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]

---

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.

---

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.

---

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.

---

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.

---

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-(phospribosyl)imidazolium carboxamide belonging to the acardixin family, with its 5' hydroxyl group converted to a monophosphate derivative. It is used as a cardiovascular drug and is a metabolite in plants, humans, E. coli, Saccharomyces cerevisiae, and mice. It is a 1-(phospribosyl)imidazolium carboxamide and aminoimidazolium compound functionally related to acardixin. It is the conjugate acid of 5-amino-1-(5-phospho-D-ribosyl)imidazolium-4-carboxamide (2-). 5-Aminoimidazolium-4-carboxamide ribonucleotide (AICAR) is an intermediate in the formation of inosinic acid and an analogue of adenosine monophosphate (AMP), capable of stimulating the activity of AMP-dependent protein kinase (AMPK). AICAR has been used clinically to treat and prevent ischemic myocardial injury. Originally used in the 1980s to maintain cardiac blood flow during surgery, it is currently attracting attention as a potential treatment for diabetes due to its ability to enhance tissue metabolic activity by altering the physical composition of muscle.
AICA ribonucleotides have been reported to exist in Arabidopsis thaliana, humans, and other organisms with relevant data.
5-Amino-1-(5-phosphate-D-ribosyl)imidazolium-4-carboxamide is a metabolite found or produced in Saccharomyces cerevisiae.
This study aimed to investigate the effects of metabolic stress and the exogenous activator AICA nucleoside (acardixin) on AMPK activation in the mammalian brain. Furthermore, given previous reports of AICA nucleoside's protective effects on peripheral organs and the brain, we sought to determine the role of AICA nucleoside in a physiological context, specifically in the context of excitatory synaptic transmission in the hippocampus of rodents.
Two aspects of AICA nucleoside—its role as an adenosine regulator (Mullane 1993) and its (undefined) role as an AMPK (a key intracellular enzyme involved in cellular metabolic stress responses) activator (Corton et al. 1995)—suggest that the protective effect of AICA nucleoside may reflect its influence on extracellular adenosine and AMPK activation. One or more of these effects, or a combination thereof, can explain how peripheral organs and tissues are protected in experimental models of various ischemia/reperfusion injuries (Nawarskas 1999; Matot and Jurim 2001), trauma (Davis et al. 2000), shock (Ragsdale and Proctor 2000), and sepsis (Melton et al. 1999), and how AICA nucleosides improve outcomes after coronary artery bypass grafting and reduce the incidence of early cardiac death and myocardial infarction in humans (Mangano 1997). However, it is clear that AMPK mediates improved glucose homeostasis in AICA-induced diabetic animal models (Halseth et al., 2002; Song et al., 2002), as the antidiabetic drug metformin also activates AMPK in vivo (Zhou et al., 2001; Fryer et al., 2002; Hawley et al., 2002) and in the skeletal muscle of diabetic patients (Musi et al., 2002). Similarly, in the brain, AICA-induced neuropathological changes and motor dysfunction induced by 5 minutes of bilateral carotid artery occlusion in gerbils (Clough-Helfman and Phillis, 1990) and specifically inhibited homocysteine-thiolactone-induced seizures in mice (Marangos et al., 1990). This was not the case for seizures induced by pentylenetetrazole, caffeine, bitter substances, or biscoline iodide (Marangos et al., 1990; Zhang et al., 1993). Its protective effects are attributed to activation of adenosine receptors, inhibition of adenosine uptake or metabolism, or scavenging of free radicals, all of which have neuroprotective effects (Lipton, 1999; de Mendonça et al., 2000). Indeed, low concentrations of AICA nucleotides (20 µM) have been shown to enhance adenosine release from rat cortical sections induced by eosin receptor activation, but without this effect on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors (White 1996). However, the role of AMPK in brain tissue protection cannot be ruled out, as prolonged (48 hours) exposure to 200 µM AICA nucleotides activating AMPK reduced ceramide-induced apoptosis in cultured cortical astrocytes (Blazquez et al., 2001). Furthermore, studies have shown that pretreatment of hippocampal neuronal cultures with very low concentrations (10–100 µM) rather than higher concentrations (0.5–1 mM) of AICA nucleosides for 1 hour completely protects neurons from subsequent 24-hour glucose deprivation, cyanide, glutamate, and β-amyloid-induced toxic damage (Culmsee et al., 2001). This protective effect occurs concurrently with AMPK activation, which can be confirmed by elevated α-subunit phosphorylation levels detected by phosphorylation-specific antibodies. Interestingly, protection against glucose deprivation was still observed in the presence of the competitive adenosine A1 receptor antagonist DPCPX; however, this protection was blocked by the use of antisense oligonucleotides targeting the α1 and α2 subunits, indicating that AMPK is directly involved in this process (Culmsee et al., 2001). Clearly, AICA nucleosides function in intact