PDE5/phosphodiesterase type 5 (IC50 = 0.23 nM)

T-0156 at 10 and 100 nM increases cGMP levels, causing relaxation of the tissue in the isolated rabbit corpus cavernosum[1].

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Inhibition of various phosphodiesterase isozymes by T-0156 [1]
T-0156 exhibited strong inhibition of the cGMP hydrolytic activity of phosphodiesterase type 5 (Fig. 2A), with an IC50 of 0.23 nM (Table 1). T-0156 also inhibited phosphodiesterase type 6 with IC50 value of 56 nM, which was 240-fold higher than that for inhibition of phosphodiesterase type 5. The cAMP hydrolytic activity of phosphodiesterase type 4 was inhibited weakly by T-0156 with IC50 value of 63 μM. T-0156 showed weak inhibitions of the cGMP hydrolytic activity of phosphodiesterase types 1 and 2 as well as the cAMP hydrolytic activity of phosphodiesterase type 3 (IC50>100 μM).

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Kinetic analysis of the effect of T-0156 on phosphodiesterase type 5 [1]
To elucidate the mechanism underlying the inhibition by T-0156, Lineweaver–Burk plots were constructed at concentrations of 0.51 to 6.01 μM cGMP substrate (Fig. 2B). The phosphodiesterase type 5 partially purified from canine lung had a cGMP Km value of 6.2 μM, and T-0156 inhibited the phosphodiesterase type 5 activity in a competitive manner with respect to cGMP hydrolysis. The inhibition constant (Ki value) of T-0156 for phosphodiesterase type 5, calculated by drug concentration vs. slope replots, was 0.22±0.08 nM.

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Effects of T-0156 on radioligand binding and on enzymes activities [1]
T-0156 at a high concentration of 10 μM inhibited [3H]CGP-39653 binding to the glutamate NMDA receptor and the activity of calpain, a Ca2+-activated neutral protease, (percentage of inhibition: 32% and 24%, respectively). T-0156 at 10 or 100 μM did not inhibit either binding of several radioligands bindings or activities of several enzymes as summarized in Table 2.

Effects of T-0156 on isometric tension and cyclic nucleotide levels in phenylephrine-precontracted rabbit corpus cavernosum [1]
T-0156 at 10 or 100 nM caused an increment in cGMP levels. The isometric tension of corpus cavernosum decreased with cGMP elevation (Fig. 3). The increase in cGMP levels by T-0156 at a concentration of 100 nM was approximately five-fold higher than that of vehicle (T-0156-treated: 6.0±1.5 pmol/mg protein, vehicle-treated: 1.1±0.4 pmol/mg protein, P<0.05). In contrast, cAMP levels were not altered by T-0156 (100 nM T-0156-treated: 25.0±8.7 pmol/mg protein, vehicle-treated: 13.3±3.7 pmol/mg protein, P=0.48).

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The potentiation of electrical field stimulation-induced relaxation by T-0156 in phenylephrine-precontracted rabbit corpus cavernosum [1]
Fig. 4 shows typical tracings of the influence of T-0156 on electrical field stimulation-induced relaxation in the rabbit corpus cavernosum. T-0156 at 1 to 100 nM produced a concentration-dependent potentiation of electrical field stimulation-induced relaxation (Fig. 5). The potentiation by T-0156 at a concentration of 100 nM was statistically significant (T-0156-treated: 76.9±19.8%, vehicle-treated: 12.3±10.1%, P<0.05).

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The potentiation of pelvic nerve stimulation-induced tumescence by T-0156 in anesthetized dogs [1]
Intraduodenal administration of T-0156 at 100 to 1000 μg/kg potentiated a pelvic nerve stimulation-induced tumescence in a dose-dependent manner and the potentiations at 300 and 1000 μg/kg were statistically significant (Fig. 6). The maximal potentiating effect of T-0156 was observed 10 to 30 min after the intraduodenal administration, and the potentiation was sustained at least for 90 min.

Receptor binding and enzyme assays [1]
The effects of T-0156 (10 or 100 μM) on radioligand bindings and on enzyme activities except for phosphodiesterase were assessed by MDS Pharma Services.

