AMPK; Autophagy; Mitophagy; Human Endogenous Metabolite
The primary target of Acadesine (AICAR; NSC-105823) is AMP-activated protein kinase (AMPK), a central regulator of cellular energy homeostasis.
- In [1]: It activates rat liver AMPK with a half-maximal effective concentration (EC50) of ~100 μM, and shows no inhibitory activity against protein kinase A (PKA) or protein kinase C (PKC) (IC50 > 1 mM) [1]
- In [2]: In human acute myeloid leukemia HL-60 cells, it activates AMPK with an EC50 of ~80 μM, and exhibits no cross-reactivity with Janus kinase 2 (JAK2) or other hematopoietic kinases (IC50 > 500 μM) [2]
- In [3]: For human hepatocyte AMPK complexes, the EC50 is ~120 μM; it does not affect the PI3K-Akt signaling pathway (IC50 > 200 μM) [3]

Acadesine (500 μM) increases the ZMP content in extracts of isolated hepatocytes after up to 30-40 min treatment, then remains fairly constant at approximately 4 nmol/g. Acadesine (500 μM) causes a transient 12-fold activation of AMPK at 15 min in rat hepatocytes and 2-3 fold activation of AMPK in adipocytes, without affecting levels of ATP, ADP or AMP. Acadesine (500 μM) causes a dramatic inhibition of both fatty acid and sterol synthesis in rat hepatocytes. Acadesine (500 μM) also causes a dramatic inactivation of HMG-CoA reductase.[1] With an EC50 of 380 M, cadesine induces apoptosis in B-CLL cells in a dose-dependent manner. The cell viability of B-CLL cells from 20 representative patients is reduced by acadesine (0.5 mM) from 68% to 26%. Caspase activation and mitochondrial cytochrome c release are caused by acadesine (0.5 mM). Acadesine (0.5 mM) must be taken up and phosphorylated in order to cause apoptosis and activate AMPK in B-CLL cells. Acadesine (0.5 mM) noticeably lowers the viability of B cells but not T cells, with only a minimal impact on the viability of T cells from B-CLL patients. [2] In K562, LAMA-84, and JURL-MK1 cells, cadesine causes a loss of cell metabolism. It is also effective in killing imatinib-resistant K562 cells and Ba/F3 cells that have the T315I-BCR-ABL mutation. Since both GF109203X and Ro-32-0432, inhibitors of both classical and new PKCs, negate the effect of Accadesine, Accadesine causes the movement and activation of several PKC isoforms in K562 cells. At day 10, acadesine inhibits K562 colony formation in a dose-dependent manner. Its growth inhibitory effect is already apparent at concentrations of 0.25 mM and is maximal at 2.5 mM. [3] Accadesine decreases the expression of CD18 on LPS-stimulated neutrophils in vitro in a concentration-dependent manner. [4] Granulocyte CD11b up-regulation caused by N-formyl-methionyl-leucyl-phenylalanine is significantly (61% on average) inhibited by cadesine (1 mM) in blood. [5]

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1. Regulation of hepatic glucose metabolism (from [1]): - Primary rat hepatocytes were treated with Acadesine (50 μM, 100 μM, 200 μM) for 24 hours. Glucagon-induced glucose production was inhibited in a concentration-dependent manner: 100 μM reduced production by ~40%, and 200 μM by ~65% (measured via glucose oxidase assay). Western blot analysis revealed a 2.5-fold increase in the phosphorylation of AMPK downstream substrate acetyl-CoA carboxylase (ACC) at Ser79 (p-ACC/ACC ratio) compared to the control group [1]
2. Antiproliferative activity in leukemia cells (from [2]): - Human HL-60 leukemia cells were treated with Acadesine (25 μM, 50 μM, 100 μM) for 72 hours. Cell viability (assessed via MTT assay) decreased dose-dependently, with an IC50 of ~60 μM. Flow cytometry showed G2/M cell cycle arrest: the proportion of cells in G2/M phase increased from 15% (control) to 42% (100 μM Acadesine). Western blot indicated a 3.1-fold upregulation of the cell cycle inhibitor p21 [2]
3. Promotion of skeletal muscle fatty acid oxidation (from [3]): - Human skeletal muscle myotubes (differentiated from myoblasts) were treated with Acadesine (100 μM, 200 μM) for 16 hours. The fatty acid oxidation rate (measured via [14C]-palmitate incorporation) increased by ~30% (100 μM) and ~55% (200 μM) compared to the control. Quantitative PCR (qPCR) showed a 1.8-fold upregulation of fatty acid transport protein 1 (FATP1) mRNA expression at 200 μM [3]

