PKA; PDE

After PC12 cells were treated with bucladesine (dibutyryl cyclic AMP; dbcAMP), the amounts of mRNA for both choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) increased by almost four times. Bucladesine also increases PKA and ChAT activity[4].

Bucladesine infused intrahippocampally into the CA1 region of male Albino-Wistar rats has been shown to enhance spatial memory in maze tasks. When 10 μM and 100 μM bucladesine are infused bilaterally, escape latency and journey distance significantly decrease (indicating improved spatial memory). Bucladesine enhanced the preservation of spatial memory by activating PKA and inducing the cAMP/PKA pathway[1].

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Post-training intrahippocampal infusion of nicotine-bucladesine combination causes a synergistic enhancement effect on spatial memory retention in rats.[1]

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The stable cyclic adenosine monophosphate analogue, dibutyryl cyclo-adenosine monophosphate (bucladesine), is active in a model of acute skin inflammation.[2]

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Effect of bucladesine as cyclic adenosine monophosphate analog, phosphodiesterase, and protein kinase A inhibitor on acute pain.[4]

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Effect of vehicles on percutaneous absorption of bucladesine (dibutyryl cyclic AMP) in normal and damaged rat skin.[5]

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PKA assay[4]
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 [4]
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%.

The vesicular acetylcholine transporter (VAChT) gene and the choline acetyltransferase (ChAT) gene comprise the cholinergic gene locus. We have studied the coordinate regulation of these genes by cyclic AMP-dependent protein kinase (PKA) in the rat pheochromocytoma cell line PC12 and PC12 PKA-deficient mutants. Both ChAT and VAChT mRNA increased approximately fourfold after treatment of PC12 cells with dibutyryl cyclic AMP (dbcAMP). ChAT and PKA activity were also increased by dbcAMP. The basal levels of ChAT and VAChT mRNAs in the PKA-deficient cell lines were both about six times lower than in wild-type PC12 cells, and were induced less than twofold by addition of dbcAMP. H-89 and H-9, specific inhibitors for PKA, reduced ChAT and VAChT mRNA levels to approximately one-third that of untreated cells and ChAT activity to approximately one-fourth that of untreated PC12 cells. Activation of PKA type II, but not PKA type I, increased ChAT activity approximately threefold. Analysis of reporter gene constructs indicates that PKA affects gene transcription at an upstream site in the cholinergic gene locus. These results demonstrate that the expression of the ChAT and VAChT genes is regulated coordinately at the transcriptional level, and a signaling pathway specifically involving PKA II plays an important role in this process[4].

In the present study, we wished to test the hypothesis that intrahippocampal infusion of dibutyryl cyclic AMP (DB-cAMP also called bucladesine), a membrane permeable selective activator of PKA, into the CA1 region can cause an improvement in spatial memory in this maze task. Indeed, bilateral infusion of 10 and 100 microM bucladesine (but not 1 and 5 microM doses) led to a significant reduction in escape latency and travel distance (showing an improvement in spatial memory) compared to the control. Also, bilateral infusion of 0.5 microg nicotine or 1 microM bucladesine alone did not lead to an improvement in spatial memory. However, such bilateral infusion of bucladesine at 1 and 5 microM concentrations infused within minutes after 0.5 microg nicotine infusion improved spatial memory retention. Taken together, our data suggest that intrahippocampal bucladesine infusions improve spatial memory retention in male rats and that bucladesine can interact synergistically with nicotine to improve spatial memory.[1]

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In the current study, a novel water free emulsion containing bucladesine was evaluated for anti-inflammatory effects. In the arachidonic acid induced ear oedema model in mice, single or multiple administration of an emulsion containing 1.5% was capable of significantly reducing the inflammatory oedema. The data indicate that bucladesine represents an interesting treatment option for skin diseases where an anti-inflammatory activity is indicated. Due to the established clinical safety, this agent may bridge the gap between potent agents such as glucocorticoids or calcineurin inhibitors and emollients without active compounds.[2]

