本文關(guān)鍵詞： 5型磷酸二酯酶抑制劑 藥效學(xué)研究 成藥性評價　出處：《浙江大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：研究目的： 對西地那非進行結(jié)構(gòu)改造所得化合物進行成藥性評價,獲得具有自主知識產(chǎn)權(quán)的新藥候選化合物TPN729,并對其進行系統(tǒng)的臨床前藥效學(xué)及相關(guān)機制研究。 研究方法： 1.TPN729的成藥性評價方法： 1)放射性同位素SPA法測定化合物對分離、純化得到的PDE1-PDE6的抑制活性；2)陰莖海綿體內(nèi)壓(ICP)監(jiān)測法測試化合物對電刺激海綿體神經(jīng)誘導(dǎo)的大鼠勃起的影響；3)LC/MS/MS法測試化合物在大鼠及比格犬體內(nèi)的藥代動力學(xué)；4). hERG心血管安全性測試,mini Ames遺傳毒性測試及小鼠急性毒性實驗對化合物進行安全性早期評價。 2.TPN729的臨床前藥效學(xué)研究方法： 1)放射性同位素SPA法測定TPN729MA (TPN729馬來酸鹽)對重組人PDE1-PDE11的抑制活性；2)離體血管環(huán)實驗測試TPN729MA對大鼠主動脈血管環(huán)平滑肌的舒張作用；3)ICP監(jiān)測法測試TPN729MA對電刺激海綿體神經(jīng)誘導(dǎo)的大鼠和比格犬的勃起的影響以及海綿體內(nèi)注射硝普鈉誘導(dǎo)比格犬勃起的影響；4)陰莖長度測量法測量TPN729MA對清醒家兔的勃起的影響；5)右心導(dǎo)管法檢測TPN729MA對野百合堿誘導(dǎo)的肺動脈高壓大鼠及對低氧誘導(dǎo)的肺動脈高壓比格犬的治療作用。 研究結(jié)果： 1.TPN729具有良好成藥性 1) TPNE01(TPN729)抑制PDE5的IC50為4.21nM,相對于PDE1、2、3、4、6的選擇性倍數(shù)分別為109、2375、8994、967、51倍,活性及選擇性均好于西地那非；TPNE01對電刺激誘導(dǎo)的大鼠勃起的ICP/BP相對值在第60分鐘大于西地那非(167.4%vs143.1%)；大鼠及比格犬灌胃給于TPNE0120mg/kg及5mg/kg后生物利用度分別為33.9%及57.6%,暴露量(AUC0-t)分別為739μg/L·h和1547μg/L·h,注射給藥的消除半衰期分別為3.76及4.12小時；TPNE01對hERG的IC5010μM, mini Ames實驗結(jié)果陰性,小鼠經(jīng)口灌胃1.0g/kg無動物死亡。TPNE01綜合成藥性最好,作為新藥候選化合物,即TPN729(后成鹽研究確定采用馬來酸為藥用形式,即TPN729MA)。 2) TPNE08除急性毒性稍強外,其他性質(zhì)良好,作為新藥備選化合物。 2. TPN729為高選擇性PDE5抑制劑,藥效作用時間長 1) TPN729MA、西地那非及他達那非對重組人PDE5的IC50分別為2.28,5.22和2.35nM; TPN729MA對PDE1和PDE6的選擇性好于西地那非(248vs88倍,20vs8倍),對PDE11的選擇性明顯好于他達拉非(2671vs5倍),TPN729MA對PDE2、3、7、8、9和10表現(xiàn)出極好的選擇性(10,000倍)。2)TPN729MA單用在1μM濃度時可以使血管環(huán)的張力恢復(fù)到去氧腎上腺素(PE)預(yù)處理前的水平,與西地那非作用相當,與硝普鈉(SNP)聯(lián)用0.1nM即可使血管環(huán)的張力恢復(fù)到PE預(yù)處理前的水平,在10-12M時對血管環(huán)的舒張作用顯著強于西地那非(60.9%vs34.9%)。3) TPN729MA2.5mg/kg可顯著增加大鼠的ICP及ICP/BP,相同劑量西地那非只可顯著增強ICP/BP;與生理鹽水組相比,TPN729MA2.5mg/kg對ICP/BP的增強在給藥后75-120分鐘具有顯著意義,而西地那非(2.5mg/kg)只在75-90分鐘內(nèi)具有顯著意義,TPN729AM比西地那非具有更長的作用時間。4) TPN729MA3.75-15.00mg/kg可使電刺激勃起神經(jīng)引起的比格犬ICP值和ICP/BP值顯著增加,TPN729MA15mg/kg對ICP/BP的增強作用與同劑量陽性對照西地那非比較有明顯的提高,在45分鐘分別為2.04和1.26,但無統(tǒng)計學(xué)差異。5)與生理鹽水組相比,TPN729MA及西地那非5μg/kg (iv)均可顯著增加SNP誘導(dǎo)的大鼠勃起的ICP相對值及ICP/BP相對值,ICP/BP相對值分別為1.93±0.08,1.67±0.17.6) TPN729MA10,30mg/kg及西地那非10mg/kg在給藥1小時后注射SNP,均可顯著增加清醒家兔陰莖勃起長度及長度-時間曲線下面積(AUC),相同劑量的TPN729MA的AUC比西地那非大36%。7)TPN729MA10、30mg/kg可以顯著降低野百合堿造模引起的肺動脈壓和平均右心室壓升高,緩解肺動脈高壓引起的右心肥厚和肺腫大,表現(xiàn)出對肺動脈高壓的治療作用,10mg/kg作用強度與同劑量西地那非相當,平均肺動脈壓分別為50.26,50.96mmHg。8) TPN729MA60、120及240μg/kg/min可明顯降低低氧引起的比格犬急性肺動脈高壓,低氧后8分鐘,與給藥前相比的降低百分率分別為13.4、11.9、12.2%,西地那非60、120及240μg/kg/min的降低百分率分別為7.0、5.6、10.8%,而生理鹽水平均增加百分率為12.2%。 結(jié)論： 1.通過成藥性評價確定了具有良好成藥前景的候選化合物TPN729MA. 2. TPN729MA為高選擇性PDE5抑制劑,其對PDE1及PDE6的選擇性好于西地那非,對PDE11的選擇性好于他達那非。 3. TPN729MA通過作用于NO→cGMP通路,抑制cGMP的水解,維持cGMP的高水平發(fā)揮擴血管作用而發(fā)揮藥效。 4. TPN729MA在多種動物體內(nèi)表現(xiàn)出明顯的增強勃起功能以及降低肺動脈高壓的作用,其作用強度與西地那非相當或更強,作用時間比西地那非更長。
[Abstract]:The purpose of the study is:
Sildenafil, a new drug candidate compound TPN729 with independent intellectual property rights, was evaluated for its pharmacology evaluation, and its preclinical pharmacodynamics and related mechanisms were studied.
Research methods:
The methods of evaluation of 1.TPN729 in adult medicine:
