蛋白质--多肽类药物传递的研究进展

RecentAdvancesinR/DProteinandPeptideDrugDelivery蛋白质/多肽类药物传递的研究进展一.背景二.蛋白质/肽类药物传递存在的问题三.新型蛋白质/肽类药物与制剂的研发四.蛋白质/肽类药物进入人体的新给药途径人类基因组计划(HumanGenomeProject)•1990.10----2003.3•美国,英国,法国,德国,中国,日本•完成人类基因组DNA的30亿个碱基对(bases)的测序•中国承担1%人类基因测序任务(“中国卷”)•人类第一次在分子水平上全面地认识自我人类生命科学研究进入一个崭新的时代---------后基因组时代(post-genomeera)二十一世纪的生物医药领域将发生革命性的变化以蛋白质/肽为基础的创新药物------------基因治疗药物基因工程药物基因工程疫苗(genevaccine,DNAvaccine)反义寡核酸药物将被迅速地研究开发FDA已批准417个protein/peptide药物/疫苗干扰素Interferon白细胞介素Interleukin-2生长因子KGF集落刺激因子GM-CSF凝血因子BeneFIX重组胰岛素Humulin重组人生长激素rhFSH单克隆抗体Zenapax多克隆抗体Thymoglobulin基因工程药物的化学本质是蛋白质或肽蛋白质和肽ProteinandPeptideProteinsandPeptide蛋白质和肽•Polypeptidescomposedofcovalentlylinkedaminoacids肽由氨基酸以共价键结合而成•Polypeptideswith40aminoacidsarecalledpeptides40个氨基酸：肽（多肽）•Polypeptideswith40aminoacidsarecalledproteins40个氨基酸：蛋白质•Functionofaproteindeterminedbyitsnon-covalent3Dstructure蛋白质的功能：三维结构AminoAcidsH3N+OORH氨基羧基侧链PolypeptidesOR3HNHOR4HH3N+OR1HNHOR2HNHO40aminoacid肽键肽键ProteinStructureFoldβMixedα/βHelixαα/β混合型β折叠型免疫球蛋白螺旋型血红蛋白ClassesofProtein/PolypeptidePharmacueticals•Vaccines疫苗(peptides,partsofproteins,killedbacteria,etc.)•Polypeptides多肽(oxytocin脑垂体激素,pitocin催产素)•Bloodproducts(FactorX,FactorVIII凝血因子,基因疗治血友病,gammaglobulin,serumalbumin)•Recombinanttherapeuticproteins重组治疗蛋白质(herceptin单克隆抗体,humulin重组胰岛素,alferon干扰素,etc.)蛋白质/多肽类药物分类Vaccines疫苗•Diptheria白喉杆菌(Corynebacteriumdiphtheriae)-diptheriatoxin•Tetanus破伤风(Clostridiumtetani)-tetanustoxin•Whoopingcough百日咳(Bordetellapertussis,)-acullelarextractTetanusToxin破伤风毒素片段β折叠型TherapeuticProtein/Peptide•Insulin(diabetes)胰岛素(糖尿病51aminoacid)•Interferonb干扰素b(relapsingMS,多发性硬化症)•Interferong干扰素g(granulomatous,肉芽瘤)•TPA组织多肽抗原Activase(heartattack,心脏病发作)TherapeuticProtein/Peptide•Actimmune(Ifg干扰素)•Activase(TPA,组织多肽抗原)•BeneFix(FIX,凝血因子IX,血友病B型治疗)•Betaseron(Ifb,干扰素,多发性硬化症缓和期,复发期)•Humulin重组胰岛素•Novolin重组胰岛素•Regranex(PDGF,血小板衍生生长因子)•Novoseven(FVIIa重组凝血因子)•Epogen红细胞生成素•Intron-A(干扰素,毛细胞白血病,生殖器疣,乙肝,丙肝,AIDS)•Neupogen白细胞介素•Pulmozyme重组脱氧核糖核酸酶•Infergen干扰素,慢性丙肝WhatisGeneDelivery?什么是基因传递?TodeliverDNAplasmid传递质粒intocellstoarisetherapeuticeffectssuchasinduction诱导enhancement促进（抗体的生成）orblockingofproteinexpression阻碍蛋白质基因表达（阻断复制）WhyisaGeneCarrierEssentialforGeneDelivery?