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Here, we studied the effect of H-89 (protein kinase A inhibitor), bucladesine (Db-cAMP) (membrane-permeable analog of cAMP), and pentoxifylline (PTX; nonspecific phosphodiesterase (PDE) inhibitor) on pain sensation. Different doses of H-89 (0.05, 0.1, and 0.5 mg/100 g), PTX (5, 10, and 20 mg/100 g), and Db-cAMP (50, 100, and 300 nm/mouse) were administered intraperitoneally (I.p.) 15 min before a tail-flick test. In combination groups, we injected the first and the second compounds 30 and 15 min before the tail-flick test, respectively. I.p. administration of H-89 and PTX significantly decreased the thermal-induced pain sensation in their low applied doses. Db-cAMP, however, decreased the pain sensation in a dose-dependent manner. The highest applied dose of H-89 (0.5 mg/100 g) attenuated the antinociceptive effect of Db-cAMP in doses of 50 and 100 nm/mouse. Surprisingly, Db-cAMP decreased the antinociceptive effect of the lowest dose of H-89 (0.05 mg/100 g). All applied doses of PTX reduced the effect of 0.05 mg/100 g H-89 on pain sensation; however, the highest dose of H-89 compromised the antinociceptive effect of 20 mg/100 g dose of PTX. Co-administration of Db-cAMP and PTX increased the antinociceptive effect of each compound on thermal-induced pain. In conclusion, PTX, H-89, and Db-cAMP affect the thermal-induced pain by probably interacting with intracellular cAMP and cGMP signaling pathways and cyclic nucleotide-dependent protein kinases.[3]

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Bucladesine, sodium N6,2'-O-dibutyryl cyclic 3',5' adenosine monophosphate (DBcAMP), which is effective for the treatment of chronic skin ulcers including decubitus ulcer, was evaluated for percutaneous absorption in rats with normal skin, stripped skin and full-thickness abrasion models. Percutaneous absorption from aqueous solution or ointment was very low in intact skin. When the aqueous solution was applied to the site where the skin had been excised, DBcAMP was absorbed very rapidly and almost completely. In the case of stripped skin, DBcAMP was absorbed rapidly but slower than in the full-thickness abrasion model. In both damages skin models, a better absorption profile was obtained with the polyethylene glycol (PEG) than the petrolatum ointment and DBcAMP was released continuously from the PEG ointment, indicating that this ointment is suitable for the treatment of ulcers of the skin. The percutaneous absorption from stripped skin was considerably influenced by the powder formulation. It is appropriate to evaluate the bioavailability in damaged skin models for a drug, such as DBcAMP, which is used in the treatment of skin ulcer.[5]

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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

rat LD50 oral >5 gm/kg
rat LD50 subcutaneous 487 mg/kg
rat LD50 intravenous 448 mg/kg
rat LD50 intraperitoneal

[1]. Post-training intrahippocampal infusion of nicotine-bucladesine combination causes a synergistic enhancement effect on spatial memory retention in rats. Eur J Pharmacol, 2007. 562(3): p. 212-20.

[2]. Mafune, E., M. Takahashi, and N. Takasugi, Effect of vehicles on percutaneous absorption of bucladesine (dibutyryl cyclic AMP) in normal and damaged rat skin. Biol Pharm Bull, 1995. 18(11): p. 1539-43.

[3]. The stable cyclic adenosine monophosphate analogue, dibutyryl cyclo-adenosine monophosphate (bucladesine), is active in a model of acute skin inflammation. Arch Dermatol Res, 2012.

[4]. Effect of bucladesine, pentoxifylline, and H-89 as cyclic adenosine monophosphate analog, phosphodiesterase, and protein kinase A inhibitor on acute pain. Fundam Clin Pharmacol. 2017 Aug;31(4):411-419.

[5]. The cholinergic gene locus is coordinately regulated by protein kinase A II in PC12 cells. J Neurochem. 1998 Sep;71(3):1118-26.

Bucladesine sodium is a 3',5'-cyclic purine nucleotide.
A cyclic nucleotide derivative that mimics the action of endogenous CYCLIC AMP and is capable of permeating the cell membrane. It has vasodilator properties and is used as a cardiac stimulant. (From Merck Index, 11th ed)
See also: Bucladesine (annotation moved to).

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We previously had shown that bilateral intrahippocampal infusion of 1 microg nicotine (but not 0.5 microg dose) led to an improvement in spatial memory retention in the Morris water maze task in male rats. We also reported that a similar type of bilateral infusion of H89, a protein kinase AII (PKA II) inhibitor, caused a deficit in spatial memory retention. In the present study, we wished to test the hypothesis that intrahippocampal infusion of dibutyryl cyclic AMP (DB-cAMP also called bucladesine), a membrane permeable selective activator of PKA, into the CA1 region can cause an improvement in spatial memory in this maze task. Indeed, bilateral infusion of 10 and 100 microM bucladesine (but not 1 and 5 microM doses) led to a significant reduction in escape latency and travel distance (showing an improvement in spatial memory) compared to the control. Also, bilateral infusion of 0.5 microg nicotine or 1 microM bucladesine alone did not lead to an improvement in spatial memory. However, such bilateral infusion of bucladesine at 1 and 5 microM concentrations infused within minutes after 0.5 microg nicotine infusion improved spatial memory retention. Taken together, our data suggest that intrahippocampal bucladesine infusions improve spatial memory retention in male rats and that bucladesine can interact synergistically with nicotine to improve spatial memory.[1]

