| Objective:To assess and evaluate the druggability of the structure modified derivatives of sildenafil to obtaind drug candidate TPN729with independent intellectual property rights, and conduct pre-clinical pharmacodynamic and related mechanisms study of it.Methods:1. Draggability evaluation of TPN7291) SPA radioimmunoassay was used to test the inhibiton activties of the compounds against PDE1-PDE6isolated and purified from tissues.2) Intracavemous pressure (ICP) monitoring method was used to test the effect on erectile function of the compounds in a rat model of erection induced by electric stimulation.3)LC/MS/MS method was used to test the pharmacokinetics of the compounds in rats and beagle dogs.4) hERG inhibiton test, mini Ames test and acute toxicity test were used to evaluate the safety of the compounds in the early stage.2. Pre-clinical pharmacodynamic study of TPN7291) SPA radioimmunoassay Was used to test the inhibiton activties of TPN729MA (TPN729Maleate) against recombinant human PDE1-PDE11.2) Isolated rat aortic rings was used to test the vasorelaxation effects of TPN729MA.3) ICP monitoring method was used to test the effect on erectile function of TPN729MA in a rat model and a dog model of erection induced by electric stimulation, and in a dog model of erection induced by sodium nitroprusside injection (SNP).4) The length of uncovered penile mucosa was measured to test the effect on erectile function of TPN729MA in a conscious-rabbit model.5) Right heart catheterization was used to test the therapeutic effect of TPN729MA on pulmonary hypertension in a MCT-induced rat model and in a hypoxia-induced dog model.Results:1. TPN729showed good druggability1) TPNE01(TPN729) inhibited PDE5with an IC50value of4.21nM, the selectivity against PDE1,2,3,4and6was109,>2375,8994,967and51folds respectively, showing higher potenty and selectivity than sildenafil. In the rat model of erection, the ICP/BP value of TPNE01wes greater than that of sildenafil (167.4%vs143.1%). After Oral administration of TPNE01in rat(20mg/kg) and in dog(5mg/kg), the bioavailability of TPNE01was33.9%and57.6%respectively, and the AUC0-t was739μg/L·h and1547μg/L·h respectively. The terminal elimination half-life of TPNE01was3.76and4.12h after intravenous administration in rats and in dogs. TPNE01inhibited hERG with an IC50value of more thanlOμM, and showed negtive effect in mini Ames test and a minimum lethal dose of more than1.0g/kg. TPNE01showed the best druggability and was selected as the drug candidate (Research Code:TPN729MA, maleate of TPN729, after salt selection study).2) TPNE08showed good druggability except in the acute toxity study, and was selected as the back-up drug candidate.2. TPN729MA was a high selective PDE5inhibitor with long lasting effect1) The IC50of TPN729MA, sildenafil and tadalafil was2.28,5.22and2.35nM respectively; The selectivity of TPN729MA against PDE1and PDE6was higher than sildenafil (248vs.88-fold and20vs.8-fold); TPN729MA showed excellent selectivity against PDE11compared with tadalafil (2671vs. fivefold). TPN729MA also showed excellent selectivity against PDE2,3,7,8,9, and10(>10,000-fold).2) When added to phenylephrine(PE)-precontracted rat aortic rings, TPN729MA(1μM) and sildenafil (1μM) completely relaxed the PE-precontracted rings; when added together with SNP, TPN729MA completely relaxed the PE-precontracted rings at0.1nM. The relaxation effect of TPN729MA at10-12M was geater than sildenafil(60.9%vs34.9%).3) TPN729MA(2.5mg/kg) showed a significant effect on ICP at120minutes and on ICP/BP between75and120minutes, while sildenafil only showed a significant effect on ICP/BP between75and90minutes.The effect of TPN729MA is longer lasting than sildenafil.4) TPN729MA3.75-15.00mg/kg showed a significant effect on ICP and ICP/BP; the effect of TPN729MA15mg/kg on ICP/BP was greater than sildnafil15mg/kg (2.04vs.1.24,45mins).5) TPN729MA (5.0μg/kg, i.v.) and sildenafil (5.0μg/kg, i.v.) significantly increased ICP and ICP/BP compared with vehicle with ICP/BP value of1.93±0.08and1.67±0.17, respectively.6) When administrated to conscious rabbits, TPN729MA(10,30mg/kg) and sildenafil(10mg/kg) significantly increase the aera of length of uncovered penile mucosa-time curve (AUC), the AUC of TPN729MA(10mg/kg) was36%greater than sildenafil(10mg/kg).7) TPN729MA(10,30mg/kg) significantly decrease the mean pulmonary artery pressure (mPAP) and right ventricular pressure (RVP), relieving the right ventricular hypertrophy and pulmonary swelling cause by MCT-induced pulmonary hypertension, showing the therapeutic effect of pulmonary hypertension. The effect of TPN729MA (10mg/kg) was comparable to sildenafil (10mg/kg) with mPAP50.26and50.96mmHg, respectively.8) TPN729MA (60,120and240μg/kg/min) significantly decrease the mPAP in the hypoxia-induced pulmonary hypertension dog model,8mins after the hypoxia, the percentage decrease of mPAP was13.4,11.9and12.2%, respectively. The percentage decrease of mPAP of sildenafil (60,120and240μg/kg/min) was7.0,5.6, and10.8%, respectively; while the percentage increase of mPAP of natural saline was12.2%.Conclusion:1. TPN729MA was selected as the drug candidate through the druggability assessment and evaluation, and showing a good druggability prospects.2. TPN729MA was a high selective PDE5inhibitor, its selectivity against PDE1and PDE6was higher than sildenafil, and its selectivity against PDE11was higher than tadalafil. 3. TPN729MA exert its effect through the NO-cGMP signaling pathway, it inhibit the hydrolysis of cGMP and maintain high levels of cGMP to play vasodilator effect.4. TPN729MA increased erectile function and decreased PAP in various animal model. The effect of TPN729MA was comparable to or better than sildenafil, but the duration of TPN729MA was longer than sildenafil. |
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