1) determination of compounds on the separation of radioactive isotopes by SPA, inhibit the activity of the purified PDE1-PDE6; 2) intracavernous pressure (ICP) in rats with erectile monitoring method test compounds on the induced electrical stimulation of the cavernous nerve; 3) LC/MS/MS test compounds in rats and beagle dogs in vivo pharmacokinetics; 4 hERG). Cardiovascular safety testing, mini Ames genetic toxicity test and acute toxicity test of mice safety early evaluation of compounds.
2.TPN729 preclinical pharmacodynamic study methods:
1) determination of TPN729MA radioactive isotope SPA method (TPN729 maleate) inhibitory activity on recombinant human PDE1-PDE11; 2) from the relaxation of aortic rings test TPN729MA on rat aortic smooth muscle; 3) ICP monitoring method test the influence of TPN729MA on electrical stimulation of the cavernous nerve in rats and beagle dogs induced the erection and intracavernous injection of sodium nitroprusside induced canine erection; 4) affect the measurement of TPN729MA penis length measurement on erectile conscious rabbits; 5) rats pulmonary hypertension detection TPN729MA right heart catheterization on monocrotaline induced and on hypoxia induced pulmonary hypertension in beagle dogs.
The results of the study:
1.TPN729 has good drug resistance
1) TPNE01 (TPN729) IC50 inhibited PDE5 4.21nM, compared to the selective multiple of PDE1,2,3,4,6 were 10923758994967,51 times, the activity and selectivity were better than sildenafil; TPNE01 rats induced by electrical stimulation of the erection of the relative value of ICP/BP in sixtieth minutes than sildenafil (167.4%vs143.1%); rats and beagle dogs fed on TPNE0120mg/kg and after 5mg/kg bioavailability were 33.9% and 57.6%, respectively (AUC0-t) exposure to 739 g/L h and 1547 g/L h, injected the elimination half-life was 3.76 and 4.12 hours; TPNE01 hERG IC5010 M, mini Ames negative results, mice by gavage 1.0g/kg the death of the animal.TPNE01 integrated into the best drug, as new drug candidate compounds, namely TPN729 (after the salt of maleic acid was determined by medicinal form, namely TPN729MA).
2) TPNE08 in acute toxicity slightly, other good properties, as a new drug candidate compounds.
2. TPN729 is a high selective PDE5 inhibitor with long time of effect
1) TPN729MA, IC50 and sildenafil tadalafil on recombinant human PDE5 were 2.28,5.22 and 2.35nM; PDE1 and TPN729MA on the selectivity of PDE6 is better than sildenafil (248vs88 times, 20vs8 times), the selectivity for PDE11 was significantly better than that of tadalafil (2671vs5 times), TPN729MA of PDE2,3,7,8,9 and 10 showed excellent selectivity (10000 times).2) TPN729MA alone at 1 M concentration can make the vascular ring tension to phenylephrine (PE) pretreatment level, and sildenafil, and sodium nitroprusside (SNP) recovery combined with 0.1nM can make the blood vessel tension to PE pre-treatment level in 10-12M the effect of relaxation of vascular rings was significantly stronger