AMPK

AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution. [1]

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5-Aminoimidazole-4-carboxamide riboside (AICA riboside; Acadesine) activates AMP-activated protein kinase (AMPK) in intact cells, and is reported to exert protective effects in the mammalian CNS. In rat cerebrocortical brain slices, AMPK was activated by metabolic stress (ischaemia > hypoxia > aglycaemia) and AICA riboside (0.1-10 mm). Activation of AMPK by AICA riboside was greatly attenuated by inhibitors of equilibrative nucleoside transport. AICA riboside also depressed excitatory synaptic transmission in area CA1 of the rat hippocampus, which was prevented by an adenosine A1 receptor antagonist and reversed by application of adenosine deaminase. However, AICA riboside was neither a substrate for adenosine deaminase nor an agonist at adenosine receptors. We conclude that metabolic stress and AICA riboside both stimulate AMPK activity in mammalian brain, but that AICA riboside has an additional effect, i.e. competition with adenosine for uptake by the nucleoside transporter. This results in an increase in extracellular adenosine and subsequent activation of adenosine receptors. Neuroprotection by AICA riboside could be mediated by this mechanism as well as, or instead of, by AMPK activation. Caution should therefore be exercised in ascribing an effect of AICA riboside to AMPK activation, especially in systems where inhibition of adenosine re-uptake has physiological consequences [2].

pH changes associated with the action of adenosine deaminase [2]
Adenosine (10, 30 and 100 µm) and AICA riboside (1, 3 and 10 mm) were prepared as 20 mL solutions in physiological saline (0.9% NaCl; pH 5.72). Adenosine deaminase (0.02 units) was added to each in triplicate and the resultant pH change measured with a Jenway 3310 pH meter. In experiments to examine the influence of AICA riboside on the ability of adenosine deaminase to metabolise adenosine, 30 µm adenosine, from a stock solution of 5 mm was added, in triplicate to solutions of 1, 3 and 10 mm AICA riboside containing 0.02 Units of adenosine deaminase.

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Purity of commercially supplied AICA riboside [2]
We examined the AICA riboside used in this study for signs of possible contamination with either adenosine or adenine nucleotides that could be broken down to adenosine. Two independent HPLC techniques were used, i.e. the one described above for tissue nucleotide determination, and a second in which AICA riboside solutions were ethenoderivatized and constituents detected with a ThermoFinnigan FL3000 scanning fluorescence detector. There were no detectable contaminants in solutions of AICA riboside as measured with either technique (data not shown) excluding the possibility that the results described can be attributable to the presence of adenosine or adenine nucleotides.

Determination of mitochondrial oxygen consumption rate in intact and permeabilized hepatocytes, and isolated mitochondria [1]
Rat or mouse hepatocytes (7–8 mg of dry cells·ml−1) were incubated in a shaking water bath at 37°C in closed vials containing 2 ml of Krebs–Ringer bicarbonate calcium buffer [120 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24 mM NaHCO3 and 1.3 mM CaCl2 (pH 7.4)] supplemented with the indicated concentrations of substrates and AICA riboside, and in equilibrium with a gas phase containing O2/CO2 (19:1). At the indicated times, the cell suspension was saturated again with O2/CO2 for 1 min and immediately transferred into a stirred oxygraph vessel equipped with a Clark oxygen electrode. The JO2 (oxygen consumption rate) was measured at 37°C before and after successive addition of 0.5 μM oligomycin, 150 μM DNP (2,4 dinitrophenol), 0.15 μg/ml antimycin and 1 mM TMPD (N,N,N′,N′-tetramethyl-1,4-phenylenediamine) plus 5 mM ascorbate.
For mitochondrial experiments, isolated rat mitochondria (1 mg of protein·ml−1) were incubated at 37°C in the oxygraph vessel containing 2 ml of the KCl medium (described above) in the presence of 1 mM AICA riboside or the indicated concentrations of ZMP and ZTP. After equilibration for 30 s, the mitochondrial respiratory rate was monitored in the presence of glutamate/malate or succinate/malate/rotenone, as described above for permeabilized hepatocytes.

