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 active transport mechanisms. Metabolism/Metabolites Liver

Toxicity Summary
In the central nervous system, there are both strychnine-sensitive and strychnine-insensitive glycine-binding sites. The strychnine-insensitive glycine-binding site is located on the NMDA receptor complex. The strychnine-sensitive glycine receptor complex consists of chloride channels, belonging to the ligand-gated ion channel superfamily. The potential anticonvulsant activity of glycine supplementation may be mediated through glycine binding to the strychnine-sensitive binding site in the spinal cord. This leads to an increase in chloride ion conductance, thereby enhancing inhibitory neurotransmission. Glycine's ability to enhance NMDA receptor-mediated neurotransmission makes it potentially useful for treating negative symptoms of schizophrenia resistant to neuroleptic blockers. Animal studies have shown that glycine supplementation can prevent endotoxin-induced death, hypoxia-reperfusion injury after liver transplantation, and D-galactosamine-mediated liver injury. Neutrophils are thought to participate in these pathological processes by invading tissues and releasing reactive oxygen species such as superoxide. In vitro studies have shown that neutrophils contain glycine-gated chloride channels, which can attenuate increases in intracellular calcium ion concentration and reduce oxidant production in neutrophils. This research is still in its early stages, but suggests that glycine supplementation may be beneficial for diseases caused by neutrophil infiltration leading to toxicity, such as acute respiratory distress syndrome.

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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
Glyceryl is occasionally used to treat myasthenia gravis, but most researchers doubt its efficacy for this disease. …It is also used as an antacid, sometimes in the form of a compound salt. However…its buffering capacity is limited… Glyceryl is used as an irrigation solution in transurethral resection of the prostate (TURP) as a 1.1% solution. While a 2.1% solution is isotonic, studies have found that the 1.1% solution does not cause hemolysis. During TURP, 10–15 liters of glyceryl solution are typically required for irrigation. Pharmaceutical Use (Veterinary): Intravenous injection in dogs can improve their tolerance to certain toxic parenteral amino acid mixtures. For more complete data on the therapeutic uses of glycine (8 in total), please visit the HSDB records page. Pharmacodynamics Glycine helps trigger oxygen release to meet the needs of cellular energy production processes; it plays an important role in the synthesis of hormones required to maintain a strong immune system.

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This study is the first to demonstrate the direct effect of oral glycine on angiogenesis in an in vivo model of CRC. In vitro experiments showed that glycine receptor (GlyR)-mediated antagonism can act on endothelial cells after stimulation with VEGF or CRC conditioned medium. In summary, the effects described in this study may lead to the future use of glycine as an inexpensive and readily available adjuvant therapy for conventional and targeted therapies against highly vascularized metastatic tumors, and to clinical trials. [3]

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The mechanism by which glycine interferes with NINJ1 aggregation may be direct or indirect. Our in vitro circular dichroism (CD) studies and liposome rupture system experiments showed that glycine does not interfere with the secondary structure or dissolution function of the NINJ1 α-helix. Glycine may still directly interact with the intracellular NINJ1 N-terminal domain or other components, thereby interfering with NINJ1 aggregation, which is consistent with our current data. Another possibility is that glycine acts on some unknown intermediate that can regulate the aggregation of NINJ1 in the plasma membrane. Our data in pyroptosis cells suggest that this intermediate may lie between active GSDMD and NINJ1. In the indirect model, any plasma membrane-associated protein, membrane lipid, or cellular metabolic pathway could be a target. Furthermore, it remains unclear whether the cytoprotective activity of glycine occurs intracellularly or extracellularly. To our knowledge, there is currently no direct evidence that the cytoprotective effects of glycine are specifically mediated intracellularly or extracellularly. Two sites of action have been proposed: extracellular (Weinberg et al., 2016) and intracellular (Rühl and Broz, 2022). Our in vitro data suggest that glycine does not act directly on the N-terminal α-helix (which is thought to be extracellular); however, we have not further elucidated the topological structure of glycine's mechanism of action. Clarifying its site of action will contribute to a deeper understanding of the molecular mechanism by which glycine inhibits NINJ1 aggregation on the plasma membrane and ultimately help determine whether glycine interacts with NINJ1 directly or indirectly. In fact, how NINJ1 is activated by the cytolytic death pathway, its aggregation triggering mechanism, and the way the plasma membrane is damaged remain important and unresolved questions in this field. Given the prevalence of the cytolytic death pathway in human health and disease, and the powerful cytoprotective effects of glycine, answers to these questions will greatly facilitate the development of treatments for pathological conditions associated with abnormal cytolytic death pathways. [4] Lead can affect other organs besides the kidneys, such as the reproductive system and the liver. Therefore, it is necessary to further investigate the mechanisms of lead toxicity and the protective effects of chemicals such as glycine on these organs. Glycine is an endogenous and safe chemical. Therefore, this amino acid may have clinical value for the treatment of a variety of complications, such as lead-induced kidney damage. Further research is needed to better understand the findings of this study in a clinical setting. According to the results, glycine therapy significantly reduced kidney damage in lead-exposed mice, possibly by alleviating oxidative stress. [5] The serotonin and NMDA signaling pathways in the prefrontal cortex (PFC) are both associated with mental illnesses, including schizophrenia, sleep disorders, epilepsy, depression, and anxiety. Breier41 proposed the cortical-subcortical imbalance hypothesis, suggesting that enhanced subcortical serotonin (5-HT) function leads to positive symptoms of schizophrenia, while weakened prefrontal 5-HT function leads to negative symptoms of schizophrenia. Autopsy brain tissue analysis, cerebrospinal fluid studies, and pharmacological challenges have shown that serotonin (5-HT) function is deficient in the cortex of patients with schizophrenia. ^42,43 One possible explanation for the effect of glycine on negative symptoms of schizophrenia is that glycine treatment may not only directly improve NMDA receptor dysfunction, but may also indirectly and temporarily compensate for 5-HT dysfunction in the prefrontal cortex (PFC). In the near future, in-depth research should be conducted to explore the interaction between glycine and serotonin in the prefrontal cortex to study the pathophysiological mechanisms 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.)