than sildenafil (60.9%vs34.9%).3) TPN729MA2.5mg/kg significantly increased ICP and ICP/BP rats, the same dose of Sildenafil can significantly increase ICP/BP; compared with the saline group, the TPN729MA2.5mg/kg of ICP/B P increased after the administration of 75-120 minutes is significant, while sildenafil (2.5mg/kg) was significant only in 75-90 minutes,.4 time TPN729AM TPN729MA3.75-15.00mg/kg) has more than Sildenafil can make erectile nerve by electrical stimulation of beagle dogs ICP and ICP/BP values increased significantly, TPN729MA15mg/kg of ICP/BP enhanced with the same dose compared with the positive control of sildenafil significantly improved respectively 2.04 and 1.26 in 45 minutes, but the difference was not statistically significant.5) compared with the normal saline group, TPN729MA and sildenafil 5 g/kg (IV) significantly increased SNP of rats induced by the erection of the relative value of ICP and ICP/BP relative value, relative value of ICP/BP was 1.93. 0.08,1.67 + 0.17.6 TPN729MA10,30mg/kg) and 10mg/kg injection in sildenafil administration 1 hours after SNP, significantly increased in conscious rabbit penile erection length and the length of time. The line area (AUC), AUC 36%.7 than sildenafil TPN729MA with the same dose) TPN729MA10,30mg/kg can significantly reduce the monocrotaline induced pulmonary arterial pressure model and the mean right ventricular pressure, alleviate pulmonary hypertension caused by right ventricular hypertrophy and pulmonary enlargement, showed therapeutic effect on pulmonary arterial pressure, the intensity of 10mg/kg effect with the same dose of sildenafil, mean pulmonary arterial pressure were 50.26,50.96mmHg.8 TPN729MA60120 and 240 g/kg/min) can significantly reduce the canine acute pulmonary hypertension induced by hypoxia, hypoxia after 8 minutes, compared with before administration of the lower percentage of 13.4,11.9,12.2%, sildenafil 60120 and 240 mu g/kg/min reduced the percentage of 7.0,5.6,10.8% respectively, and the physiological the average percentage increase in 12.2%. saline
Conclusion:
1. the candidate compound TPN729MA., which has a promising future for the medicine, was determined by the drug resistance evaluation
2. TPN729MA is a highly selective PDE5 inhibitor, on PDE1 and PDE6 in good selectivity of sildenafil, selectivity to PDE11 is better than that of tadalafil.
3. TPN729MA plays an effective role by acting on the NO - cGMP pathway, inhibiting the hydrolysis of cGMP and maintaining the high level of cGMP to play the role of vasodilator.
4. TPN729MA in a variety of animal in vivo showed significantly enhanced erectile function and reduce pulmonary artery pressure effect, the strength of effect and sildenafil equivalent or stronger, longer duration than sildenafil.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R969

【參考文獻】

相關(guān)期刊論文 前2條

1 陳巧云,王楠,姚峰;磷酸二酯酶5抑制劑治療勃起功能障礙的療效和安全性比較[J];中華男科學(xué);2004年04期

2 宋潔梅;溫小安;孫宏斌;;肺動脈高壓發(fā)生機制及治療藥物的研究進展[J];中國藥科大學(xué)學(xué)報;2013年01期

，

本文編號：1453883