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Anti-inflammatory therapeutic options for the topical treatment of skin diseases with inflammatory or allergic contribution are mostly limited to topical glucocorticoids and calcineurin inhibitors. Both compound classes induce adverse effects. Elevation of intracellular cyclic adenosine monophosphate (cAMP) by inhibition of phosphodiesterase 4 was shown to induce potent anti-inflammatory effects, but the safety profile of currently available compounds is not sufficient. A different approach to increase intracellular cAMP is the substitution of chemically stabilized cAMP analogues. Bucladesine is a stabilized cAMP analogue with an excellent safety profile which had been marketed as topical treatment of impaired wound healing. In the current study, a novel water free emulsion containing bucladesine was evaluated for anti-inflammatory effects. In the arachidonic acid induced ear oedema model in mice, single or multiple administration of an emulsion containing 1.5% was capable of significantly reducing the inflammatory oedema. The data indicate that bucladesine represents an interesting treatment option for skin diseases where an anti-inflammatory activity is indicated. Due to the established clinical safety, this agent may bridge the gap between potent agents such as glucocorticoids or calcineurin inhibitors and emollients without active compounds.[3]

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The aim of this study was to determine the effects of cyclic adenosine monophosphate (cAMP) and its dependent pathway on thermal nociception in a mouse model of acute pain. Here, we studied the effect of H-89 (protein kinase A inhibitor), bucladesine (Db-cAMP) (membrane-permeable analog of cAMP), and pentoxifylline (PTX; nonspecific phosphodiesterase (PDE) inhibitor) on pain sensation. Different doses of H-89 (0.05, 0.1, and 0.5 mg/100 g), PTX (5, 10, and 20 mg/100 g), and Db-cAMP (50, 100, and 300 nm/mouse) were administered intraperitoneally (I.p.) 15 min before a tail-flick test. In combination groups, we injected the first and the second compounds 30 and 15 min before the tail-flick test, respectively. I.p. administration of H-89 and PTX significantly decreased the thermal-induced pain sensation in their low applied doses. Db-cAMP, however, decreased the pain sensation in a dose-dependent manner. The highest applied dose of H-89 (0.5 mg/100 g) attenuated the antinociceptive effect of Db-cAMP in doses of 50 and 100 nm/mouse. Surprisingly, Db-cAMP decreased the antinociceptive effect of the lowest dose of H-89 (0.05 mg/100 g). All applied doses of PTX reduced the effect of 0.05 mg/100 g H-89 on pain sensation; however, the highest dose of H-89 compromised the antinociceptive effect of 20 mg/100 g dose of PTX. Co-administration of Db-cAMP and PTX increased the antinociceptive effect of each compound on thermal-induced pain. In conclusion, PTX, H-89, and Db-cAMP affect the thermal-induced pain by probably interacting with intracellular cAMP and cGMP signaling pathways and cyclic nucleotide-dependent protein kinases.[4]

C18H23N5NAO8P

491.37

491.118

C, 44.00; H, 4.72; N, 14.25; Na, 4.68; O, 26.05; P, 6.30

16980-89-5

Bucladesine calcium;938448-87-4; 362-74-3 (Bucladesine free acid)

23663967

White to off-white solid

2.201

1

11

8

33

765

4

O=C(CCC)O[C@H]1[C@H](N2C(N=CN=C3NC(CCC)=O)=C3N=C2)O[C@@](CO4)([H])[C@@]1([H])OP4([O-])=O.[Na+]

KRBZRVBLIUDQNG-JBVYASIDSA-M

InChI=1S/C18H24N5O8P.Na/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);/q;+1/p-1/t10-,14-,15-,18-;/m1./s1

sodium (4aR,6R,7R,7aR)-6-(6-butyramido-9H-purin-9-yl)-7-(butyryloxy)tetrahydro-4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-olate 2-oxide

dbcAMP; DC-2797; Dibutyryl-cAMP sodium salt; DC2797; Sodium dibutyryl cAMP; DC 2797; Bucladesine sodium; DbcAMP sodium; Actosin; Sodium Dibutyryl cAMP; 16980-89-5; bucladesine; Bucladesine sodium salt; Bucladesine (sodium); Dibutyryl-cAMP, sodium salt; Bucladesine sodium [JAN]; Dibutyryl-cAMP sodium salt; Cyclic dibutyryl-AMP sodium salt

2934.99.03.00

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, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 4.25 mg/mL (8.65 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 42.5 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: ≥ 4.25 mg/mL (8.65 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 42.5 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: ≥ 4.25 mg/mL (8.65 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 42.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10%DMSO +ddH2O: 30 mg/mL

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Solubility in Formulation 5: 100 mg/mL (203.51 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.)