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Determination of nucleotides and Pi concentrations, and of gluconeogenesis and ketogenesis [1]
After 30 min of incubation, samples of the cell suspension were quenched in ice-cold 25 mM HClO4 [5% (w/v)]/25 mM EDTA and centrifuged (13000 g for 2 min). The supernatants were neutralized and adenine nucleotides, ZMP and AICA riboside concentrations were measured by HPLC. To measure intracellular Pi, a 0.7 ml sample of the cell suspension was centrifuged (13000 g for 1 min) through a silicon oil layer into 0.25 ml of HClO4 [10% (w/v)]/25 mM EDTA. Pi was measured colorimetrically. For gluconeogenesis and ketogenesis measurements, samples of the cell suspension were quenched in ice-cold HClO4 [5% (w/v)], centrifuged (13000 g for 2 min) and the supernatants were neutralized. Glucose, acetoacetate and β-hydroxybutyrate concentrations were measured using enzymatic methods coupled with spectrophotometric determinations of NADH, as described previously.

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Treatment of cerebrocortical slices [2]
Cerebrocortical slices were transferred to incubation chambers placed in a water bath where they were warmed to 33–34°C. After approximately 2.5–3 h, some slices were transferred to identical experimental chambers that contained either AICA riboside (0.1–10 mm) or AICA riboside plus a combination of uptake inhibitors dipyridamole and nitrobenzylthioinosine (DIPY/NBTI; 5 µm and 1 µm, respectively). Additionally, to induce metabolic stress, slices were transferred to chambers that were devoid of glucose (aglycaemia; glucose substituted with 10 mm sucrose) or oxygen (hypoxia; bubbled with 95% N2/5% CO2) or devoid of both glucose and oxygen (ischaemia). Slices used as time controls were transferred to identical chambers containing standard aCSF. At appropriate times, slices were snap-frozen in liquid nitrogen and stored at − 80°C.

Slice preparation [2]
Sprague–Dawley rats of either sex, aged 16–23 days, were killed by cervical dislocation. The brain was rapidly removed and placed in ice-cold artificial cerebro-spinal fluid (aCSF) containing 11 mm Mg2+ wherein 400-µm cerebrocortical slices, comprising sagittal sections of hippocampus and overlying neocortex, were cut with a Vibratome, as previously described (Dale et al. 2000). Slices were placed in an incubation chamber comprising a nylon mesh within a beaker of continuously circulating, oxygenated (95% O2/5% CO2) standard aCSF (1 mm Mg2+) and kept at room temperature for at least 1 h before use. The composition of the aCSF solution was: NaCl 124 mm; KCl 3 mm; CaCl2 2 mm; NaHCO3 26 mm; NaH2PO4 1.25 mm; d-glucose 10 mm; MgSO4 1 mm; pH 7.4 with 95% O2/5% CO2.

Treatment of cerebrocortical slices [2]
Cerebrocortical slices were transferred to incubation chambers placed in a water bath where they were warmed to 33–34°C. After approximately 2.5–3 h, some slices were transferred to identical experimental chambers that contained either AICA riboside (0.1–10 mm) or AICA riboside plus a combination of uptake inhibitors dipyridamole and nitrobenzylthioinosine (DIPY/NBTI; 5 µm and 1 µm, respectively). Additionally, to induce metabolic stress, slices were transferred to chambers that were devoid of glucose (aglycaemia; glucose substituted with 10 mm sucrose) or oxygen (hypoxia; bubbled with 95% N2/5% CO2) or devoid of both glucose and oxygen (ischaemia). Slices used as time controls were transferred to identical chambers containing standard aCSF. At appropriate times, slices were snap-frozen in liquid nitrogen and stored at − 80°C.

