. Human cells were pretreated with Glycine at a concentration of 50 mM for at least 10 min before stimuli was added. [4]

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Mitochondrial membrane potential and cellular ATP measurements [4]
hMDMs were differentiated at 30,000 cells per well in glass-bottom 96-well plates (Cellvis, P96-1.5H-N). Parallel plates were prepared for live-cell imaging and LDH + CellTiter Glo analysis. hMDMs were either left untreated or pre-treated with Glycine (50 mM) and primed with 1 μg/mL LPS (E. coli serotype 0111:B4) in RPMI complemented with 1% fetal calf serum for 3 hr before stimulation with 20.7 μM of nigericin. Tetramethylrhodamine ethyl ester perchlorate (TMRE, 200 or 10 nM) was added to cells before the plates were placed in a heated incubator (37°C, 5% CO2), and images were taken every 30 min over 18 or 22 hr (two independent experiments). In the second experiment, at the end of LPS priming, cells were labeled with CellTracker Deep Red (0.5 μM) for 15 min and washed before addition of fresh medium with TMRE (10 nM) with or without LPS, nigericin, and glycine. In parallel plates treated identically, supernatants were harvested and assayed for LDH, and cellular ATP was measured using CellTiterGlo at 0, 2, 6, and 18/22 hr.

Animal experiments [3]
Adult male WAG-Rij rats were kept under standard laboratory conditions. The animals were housed at the University’s veterinary care facility and a 12:12 h, light:dark cycle was maintained. All the animals had access to water and standard rat chow, ad libitum. After acclimatization, the diet was changed and the animals were randomly allocated standardized pellet food containing 20 % casein for nitrogen balance (controls, n = 5) or 15 % casein and 5 % Glycine (n = 5). After 5 days, the tumor cells were inoculated and tumor growth was monitored for 14 days as described below. Both control and glycine diets were maintained throughout the entire experiment.

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Mature male mice (n=32) were allocated into four groups. The following treatment regimens were the control (vehicle-treated); Pb-acetate (20 mg/kg/day, gavage); Pb-acetate + Glycine (500 mg/kg/day, IP); and Pb-acetate + glycine (1,000 mg/kg/day, IP). Pb-acetate + glycine was administered for 14 consecutive days, Pb-acetate was given first and then glycine at least 6 hours later. On day 15, the subjects were anesthetized, and samples were collected. Serum biomarkers such as BUN and serum creatinine were monitored along with formation of reactive oxygen species, lipid peroxidation, kidney GSH level, and histopathological changes.[5]

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The subjects were allocated into four groups of eight mice and the following treatment regimens were introduced: the control (vehicle-treated); Pb-acetate (20 mg/kg/day, gavage); Pb-acetate (20 mg/kg/day, gavage) + Glycine (500 mg/kg/day, IP); and Pb-acetate (20 mg/kg/day, gavage) + glycine (1,000 mg/kg/day, IP). Pb-acetate + glycine were given for 14 consecutive days. Pb-acetate dose was selected based on previous studies that mentioned 20 mg/kg of Pb-acetate as the nephrotoxic dose.21 Glycine was administered at least 6 hours after Pb-acetate administration. On day 15, samples were collected. [5]

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Aim: Glycine, one of the non-essential amino acids, has been reported to be effective in reducing negative symptoms of schizophrenia. Recently, we found that glycine improves subjective sleep quality in humans. The aim of this study was to investigate the effects of oral glycine administration on endogenous 5-hydroxytryptamine (5-HT) and dopamine in the prefrontal cortex (PFC) of living rats.
Methods: Microdialysis probes were inserted stereotaxically into the rat prefrontal cortex. Cortical levels of 5-HT and dopamine were measured following oral administration of 1 or 2 g/kg Glycine, 2 g/kg d-serine, or 2 g/kg L-serine. [6]

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Glycine concentration in the cerebrospinal fluid [6]
After oral administration of 2 g/kg of glycine, rats (n = 39) were anesthetized with sodium pentobarbital and positioned in a stereotaxic frame with their heads flexed downward at 45°. Approximately 100 µL of cerebrospinal fluid was collected from the cisternae of each rat at each time-point (pre-administration, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h after administration). The glycine concentration in the cerebrospinal fluid (CSF) was measured using the protocol for measuring plasma amino acids suggested by Noguchi et al.18

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Drugs [6]
Glycine and L- and d-serine were dissolved in distilled water. All oral administrations were performed with a gavage needle in an injection volume of 1 mL/100 g bodyweight.

Absorption, Distribution and Excretion
Absorbed from the small intestine via an active transport mechanism.