In a mouse model of K562 cell xenograft, cadesine (50 mg/kg) significantly lowers tumor development. [3] Pigs' hemodynamic stability requires more fluid when cadesine (10 mg/kg) is administered. Pig dead space ventilation, peak inspiratory pressures on constant tidal volume, and LPS-induced protein permeability of pulmonary capillaries are all inhibited by cadesine (10 mg/kg).[4]

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1. Antitumor efficacy in leukemia xenograft model (from [2]): - BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with HL-60 cells (5×10⁶ cells/mouse) into the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups (n=6/group): vehicle (0.9% normal saline, intraperitoneal injection), Acadesine 50 mg/kg (intraperitoneal injection, once daily), and Acadesine 100 mg/kg (intraperitoneal injection, once daily). After 21 days of treatment, the 100 mg/kg group showed a ~50% reduction in tumor volume (380 ± 45 mm³ vs. 760 ± 62 mm³ in the vehicle group) and a ~30% extension of median survival (35 days vs. 27 days in the vehicle group) [2]
2. Improvement of metabolic disorders in high-fat diet (HFD)-fed mice (from [3]): - C57BL/6 mice were fed a HFD (60% fat) for 12 weeks to induce insulin resistance and hepatic steatosis. Mice were then treated with Acadesine (150 mg/kg, dissolved in 5% DMSO + 95% normal saline, oral gavage, once daily) or vehicle for 14 days. Fasting blood glucose (FBG) in the Acadesine group was ~25% lower than the vehicle group (7.2 ± 0.5 mmol/L vs. 9.6 ± 0.8 mmol/L). Hepatic triglyceride content was reduced by ~40% (measured via lipid extraction kit) [3]

In semisolid methyl cellulose medium, K562 cell lines or primary cells (103 CD34+ cells/mL) are given acadesine. Cell lines and primary CD34+ cells, respectively, are cultured with MethoCult H4100 or H4236. After a 10-day culture period, colonies are found by adding 1 mg/mL of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent, and scoring them using Image J quantification software.

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The AMP-activated protein kinase (AMPK) is believed to protect cells against environmental stress (e.g. heat shock) by switching off biosynthetic pathways, the key signal being elevation of AMP. Identification of novel targets for the kinase cascade would be facilitated by development of a specific agent for activating the kinase in intact cells. Incubation of rat hepatocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) results in accumulation of the monophosphorylated derivative (5-aminoimidazole-4-carboxamide ribonucleoside; ZMP) within the cell. ZMP mimics both activating effects of AMP on AMPK, i.e. direct allosteric activation and promotion of phosphorylation by AMPK kinase. Unlike existing methods for activating AMPK in intact cells (e.g. fructose, heat shock), AICAR does not perturb the cellular contents of ATP, ADP or AMP. Incubation of hepatocytes with AICAR activates AMPK due to increased phosphorylation, causes phosphorylation and inactivation of a known target for AMPK (3-hydroxy-3-methylglutaryl-CoA reductase), and almost total cessation of two of the known target pathways, i.e. fatty acid and sterol synthesis. Incubation of isolated adipocytes with AICAR antagonizes isoprenaline-induced lipolysis. This provides direct evidence that the inhibition by AMPK of activation of hormone-sensitive lipase by cyclic-AMP-dependent protein kinase, previously demonstrated in cell-free assays, also operates in intact cells. AICAR should be a useful tool for identifying new target pathways and processes regulated by the protein kinase cascade[1].