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Generation of AMPKα1α2LS−/− knockout mice [1]
To obtain a deletion of both catalytic subunits in the liver (AMPKα1α2LS−/−), we first generated a liver-specific AMPKα2-null mouse (AMPKα2−/−) by crossing floxed AMPKα2 mice and an AlfpCre transgenic line expressing the Cre recombinase under the control of the albumin and α-fetoprotein regulatory elements. We then produced a liver-specific AMPKα1α2LS−/− mouse on an AMPKα1−/− background by crossing liver-specific AMPKα2−/− mice with AMPKα1−/− mice. Mice were genotyped using PCR on DNA extracted from a tail biopsy using specific primers for the Cre transgene and for the floxed AMPKα2, the deleted AMPKα1 and the wild-type AMPKα1 gene.

[1]. AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside. Biochem J. 2007 Jun 15;404(3):499-507.
[2]. AICA riboside both activates AMP-activated protein kinase and competes with adenosine for the nucleoside transporter in the CA1 region of the rat hippocampus. J Neurochem. 2004 Mar;88(5):1272-82.

AICA ribonucleotide is a 1-(phosphoribosyl)imidazolecarboxamide that is acadesine in which the hydroxy group at the 5' position has been converted to its monophosphate derivative. It has a role as a cardiovascular drug, a plant metabolite, a human metabolite, an Escherichia coli metabolite, a Saccharomyces cerevisiae metabolite and a mouse metabolite. It is a 1-(phosphoribosyl)imidazolecarboxamide and an aminoimidazole. It is functionally related to an acadesine. It is a conjugate acid of a 5-amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide(2-).
5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) is an intermediate in the generation of inosine monophosphate and analog of adenosine monophosphate (AMP) that is capable of stimulating AMP-dependent protein kinase (AMPK) activity. AICAR has been used clinically to treat and protect against cardiac ischemic injury. The drug was first used in the 1980s as a method to preserve blood flow to the heart during surgery and is currently of interest as a potential treatment for diabetes by increasing the metabolic activity of tissues by changing the physical composition of muscle.
AICA ribonucleotide has been reported in Arabidopsis thaliana, Homo sapiens, and other organisms with data available.
5-Amino-1-(5-phospho-D-ribosyl)imidazole-4-carboxamide is a metabolite found in or produced by Saccharomyces cerevisiae.

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The present investigation was undertaken to examine the activation of AMPK in mammalian brain by both metabolic stress and the exogenous activator AICA riboside (Acadesine). Furthermore, given that AICA riboside has been reported to protect both peripheral organs and the brain we sought to determine the effects of AICA riboside in a physiological context, that of excitatory synaptic transmission in the rodent hippocampus.

Two aspects of AICA riboside, i.e. its (unspecified) action as an adenosine regulating agent (Mullane 1993) and as an activator (Corton et al. 1995) of AMPK (a key intracellular enzyme involved in the cellular response to metabolic stress), raises the possibility that the protective action of AICA riboside might reflect its effects on extracellular adenosine and activation of AMPK. One or other, or a combination, of these actions could explain the protection of peripheral organs and tissue in a wide variety of experimental models of ischaemia/reperfusion injury (Nawarskas 1999; Matot and Jurim 2001), trauma (Davis et al. 2000), shock (Ragsdale and Proctor 2000) and sepsis (Melton et al. 1999), and how, in humans, AICA riboside improved outcome after coronary artery bypass surgery, reducing the incidence of early cardiac death and myocardial infarction (Mangano 1997). However, it is clear that AMPK mediates the AICA riboside-induced improvement of glucose homeostasis in animal models of diabetes (Halseth et al. 2002; Song et al. 2002), as the antidiabetes drug, metformin, also activates AMPK in vivo (Zhou et al. 2001; Fryer et al. 2002; Hawley et al. 2002) and in skeletal muscle of diabetic humans (Musi et al. 2002).