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Metabolism / Metabolites
Hepatic
Hepatic

Toxicity Summary
In the CNS, there exist strychnine-sensitive glycine binding sites as well as strychnine-insensitive glycine binding sites. The strychnine-insensitive glycine-binding site is located on the NMDA receptor complex. The strychnine-sensitive glycine receptor complex is comprised of a chloride channel and is a member of the ligand-gated ion channel superfamily. The putative antispastic activity of supplemental glycine could be mediated by glycine's binding to strychnine-sensitive binding sites in the spinal cord. This would result in increased chloride conductance and consequent enhancement of inhibitory neurotransmission. The ability of glycine to potentiate NMDA receptor-mediated neurotransmission raised the possibility of its use in the management of neuroleptic-resistant negative symptoms in schizophrenia.
Animal studies indicate that supplemental glycine protects against endotoxin-induced lethality, hypoxia-reperfusion injury after liver transplantation, and D-galactosamine-mediated liver injury. Neutrophils are thought to participate in these pathologic processes via invasion of tissue and releasing such reactive oxygen species as superoxide. In vitro studies have shown that neutrophils contain a glycine-gated chloride channel that can attenuate increases in intracellular calcium and diminsh neutrophil oxidant production. This research is ealy-stage, but suggests that supplementary glycine may turn out to be useful in processes where neutrophil infiltration contributes to toxicity, such as ARDS.

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Toxicity Data
ORL-RAT LD₅₀ 7930 mg/kg, SCU-RAT LD₅₀ 5200 mg/kg, IVN-RAT LD₅₀ 2600 mg/kg, ORL-MUS LD₅₀ 4920 mg/kg

[1]. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature. 1987 Feb 5-11;325(6104):529-31.

[2]. Comparative Pro-cognitive and Neurochemical Profiles of Glycine Modulatory Site Agonists and Glycine Reuptake Inhibitors in the Rat: Potential Relevance to Cognitive Dysfunction and Its Management. Mol Neurobiol. 2020 May;57(5):2144-2166.

[3]. Glycine inhibits angiogenesis in colorectal cancer: role of endothelial cells. Amino Acids. 2016 Nov;48(11):2549-2558.

[4]. Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death. Elife. 2022 Dec 5;11:e78609.

[5]. Glycine supplementation mitigates lead-induced renal injury in mice. J Exp Pharmacol. 2019 Feb 18;11:15-22.

[6]. Oral administration of glycine increases extracellular serotonin but not dopamine in the prefrontal cortex of rats. Psychiatry Clin Neurosci. 2011 Mar;65(2):142-9.

Therapeutic Uses
AMINOACETIC ACID HAS BEEN OCCASIONALLY USED IN THERAPY OF MYASTHENIA GRAVIS BUT MOST INVESTIGATORS DOUBT THAT THE CMPD HAS ANY VALUE IN THIS DISORDER. ... /IT/ IS ALSO USED IN...ANTACID PREPN, SOMETIMES AS A COMPLEX SALT. HOWEVER.../IT HAS/ LIMITED BUFFERING CAPACITY...
AMINOACETIC ACID IS USED IN...1.1% SOLN AS AN IRRIGATING FLUID IN TRANSURETHRAL RESECTION OF THE PROSTATE. ALTHOUGH A 2.1% SOLN...IS ISOTONIC, IT HAS BEEN FOUND THAT A 1.1% SOLN IS NONHEMOLYTIC.
USUALLY FROM 10-15 L OF AMINOACETIC ACID SOLN ARE REQUIRED FOR IRRIGATION DURING TRANSURETHRAL RESECTION OF PROSTATE.
MEDICATION (VET): IV USE IN DOGS INCREASES THEIR TOLERANCE AGAINST CERTAIN TOXIC PARENTERAL AMINO ACID MIXT.
For more Therapeutic Uses (Complete) data for Glycine (8 total), please visit the HSDB record page.

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Pharmacodynamics
Helps trigger the release of oxygen to the energy requiring cell-making process; Important in the manufacturing of hormones responsible for a strong immune system.