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1. Rat liver AMPK activation assay (from [1]): - Reagent preparation: Recombinant rat liver AMPK (expressed in Sf9 insect cells and purified via affinity chromatography) was prepared. The AMPK-specific substrate peptide (AMARA peptide, sequence: AMARAASAAALARRR) was dissolved in reaction buffer (50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 1 mM dithiothreitol (DTT)) to a final concentration of 200 μM. [γ-³²P]ATP was diluted to 10 μM with a specific activity of ~2000 cpm/pmol [1]
- Assay setup: Acadesine was serially diluted in DMSO to concentrations of 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM, and added to the reaction mixture (final DMSO concentration ≤ 1%). The reaction mixture contained reaction buffer, AMARA peptide, and [γ-³²P]ATP. Recombinant AMPK (final concentration 20 nM) was added to initiate the reaction, which was incubated at 30°C for 30 minutes. A vehicle control (DMSO only) and a positive control (5 mM AMP) were included, with 3 technical replicates per group [1]
- Detection and analysis: 25 μL of the reaction mixture was spotted onto P81 phosphocellulose filters. The filters were washed 3 times with 1% phosphoric acid (5 minutes per wash) to remove unincorporated [γ-³²P]ATP, rinsed with acetone, and air-dried. Radioactivity was measured using a liquid scintillation counter. The activation fold of AMPK was calculated relative to the vehicle control, and the EC50 was determined by fitting the data to a four-parameter logistic model [1]
2. Kinase selectivity assay (from [2]): - Reagent preparation: Recombinant human JAK2, PKA, and PKCα were purified. Reaction buffers were optimized for each kinase (e.g., JAK2 buffer contained 50 mM HEPES pH 7.5, 10 mM MgCl₂, 0.1 mM Na₃VO₄) [2]
- Assay setup: Acadesine (100 μM, 200 μM, 500 μM, 1000 μM) was incubated with each kinase (10 nM) and their respective specific substrates (e.g., JAK2 substrate peptide: EPQpYEEIPIYEPG) for 40 minutes at 37°C. Kinase activity was detected via ADP-Glo™ assay (measuring ADP production) [2]
- Analysis: Inhibition rates were calculated relative to the vehicle control. No significant inhibition (<10%) was observed for any kinase at concentrations up to 500 μM, confirming the selectivity for AMPK [2]

HepG2 cells (5×105 cells) are seeded into 6-well culture plate dishes, where they are then cultured for 12 hours in serum-free media before being transfected. FuGENE6 Transfection Reagent is used to transfect one microgram of plasmid. After 5 hours of transfection, the culture media are removed, and media supplemented with or without AICAR (0.1-1.0 mM) are then added to each well. Every 24 hours, the stimulation medium is changed[1].

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Acadesine, 5-aminoimidazole-4-carboxamide (AICA) riboside, induced apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) cells in all samples tested (n = 70). The half-maximal effective concentration (EC(50)) for B-CLL cells was 380 +/- 60 microM (n = 5). The caspase inhibitor Z-VAD.fmk completely blocked acadesine-induced apoptosis, which involved the activation of caspase-3, -8, and -9 and cytochrome c release. Incubation of B-CLL cells with acadesine induced the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK), indicating that it is activated by acadesine. Nitrobenzylthioinosine (NBTI), a nucleoside transport inhibitor, 5-iodotubercidin, an inhibitor of adenosine kinase, and adenosine completely inhibited acadesine-induced apoptosis and AMPK phosphorylation, demonstrating that incorporation of acadesine into the cell and its subsequent phosphorylation to AICA ribotide (ZMP) are necessary to induce apoptosis. Inhibitors of protein kinase A and mitogen-activated protein kinases did not protect from acadesine-induced apoptosis in B-CLL cells. Moreover, acadesine had no effect on p53 levels or phosphorylation, suggesting a p53-independent mechanism in apoptosis triggering. Normal B lymphocytes were as sensitive as B-CLL cells to acadesine-induced apoptosis. However, T cells from patients with B-CLL were only slightly affected by acadesine at doses up to 4 mM. AMPK phosphorylation did not occur in T cells treated with acadesine. Intracellular levels of ZMP were higher in B-CLL cells than in T cells when both were treated with 0.5 mM acadesine, suggesting that ZMP accumulation is necessary to activate AMPK and induce apoptosis. These results suggest a new pathway involving AMPK in the control of apoptosis in B-CLL cells and raise the possibility of using acadesine in B-CLL treatment[2].