为什么基因载体对基因传递是重要的?DNAitselfcannotdiffuse扩散throughcellmembranebecauseofitsnegativecharge负电荷cellmembranealsohasnegativechargeNaked无保护的DNAisrapidlydegradedbyDNaseinbloodorcellularlysosome血或细胞中的溶酶体TheProblemwithProtein/peptide蛋白质/肽存在的问题TheProblemwithProtein/peptide(1)(invivo-inthebody)•Verylargeandunstablemolecules分子大,不稳定•Easilydestroyed破坏/eliminated消除bythebody•Easilydestroyed破坏byrelativelymildstorageconditions不严格的储藏条件•Hardtoobtaininlargequantities大量制备困难TheProblemwithProtein/Peptide(2)(invivo-inthebody)•Elimination消除byBandTcells•Proteolysis蛋白质水介byendo/exopeptidases肽酶•Smallproteins(30kD)filteredoutbythekidneys肾veryquickly•Unwantedallergicreactions过敏反应maydevelop(eventoxicity)TheProblemwithProtein/Peptide(3)(invitro-inthebottle瓶子)•Denaturation变性•Aggregation聚合•Precipitation沉淀•Adsorption吸附•Deamidation脱酰胺•Oxidation氧化•Disulfideexchange二硫化物交换•Proteolysis蛋白质水介Noncovalent非共价的Covalent共价的Protein/PeptideNewFormulation•Proteinsequence序列modification(sitedirectedmutagenisis诱变)解决稳定性问题•PEGylationPEG化延长T1/2•Proteinylation蛋白质化延长T1/2靶向•Microsphere微球/Nanosphereencapsulation微囊•Micelles胶束控制释放靶向•Formulatingwithpermeabilizers渗透剂提高生物利用度新型蛋白质/肽类药物与制剂的研发SiteDirectedMutagenesis诱变•Allowsaminoacidsubstitutions氨基酸取代•atspecificsitesinaprotein•Proteinengineering蛋白质工程(size,shape,etc.)SiteDirectedMutagenesis诱变E343H亚铁螯合酶在特殊部位用半胱氨酸取代(置换)形成二硫化物提高稳定性PEGylationPEG化•PEGisanon-toxic,hydrophilic亲水性FDAapproved,unchargedpolymer没有负荷的聚合物•Increasesinvivohalflife(4-400X)•肺递释PEG–GCSF(粒细胞集落刺激因子：化疗，血液病)生物利用度3.0%→11.5%•Decreasesimmunogenicity降低免疫性•Increasesproteaseresistance增加对蛋白酶的抵抗•Increasessolubility&stability提高溶解度和稳定性聚乙二醇(PEG)分子中一个或多个分子与蛋白质或肽发生化学结合Peptide-PEGmonomers单体PeptidePEGHydrophobicblock亲脂性区Hydrophilicblock亲水性区OR3HNHOR4HH3N+OR1HNHOR2HNHOCH-CH-CH-CH-CH-CH-CH-CH-CH-CH||||||||||OHOHOHOHOHOHOHOHOHOH聚乙二醇(PEG)分子中一个或多个分子与蛋白质或肽发生化学结合→长循环PEGylation(PEG化)+CH-CH-CH-CH-CH-CH-CH-CH-CH-CH||||||||||OHOHOHOHOHOHOHOHOHOH聚乙二醇(PEG)分子中一个或多个分子与蛋白质或肽发生化学结合→长循环Mixedα/βPulmonaryDeliveryPEG-GCSFCompoundBioavailabilityGCSF(18.8kDa)3.0%PEG-G-CSF(81.5kDa)11.5%PEG-G-CSF(146.8kDa)3.4%PEG-GCSF粒细胞集落刺激因子肺部传递PEGBiopharmaceuticalsADAGENONCASPARPEG-INTRON干扰素PEGASYS干扰素NEULASTPEGVISOMANTMACUGENCDP870CDP860CERAProteinylation蛋白质化•Attachment配置ofadditionalorsecondary(nonimmunogenic不会产生免疫性的)proteinforinvivoprotection体内保护increasesinvivohalflife(10X)延长T1/2•Cross-linkingwithSerumAlbumin血清白蛋白•Cross-linkingorconnectingbyproteinengineering蛋白质工程withantibodyfragments抗体片段Proteinylation蛋白质化+ProteinDrugScFv(antibody)主动靶向PeptideSynthesis激活未保护的肽肽键MicrosphereEncapsulation微囊提高稳定性控制释放速度Encapsulation•Processinvolvesencapsulatingprotein蛋白质•orpeptide肽drugsinsmallporousparticles多孔性•粒子forprotectionfrom“insults”避免损害andforsustainedrelease缓释•Twotypesofmicrospheres–Nonbiodegradable非生物可降介–Biodegradable生物可降介PLGA(polylactic-co-glycolicacid)MicrosphereRelease微球的释放•Hydrophilic亲水(i.e.gelatin)–