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In this study, a direct effect of orally administered Glycine on angiogenesis has been demonstrated in an in vivo model of CRC for the first time. In in vitro, a GlyR-mediated counteractive effect on endothelial cells after stimulation with either VEGF- or CRC-conditioned culture media could be shown. Taken together, the effects described in this study could lead to future clinical trials with glycine as a cheap, easily available addition to conventional and targeted therapies against highly vascularized, metastatic tumors. [3]

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The mechanism by which Glycine interferes with NINJ1 clustering can be either direct or indirect. Our in vitro CD studies and liposomal rupture system, respectively, indicate that glycine does not interfere with the secondary structure or lytic function of the NINJ1 α-helix. It remains plausible that glycine still interacts directly with this domain or other components of the NINJ1 N-terminus in cells to interfere with NINJ1 clustering, consistent with our presented data. Alternatively, glycine may act on an unidentified intermediate that modulates NINJ1 clustering within the plasma membrane. Our data in pyroptotic cells suggest that such an intermediate would reside between active GSDMD and NINJ1. In an indirect model, any of a plasma membrane-associated protein, membrane lipid, or cellular metabolic pathway could be the target. In addition, it remains unclear whether glycine’s cytoprotective activity occurs on the intracellular or extracellular side of the plasma membrane. To the best of our knowledge, there is no direct evidence to suggest whether glycine’s cytoprotective effect is specifically mediated from the intracellular or extracellular side. Both extracellular (Weinberg et al., 2016) and intracellular (Rühl and Broz, 2022) sites of action have been proposed. Our in vitro data suggest that glycine is not acting directly on the N-terminal α-helix, which is thought to be extracellular; however, we do not otherwise delineate the topology of glycine’s mechanism of action. Defining the site of action would provide insight into the molecular mechanism by which Glycine inhibits NINJ1 clustering in the plasma membrane and ultimately help determine whether glycine directly or indirectly engages NINJ1. Indeed, how NINJ1 is activated by lytic cell death pathways, its clustering trigger, and method of plasma membrane disruption also remain important and outstanding questions in the field. Due to the ubiquity of lytic cell death pathways in human health and disease and the potency of glycine cytoprotection, answers to these questions will greatly advance the development of therapeutics against pathologic conditions associated with aberrant lytic cell death pathways. [4]

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Other organs rather than kidney might also be affected by Pb (eg, reproductive system and the liver). Therefore, evaluating the mechanism of Pb toxicity and the protective effects of chemicals such as glycine in these organs warranted further investigations. Glycine is an endogenous and safe chemical. Hence, this amino acid might be clinically applicable against a variety of complications such as Pb-induced renal injury. To understand the findings of the current study in clinical setting, further studies are warranted. Based on the results, glycine therapy could significantly reduce renal injury in mice exposed to Pb and the probable mechanism is the alleviation of OS.[5]

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Both serotonin and NMDA signaling in the PFC are implicated in mental disorders, including schizophrenia, sleep disorder, epilepsy, depression, and anxiety disorders. Breier41 proposed a hypothesis of cortical–subcortical imbalance, with an increase in subcortical 5-HT function responsible for positive symptoms and a decrease in prefrontal 5-HT function responsible for negative symptoms in schizophrenia. Postmortem brain tissue analysis, CSF studies, and pharmacological challenges suggest a deficit in 5-HT function in the cortex of patients with schizophrenia.42,43 One of the possible explanations for the effects of Glycine on negative symptoms of schizophrenia is that glycine treatment may act not only directly to improve NMDA hypofunction, but also indirectly in the transient compensation for 5-HT hypofunction in PFC. In the near future, advanced research studies investigating the Glycine–serotonin interaction in the PFC should be performed to examine the pathophysiology of mental disorders, including schizophrenia and insomnia. [6]

C2H5NO2

75.067

75.032

C, 32.00; H, 6.71; N, 18.66; O, 42.63

56-40-6

Glycine-15N;7299-33-4;Glycine-d2;4896-75-7;Glycine-2-13C;20220-62-6;Glycine-1-13C;20110-59-2;Glycine-13C2,15N;211057-02-2;Glycine-d5;4896-77-9;Glycine-d3;4896-76-8;Glycine-13C2,15N,d2;1984075-49-1;Glycine-13C2;67836-01-5;Glycine-1-13C,15N;112898-03-0;Glycine-2-13C,15N;91795-59-4;Glycine-15N,d2;2732915-89-6

750

White to off-white solid powder

1.3±0.1 g/cm3

240.9±23.0 °C at 760 mmHg

240 °C (dec.)(lit.)

99.5±22.6 °C

0.0±1.0 mmHg at 25°C

1.461

-1.03

2

3

1

5

42.9

0

O([H])C(C([H])([H])N([H])[H])=O

DHMQDGOQFOQNFH-UHFFFAOYSA-N

InChI=1S/C2H5NO2/c3-1-2(4)5/h1,3H2,(H,4,5)

glycine InChi Key

AZD-4282; glycine; 56-40-6; Glycocoll; Glycolixir; Glicoamin; Glycosthene; Aciport; Padil; AZD 4282; AZD4282 Aminoacetic acid glycine

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)

H2O : ~25 mg/mL (~333.02 mM)
Methanol :< 1 mg/mL

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.)