1、Chapter 17 Delivery of BiopharmaceuticalsBiotechnology-derived Products课程要求掌握生物技术药物的概念、特点；熟悉多掌握生物技术药物的概念、特点；熟悉多肽蛋白质类药物注射剂的处方设计、制备肽蛋白质类药物注射剂的处方设计、制备工艺与质量评价。工艺与质量评价。了解多肽蛋白质类药物的其它非注射剂的了解多肽蛋白质类药物的其它非注射剂的类型；了解基因传递系统的概念、类型。类型；了解基因传递系统的概念、类型。Introduction生物技术药物生物技术药物（Biopharmaceuticals,Biotechnology-derived
2、 products）是通是通过细胞融合、重组过细胞融合、重组DNADNA或转基因等技术，利或转基因等技术，利用细菌、酵母、昆虫、植物或哺乳动物细用细菌、酵母、昆虫、植物或哺乳动物细胞等各种表达系统制备而成的，用于预防、胞等各种表达系统制备而成的，用于预防、诊断或治疗疾病的药物。诊断或治疗疾病的药物。Biopharmaceuticals are medical drugs produced using biotechnology.Biotechnology 生物技术生物技术Any technological application that uses biological systems,liv
3、ing organisms,or derivatives,to make or modify products or processes for specific use.Biotechnology is often used to refer to genetic engineering technology of the 21st century.Biopharmaceuticals:current statusBiopharmaceuticals:current statusApproximately one in every three new drugs now coming on
4、the market is a biopharmaceutical.Over 200 biopharmaceutical products had gained marketing approval in the USA and/or EU.All but three are protein-based therapeutic agents.Classification of biopharmaceuticals Blood factors 血液因子血液因子(Factor VIII and Factor IX)Thrombolytic agents 溶栓类溶栓类(tissue plasmino
5、gen activator)Hormones 激素类激素类(insulin,glucagon,growth hormone,gonadotrophins)Haematopoietic growth factors 造血生长因子造血生长因子(Erythropoietin,colony stimulating factors)Interferons 干扰素干扰素(Interferons-,-,-)Interleukin-based products 白介素类白介素类(Interleukin-2)Vaccines 疫苗疫苗(Hepatitis B surface antigen)Monoclonal
6、 antibodies 单克隆抗体单克隆抗体Additional products(tumor necrosis factor,therapeutic enzymes)Comparing Traditional Medical Discovery with BiotechnologyTraditional MedicinesTraditional MedicinesSynthetic small molecules produced through chemical processesIdentified through high-throughput screens of 10,000 ra
7、ndomly generated compoundsFunction predominantly intracellularlyLow specificity for targetsBiopharmaceuticalsBiopharmaceuticalsBiological macromolecules produced by living systems Created from amino acids and nucleic acidsRely on defined biological pathwaysFunction predominantly extracellularlyHave
8、a targeted mechanism of action生物技术药物的特点大多数是蛋白质或多肽类药物大多数是蛋白质或多肽类药物药理活性高，剂量低；药理活性高，剂量低；结构复杂，理化性质不稳定；结构复杂，理化性质不稳定；口服给药易受胃肠道口服给药易受胃肠道pHpH、菌群及酶系统破坏；、菌群及酶系统破坏；Most are administered by injection rather than via the oral route.生物半衰期短，体内清除率高；生物半衰期短，体内清除率高；具有多功能性，作用广泛具有多功能性，作用广泛,免疫原性的问题免疫原性的问题；检测存在诸多困难和不便。检测存
9、在诸多困难和不便。Delivery of BiopharmaceuticalsProtein/peptide deliveryVaccine deliveryDelivery of nucleic acid-based therapeuticsProtein/peptide deliveryProteinsPolypeptides composed of covalently linked amino acidsPolypeptides with 40 amino acids are called proteinsFunction of a protein determined by its
10、non-covalent 3D structureAmino AcidsH3N+OORHPolypeptidesOR3HNHOR4HH3N+OR1HNHOR2HNHOLevels of Protein StructureThe Problem with ProteinsVery large and unstable moleculesStructure is held together by weak noncovalent bonds 非共价键非共价键Easily destroyed by relatively mild storage conditionsHard to obtain in
11、 large quantitiesEasily destroyed/eliminated by the bodyThe Problem with Proteins(in vivo)Elimination清除清除 by B and T cellsProteolysis by peptidases 在肽酶的作用下水解在肽酶的作用下水解Small proteins(30 kD)filtered out by the kidneys very quicklyUnwanted allergic reactions 过敏反应过敏反应 may develop(even toxicity)Loss due t
12、o insolubility/adsorptionThe Problem with Proteins(in vitro-in the bottle)Denaturation 变性变性Aggregation 聚集聚集Precipitation 沉淀沉淀Adsorption 吸附吸附Deamidation 脱酰胺脱酰胺Oxidation 氧化氧化Disulfide exchange 二硫键交二硫键交换换Proteolysis 蛋白水解蛋白水解Noncovalent CovalentHow to Deal with These Problems?StorageFormulationDeliveryP
