PKA; PDE
PKA (cAMP-dependent protein kinase), PDE (phosphodiesterase).

Bucladesine sodium (also known as Dibutyryl-cAMP) is a cell-permeable PKA activator and a cAMP analog that mimics the action of endogenous cAMP. It is a cyclic nucleotide derivative (structurally similar to cAMP) and is also a phosphodiesterase inhibitor. Dibutyryl-cAMP preferentially activates cAMP-dependent protein kinase. The products releaes butyrate due to intracellular and extracellular esterase action. Butyrate was shown to have distinct biological effects. The compound is used in a wide variety of research applications because it mimics cAMP and can induce normal physiological responses when added to cells in experimental conditions.

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Kinase Assay: Bucladesine sodium (also known as Dibutyryl-cAMP) is a cell-permeable PKA activator and a cAMP analog that mimics the action of endogenous cAMP. It is a cyclic nucleotide derivative (structurally similar to cAMP) and is also a phosphodiesterase inhibitor. Dibutyryl-cAMP preferentially activates cAMP-dependent protein kinase.

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Bucladesine is a potent and cell-permeable PKA activator and a cAMP analog that mimics the action of endogenous cAMP. It preferentially activates cAMP-dependent protein kinase. It is also a phosphodiesterase inhibitor, preventing the breakdown of cAMP. The compound's in vitro activity is characterized by its ability to activate PKA and increase intracellular cAMP levels, leading to downstream signaling effects.

Experimentally naïve male Albino Swiss mice (Laboratory Animals Breeding, Słaboszów, Poland) weighing 20–30 g were used in all experiments. The animals were housed in polycarbonate cages at a controlled temperature (23–25°C) and humidity (50–60%) with 12 h light/dark cycle (lights on at 6 h). Tap water and food pellets were available ad libitum. All experiments were performed after at least 7 days of acclimatization. The experimental protocols were approved by the Ethical Committee of the Medical University in Lublin (license number 40/2010). To allow for skin penetration of the cream formulations and based on preliminary experiments with ketoprofen cream in this model (data not presented), a pre-treatment time of 3 h was selected. To evaluate the effect of 0.5 or 1.5% bucladesine cream, an amount of 10 ± 1 mg cream each was spread onto the outer surface of both, left and right ears using small spatula. A third group of mice was administered with 2.5% ketoprofen gel. Corresponding vehicle treatment groups were administered either the respective cream base without bucladesine or, as reference for ketoprofen, a cosmetic night cream. To evaluate the effect of repeated administration, four separate groups of mice were dosed twice, i.e., 7 and 3 h before administration of arachidonic acid with the 0.5 and 1.5% cream or corresponding vehicle. Arachidonic acid or actetone were administered 3 h after the second cream administration onto left and right ears, respectively. The ear thickness was measured with a precise spring-loaded caliper (Mitutoyo, type 7309, Kawasaki, Japan) before and 60 min after application of arachidonic acid or vehicle.

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Bucladesine has vasodilator properties and is used as a cardiac stimulant. By activating PKA and increasing cAMP levels, it modulates various physiological processes including smooth muscle relaxation and cardiac contractility. It has been used in research to study cAMP-mediated signaling pathways and their effects on cellular function.

PKA assay[2]
Cells were washed twice with 10 mM sodium phosphate buffer, pH 7.4, 0.15 M NaC1, and then scraped from the culture plate in 1 ml of the same buffer. The cells were collected by centrifugation, and then homogenized by brief sonication in cell homogenization buffer [50 mMTris-HC1, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol (DTT), 50 mM leupeptin, and 0.1 mM phenylmethylsulfonyl fluorideI. The particulate fraction was removed by centrifugation in a microcentrifuge at 14,000 rpm at 4°Cfor 20 mm. PKA activity was measured in the supernatant by the method ofRoskoski (1983), using the synthetic peptide substrate Leu-Arg-ArgAla-Ser-Leu-Gly (Kemptide). The reaction mixture of 50 ~.tlcontained cell lysate and a final concentration of 25 mM Tris-HC1 buffer (pH7.4), 5 mM magnesium acetate, 5 mM DTT, 5 mM cAMP, 20 ,~iMKemptide, 0.25 mM isobutylmethylxanthine, and 0.1 mM [y- 32P I ATP (200 cpm/pmol), and, when added, 20 ,uM PKA peptide inhibitor 5-24. Reactions were incubatedfor 10 mm at 30°Candterminated by addition of 50 j.tl of 7.5 mM phosphoric acid. Fifty microliters of the reaction mixture was spotted onto a P81 filter and washed five times with 75 mM phosphoric acid and counted as previously described. The difference in activity in the presence versus absence of PKA peptide inhibitor 5-24 was used to calculate PKA activity.

