AMPK; Autophagy; Mitophagy; Human Endogenous Metabolite

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]

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]

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].

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].

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.

Biological Half-Life
1 week

Protein Binding
Negligible (approximately 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
Acadesine has been shown to induce cell death apoptosis selectively in B-cells taken from healthy subjects and patients with B-CLL, with little effect on T-cells. As T-cells have an important role in fighting infection, it is anticipated that patients treated with acadesine will have a reduced risk of serious infections compared to those on current chemotherapies.

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.)