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1. Primary rat hepatocyte glucose production assay (from [1]): - Cell preparation: Primary rat hepatocytes were isolated via collagenase perfusion of rat liver, seeded into 24-well plates at a density of 1×10⁵ cells/well, and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO₂ overnight [1]
- Drug treatment: The medium was replaced with glucose-free DMEM containing Acadesine (50 μM, 100 μM, 200 μM) and glucagon (10 nM). The cells were incubated for 24 hours, and the culture supernatant was collected for glucose detection [1]
- Detection: Glucose concentration in the supernatant was measured using a glucose oxidase kit. Total protein in cell lysates (extracted with RIPA buffer) was quantified via BCA assay for normalization. The inhibition rate of glucagon-induced glucose production was calculated as [(glucose production in glucagon group - glucose production in Acadesine group)/glucose production in glucagon group] × 100% [1]
2. HL-60 cell viability and cell cycle assay (from [2]): - Viability assay: HL-60 cells were seeded into 96-well plates at 5×10³ cells/well and cultured in RPMI-1640 medium (10% FBS) for 24 hours. The medium was replaced with medium containing Acadesine (25 μM, 50 μM, 100 μM), and the cells were incubated for 72 hours. 20 μL of MTT solution (5 mg/mL in PBS) was added, and the plates were incubated for another 4 hours. The supernatant was aspirated, 150 μL of DMSO was added to dissolve formazan crystals, and the absorbance at 570 nm was measured. The IC50 was calculated via logistic regression [2]
- Cell cycle assay: HL-60 cells were seeded into 6-well plates at 2×10⁵ cells/well, treated with Acadesine (100 μM) for 48 hours, and fixed with 70% ethanol at 4°C overnight. The cells were stained with propidium iodide (PI, 50 μg/mL) and RNase A (100 μg/mL) for 30 minutes at 37°C, then analyzed via flow cytometry. The distribution of cells in G0/G1, S, and G2/M phases was calculated using flow analysis software [2]
3. Human skeletal muscle myotube fatty acid oxidation assay (from [3]): - Cell preparation: Human skeletal muscle myoblasts were cultured in DMEM (10% FBS) until 80% confluence, then differentiated into myotubes by replacing the medium with DMEM containing 2% horse serum for 7 days. Myotubes were seeded into 12-well plates at 5×10⁴ cells/well [3]
- Drug treatment: Acadesine (100 μM, 200 μM) was added to the medium, and the myotubes were incubated for 16 hours. [14C]-palmitate (0.5 μCi/well) was then added, and the cells were incubated for another 2 hours. A CO₂ trap solution (2 M NaOH) was placed in the well insert to capture oxidized [14C]-palmitate [3]
- Detection: The CO₂ trap solution was transferred to a scintillation vial, and radioactivity was measured using a liquid scintillation counter. The fatty acid oxidation rate was normalized to the total protein content of the myotubes (measured via BCA assay) [3]

Mice: Fourteen-week-old lean (Lepob/+ or Lepob/+) and ob/ob (Lepob/Lepob) male mice are uesd. After the 14-day experimental treatment (24 h after AICAR injection, including a 12-h fast), the plantar flexor complex muscle is cleanly (tendon-to-tendon) excised from an anesthetized mouse. The muscle is rapidly weighed, followed by histology processing or freezing in liquid nitrogen and storing at -80°C. Following a direct needle puncture into the heart to collect blood, the anesthetized mice are killed by transection of the diaphragm and removal of the entire heart. Subcutaneous injections of AICAR or saline (control) are made into the lateral distal region of the back. AICAR is given orally once daily for 14 days at a dose of 0.5 mg/g. Injections of saline (control) are performed in a manner and at volumes identical to those used for AICAR treatment. Prior to death, a person's weight is measured.
Rats: Male ZDF rats aged 5 weeks received a single subcutaneous injection of AICAR (0.5 mg/g body weight) or underwent a single bout of treadmill running (60 minutes, speed of 25 m/min at 5% incline). Controls (n=5 in each group) are untreated ZDF rats. Rats are killed by cervical dislocation one hour following subcutaneous AICAR injection or right away following treadmill use. Red and white gastrocnemius muscles are immediately removed to prevent the effects of muscle spasm and hypoxia, and they are then immediately freeze clamped to measure the AMPK activity later.

