选择性PDE5抑制剂TPN729的成药性评价及临床前药效学研究

发布时间：2018-01-22 05:58

本文关键词： 5型磷酸二酯酶抑制剂药效学研究成药性评价　出处：《浙江大学》2014年博士论文　论文类型：学位论文

【摘要】：研究目的：对西地那非进行结构改造所得化合物进行成药性评价,获得具有自主知识产权的新药候选化合物TPN729,并对其进行系统的临床前药效学及相关机制研究。研究方法： 1.TPN729的成药性评价方法： 1)放射性同位素SPA法测定化合物对分离、纯化得到的PDE1-PDE6的抑制活性；2)阴茎海绵体内压(ICP)监测法测试化合物对电刺激海绵体神经诱导的大鼠勃起的影响；3)LC/MS/MS法测试化合物在大鼠及比格犬体内的药代动力学；4). hERG心血管安全性测试,mini Ames遗传毒性测试及小鼠急性毒性实验对化合物进行安全性早期评价。 2.TPN729的临床前药效学研究方法： 1)放射性同位素SPA法测定TPN729MA (TPN729马来酸盐)对重组人PDE1-PDE11的抑制活性；2)离体血管环实验测试TPN729MA对大鼠主动脉血管环平滑肌的舒张作用；3)ICP监测法测试TPN729MA对电刺激海绵体神经诱导的大鼠和比格犬的勃起的影响以及海绵体内注射硝普钠诱导比格犬勃起的影响；4)阴茎长度测量法测量TPN729MA对清醒家兔的勃起的影响；5)右心导管法检测TPN729MA对野百合碱诱导的肺动脉高压大鼠及对低氧诱导的肺动脉高压比格犬的治疗作用。研究结果： 1.TPN729具有良好成药性 1) TPNE01(TPN729)抑制PDE5的IC50为4.21nM,相对于PDE1、2、3、4、6的选择性倍数分别为109、2375、8994、967、51倍,活性及选择性均好于西地那非；TPNE01对电刺激诱导的大鼠勃起的ICP/BP相对值在第60分钟大于西地那非(167.4%vs143.1%)；大鼠及比格犬灌胃给于TPNE0120mg/kg及5mg/kg后生物利用度分别为33.9%及57.6%,暴露量(AUC0-t)分别为739μg/L·h和1547μg/L·h,注射给药的消除半衰期分别为3.76及4.12小时；TPNE01对hERG的IC5010μM, mini Ames实验结果阴性,小鼠经口灌胃1.0g/kg无动物死亡。TPNE01综合成药性最好,作为新药候选化合物,即TPN729(后成盐研究确定采用马来酸为药用形式,即TPN729MA)。 2) TPNE08除急性毒性稍强外,其他性质良好,作为新药备选化合物。 2. TPN729为高选择性PDE5抑制剂,药效作用时间长 1) TPN729MA、西地那非及他达那非对重组人PDE5的IC50分别为2.28,5.22和2.35nM; TPN729MA对PDE1和PDE6的选择性好于西地那非(248vs88倍,20vs8倍),对PDE11的选择性明显好于他达拉非(2671vs5倍),TPN729MA对PDE2、3、7、8、9和10表现出极好的选择性(10,000倍)。2)TPN729MA单用在1μM浓度时可以使血管环的张力恢复到去氧肾上腺素(PE)预处理前的水平,与西地那非作用相当,与硝普钠(SNP)联用0.1nM即可使血管环的张力恢复到PE预处理前的水平,在10-12M时对血管环的舒张作用显著强于西地那非(60.9%vs34.9%)。3) TPN729MA2.5mg/kg可显著增加大鼠的ICP及ICP/BP,相同剂量西地那非只可显著增强ICP/BP;与生理盐水组相比,TPN729MA2.5mg/kg对ICP/BP的增强在给药后75-120分钟具有显著意义,而西地那非(2.5mg/kg)只在75-90分钟内具有显著意义,TPN729AM比西地那非具有更长的作用时间。4) TPN729MA3.75-15.00mg/kg可使电刺激勃起神经引起的比格犬ICP值和ICP/BP值显著增加,TPN729MA15mg/kg对ICP/BP的增强作用与同剂量阳性对照西地那非比较有明显的提高,在45分钟分别为2.04和1.26,但无统计学差异。5)与生理盐水组相比,TPN729MA及西地那非5μg/kg (iv)均可显著增加SNP诱导的大鼠勃起的ICP相对值及ICP/BP相对值,ICP/BP相对值分别为1.93±0.08,1.67±0.17.6) TPN729MA10,30mg/kg及西地那非10mg/kg在给药1小时后注射SNP,均可显著增加清醒家兔阴茎勃起长度及长度-时间曲线下面积(AUC),相同剂量的TPN729MA的AUC比西地那非大36%。7)TPN729MA10、30mg/kg可以显著降低野百合碱造模引起的肺动脉压和平均右心室压升高,缓解肺动脉高压引起的右心肥厚和肺肿大,表现出对肺动脉高压的治疗作用,10mg/kg作用强度与同剂量西地那非相当,平均肺动脉压分别为50.26,50.96mmHg。8) TPN729MA60、120及240μg/kg/min可明显降低低氧引起的比格犬急性肺动脉高压,低氧后8分钟,与给药前相比的降低百分率分别为13.4、11.9、12.2%,西地那非60、120及240μg/kg/min的降低百分率分别为7.0、5.6、10.8%,而生理盐水平均增加百分率为12.2%。结论： 1.通过成药性评价确定了具有良好成药前景的候选化合物TPN729MA. 2. TPN729MA为高选择性PDE5抑制剂,其对PDE1及PDE6的选择性好于西地那非,对PDE11的选择性好于他达那非。 3. TPN729MA通过作用于NO→cGMP通路,抑制cGMP的水解,维持cGMP的高水平发挥扩血管作用而发挥药效。 4. TPN729MA在多种动物体内表现出明显的增强勃起功能以及降低肺动脉高压的作用,其作用强度与西地那非相当或更强,作用时间比西地那非更长。
[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.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R969

【参考文献】