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PKC assay [2]
Cell lysates were prepared as described for thePKA assay. The reaction mixture of 50 j.el contained cell lysate and a final concentration of 25 mM Tris-HC1 buffer (pH 7.4), 5 mM magnesium acetate, 5 mM DTT, 20 ~.tM synthetic substrate (Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-LysLys), 0.25 mM isobutylmethyixanthine, and 0.1 mM [y32p] ATP (200 cpm/pmol). Reactions were incubated for 10 mm at 30°C,terminated with phosphoric acid, and analyzed as described for the PKA assay. As a control, the specific PKC peptide inhibitor 19-36, at 20 1.tM was used and shown to inhibit the activity in cell extracts by >90%.

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In vitro assays for studying PKA activation are performed using purified PKA enzyme and a peptide substrate to measure kinase activity. cAMP levels can be measured in cell lysates using ELISA or radioimmunoassay. Phosphodiesterase inhibition assays are performed using purified PDE enzymes and a fluorescent or radioactive substrate to determine IC50 values for PDE inhibition.

Cell-based assays are conducted to evaluate Bucladesine's effects on cAMP signaling. Cells are treated with the compound, and intracellular cAMP levels are measured. PKA activity is assessed by measuring the phosphorylation of PKA substrates by Western blot. The compound's effects on cell proliferation, differentiation, and gene expression are evaluated in various cell types.

For topical administration of bucladesine as 5% solution, 20 μl of drug or vehicle solution was administered onto the outer surface of both, left and right ears each, 60 min prior to arachidonic acid challenge. The inflammatory response was induced by administration of 20 μl arachidonic acid (Sigma-Aldrich, Munich, Germany; 5% in acetone) on the outer surface of left ears. The right ears were treated with acetone only to determine the individual differences in ear thicknesses.Na ve male Albino Swiss mice

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In vivo studies have been conducted to evaluate Bucladesine's vasodilator and cardiac stimulant effects. Animal models of cardiovascular disease are used to assess the compound's effects on blood pressure, heart rate, and cardiac function. The compound is typically administered via intravenous or intraperitoneal routes. Detailed in vivo protocols are not provided in the available literature.

[1]. Exp Neurol.2001 Jan;167(1):59-64.
[2]. J Neurochem. 1998 Sep;71(3):1118-26.

Buchadesine is a 3',5'-cyclic purine nucleotide, a 2'-butyrate and 6-n-butyryl derivative of 3',5'-cyclic adenosine monophosphate (cAMP). It has agonist, vasodilator, and cardiotonic effects. It is a butyrate nucleotide, a 3',5'-cyclic purine nucleotide, belonging to the butyramide class of compounds. Its function is related to 3',5'-cyclic adenosine monophosphate (cAMP). It is a cyclic nucleotide derivative that mimics the effects of endogenous cAMP and can cross cell membranes. It has vasodilatory effects and can be used as a cardiac stimulant. (Excerpt from Merck Index, 11th edition)

C18H24N5O8P

469.39

469.136

C, 46.06; H, 5.15; N, 14.92; O, 27.27; P, 6.60

362-74-3

16980-89-5 (sodium);362-74-3;

9687

Typically exists as solids at room temperature

1.7±0.1 g/cm3

203 - 205 °C

1.717

1.763

173.8

2

11

8

32

759

4

CCCC(NC1N=CN=C2N(C3O[C@@H]4COP(O[C@H]4[C@H]3OC(CCC)=O)(O)=O)C=NC=12)=O

CJGYSWNGNKCJSB-YVLZZHOMSA-N

InChI=1S/C18H24N5O8P/c1-3-5-11(24)22-16-13-17(20-8-19-16)23(9-21-13)18-15(30-12(25)6-4-2)14-10(29-18)7-28-32(26,27)31-14/h8-10,14-15,18H,3-7H2,1-2H3,(H,26,27)(H,19,20,22,24)/t10-,14-,15-,18-/m1/s1

[(4aR,6R,7R,7aR)-6-[6-(butanoylamino)purin-9-yl]-2-hydroxy-2-oxo-4a,6,7,7a-tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-yl] butanoate

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)