10 years EU regulation of pediatric medicines – impact on cardiovascular drug formulations

Introduction: Child-appropriate drug formulations are mandatory for an efficient and safe drug therapy in children. Since the implementation of supportive legislations development of novel drug formulations has significantly been enforced despite the fact that children are a heterogeneous group of patients with varying needs according to age, maturation and disease.

Areas covered: In this review, recent advances and current strategies are evaluated how to overcome the specific hurdles in pediatric drug development. For cardiovascular diseases as an example, EMA’s decisions on pediatric investigation plans (PIPs) have been evaluated. New developments with innovative platform technologies such as mini-tablets and orodispersible preparations have been identified indicating a clear shift from liquid preparations to small-sized solid (multiparticulate) or orodispersible dosage forms. Reasons for this shift of paradigm are discussed.

Expert opinion: Innovative platform technologies for solid drug dosage forms such as mini-tablets, orodispersible tablets or film preparations will continue to conquer the pharmaceutical market. Still, there are some major issues to be resolved, e.g. how to ensure quality of the new dosage forms and dose accuracy in flexible dosing, but the governmental incentives will continue to accelerate development of pediatric medicines and will bridge the still existing gaps in the near future.     

PUMA、caspase-3蛋白在胃癌组织中的表达及其临床意义

目的通过检测PUMA、caspase-3蛋白在胃癌组织和癌旁正常组织中的表达水平,从而探讨两者在胃癌发生、发展中的作用及其临床意义。方法针对40例胃癌组织标本及30例癌旁正常组织标本,通过免疫组化方法对其PUMA、caspase-3蛋白的表达进行测定。结果 PUMA蛋白在胃癌组织中的表达阳性率为22.5%(9/40),在癌旁正常胃组织中表达阳性率为66.7%(20/30),两者比较差异具有统计学意义;caspase-3在胃癌组织中的表达阳性率为25.0%(10/40),在癌旁正常胃组织中表达阳性率为70.0%(21/30),两者比较差异具有统计学意义。PUMA的表达与临床病理分期、肿瘤病理分化程度、是否伴有淋巴结转移有关,PUMA与caspase-3蛋白在胃癌组织中表达呈正相关。结论 PUMA与caspase-3蛋白在胃癌的发生发展中起重要作用,可能会成为胃癌的分子标记物或胃癌治疗的新分子靶点。     

PUMA、Bcl-2在喉鳞状细胞癌组织中的表达及临床意义

目的观察喉鳞状细胞癌组织中PUMA、Bcl-2的表达,探讨其与喉癌发生发展及预后的关系。方法运用免疫组化sP法检测48例喉鳞癌组织及30例癌旁组织中PUMA、Bcl-2蛋白的表达,并分析二者的表达与临床病理参数的关系。结果PUMA和Bcl-2在喉癌组织中的表达阳性率分别为62．5％、43．8％,在喉鳞癌组织中的表达较喉癌旁组织低（P〈0．05）;PUMA的表达与肿瘤的组织学分化程度、有无颈淋巴结转移及术后生存期显著相关（P均〈0．05）,Bcl-2的表达与肿瘤的组织学分化程度、TNM分期及术后生存期显著相关（P〈0．05）;PUMA蛋白阳性组与PUMA蛋白阴性组的Bcl-2蛋白阳性率有统计学差异（P〈0．05）。结论PUMA、Bcl-2在细胞凋亡及肿瘤发生中起重要作用,对喉鳞癌的早期诊断和预后判断有重要意义。     

促凋亡蛋白PUMA对小鼠肝细胞凋亡的影响及其分子机制

目的：通过腺病毒介导puma在小鼠肝细胞BNL.CL2内过表达,观察其对周期和凋亡的影响及其分子机制.方法：用HEK293 low passage（LP）细胞包装制备重组PUMA腺病毒（Ad-puma）和GFP（Ad-GFP）腺病毒,PCR,Western Blot检测鉴定包装的腺病毒.腺病毒感染BNL.CL2细胞后,流式细胞仪检测感染Ad-PUMA后的BNL.CL2细胞的周期和凋亡.免疫共沉淀实验分析BCL-2家族成员间在PUMA介导的凋亡中的相互作用.结果：①用HEK293包装制备了目的细胞中有效高表达Ad-PUMA和Ad-GFP浓缩病毒存储液.所制备的病毒浓缩液Ad-PUMA的滴度为2.0×1011ifu/L;Ad-GFP滴度为2.1×1012ifu/L.②Ad-PUMA感染BNL CL.2细胞后24,36 h与对照组相比细胞凋亡增加;36 h S期细胞明显增多细胞周期中的S期的细胞则明显增多,G2期细胞相对减少.③小鼠肝细胞中PUMA介导的凋亡不仅可能与PUMA,Bax与Bcl-XL的竞争性结合作用有关,也存在PUMA与Bax的直接激活作用.结论：PUMA可有效地促进鼠肝细胞BNL.CL2的凋亡.可能Ad-PUMA介导的PUMA过表达后使细胞从S期到G2期时发生停留或阻滞.在BNL.CL2细胞中,PUMA促凋亡的分子机制既与PUMA,Bax与Bcl-XL的竞争性结合作用有关,也存在PUMA与Bax的直接相互作用.     