Similarly, in the brain, AICA riboside exerts protective effects in attenuating neuropathology and locomotor deficits associated with 5-min bilateral carotid artery occlusion in the gerbil (Clough-Helfman and Phillis 1990) and specifically inhibiting homocysteine thiolactone-induced seizures in mice (Marangos et al. 1990), although not pentylenetetrazol, caffeine, picrotoxin or bicuculline methiodide-induced seizures (Marangos et al. 1990; Zhang et al. 1993). Its protective influences have been attributed to activation of adenosine receptors, inhibition of adenosine uptake or metabolism, or to scavenging of free radicals, all of which are known to be neuroprotective (Lipton 1999; de Mendonça et al. 2000). Indeed, a low concentration of AICA riboside (20 µm) has also been shown to potentiate adenosine release from rat cortical slices induced by activation of kainate receptors, but not by α-amino-3-hydroxy-5-methyl-4-isoxasole propionic acid (AMPA) or N-methyl-d-aspartate (NMDA) receptors (White 1996).

However, a role for AMPK in the protection of brain tissues cannot be excluded since activation of AMPK via chronic exposure (48 h) to 200 µm AICA riboside reduced ceramide-induced apoptosis in cultured cortical astrocytes (Blazquez et al. 2001). Moreover, 1 h pre-treatment of hippocampal neuronal cultures with very low (10–100 µm), but not higher (0.5–1 mm) concentrations of AICA riboside has been shown to offer complete protection against toxicity induced by subsequent 24 h exposure to glucose deprivation, cyanide, glutamate and amyloid β peptide (Culmsee et al. 2001). This protection was concomitant with an activation of AMPK as measured by increased phosphorylation of the α subunit using a phospho-specific antibody. Interestingly, protection against glucose withdrawal was observed in the presence of DPCPX, a competitive adenosine A1 receptor antagonist, and could be prevented with antisense oligonucleotides directed against the catalytic α1 and α2 subunits, which suggested a direct involvement of AMPK (Culmsee et al. 2001). Clearly, the combined actions of AICA riboside via adenosine A1 receptors in intact neuronal tissue, which can limit seizure activity (Dunwiddie 1999) and ischaemic brain damage (de Mendonça et al. 2000), and the activation of AMPK, which may additionally provide astrocytic ketone bodies for neuronal oxidative metabolism (Blazquez et al. 1999), endows AICA riboside with several attractive qualities as a potential agent for use in acute or chronic dysfunctions of the mammalian CNS.

In the present study we have shown that AMPK can be activated by metabolic stress induced by hypoxia, aglycaemia and ischaemia in intact brain tissue, with the extent of activation reflecting the severity of the insult. The apparent decline in the activity of AMPK after prolonged ischaemia, may reflect widespread neuronal pathology and/or severe depletion of intracellular ATP, which showed little signs of recovery even after 30 min in oxygen/glucose-rich aCSF. Reductions in ATP levels would render the upstream kinase (AMPKK) less able to phosphorylate AMPK and contribute to a decline in AMPK activity. Indeed, transient increases in AMPK activity followed by marked decreases have also been observed during ischaemia in intact rat hearts perfused in vitro (Marsin et al. 2000) or hypoxia or treatment with the ATP synthesis inhibitor, oligomycin, in monocytes (Marsin et al. 2002).

As in non-neuronal tissue, AICA riboside activated AMPK via intracellular production of ZMP whilst not affecting ATP/ADP ratios. That conversion of AICA riboside to ZMP requires transport of AICA riboside via the equilibrative adenosine transporter is a novel finding of this study clearly indicated by the inhibitory effects on ZMP accumulation and AMPK activity using the nucleoside transport inhibitors DIPY and NBTI. Moreover, the functional implications of AICA riboside transport for neuronal activity is to result in an increase in extracellular adenosine, a process reversed by exogenous adenosine deaminase, which may in itself be inhibited by AICA riboside, thereby further increasing levels of extracellular adenosine. The accumulation of extracellular adenosine is capable of causing strong inhibition of excitatory synaptic transmission, likely via inhibition of glutamate release via pre-synaptic adenosine A1 receptors. We addressed whether AICA riboside was an agonist at adenosine receptors using two different reporter gene assays which showed that AICA riboside is devoid of efficacy at A1 receptors (as well as A2A and A2B receptors) in yeast and against A3 receptors expressed in CHO cells. Given the use of all four adenosine receptors and two assay systems, it makes it unlikely, although not impossible, that the lack of effect of AICA riboside in both could be attributable to inactivation of all four adenosine receptors by some phosphorylation event initiated by AMPK activation. Instead, the simplest interpretation of the reporter gene assay data is that AICA riboside is not an agonist at adenosine receptors.