neural tissue via adenosine A1 receptors, limiting seizures (Dunwiddie 1999) and ischemic brain injury (de Mendonça et al. 2000), and activating AMPK, potentially providing astrocyte ketone bodies for neuronal oxidative metabolism (Blazquez et al. 1999). These effects endow AICA nucleosides with a number of attractive properties, making them potential drugs for treating acute or chronic dysfunction of the mammalian central nervous system. In this study, we found that in intact brain tissue, metabolic stress induced by hypoxia, hypoglycemia, and ischemia can activate AMPK, and the degree of activation reflects the severity of injury. Prolonged ischemia resulted in a significant decrease in AMPK activity, possibly reflecting widespread neuronal lesions and/or severe intracellular ATP depletion; even after 30 minutes of immersion in oxygen-rich/glucose-rich artificial cerebrospinal fluid (aCSF), ATP levels showed almost no signs of recovery. The decrease in ATP levels weakens the ability of upstream kinase (AMPKK) to phosphorylate AMPK, leading to a decrease in AMPK activity. In fact, a transient increase followed by a significant decrease in AMPK activity has been observed during in vitro perfusion of intact rat heart ischemia (Marsin et al., 2000), monocyte hypoxia, or treatment with the ATP synthesis inhibitor oligomycin (Marsin et al., 2002). Similar to non-neuronal tissues, AICA nucleosides activate AMPK through the production of intracellular ZMP without affecting the ATP/ADP ratio. A novel finding of this study is that the conversion of AICA nucleosides to ZMP requires transport via balanced adenosine transporters, a finding clearly confirmed by the inhibitory effects of nucleoside transport inhibitors DIPY and NBTI on ZMP accumulation and AMPK activity. Furthermore, the functional significance of AICA nucleoside transport for neuronal activity lies in leading to increased extracellular adenosine levels. While exogenous adenosine deaminase can reverse this process, AICA nucleosides themselves may inhibit adenosine deaminase activity, thereby further increasing extracellular adenosine levels. The accumulation of extracellular adenosine can strongly inhibit excitatory synaptic transmission, likely through the inhibition of glutamate release mediated by presynaptic adenosine A1 receptors. We investigated whether AICA nucleotides are adenosine receptor agonists using two different reporter gene assays. The results showed that AICA nucleotides were inactive against A1 receptors (as well as A2A and A2B receptors) in yeast and against A3 receptors expressed in CHO cells. Given that we used all four adenosine receptors and two assay systems, while not entirely impossible, the inactivity of AICA nucleotides in both systems is unlikely to be due to some phosphorylation event triggered by AMPK activation leading to the inactivation of all four adenosine receptors. Instead, the simplest interpretation of the reporter gene assay data is that AICA nucleotides are not adenosine receptor agonists. The consequences of AICA nucleotides inhibiting adenosine reuptake are not limited to A1 receptors but may activate major adenosine receptors in specific brain regions (such as A2A receptors in the striatum). This could lead to interactions between A1, A2A, and A3 receptors, resulting in A1 receptor-mediated desensitization to inhibitory effects (Dunwiddie et al., 1997; Lopes et al., 2002). These interactions may have detrimental consequences (Ribeiro et al., 2002) and may explain mixed outcomes obtained in different epilepsy models. However, previous studies have shown that inhibiting adenosine uptake can increase extracellular adenosine levels and alleviate neuropathological damage in experimental models of various central nervous system diseases (de Mendonça et al. 2000). For example, intraperitoneal injection of prazolam after hypoxia/ischemia-induced injury in newborn rat pups reduced infarct volume and improved histological parameters of the striatum, thalamus, hippocampus, and cortex (Gidday et al. 1995), and also enhanced neuroprotective ischemic preconditioning in the hippocampus of gerbils (Kawahara et al. 1998). In fact, in a recent clinical trial, the combined use of aspirin and DIPY (a commonly used antiplatelet drug) reduced the recurrence rate of stroke, and DIPY has been shown to increase human plasma adenosine levels, which may be a factor contributing to a good prognosis (Picano and Abbracchio, 1998). Therefore, the neuroprotective potential of AICA nucleosides also seems to lie in their ability to increase extracellular adenosine levels. In conclusion, our results suggest that AICA nucleosides (while activating AMPK) can also exert their effects by increasing extracellular adenosine levels in intact nerve tissue, which suggests that we should be cautious in interpreting the results obtained from the use of this drug. Which of the two mechanisms of action of AICA nucleosides is more important in vivo for neuroprotection remains to be further investigated. Nevertheless, the existence of these two mutually beneficial properties may provide a valuable synergistic effect for the treatment of central nervous system diseases in mammals. [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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

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