PFT-α对脑缺血后神经干细胞移植中PUMA的调控研究

目的探索p53抑制剂(PET-α)对脑缺血后神经干细胞(NSCs)中p53下游促凋亡基因(PuMA)的表达影响。方法利用SD仔鼠分离NSCs,原代及传代培养,经氧糖剥夺(OGD)处理6 h后分为OGD+二甲基亚砜(DMSO)组和OGD+PFT-α组,采用免疫印迹技术比较2组p53蛋白及PUMA的表达变化。体内实验中采用Longa法建立经CT证实的大脑中动脉栓塞模型大鼠48只,随机分为PFT-α+NSCs移植组(n=24)和DMSO+NSCs移植组(n=24);采用免疫荧光技术检测不同移植组中PUMA的表达。结果 (1)体外实验中氧糖剥夺6 h后OGD+PFT-α组中p53蛋白入核水平下调,胞浆内表达上调,而OGD+DMSO组主要表达位于胞核;PUMA的表达水平明显低于OGD+DMSO组(P<0.05);(2)体内实验中,与DMSO+NSCs组相比,PIT-α+NSCs移植组中PUMA表达阳性的细胞明显减少[(38.9±4.3)%vs.(29.4±5.6)%,P<0.01]。结论 PFT-α与神经干细胞联合应用后,可明显抑制PUMA的表达,这可能是PFT-α促进移植后神经干细胞在脑缺血区存活的重要机制。     

Phosphorylation of Tip60 by p38α regulates p53-mediated PUMA induction and apoptosis in response to DNA damage

Tip60 is a multifunctional acetyltransferase involved in multiple cellular functions. Acetylation of p53 at K120 by Tip60 promotes p53-mediated apoptosis after DNA damage. We previous showed that Tip60 activity is induced by phosphorylation at T158 by p38. In this study, we investigated the role of p38-mediated Tip60 phosphorylation in p53-mediated, DNA damage-induced apoptosis. We found that DNA damage induces p38 activation, Tip60-T158 phosphorylation, and p53-K120 acetylation with similar kinetics. p38α is essential for DNA damage-induced Tip60-T158 phosphorylation. In addition, both p38α and Tip60 are essential for p53-K120 acetylation, binding of p53 to PUMA promoter, PUMA expression and apoptosis induced by DNA damage. Moreover, DNA damage induces protein kinase activity of p38α towards Tip60-T158, and constitutive activation of p38 in cells leads to increases in Tip60-T158 phosphorylation, p53-K120 acetylation, PUMA expression and apoptosis. Furthermore, the Tip60-T158A mutant that cannot be phosphorylated by p38 fails to mediate p53-K120 acetylation, PUMA induction, and apoptosis following DNA damage. These results establish that Tip60-T158 phosphorylation by p38 plays an essential role in stimulating Tip60 activity required for inducing the p53-PUMA pathway that ultimately leads to apoptosis in response to DNA damage, which provides a mechanistic basis for the tumor-suppressing function of p38 and Tip60.     

TRPC通道调节PUMA的表达参与NO诱导海马神经元凋亡的研究

目的探讨TRPC离子通道通过p53通路参与一氧化氮供体硝普钠（SNP）诱导的原代培养海马神经元凋亡。方法原代培养的海马神经元,经RT-PCR检测TRPC1-6的表达;给予SNP处理后,通过Real-time RT-PCR检测p53下游靶基因Bax、Apaf-1、PUMA和Survivin的表达;给予TRPC通道阻断剂和激活剂,检测其是否影响SNP诱导的p53下游靶基因表达。结果 TRPC在原代培养海马神经元上广泛表达。SNP处理神经元4、8、24h后,p53下游靶基因Bax、Apaf-1和Survivin表达无明显变化（P〉0.05）。而PUMA则在3个时间点分别增加至1.94、1.86和1.90倍,与对照组比较差异有统计学意义（P〈0.01）。TRPC通道阻断剂2-APB,可降低SNP诱导的PUMA上调（1.91vs1.12）（P〈0.01）。结论 TRPC通道可以通过调节PUMA的表达参与SNP诱导的原代培养海马神经元凋亡。     

黄芩苷对肝星状细胞iNOS/PUMA信号通路的影响

目的研究黄芩苷通过iNOS/PUMA信号通路促进肝星状细胞凋亡的作用。方法MTT检测细胞增殖抑制率,Western Blot检测iNOS、PUMA蛋白表达,real-time PCR检测PUMA mRNA表达,一氧化氮检测试剂盒检测NO含量,iNOS抑制剂L-NAME预处理后检测细胞增殖抑制率和PUMA表达,Hoechst 33342核染色检测细胞凋亡。结果50 mM黄芩苷引起肝星状细胞活化诱导性死亡;黄芩苷促进肝星状细胞PUMA mRNA、蛋白表达;iNOS抑制剂L-NAME预处理后PUMA表达降低、细胞凋亡减少。结论黄芩苷能通过iNOS/PUMA信号通路促进肝星状细胞凋亡。