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Phosphodiesterase assay [1]
The phosphodiesterase assay was performed by the radiolabeled nucleotide method (Thompson et al., 1979). Assay buffer contained 50 mM Tris–HCl, pH 8.0, 5 mM MgCl2, 4 mM 2-mercaptoethanol, 0.33 mg/ml of bovine serum albumin, 0.1 to 30 μl of enzyme solution, unlabeled cGMP or cAMP, and 12.5 nM [3H]cGMP or 4.88 nM [3H]cAMP. The reaction was started by mixing the substrate into 500 μl of the assay buffer, and tubes were incubated at 37 °C for 30 min. After boiling for 1.5 min, the mixtures were added to 100 μl of a 1 mg/ml solution of Crotalus atrox snake venom and incubated at 37 °C for 30 min. The reaction was stopped by the addition of 500 μl methanol, and the resultant solutions were applied to a Dowex (1×8–400) column (volume 0.25 ml). Aqueous scintillation fluid was added to each eluate, and the radioactivity was measured. For studies of inhibition of phosphodiesterase activities, inhibitor was added to the assay buffer containing enzyme and preincubated for 5 min before reactions were initiated by the addition of substrate.

Relaxation and cyclic nucleotide levels in rabbit corpus cavernosum [1]
\nThe preparation of rabbit corpus cavernosum in the organ bath was contracted with phenylephrine (5 μM). T-0156 (10 or 100 nM) was then added to the preparation. After attainment of the maximal relaxant response to T-0156, the preparation was frozen immediately by liquid N2. The frozen preparation was homogenized with in 1 ml of 6% trichloroacetic acid containing EDTA (1 mM). After centrifugation (5000 rpm, for 15 min, 4 °C), the supernatant was extracted with water-saturated diethyl ether, and aliquots of the aqueous phase were lyophilized to dryness and then reconstituted in 1 ml of 50 mM sodium acetate buffer (pH 6.2). The pellet was dissolved in 0.5 ml of 2 N NaOH, and used in the measurement of protein content. The cyclic nucleotide and protein contents in each solution were measured with commercially available cGMP and cAMP immunoassay kits and BCA protein assay kit, respectively. The relaxation of the preparation induced by T-0156 was expressed as the percentage of the magnitude of phenylephrine-induced contraction. The cyclic nucleotide levels were expressed in pmol per mg protein.\n

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\n\nElectrical field stimulation-induced relaxation [1]
\nElectrical field stimulation-induced relaxation of the preparation of rabbit corpus cavernosum was performed according to the method of Takagi et al. (2001). Atropine and guanethidine were added to each organ bath chamber (1 and 5 μM, respectively) before contraction of the preparations by adding phenylephrine (5 μM). After the phenylephrine contractile response was stabilized, the preparation was subjected to electrical field stimulation (20 V, 0.2-ms pulse duration, for 40 s)-induced relaxation at intervals of 10 min. The stimulatory condition was selected by changing the frequency (1 to 16 Hz) in order to obtain approximately 10% relaxation of the phenylephrine-precontracted preparation. Electrical field stimulation was performed with the platinum electrode set up on both sides of the tissue strips and an electrical stimulator and a power booster (PB 401; Physio-Tech; Tokyo, Japan). After the electrical field stimulation-induced relaxation at an interval of 10 min was stabilized, T-0156 (1 to 100 nM) or vehicle was added to the preparation at intervals of 30 min. Papaverine was added to each organ bath chamber (100 μM) to confirm the maximal relaxation of the preparation at the end of experiment. The potentiation was expressed as percentage of the amplitude of the relaxation response before treatment with T-0156 or vehicle.\n

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\n\nPelvic nerve stimulation-induced tumescence in anesthetized dogs [1]
\nPelvic nerve stimulation-induced tumescence in anesthetized dogs was performed according to the methods of Noto et al. (2000). Dogs weighting 11.4 to 18.7 kg were anesthetized with sodium pentobarbital (30 mg/kg i.v. bolus injection, followed by 4.5 mg/kg/h i.v. infusion). An endotracheal tube was placed to ventilate (15 ml/kg/stroke, 20 strokes/min) with room air. The abdomen was opened through a midline abdominal incision. A polythene catheter was inserted into the duodenum and held in place by a ligature. The left pelvic nerve, located superior and lateral to the prostate was carefully isolated and placed on a bipolar electrode. A 21-gauge venous needle was placed in the corpus cavernosum on the left side and used for recording intracavernous pressure. The pelvic nerve was stimulated by electrical square pulse (10 V, 0.2-ms pulse duration, for 40 s) at frequencies from 2.5 to 20 Hz at intervals of 20 min. All experiments were started when the submaximal nerve stimulation evoked consistent responses. T-0156 or vehicle was administered intraduodenally (100, 300 or 1000 mg/kg) and the effect of T-0156 on the pelvic nerve stimulation-induced tumescence was assessed 10, 30, 50, 70 and 90 min after T-0156-treatment. For quantitative determination of the tumescence, we measured the area under the curve and expressed it as millimeters of mercury multiplied by minutes. The potentiation was expressed as percentage of the pelvic nerve stimulation-induced tumescence before treatment with T-0156 or vehicle.\n\n

[1]. Enzymological and pharmacological profile of T-0156, a potent and selective phosphodiesterase type 5 inhibitor. Eur J Pharmacol. 2002 Dec 5;456(1-3):91-8.