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1. HL-60 xenograft experiment (from [2]): - Animal housing: BALB/c nude mice (6–8 weeks old, male) were housed in a specific pathogen-free (SPF) environment with a 12-hour light/dark cycle, constant temperature (22±2°C), and constant humidity (50±5%). Mice had free access to standard rodent chow and sterile water [2]
- Tumor inoculation: HL-60 cells were cultured to logarithmic growth phase, trypsinized, and resuspended in PBS at a concentration of 5×10⁷ cells/mL. Each mouse was subcutaneously injected with 0.1 mL of the cell suspension (5×10⁶ cells) into the right flank [2]
- Grouping and dosing: When tumors grew to an average volume of ~100 mm³, mice were randomly divided into 3 groups (n=6/group): (1) Vehicle group: 0.9% normal saline, 0.2 mL/mouse, intraperitoneal injection, once daily; (2) Low-dose group: Acadesine 50 mg/kg, dissolved in 0.9% normal saline, 0.2 mL/mouse, intraperitoneal injection, once daily; (3) High-dose group: Acadesine 100 mg/kg, same solvent and administration route/frequency as the low-dose group. Treatment lasted for 21 days [2]
- Monitoring and sampling: Tumor volume (calculated as length × width² / 2) and body weight were measured every 3 days. Mice were monitored for survival until the endpoint (tumor volume > 1500 mm³ or severe morbidity). At the endpoint, tumors were excised for histological analysis (H&E staining) [2]
2. HFD-fed mouse metabolic experiment (from [3]): - Animal model: C57BL/6 mice (6 weeks old, male) were fed a HFD (60% fat) for 12 weeks to induce metabolic disorders. Mice were fasted for 12 hours before the experiment to measure baseline FBG [3]
- Drug formulation and dosing: Acadesine was dissolved in 5% DMSO + 95% normal saline to a concentration of 15 mg/mL. Mice were divided into 2 groups (n=6/group): (1) Vehicle group: 5% DMSO + 95% normal saline, 10 mL/kg, oral gavage, once daily; (2) Acadesine group: 150 mg/kg, 10 mL/kg, oral gavage, once daily. Treatment lasted for 14 days [3]
- Efficacy assessment: FBG was measured via tail vein blood using a glucose meter every 3 days. At the end of treatment, mice were euthanized, and liver tissue was collected to measure triglyceride content via a lipid extraction kit [3]

Biological half-life
1 week

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1. Rat pharmacokinetic parameters (cited from [3]): - Study design: Male Sprague-Dawley (SD) rats (250–300 g) were divided into two groups (n=4 per group): oral (150 mg/kg) and intravenous (50 mg/kg) Acadicine[3]
- Sample collection: Blood samples were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 12 hours after administration. Plasma was separated by centrifugation (3000×g, 4°C for 10 minutes) [3] - Key parameters: (1) Oral bioavailability: ~40%; (2) Half-life (t1/2): about 2.8 hours after oral administration and about 1.9 hours after intravenous injection; (3) Peak concentration (Cmax): about 2.5 μg/mL after oral administration (reached in 1 hour); (4) Area under the curve (AUC₀-∞): about 19.2 μg·h/mL after oral administration and about 24.5 μg·h/mL after intravenous injection [3] 2. Metabolism profile (cited from [1]): - In rat liver microsomes, acaldecine (100 μM) was metabolized to the active form 5-aminoimidazolium-4-carboxamide nucleotide (ZMP) with a conversion of about 85% after 2 hours of incubation. No other major metabolites were detected by HPLC analysis [1]

Protein Binding

Negligible (approximately 1%)

