Phosphodiesterase 5 (PDE5)

The previous studies showed that the phosphodiesterase-5 (PDE5) inhibitor sildenafil inhibited the microglial activation induced by lipopolysaccharide (LPS). However, whether yonkenafil, a novel PDE5 inhibitor, also inhibits microglial activation and the underlying mechanism of inhibition remain elusive. Here we found that yonkenafil significantly suppressed the production of NO, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) and the protein expression of inducible NO synthase (iNOS) induced by LPS in microglial cells in a concentration-dependent manner. Knockdown of PDE5 inhibits NO and iNOS protein expression in LPS-stimulated N9 microglia. Moreover, we observed that the nuclear factor-κB (NF-κB) transcriptionally upregulated PDE5 expression, which was inhibited by sildenafil and yonkenafil in LPS-stimulated N9 microglia. Therefore, sildenafil and yonkenafil may exert their inhibitory effects on microglial activation by reducing the expression of PDE5. Furthermore, sildenafil and yonkenafil increased the cyclic guanosine monophosphate (cGMP) level in N9 microglia, and 8-Br-cGMP, an analogue of cGMP, downregulates extracellular signal-regulated kinases 1 and 2 (ERK1/2)/the NF-κB pathway, suggesting that sildenafil and yonkenafil inhibit microglial activation by decreasing PDE5 expression and increasing the cGMP level. Importantly, sildenafil and yonkenafil significantly alleviated the death of SH-SY5Y neuroblastoma cells and primary cortical neurons induced by the conditioned medium from activated microglia. Together, these findings position PDE5 as a potential therapy target for the treatment of neuroinflammation accompanied by microglial activation. [2]

Yonkenafil (4–32 mg/kg, intravenously administered daily for seven days) has been shown to improve post-stroke behavioral outcomes, decrease the amount of cerebral infarct, prevent neuronal death, and markedly increase the expression deficits of NGF/TrkA and BDNF/TrkB. Blood brain synaptic function[1].
Yonkenafil is a novel phosphodiesterase type 5 (PDE5) inhibitor. Here we evaluated the effect of yonkenafil on ischemic injury and its possible mechanism of action. Male Sprague–Dawley rats underwent middle cerebral artery occlusion, followed by intraperitoneal or intravenous treatment with yonkenafil starting 2 h later. Behavioral tests were carried out on day 1 or day 7 after reperfusion. Nissl staining, Fluoro-Jade B staining and electron microscopy studies were carried out 24 h post-stroke, together with an analysis of infarct volume and severity of edema. Levels of cGMP-dependent Nogo-66 receptor (Nogo-R) pathway components, hsp70, apaf-1, caspase-3, caspase-9, synaptophysin, PSD-95/neuronal nitric oxide synthases (nNOS), brain-derived neurotrophic factor (BDNF)/tropomyosin-related kinase B (TrkB) and nerve growth factor (NGF)/tropomyosin-related kinase A (TrkA) were also measured after 24 h. Yonkenafil markedly inhibited infarction and edema, even when administration was delayed until 4 h after stroke onset. This protection was associated with an improvement in neurological function and was sustained for 7 d. Yonkenafil enlarged the range of penumbra, reduced ischemic cell apoptosis and the loss of neurons, and modulated the expression of proteins in the Nogo-R pathway. Moreover, yonkenafil protected the structure of synapses and increased the expression of synaptophysin, BDNF/TrkB and NGF/TrkA. In conclusion, yonkenafil protects neuronal networks from injury after stroke. [1]

cGMP Formation [2]
In order to determine whether sildenafil or yonkenafil promotes cGMP formation, intracellular cGMP was measured using an enzyme-linked immunoassay kit. N9 microglial cells were cultured in six-well plates and upon reaching confluence were treated with sildenafil (10–100 μM) or yonkenafil (3–10 μM) for 16 h. After incubation, media were removed and 200 ml of 0.1 NHCl containing IBMX added to the cells and incubated for 20 min to extract cGMP. cGMP concentrations were then measured according to the manufacturer’s instructions. The protein content was determined by the bicinchoninic acid (BCA) protein assay kit and cGMP levels expressed as femtomole milligram per protein.