This study investigated the enzymatic and pharmacological properties of a novel phosphodiesterase type 5 inhibitor, methyl 2-(2-methylpyridin-4-yl)methyl-4-(3,4,5-trimethoxyphenyl)-8-(pyrimidin-2-yl)methoxy-1,2-dihydro-1-oxo-2,7-naphthyl-3-carboxylic acid hydrochloride (T-0156), both in vitro and in vivo. The inhibitory effects of T-0156 on six phosphodiesterase isoenzymes isolated from canine tissues were studied. The results showed that T-0156 competitively and specifically inhibited the hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase type 5 at low concentrations (IC50 = 0.23 nM). T-0156 also exhibited strong inhibitory activity against phosphodiesterase type 6, with an IC50 of 56 nM, which was 240 times higher than its inhibitory activity against phosphodiesterase type 5. T-0156 exhibited low inhibitory activity against phosphodiesterases types 1, 2, 3, and 4 (IC50 > 10 μM). In isolated rabbit corpora cavernosa, 10 nM and 100 nM T-0156 increased cGMP levels (100 nM T-0156 treatment group: 6.0 ± 1.5 pmol/mg protein, solvent control group: 1.1 ± 0.4 pmol/mg protein, P < 0.05), leading to tissue relaxation. Concentrations of T-0156 from 1 to 100 nM enhanced the electric field-induced relaxation of isolated rabbit corpora cavernosa in a concentration-dependent manner (100 nM T-0156 treatment group: 76.9 ± 19.8%, solvent control group: 12.3 ± 10.1%, P < 0.05). Intraduodenal administration of 100 to 1000 μg/kg of T-0156 enhanced penile erection induced by pelvic nerve stimulation in anesthetized dogs (1000 μg/kg T-0156 treatment group: 279.0±38.4%, solvent control group: 9.8±4.5%, P<0.05). These results indicate that T-0156 may enhance the nitric oxide (NO)/cGMP pathway under in vitro and in vivo experimental conditions by blocking phosphodiesterase type 5. This study clearly demonstrates that T-0156 is a highly effective and selective phosphodiesterase type 5 inhibitor and an effective tool for in vitro and in vivo pharmacological studies. [1]