The consequences of AICA riboside inhibition of adenosine re-uptake would not be specific to A1 receptors, but instead would result in activation of the predominant adenosine receptors in that particular region of the brain (for example A2A receptors in the striatum). This could lead to the situation where interactions between A1, A2A and A3 receptors might result in the desensitisation of A1 receptor-mediated inhibition (Dunwiddie et al. 1997; Lopes et al. 2002). These interactions may have deleterious consequences (Ribeiro et al. 2002) and might explain the mixed results obtained in different models of seizure activity. Nonetheless, previous studies have shown that inhibition of adenosine uptake can raise extracellular adenosine and reduce neuropathology in a variety of experimental models of CNS disorders (de Mendonça et al. 2000). For example, propentofylline, given intraperitoneally after combined hypoxia/ischaemia in newborn rat pups, reduced the volume of infarcted tissue and resulted in better histological measures in striatum, thalamus, hippocampus and cortex (Gidday et al. 1995) and potentiated the neuroprotective ischaemic preconditioning phenomenon in gerbil hippocampus (Kawahara et al. 1998). Indeed, in a recent clinical trial a combination of aspirin and DIPY (a clinically used antiplatelet drug), reduced the recurrence of stroke, whilst DIPY has been shown to elevate plasma levels of adenosine in humans, a factor which may have contributed to the favourable outcome (Picano and Abbracchio 1998). Thus it would seem that an additional facet of the neuroprotective potential of AICA riboside may reside in its ability to elevate extracellular adenosine.

In conclusion, our findings that AICA riboside (while activating AMPK) also exerts effects by elevating extracellular levels of adenosine in intact neuronal tissue, should encourage caution in the interpretation of results obtained with this agent. It remains to be determined which of these two mechanisms of AICA riboside action is more important in providing neuroprotection in vivo. Nonetheless, the existence of these two mutually beneficial properties potentially provides a valuable synergism that could be exploited therapeutically in disorders of the mammalian CNS.[2]

C9H15N4O8P

338.2112

338.063

C, 31.96; H, 4.47; N, 16.57; O, 37.84; P, 9.16

3031-94-5

3031-94-5; 681006-28-0 (phosphate)

65110

White to light brown solid powder

2.3g/cm3

845.3ºC at 760 mmHg

198-202ºC dec.

465ºC

1.831

-3.8

6

10

5

22

475

4

C1=NC(=C(N1[C@H]2[C@@H]([C@@H]([C@H](O2)COP(=O)(O)O)O)O)N)C(=O)N

NOTGFIUVDGNKRI-UUOKFMHZSA-N

InChI=1S/C9H15N4O8P/c10-7-4(8(11)16)12-2-13(7)9-6(15)5(14)3(21-9)1-20-22(17,18)19/h2-3,5-6,9,14-15H,1,10H2,(H2,11,16)(H2,17,18,19)/t3-,5-,6-,9-/m1/s1

[(2R,3S,4R,5R)-5-(5-amino-4-carbamoylimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate

AICA ribonucleotide; 3031-94-5; Z-nucleotide; aminoimidazole carboxamide ribonucleotide; 5'-Phosphoribosyl-5-amino-4-imidazolecarboxamide; 5-amino-4-imidazolecarboxamide ribotide; Acadesine 5'-monophosphate; AICA-ribonucleotide;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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.)