Microglial-Conditioned Medium Preparation, Treatments, and Detection [2]
N9 microglial cells were pretreated with different concentrations of sildenafil (10–100 μM) or yonkenafil (3–30 μM) for 2 h then stimulated with LPS (1 μg/ml) for 48 h. The culture medium was collected as conditioned medium (CM) and clarified by centrifugation at 12,000 g for 5 min to remove cellular debris. The conditioned medium was then transferred to neuronal cells, which were further incubated at 37 °C for 24 h. Cell viability was measured by MTT assay.

Animal/Disease Models: Male SD (Sprague-Dawley) Rat[1]
Doses: 4, 8, 16 and 32 mg/kg
Route of Administration: iv daily for 7 days
Experimental Results: Induced a dose-dependent decrease in infarct volume, with an ED50 of 12.27 mg/kg. Increased hsp70 expression, diminished apaf-1 expression, and inhibited caspase-3 and caspase-9 cleavage. Dramatically prevented neuronal damage and increases the number of surviving neurons after stroke. Prevented decrease in synaptophysin levels and increase in PSD-95 and nNOS levels.

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Yonkenafil (yonkenafil hydrochloride) was dissolved in normal saline and administered by the intraperitoneal (i.p.) or intravenous (i.v.) route. [1]
For the beam walking and rotarod tests, animals were randomly assigned to the following experimental treatment groups (Table 1): a sham-operated group (natural saline, 10 ml/kg daily for 7 d); an MCAO group (rats suffered I/R and normal saline daily for 7 d); and an MCAO + yonkenafil group (rats suffered I/R, followed by i.p. yonkenafil 16 mg/kg daily for 7 d). [1]
For the other tests, the remaining animals were randomly assigned to the following experimental treatment groups (Table 1): a sham-operated group (natural saline, 10 ml/kg); an MCAO group (rats suffered I/R and normal saline); MCAO + yonkenafil groups (rats suffered I/R, followed by i.v. yonkenafil 4, 8, 16 and 32 mg/kg starting 2 h later in the dose–response experiment; rats suffered I/R, followed by i.v. yonkenafil 16 mg/kg starting 2 h, 4 h or 6 h later in the therapeutic-time window experiment; rats suffered I/R, followed by i.v. yonkenafil 16 mg/kg starting 2 h later in the transmission electron microscopy experiment; rats suffered I/R, followed by i.v. yonkenafil 8, 16 and 32 mg/kg starting 2 h later for the other experiments); and MCAO + inhibitor + yonkenafil groups (rats suffered I/R and inhibitors were administered immediately after MCAO, followed by yonkenafil 2 h later).

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Behavioral testing [1]
Neurological functional deficits were evaluated 22 h after reperfusion using a modified six-point scoring method applied by an investigator who was blinded as to the experimental treatment groups (Minematsu et al., 1992). The scale was: 0: no neurological deficit; 1: failure to extend the forepaw fully; 2: circling; 3: falling to one side; 4: no spontaneous walking with a depressed level of consciousness; and 5: dead. Two tests were used to evaluate the behavioral outcome: a beam walking test and a rotarod test after administration of yonkenafil (or vehicle) for 7 d consecutively.

