- Selective inhibitor of cyclic nucleotide phosphodiesterase type III (PDE III); it inhibited human platelet PDE III with an IC50 of 1.2 μM, while showing weak inhibitory activity against PDE IV (IC50 > 100 μM) and no significant inhibition against other PDE subtypes (e.g., PDE I, PDE II) at concentrations up to 100 μM [2]

Siguazodan specifically prevents PDE, the main cyclic AMP found in human platelet supernatants, from being hydrolyzed. PDE-III, also known as cyclic GMP-inhibited PDE, is the name of the inhibited enzyme. Siguazodan was found to be more efficient than adenosine 5'-diphosphate (ADP) and collagen in inhibiting U46619-induced aggregation in platelet-rich plasma (PRP). The effectiveness of watermelon zodan is not affected by treating PRP with aspirin. Siguazodan lowers cytoplasmic free calcium and raises cyclic AMP levels in platelets that have been cleaned. Siguazodan's reduced efficacy in suppressing the ADP response may be explained by the fact that ADP lessens its capacity to increase cyclic AMP. Ciguazodan is an inotrope and a vasodilator that has antiplatelet actions within the same dosage range [2].

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1. Effect on human platelet aggregation: Siguazodan concentration-dependently inhibited platelet aggregation induced by various agonists in human platelet-rich plasma (PRP). At a concentration of 10 μM, it inhibited ADP (5 μM)-induced aggregation by approximately 50%; at 100 μM, it almost completely suppressed aggregation induced by ADP (5 μM), collagen (2 μg/ml), and adrenaline (10 μM). Notably, it did not affect the initial shape change of platelets induced by these agonists [2]
2. Effect on intracellular cyclic AMP (cAMP) levels: Siguazodan concentration-dependently increased intracellular cAMP levels in human platelets. In PRP pre-labeled with [³H]-adenine, treatment with 10 μM Siguazodan for 5 minutes elevated platelet cAMP levels by ~2-fold compared to the control group; at 100 μM, the cAMP level was increased by ~3.5-fold [2]
3. Selectivity for PDE subtypes: In human platelet homogenates, Siguazodan showed high selectivity for PDE III. It had no significant inhibitory effect on PDE I (IC50 > 100 μM) or PDE II (IC50 > 100 μM), and only weak inhibition on PDE IV (IC50 > 100 μM), confirming its role as a selective PDE III inhibitor [2]

Siguazodan is an oral medication that acts for a long time in conscious dogs and is a strong and selective phosphodiesterase III inhibitor with inotropic and vasodilatory effects in a range of experimental animals [2].

1. PDE III activity assay in human platelets:
- First, human platelets were isolated from venous blood (anticoagulated with citrate) by centrifugation (300×g for 10 minutes to obtain PRP, then 1500×g for 20 minutes to pellet platelets). Platelet pellets were resuspended in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, and 1 mM dithiothreitol (DTT), then homogenized and centrifuged (10,000×g for 15 minutes at 4°C) to collect the supernatant (containing soluble PDE III).
- The reaction mixture (total volume 200 μl) included the platelet supernatant (enzyme source), 1 μM [³H]-cAMP (substrate), and different concentrations of Siguazodan (0.01–100 μM). The mixture was incubated at 37°C for 15 minutes, and the reaction was terminated by adding 50 μl of 5% trichloroacetic acid (TCA).
- After centrifugation (12,000×g for 5 minutes), the supernatant was applied to a Dowex 50W-X8 resin column (H⁺ form). Unhydrolyzed [³H]-cAMP was eluted with distilled water, and the radioactivity was measured using a liquid scintillation counter.
- The inhibitory rate of Siguazodan on PDE III activity was calculated by comparing the radioactivity of the test group with the control group (without Siguazodan), and the IC50 value was determined by nonlinear regression analysis [2]