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1. Acute toxicity in mice (cited from [3]): - Female ICR mice were given a single oral dose of acadecine (150 mg/kg, 300 mg/kg, 600 mg/kg). Mice were monitored for 7 days. No deaths were observed in the 150 mg/kg and 300 mg/kg dose groups; the 600 mg/kg dose group resulted in a 20% mortality rate (1/5 of mice). Mild toxic symptoms (drowsiness, decreased appetite) in the 300 mg/kg dose group subsided within 48 hours. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels remained within the normal range [3]
2. Subchronic toxicity in rats (cited from [2]): - Male SD rats were given acadecine (50 mg/kg, 100 mg/kg) once daily via intraperitoneal injection for 28 days. No significant changes were observed in body weight, food intake, or organ weight (liver, kidney, heart). Histological examination of major organs showed no abnormal lesions, and serum blood urea nitrogen (BUN) and creatinine (Cr) levels were normal [2]. 3. Plasma protein binding rate (cited from [1]): - The plasma protein binding rate of acardine was determined by ultrafiltration (30 kDa filtration membrane). Acardine (0.1 μM, 1 μM, 10 μM) was added to rat plasma. After ultrafiltration, the concentration of acardine in the filtrate and plasma was determined by high performance liquid chromatography (HPLC). The binding rate at all concentrations was <10%, indicating that the plasma protein binding rate was extremely low [1].

[1]. Eur J Biochem. 1995 Apr 15;229(2):558-65.

[2]. Blood. 2003 May 1;101(9):3674-80.

[3]. PLoS One. 2009 Nov 18;4(11):e7889.

Pharmacodynamics
Studies have shown that acardixin selectively induces apoptosis in B cells in healthy subjects and patients with B-cell chronic lymphocytic leukemia (B-CLL), with minimal effect on T cells. Since T cells play an important role in fighting infection, it is expected that patients treated with acardixin will have a lower risk of serious infection compared to those receiving existing chemotherapy. 1. Mechanism of action (cited from [1]): Acardixin is a nucleoside analog that is converted into ZMP (5-aminoimidazole-4-carboxamide nucleotide) in cells. ZMP mimics AMP, allosterically activates AMPK, and AMPK then phosphorylates downstream substrates (e.g., ACC), thereby regulating glucose and lipid metabolism. It does not bind directly to AMPK but exerts its effects through the accumulation of intracellular ZMP [1]. 2. Applications in hematology (cited from [2]): Acardixin has been used as a tool compound to study the role of AMPK in the proliferation and survival of leukemia cells. It can induce G2/M phase arrest in HL-60 cells, which suggests its potential to enhance antitumor efficacy when used in combination with other chemotherapeutic drugs, but it has not yet entered the clinical trial stage for leukemia [2]
3. Value in metabolic disease research (cited from [3]): Because acardixin can activate AMPK and improve glucose/lipid metabolism, it is widely used in preclinical studies of type 2 diabetes and non-alcoholic fatty liver disease (NAFLD). It can be used as a positive control for AMPK activators and helps to verify the effectiveness of AMPK as a therapeutic target for metabolic disorders [3]
4. Current status of research and development (cited from [1][2][3]): Acardixin has not yet been approved for human clinical use. Due to its relatively low potency and short half-life, it is mainly used as a research tool to study AMPK-mediated signaling pathways and cellular energy metabolism, rather than as a candidate drug [1][2][3]

C9H14N4O5

258.2313

258.096

C, 41.86; H, 5.46; N, 21.70; O, 30.98

2627-69-2

AICAR phosphate;681006-28-0;AICAR-13C2,15N;1609374-70-0

17513

White to light yellow solid powder

2.1±0.1 g/cm3

726.3±60.0 °C at 760 mmHg

214-215 °C

393.1±32.9 °C

0.0±2.5 mmHg at 25°C

1.821

-2.93

5

7

3

18

330

4

O1[C@]([H])(C([H])([H])O[H])[C@]([H])([C@]([H])([C@]1([H])N1C([H])=NC(C(N([H])[H])=O)=C1N([H])[H])O[H])O[H]

RTRQQBHATOEIAF-UUOKFMHZSA-N

InChI=1S/C9H14N4O5/c10-7-4(8(11)17)12-2-13(7)9-6(16)5(15)3(1-14)18-9/h2-3,5-6,9,14-16H,1,10H2,(H2,11,17)/t3-,5-,6-,9-/m1/s1

5-amino-1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazole-4-carboxamide

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)

DMSO: ~51 mg/mL (~197.5 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.05 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (8.05 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.05 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 6mg/mL

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Solubility in Formulation 5: 110 mg/mL (425.98 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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