13、harmaceuticsStorage Refrigeration 冷藏冷藏Low temperature reduces microbial growth and metabolismLow temperature reduces thermal or spontaneous denaturationLow temperature reduces adsorptionFreezing is best for long-term storageFreeze/Thaw can denature proteinsStorage-PackagingSmooth glass walls best to
14、 reduce adsorption or precipitationAvoid polystyrene聚苯乙烯聚苯乙烯 or containers with silanyl硅烷硅烷 or plasticizer coatingsDark,opaque walls reduce oxidationAir-tight containers or argon atmosphere reduces air oxidationStorage-AdditivesAddition of stabilizing salts or ions(Zn+for insulin)Addition of polyols
15、(glycerol and/or polyethylene glycol)to solubilizeAddition of sugars or dextran to displace water or reduce microbe growthUse of surfactants(CHAPS)to reduce adsorption and aggregationFreeze DryingFreeze liquid sample in containerPlace under strong vacuumSolvent sublimates leaving only solid or nonvo
16、latile compoundsReduces moisture content to 0.1%Protein PharmaceuticsStorageFormulationDeliveryProtein FormulationProtein sequence modification 蛋白序列的蛋白序列的修饰修饰(site directed mutagenisis 点突变点突变)PEGylation PEGPEG化化Proteinylation 蛋白质化蛋白质化Microsphere/Nanosphere encapsulation 微球或纳米球包裹微球或纳米球包裹Site Directed
17、 MutagenesisAllows amino acid substitutions at specific sites in a proteini.e.substituting a Methionine甲硫氨酸甲硫氨酸 to a Leucine亮氨酸亮氨酸 will reduce likelihood of oxidationStrategic placement of cysteines半胱氨半胱氨酸酸 to produce disulfidesPEGylation+CH-CH-CH-CH-CH-CH-CH-CH-CH-CH|OH OH OH OH OH OH OH OH OH OHPE
18、GylationPEG is a non-toxic,hydrophilic,FDA approved,uncharged polymerIncreases in vivo half life(4-400X)Decreases immunogenicity 降低免疫原性降低免疫原性Improved stability towards proteolysis Increase solubility of the protein in aqueous solutionExamples of Marketed PEGylated Proteins and PeptidesProteinylation
19、Attachment of additional or secondary(nonimmunogenic)proteins for in vivo protectionIncreases in vivo half life(10X)Cross-linking with Serum AlbuminCross-linking or connecting by protein engineering with antibody fragmentsMicrosphere Encapsulation100 m mmProtein PharmaceuticsStorageFormulationDelive
20、ryRoutes of DeliveryParenteral(injection)注射给药注射给药 Route of delivery for 95%of proteinsNon-parenteral Oral Nasal delivery Pulmonary Transdermal routeParenteral DeliveryIntravenous 静脉注射静脉注射Intramuscular 肌肉注射肌肉注射Subcutaneous 皮下注射皮下注射Intradermal 皮内注射皮内注射Non-invasive routesLow bioavailabilityHigh costora
21、lnasaltransdermalpulmonaryProperties of oral peptide drugsProperties of oral peptide drugsDDAVP:Desmopressin acetate(醋酸去氨加压素）Delivery of nucleic acid-based therapeutics核酸药物传递系统核酸药物传递系统Nucleic acidbased therapeuticsGene therapyAntisense technology 反义技术反义技术RNA interference RNA干扰干扰Gene Therapy To treat
22、 or prevent diseases by introducing functional genes into the body 将限定的遗传物质转入患者的靶细胞，达到预防将限定的遗传物质转入患者的靶细胞，达到预防或改变特殊疾病状态的治疗方法。或改变特殊疾病状态的治疗方法。Therapeutic EffectMethods for gene introductionMethods for gene introduction 导入基因的方式导入基因的方式ex vivo(out of the body)离体法transduced somatic cellstransduced hematopo
23、ietic cellsin vivo(whole body)体内法direct,local administration systemic administrationEx Vivo Gene TherapyTransfected cell in culture(Integrating vector)Transplant modified cells back into patientIsolate cellsIn Vivo Gene Therapy Gene delivery systemsGene delivery systemsViral systems Viral systems 病毒载体病毒载体 Adenoviruses Retroviruses Adeno-associated VirusNonviral systems Nonviral systems 非病毒载体非病毒载体 Naked DNA Lipid based systems Polymers