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T-0156 is a novel phosphodiesterase type 5 inhibitor recently discovered in our laboratory (Kikkawa et al., 2001). This study aims to investigate the enzymatic and pharmacological properties of T-0156 in vitro and in vivo. In enzyme activity assays using six phosphodiesterase isoenzymes obtained from canine tissue, T-0156 exhibited potent inhibition only against phosphodiesterase type 5 (PDE5), with an IC50 value of 0.23 nM. T-0156's inhibitory potency against PDE5 was 240 times higher than that against PDE6 and 290,000 times higher than that against PDE4. T-0156 showed lower inhibitory potency against PDEs 1, 2, and 3 (IC50 > 100 μM). T-0156's inhibitory potency and selectivity against PDE5 were higher than those of sildenafil (a drug used to treat erectile dysfunction) (Goldstein et al., 1998). Therefore, T-0156 is a highly potent and selective PDE5 inhibitor. The structure of T-0156 is distinctly different from that of cGMP, while the structures of other type 5 phosphodiesterase inhibitors (such as zapristin and sildenafil) are cGMP-related. However, kinetic analysis indicates that T-0156 is a competitive inhibitor, similar to the cases of zapristin (Turko et al., 1998) and sildenafil (Ballard et al., 1998). Residues located in the catalytic domain of type 5 phosphodiesterases, such as Tyr602, His607, His643, and Asp754, have been reported to potentially form important interactions with sildenafil (Turko et al., 1999). Elucidating the type 5 phosphodiesterase binding site of T-0156 will be of great significance. Phosphodiesterase type 5 (PDE5) is the main cGMP hydrolase in the corpus cavernosum of the penis (Boolell et al., 1996), and inhibition of this enzyme leads to increased cGMP levels in this tissue (Jeremy et al., 1997). In this study, T-0156 increased cGMP levels in the rabbit corpus cavernosum without affecting cAMP levels, indicating that T-0156 possesses the pharmacological properties of a specific PDE5 inhibitor. T-0156 also relaxes this tissue. Nitric oxide (NO) has been reported to be released from neurons or arterioles and sinusoidal endothelial cells containing NO synthase (NOS), and can increase intracellular cGMP levels by activating guanylate cyclase, thereby causing relaxation of the smooth muscle of the corpus cavernosum (Trigo-Rocha et al., 1993a). In this study, T-0156 induced cGMP accumulation and caused relaxation in NOS-derived preparations without NO, indicating that the effect of T-0156 depends on pre-existing NO activation of guanylate cyclase, which may originate from penile corpora cavernosa cells. We also examined the effect of T-0156 on field-induced relaxation in isolated rabbit corpora cavernosa, an alternative bioassay for assessing the effects of the NO/cGMP pathway. In our previous study, pretreatment with tetrodotoxin or the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated field-induced relaxation, while excess L-arginine restored L-NAME-inhibited relaxation, suggesting that field-induced relaxation is related to NO produced by NOS-containing neurons (Takagi et al., 2001). T-0156 enhanced field-induced relaxation in a concentration-dependent manner. These results indicate that T-0156 effectively inhibits phosphodiesterase type 5 (PDE5) in vitro and exerts its pharmacological effect by enhancing the NO/cGMP pathway. Furthermore, it was observed that although a concentration of 10 nM T-0156 could inhibit PDE5 activity 100%, its enhancing effect on the NO/cGMP pathway in isolated rabbit penile corpora cavernosa did not reach saturation. One possible explanation for this is that the cell membrane permeability of T-0156 may be low, thus requiring higher concentrations to inhibit PDE5 in isolated tissues. Further research is needed to elucidate the exact mechanisms of these phenomena. Direct electrical stimulation of the pelvic nerve can induce penile erection in dogs, a process mediated by the NO/cGMP pathway (Trigo-Rocha et al., 1993b). This pelvic nerve stimulation-induced erection has been used as a model for the pharmacological evaluation of PDE5 inhibitors. In this model, intraduodenal administration of sildenafil reportedly dose-dependently enhanced intestinal swelling, indicating that sildenafil is an orally effective phosphodiesterase type 5 (PDE5) inhibitor (Noto et al., 2000). A dose-dependent enhancement of intestinal swelling was also observed when T-0156 was administered intraduodenally. These results suggest that T-0156 is an orally effective PDE5 inhibitor in dogs. Although T-0156 is 16 times more potent as a PDE5 inhibitor than sildenafil, its efficacy at a dose of 300 μg/kg is nearly identical to that of sildenafil (Noto et al., 2000). The efficacy of orally or duodenally administered drugs is generally modulated by factors such as bioavailability, serum protein binding, and cell membrane permeability. The comparable in vivo efficacy of T-0156 to sildenafil may be due to the lower bioavailability of T-0156 (approximately 9%; our preliminary data). Since T-0156 takes less time (10 to 30 minutes) to reach its maximum erectile enhancement effect than sildenafil (30 minutes), its absorption rate may be faster than that of sildenafil (Noto et al., 2000). As shown in Table 2, 10 μM of T-0156 had no significant effect on the binding of radioligands to several receptors or on the activity of several enzymes, indicating that its inhibitory selectivity against phosphodiesterase type 5 is more than 40,000 times. The results support the practicality of T-0156 as a selective phosphodiesterase type 5 inhibitor. In conclusion, T-0156 is considered a useful tool in enzymological and pharmacological studies as a potent selective phosphodiesterase type 5 inhibitor. [1]

C31H29N5O7

583.59

324572-92-1

Typically exists as solids at room temperature

779.1ºC at 760 mmHg

425ºC

4.799

CC1=NC=CC(=C1)CN2C(=C(C3=C(C2=O)C(=NC=C3)OCC4=NC=CC=N4)C5=CC(=C(C(=C5)OC)OC)OC)C(=O)OC

methyl 2-[(2-methylpyridin-4-yl)methyl]-1-oxo-8-(pyrimidin-2-ylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylate

t-0156; T0156; methyl 2-[(2-methylpyridin-4-yl)methyl]-1-oxo-8-(pyrimidin-2-ylmethoxy)-4-(3,4,5-trimethoxyphenyl)-1,2-dihydro-2,7-naphthyridine-3-carboxylate; Methyl 2-((2-methylpyridin-4-yl)methyl)-1-oxo-8-(pyrimidin-2-ylmethoxy)-4-(3,4,5-trimethoxyphenyl)-1,2-dihydro-2,7-naphthyridine-3-carboxylate; 324572-92-1; 1,2-Dihydro-2-[(2-methyl-4-pyridinyl)methyl]-1-oxo-8-(2-pyrimidinylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylic acid methyl ester hydrochloride; methyl 2-[(2-methylpyridin-4-yl)methyl]-1-oxo-8-(pyrimidin-2-ylmethoxy)-4-(3,4,5-trimethoxyphenyl)-2,7-naphthyridine-3-carboxylate; CHEMBL540294;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)