A novel method for the quantitation of yonkenafil, a new synthetic phosphodiesterase V inhibitor, in rat plasma using high-performance liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed. The analyte and internal standard (diazepam) were extracted from plasma (100 microl) by liquid-liquid extraction and separated on a C18 column using 10mM ammonium acetate buffer: methanol (15:85, v/v) as mobile phase in a run time of 3.0 min. The detector was a Q-trap mass spectrometer with an ESI interface operating in the multiple reaction monitoring (MRM) mode. The assay was linear over the concentration range 1.0-1000 ng/ml with a limit of detection of 0.20 ng/ml. Intra- and inter-day precision (as relative standard deviation) were both within 8.45% with good accuracy. The method was successfully applied to a preclinical pharmacokinetic study of yonkenafil in rat after sublingual, oral and intravenous administration. The results demonstrate that the sublingual route gives a higher bioavailablity than the oral route and may represent a useful alternative route of yonkenafil administration. J Pharm Biomed Anal. 2008 Aug 5;47(4-5):985-9.

[1]. Yonkenafil: a novel phosphodiesterase type 5 inhibitor induces neuronal network potentiation by a cGMP-dependent Nogo-R axis in acute experimental stroke. Exp Neurol. 2014 Nov;261:267-77.

[2]. NF-κB Upregulates Type 5 Phosphodiesterase in N9 Microglial Cells: Inhibition by Sildenafil and Yonkenafil. Mol Neurobiol. 2016 May;53(4):2647-58.

In conclusion, we have demonstrated that yonkenafil protects against the effects of ischemic injury, ranging from neurological deficits to edema. Yonkenafil protects neuronal networks during stroke by increasing neuronal survival and enhancing the connections between neurons, and this is mediated by cGMP-dependent Nogo-R, BDNF/TrkB, and NGF/TrkA pathways. [1]

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In summary, the present study showed that sildenafil and yonkenafil downregulated LPS-induced PDE5 expression through NF-κB transcriptional regulation to elevate cGMP levels, which suppressed the pro-inflammatory factors through the ERK1/2/NF-κB pathway. Moreover, sildenafil and yonkenafil exerted protective effects against microglial-mediated neuron injury. Considering these results, sildenafil and yonkenafil may represent a potential new source of drugs for the treatment of neuroinflammation accompanied by microglial activation. [2]

C24H33N5O4S

487.614924192429

487.225

C, 59.12; H, 6.82; N, 14.36; O, 13.12; S, 6.57

804518-63-6

Yonkenafil hydrochloride;804519-64-0;Yonkenafil-d8;Yonkenafil-d7

135489224

Off-white to light yellow solid powder

2.3

1

7

8

34

852

0

C1(C2=CC(S(N3CCN(CC)CC3)(=O)=O)=CC=C2OCC)NC(=O)C2C(C)=CN(CCC)C=2N=1

RXMDFMQMRASWOG-UHFFFAOYSA-N

InChI=1S/C24H33N5O4S/c1-5-10-28-16-17(4)21-23(28)25-22(26-24(21)30)19-15-18(8-9-20(19)33-7-3)34(31,32)29-13-11-27(6-2)12-14-29/h8-9,15-16H,5-7,10-14H2,1-4H3,(H,25,26,30)

2-[2-ethoxy-5-(4-ethylpiperazin-1-yl)sulfonylphenyl]-5-methyl-7-propyl-3H-pyrrolo[2,3-d]pyrimidin-4-one

Yonkenafil; 804518-63-6; Tunodafil; 2-(2-Ethoxy-5-((4-ethylpiperazin-1-yl)sulfonyl)phenyl)-5-methyl-7-propyl-3H-pyrrolo[2,3-d]pyrimidin-4(7H)-one; Tunodafil [INN]; L9U6QT7F76; 2-[2-ethoxy-5-(4-ethylpiperazin-1-yl)sulfonylphenyl]-5-methyl-7-propyl-3H-pyrrolo[2,3-d]pyrimidin-4-one; 2-[2-Ethoxy-5-(4-ethyl-piperazine-1-sulfonyl)-phenyl]-5-methyl-7-propyl-3,7-dihydro-pyrrolo[2,3-d]pyrimidin-4-one 804519-64-0 HCl 804520-62-5 2HCl;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO: 100 mg/mL (205.08 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.13 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 25.0 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: ≥ 2.5 mg/mL (5.13 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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