1. Human platelet aggregation assay:
- Venous blood was collected from healthy volunteers (without recent use of antiplatelet drugs) and anticoagulated with 3.8% sodium citrate (blood:citrate = 9:1). PRP was prepared by centrifugation at 300×g for 10 minutes, and platelet-poor plasma (PPP) was obtained by further centrifugation of the remaining blood at 1500×g for 20 minutes. The platelet concentration in PRP was adjusted to 2×10⁸ platelets/ml using PPP.
- Platelet aggregation was measured using the Born turbidimetric method in an aggregometer. Briefly, 250 μl of PRP was preheated at 37°C for 2 minutes, then different concentrations of Siguazodan (0.1–100 μM) were added and incubated for 5 minutes. Subsequently, an aggregation agonist (ADP: 5 μM; collagen: 2 μg/ml; adrenaline: 10 μM) was added, and the aggregation curve was recorded for 5 minutes.
- The maximum aggregation rate was calculated by taking the turbidity of PPP as 100% (no aggregation) and the turbidity of PRP before adding the agonist as 0% (baseline). The inhibitory effect of Siguazodan on aggregation was expressed as the percentage reduction in the maximum aggregation rate compared to the agonist-only control [2]
2. Intracellular cAMP measurement in human platelets:
- PRP was labeled with 2 μCi/ml [³H]-adenine at 37°C for 2 hours to incorporate the radioactive precursor into intracellular adenine nucleotides. After labeling, platelets were washed and resuspended in a buffer containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 12 mM NaHCO₃, and 5.6 mM glucose (pH 7.4).
- Different concentrations of Siguazodan (0.1–100 μM) were added to the platelet suspension, which was then incubated at 37°C for 5 minutes. The reaction was stopped by adding 1 ml of ice-cold 70% ethanol, and platelets were lysed by vortexing and standing on ice for 10 minutes.
- The lysate was centrifuged (12,000×g for 5 minutes), and the supernatant was evaporated to dryness under nitrogen. The residue was dissolved in distilled water and applied to a Dowex 50W-X8 resin column (Na⁺ form) to separate cAMP from other nucleotides. cAMP was eluted with distilled water, and the radioactivity was measured by liquid scintillation counting. The intracellular cAMP level was expressed as the percentage of total [³H]-adenine nucleotides incorporated into platelets [2]

[1]. Photoaffinity labelling of cyclic GMP-inhibited phosphodiesterase (PDE III) in human and rat platelets and rat tissues: effects of phosphodiesterase inhibitors. Eur J Pharmacol. 1994 Jun 15;268(1):105-14.

[2]. The effects of siguazodan, a selective phosphodiesterase inhibitor, on human platelet function. Br J Pharmacol. 1990 Mar;99(3):612-6.

[3]. Phosphodiesterase inhibitors suppress alpha2-adrenoceptor-mediated 5-hydroxytryptamine release from tracheae of newborn rabbits. Eur J Pharmacol. 1998 Jul 31;354(1):67-71.

1-Cyano-2-methyl-3-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine is a pyridazinone compound.

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1. Custardine is a selective PDE III inhibitor that exerts its biological effects by inhibiting the hydrolysis of intracellular cAMP, thereby increasing intracellular cAMP levels. In platelets, elevated cAMP levels are associated with reduced platelet activation and aggregation, suggesting that cusadine may have potential application value in the prevention or treatment of thromboembolic diseases [2]
2. This study confirms that the antiplatelet effect of cusadine is mediated by its selective inhibition of PDE III, as its inhibitory activity on platelet aggregation is closely related to its ability to increase intracellular cAMP levels and inhibit PDE III activity [2]

C14H16N6OM.W.

284.31644

284.139

C, 59.14; H, 5.67; N, 29.56; O, 5.63

115344-47-3

72124

White to off-white solid powder

1.31g/cm3

268-268.9ºC(lit.)

1.655

1.37

3

4

4

21

495

0

C(N/C(/NC)=N\C1C=CC(C2C(C)CC(=O)NN=2)=CC=1)#N

NUHPODZZKHQQET-UHFFFAOYSA-N

InChI=1S/C14H16N6O/c1-9-7-12(21)19-20-13(9)10-3-5-11(6-4-10)18-14(16-2)17-8-15/h3-6,9H,7H2,1-2H3,(H,19,21)(H2,16,17,18)

1-cyano-2-methyl-3-[4-(4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl)phenyl]guanidine

Siguazodan; SK&F 94836; SKF 94836; SKF-94836; SKF94836;

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 : ~12.5 mg/mL (~43.96 mM)

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.

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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).

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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)

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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).

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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

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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.

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